ERK phosphorylation is predictive of resistance to IGF-1R inhibition in Small Cell Lung Cancer

PubMed Central

Zinn, Rebekah L.; Gardner, Eric E.; Marchionni, Luigi; Murphy, Sara C.; Dobromilskaya, Irina; Hann, Christine L.; Rudin, Charles M.

2013-01-01

New therapies are critically needed to improve the outcome for patients with small cell lung cancer (SCLC). IGF-1R inhibition is a potential treatment strategy for SCLC: the IGF-1R pathway is commonly upregulated in SCLC, and has been associated with inhibition of apoptosis and stimulation of proliferation through downstream signaling pathways including PI3K-Akt and MAPK. To evaluate potential determinants of response to IGF-1R inhibition, we assessed the relative sensitivity of 19 SCLC cell lines to OSI-906, a small molecule inhibitor of IGF-1R and the closely related insulin receptor (IR). Approximately one third of these cell lines were sensitive to OSI-906, with an IC50 < 1 μM. Cell line expression of IGF-1R, IR, IGF-1, IGF-2, IGFBP3, and IGFBP6 did not correlate with sensitivity to OSI-906. Interestingly, OSI-906 sensitive lines expressed significantly lower levels of baseline phospho-ERK relative to resistant lines (p=0.006). OSI-906 treatment resulted in dose-dependent inhibition of phospho-IGF-1R and phospho-Akt in both sensitive and resistant cell lines, but induced apoptosis and cell cycle arrest only in sensitive lines. We tested the in vivo efficacy of OSI-906 using an NCI-H187 xenograft model and two SCLC patient xenografts in mice. OSI-906 treatment resulted in 50% tumor growth inhibition in NCI-H187 and 30% inhibition in the primary patient xenograft models compared to mock treated animals. Taken together our data support IGF-1R inhibition as a viable treatment strategy for a defined subset of SCLC and suggest that low pretreatment levels of phospho-ERK may be indicative of sensitivity to this therapeutic approach. PMID:23515613

Frequent biphasic cellular responses of permanent fish cell cultures to deoxynivalenol (DON)

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

Pietsch, Constanze, E-mail: constanze.pietsch@unibas.ch; Bucheli, Thomas D.; Wettstein, Felix E.

Contamination of animal feed with mycotoxins is a major problem for fish feed mainly due to usage of contaminated ingredients for production and inappropriate storage of feed. The use of cereals for fish food production further increases the risk of a potential contamination. Potential contaminants include the mycotoxin deoxynivalenol (DON) which is synthesized by globally distributed fungi of the genus Fusarium. The toxicity of DON is well recognized in mammals. In this study, we confirm cytotoxic effects of DON in established permanent fish cell lines. We demonstrate that DON is capable of influencing the metabolic activity and cell viability inmore » fish cells as determined by different assays to indicate possible cellular targets of this toxin. Evaluation of cell viability by measurement of membrane integrity, mitochondrial activity and lysosomal function after 24 h of exposure of fish cell lines to DON at a concentration range of 0-3000 ng ml{sup -1} shows a biphasic effect on cells although differences in sensitivity occur. The cell lines derived from rainbow trout are particularly sensitive to DON. The focus of this study lies, furthermore, on the effects of DON at different concentrations on production of reactive oxygen species (ROS) in the different fish cell lines. The results show that DON mainly reduces ROS production in all cell lines that were used. Thus, our comparative investigations reveal that the fish cell lines show distinct species-related endpoint sensitivities that also depend on the type of tissue from which the cells were derived and the severity of exposure. - Highlights: > DON uptake by cells is not extensive. > All fish cell lines are sensitive to DON. > DON is most cytotoxic to rainbow trout cells. > Biphasic cellular responses were frequently observed. > Our results are similar to studies on mammalian cell lines.« less

Optimization of a cAMP response element signal pathway reporter system.

PubMed

Shan, Qiang; Storm, Daniel R

2010-08-15

A sensitive cAMP response element (CRE) reporter system is essential for studying the cAMP/protein kinase A/cAMP response element binding protein signal pathway. Here we have tested a few CRE promoters and found one with high sensitivity to external stimuli. Using this optimal CRE promoter and the enhanced green fluorescent protein as the reporter, we have established a CRE reporter cell line. This cell line can be used to study the signal pathway by fluorescent microscope, fluorescence-activated cell analysis and luciferase assay. This cell line's sensitivity to forskolin, using the technique of fluorescence-activated cell sorting, was increased to approximately seven times that of its parental HEK 293 cell line, which is currently the most commonly used cell line in the field for the signal pathway study. Therefore, this newly created cell line is potentially useful for studying the signal pathway's modulators, which generally have weaker effect than its mediators. Our research has also established a general procedure for optimizing transcription-based reporter cell lines, which might be useful in performing the same task when studying many other transcription-based signal pathways. (c) 2010 Elsevier B.V. All rights reserved.

Calcium influences sensitivity to growth inhibition induced by a cell surface sialoglycopeptide

NASA Technical Reports Server (NTRS)

Betz, N. A.; Fattaey, H. K.; Johnson, T. C.; Spooner, B. S. (Principal Investigator)

1994-01-01

While studies concerning mitogenic factors have been an important area of research for many years, much less is understood about the mechanisms of action of cell surface growth inhibitors. We have purified an 18 kDa cell surface sialoglycopeptide growth inhibitor (CeReS-18) which can reversibly inhibit the proliferation of diverse cell types. The studies discussed in this article show that three mouse keratinocyte cell lines exhibit sixty-fold greater sensitivity than other fibroblasts and epithelial-like cells to CeReS-18-induced growth inhibition. Growth inhibition induced by CeReS-18 treatment is a reversible process, and the three mouse keratinocyte cell lines exhibited either single or multiple cell cycle arrest points, although a predominantly G0/G1 cell cycle arrest point was exhibited in Swiss 3T3 fibroblasts. The sensitivity of the mouse keratinocyte cell lines to CeReS-18-induced growth inhibition was not affected by the degree of tumorigenic progression in the cell lines and was not due to differences in CeReS-18 binding affinity or number of cell surface receptors per cell. However, the sensitivity of both murine fibroblasts and keratinocytes could be altered by changing the extracellular calcium concentration, such that increased extracellular calcium concentrations resulted in decreased sensitivity to CeReS-18-induced proliferation inhibition. Thus the increased sensitivity of the murine keratinocyte cell lines to CeReS-18 could be ascribed to the low calcium concentration used in their propagation. Studies are currently under way investigating the role of calcium in CeReS-18-induced growth arrest. The CeReS-18 may serve as a very useful tool to study negative growth control and the signal transduction events associated with cell cycling.

Selenium Nanoparticles Induce the Chemo-Sensitivity of Fluorouracil Nanoparticles in Breast and Colon Cancer Cells.

PubMed

Abd-Rabou, Ahmed A; Shalby, Aziza B; Ahmed, Hanaa H

2018-05-11

Drug resistance is a major challenge of breast and colon cancer therapies leading to treatment failure. The main objective of the current study is to investigate whether selenium nanoparticles (nano-Se) can induce the chemo-sensitivity of 5-fluorouracil (FU)-encapsulated poly (D, L-lactide-co-glycolide) nanoparticles (nano-FU) in breast and colon cancer cell lines. Nano-Se and nano-FU were synthesized and characterized, then applied individually or in combination upon MCF7, MDA-MB-231, HCT 116, and Caco-2 cancerous cell lines. Cytotoxicity, cellular glucose uptake, and apoptosis, as well as malondialdehyde (MDA), nitric oxide (NO), and zinc (Zn) levels, were investigated upon the different treatments. We have resulted that nano-FU induced cell death in MCF7 and Caco-2 more effectively than MDA-MB-231 and HCT 116 cell lines. Moreover, nano-FU plus nano-Se potentiate MCF7 and Caco-2 chemo-sensitivity were higher than MDA-MB-231 and HCT 116 cancerous cell lines. It is relevant to note that Se and FU nano-formulations inhibited cancer cell bioenergetics via glucose uptake slight blockage. Furthermore, nano-FU increased the levels of NO and MDA in media over cancer cells, while their combinations with nano-Se rebalance the redox status with Zn increment. We noticed that MCF7 cell line is sensitive, while MDA-MB-231 cell line is resistant to Se and nano-Se. This novel approach could be of great potential to enhance the chemo-sensitivity in breast and colon cancer cells.

Cell Penetrating Capacity and Internalization Mechanisms Used by the Synthetic Peptide CIGB-552 and Its Relationship with Tumor Cell Line Sensitivity.

PubMed

Astrada, Soledad; Fernández Massó, Julio Raúl; Vallespí, Maribel G; Bollati-Fogolín, Mariela

2018-03-30

CIGB-552 is a twenty-amino-acid novel synthetic peptide that has proven to be effective in reducing tumor size and increasing lifespan in tumor-bearing mice. Such capability is conferred by its cell-penetrating peptide character, which allows it to enter cells and elicit a pro-apoptotic effect through its major mediator, COMMD1 protein. Cell-penetrating peptides are able to use different internalization mechanisms, such as endocytosis or direct transduction through the plasma membrane. Although CIGB-552 cytotoxicity has been evaluated in several non-tumor- and tumor-derived cell lines, no data regarding the relationship between cell line sensitivity, cell penetrating capacity, the internalization mechanisms involved, COMMD1 expression levels, or its subcellular localization has yet been produced. Here, we present the results obtained from a comparative analysis of CIGB-552 sensitivity, internalization capacity and the mechanisms involved in three human tumor-derived cell lines from different origins: mammary gland, colon and lung (MCF-7, HT-29 and H460, respectively). Furthermore, cell surface markers relevant for internalization processes such as phosphatidylserine, as well as CIGB-552 target COMMD1 expression/localization, were also evaluated. We found that both endocytosis and transduction are involved in CIGB-552 internalization in the three cell lines evaluated. However, CIGB-552 incorporation efficiency and contribution of each mechanism is cell-line dependent. Finally, sensitivity was directly correlated with high internalization capacity in those cell lines where endocytosis had a major contribution on CIGB-552 internalization.

Pharmacogenomic agreement between two cancer cell line data sets.

PubMed

2015-12-03

Large cancer cell line collections broadly capture the genomic diversity of human cancers and provide valuable insight into anti-cancer drug response. Here we show substantial agreement and biological consilience between drug sensitivity measurements and their associated genomic predictors from two publicly available large-scale pharmacogenomics resources: The Cancer Cell Line Encyclopedia and the Genomics of Drug Sensitivity in Cancer databases.

The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity.

PubMed

Barretina, Jordi; Caponigro, Giordano; Stransky, Nicolas; Venkatesan, Kavitha; Margolin, Adam A; Kim, Sungjoon; Wilson, Christopher J; Lehár, Joseph; Kryukov, Gregory V; Sonkin, Dmitriy; Reddy, Anupama; Liu, Manway; Murray, Lauren; Berger, Michael F; Monahan, John E; Morais, Paula; Meltzer, Jodi; Korejwa, Adam; Jané-Valbuena, Judit; Mapa, Felipa A; Thibault, Joseph; Bric-Furlong, Eva; Raman, Pichai; Shipway, Aaron; Engels, Ingo H; Cheng, Jill; Yu, Guoying K; Yu, Jianjun; Aspesi, Peter; de Silva, Melanie; Jagtap, Kalpana; Jones, Michael D; Wang, Li; Hatton, Charles; Palescandolo, Emanuele; Gupta, Supriya; Mahan, Scott; Sougnez, Carrie; Onofrio, Robert C; Liefeld, Ted; MacConaill, Laura; Winckler, Wendy; Reich, Michael; Li, Nanxin; Mesirov, Jill P; Gabriel, Stacey B; Getz, Gad; Ardlie, Kristin; Chan, Vivien; Myer, Vic E; Weber, Barbara L; Porter, Jeff; Warmuth, Markus; Finan, Peter; Harris, Jennifer L; Meyerson, Matthew; Golub, Todd R; Morrissey, Michael P; Sellers, William R; Schlegel, Robert; Garraway, Levi A

2012-03-28

The systematic translation of cancer genomic data into knowledge of tumour biology and therapeutic possibilities remains challenging. Such efforts should be greatly aided by robust preclinical model systems that reflect the genomic diversity of human cancers and for which detailed genetic and pharmacological annotation is available. Here we describe the Cancer Cell Line Encyclopedia (CCLE): a compilation of gene expression, chromosomal copy number and massively parallel sequencing data from 947 human cancer cell lines. When coupled with pharmacological profiles for 24 anticancer drugs across 479 of the cell lines, this collection allowed identification of genetic, lineage, and gene-expression-based predictors of drug sensitivity. In addition to known predictors, we found that plasma cell lineage correlated with sensitivity to IGF1 receptor inhibitors; AHR expression was associated with MEK inhibitor efficacy in NRAS-mutant lines; and SLFN11 expression predicted sensitivity to topoisomerase inhibitors. Together, our results indicate that large, annotated cell-line collections may help to enable preclinical stratification schemata for anticancer agents. The generation of genetic predictions of drug response in the preclinical setting and their incorporation into cancer clinical trial design could speed the emergence of 'personalized' therapeutic regimens.

Application of recombinant fluorescent mammalian cells as a toxicity biosensor.

PubMed

Kim, E J; Lee, Y; Lee, J E; Gu, M B

2002-01-01

With respect to developing a more sensitive biosensor, a recombinant fluorescent Chinese Hamster Ovary cell line was used for the monitoring of various toxicants. Both cell lines, EFC-500 and KFC-A10, were able to detect toxicants sensitively. They were characterized with mitomycin C and gamma-ray as genotoxicants and bisphenol A, nonylphenol, ziram and methyl bromide as possible and known EDCs. When compared to each other, the response of KFC-A10 was generally more informative and sensitive. Compared to typical bacterial biosensor systems, these cell lines offered a sensitivity of 2- to 50-fold greater for the tested chemicals. Based on these results, the use of mammalian cells offers a sensitive biosensor system that is not only fast, cheap and reproducible but also capable of monitoring the endocrine-like characteristics of environmental toxicants.

Human neuroblastoma cells with acquired resistance to the p53 activator RITA retain functional p53 and sensitivity to other p53 activating agents

PubMed Central

Michaelis, M; Rothweiler, F; Agha, B; Barth, S; Voges, Y; Löschmann, N; von Deimling, A; Breitling, R; Wilhelm Doerr, H; Rödel, F; Speidel, D; Cinatl, J

2012-01-01

Adaptation of wild-type p53 expressing UKF-NB-3 cancer cells to the murine double minute 2 inhibitor nutlin-3 causes de novo p53 mutations at high frequency (13/20) and multi-drug resistance. Here, we show that the same cells respond very differently when adapted to RITA, a drug that, like nutlin-3, also disrupts the p53/Mdm2 interaction. All of the 11 UKF-NB-3 sub-lines adapted to RITA that we established retained functional wild-type p53 although RITA induced a substantial p53 response. Moreover, all RITA-adapted cell lines remained sensitive to nutlin-3, whereas only five out of 10 nutlin-3-adapted cell lines retained their sensitivity to RITA. In addition, repeated adaptation of the RITA-adapted sub-line UKF-NB-3rRITA10 μM to nutlin-3 resulted in p53 mutations. The RITA-adapted UKF-NB-3 sub-lines displayed no or less pronounced resistance to vincristine, cisplatin, and irradiation than nutlin-3-adapted UKF-NB-3 sub-lines. Furthermore, adaptation to RITA was associated with fewer changes at the expression level of antiapoptotic factors than observed with adaptation to nutlin-3. Transcriptomic analyses indicated the RITA-adapted sub-lines to be more similar at the gene expression level to the parental UKF-NB-3 cells than nutlin-3-adapted UKF-NB-3 sub-lines, which correlates with the observed chemotherapy and irradiation sensitivity phenotypes. In conclusion, RITA-adapted cells retain functional p53, remain sensitive to nutlin-3, and display a less pronounced resistance phenotype than nutlin-3-adapted cells. PMID:22476102

Human neuroblastoma cells with acquired resistance to the p53 activator RITA retain functional p53 and sensitivity to other p53 activating agents.

PubMed

Michaelis, M; Rothweiler, F; Agha, B; Barth, S; Voges, Y; Löschmann, N; von Deimling, A; Breitling, R; Doerr, H Wilhelm; Rödel, F; Speidel, D; Cinatl, J

2012-04-05

Adaptation of wild-type p53 expressing UKF-NB-3 cancer cells to the murine double minute 2 inhibitor nutlin-3 causes de novo p53 mutations at high frequency (13/20) and multi-drug resistance. Here, we show that the same cells respond very differently when adapted to RITA, a drug that, like nutlin-3, also disrupts the p53/Mdm2 interaction. All of the 11 UKF-NB-3 sub-lines adapted to RITA that we established retained functional wild-type p53 although RITA induced a substantial p53 response. Moreover, all RITA-adapted cell lines remained sensitive to nutlin-3, whereas only five out of 10 nutlin-3-adapted cell lines retained their sensitivity to RITA. In addition, repeated adaptation of the RITA-adapted sub-line UKF-NB-3(r)RITA(10 μM) to nutlin-3 resulted in p53 mutations. The RITA-adapted UKF-NB-3 sub-lines displayed no or less pronounced resistance to vincristine, cisplatin, and irradiation than nutlin-3-adapted UKF-NB-3 sub-lines. Furthermore, adaptation to RITA was associated with fewer changes at the expression level of antiapoptotic factors than observed with adaptation to nutlin-3. Transcriptomic analyses indicated the RITA-adapted sub-lines to be more similar at the gene expression level to the parental UKF-NB-3 cells than nutlin-3-adapted UKF-NB-3 sub-lines, which correlates with the observed chemotherapy and irradiation sensitivity phenotypes. In conclusion, RITA-adapted cells retain functional p53, remain sensitive to nutlin-3, and display a less pronounced resistance phenotype than nutlin-3-adapted cells.

Estradiol impairs the antiproliferative and proapoptotic effect of Zoledronic acid in hormone sensitive breast cancer cells in vitro

PubMed Central

Weingartshofer, Sigrid; Grunt, Thomas W.; Mairhofer, Mario; Tan, Yen; Gamper, Jutta; Singer, Christian F.

2017-01-01

Background Zoledronic acid (ZA) has antiresorptive effects and protects from bone metastasis in women with early breast cancer. In addition, in postmenopausal women with endocrine responsive breast cancer ZA prolongs DFS. The exact mechanism is still unclear. We have therefore investigated the effect of increasing concentrations of ZA in breast cancer cell lines in the absence or presence of estradiol to mimic the hormonal environment in vitro. Materials and methods Using assays for cell proliferation (EZ4U, BrdU) and cell death (Annexin/PI), we have analyzed the dose-dependent antiproliferative and pro-apoptotic effects of ZA in two hormone sensitive cell lines (MCF-7 and T47D) and a hormone insensitive, triple negative cell line (MDA-MB-231) in the presence of 0, 1 and 10 nM estradiol. Results In the absence of estradiol, ZA exerts dose-dependent antiproliferative and pro-apoptotic antitumor effects in both, hormone sensitive (MCF-7, T47D) and -insensitive (MDA-MB-231) breast cancer cell lines (p<0.0001). In the presence of estradiol, the antitumoral effect of ZA was significantly decreased only in the hormone sensitive MCF-7 and T47D cell lines (p = 0.0008 and p = 0.0008, respectively). Conclusion We have demonstrated that estradiol impairs the antiproliferative and proapoptotic effect of ZA in hormone sensitive, but not in hormone insensitive breast cancer cell lines. Our findings provide a possible explanation for the differential effect of ZA on DFS in pre- and postmenopausal patients with hormone sensitive early breast cancer, which has been demonstrated clinically. We further hypothesize that endocrine insensitive tumors such as triple negative breast cancer (TNBC) should benefit from ZA irrespective of their menopausal status. PMID:28945801

Estradiol impairs the antiproliferative and proapoptotic effect of Zoledronic acid in hormone sensitive breast cancer cells in vitro.

PubMed

Gschwantler-Kaulich, Daphne; Weingartshofer, Sigrid; Grunt, Thomas W; Mairhofer, Mario; Tan, Yen; Gamper, Jutta; Singer, Christian F

2017-01-01

Zoledronic acid (ZA) has antiresorptive effects and protects from bone metastasis in women with early breast cancer. In addition, in postmenopausal women with endocrine responsive breast cancer ZA prolongs DFS. The exact mechanism is still unclear. We have therefore investigated the effect of increasing concentrations of ZA in breast cancer cell lines in the absence or presence of estradiol to mimic the hormonal environment in vitro. Using assays for cell proliferation (EZ4U, BrdU) and cell death (Annexin/PI), we have analyzed the dose-dependent antiproliferative and pro-apoptotic effects of ZA in two hormone sensitive cell lines (MCF-7 and T47D) and a hormone insensitive, triple negative cell line (MDA-MB-231) in the presence of 0, 1 and 10 nM estradiol. In the absence of estradiol, ZA exerts dose-dependent antiproliferative and pro-apoptotic antitumor effects in both, hormone sensitive (MCF-7, T47D) and -insensitive (MDA-MB-231) breast cancer cell lines (p<0.0001). In the presence of estradiol, the antitumoral effect of ZA was significantly decreased only in the hormone sensitive MCF-7 and T47D cell lines (p = 0.0008 and p = 0.0008, respectively). We have demonstrated that estradiol impairs the antiproliferative and proapoptotic effect of ZA in hormone sensitive, but not in hormone insensitive breast cancer cell lines. Our findings provide a possible explanation for the differential effect of ZA on DFS in pre- and postmenopausal patients with hormone sensitive early breast cancer, which has been demonstrated clinically. We further hypothesize that endocrine insensitive tumors such as triple negative breast cancer (TNBC) should benefit from ZA irrespective of their menopausal status.

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.

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

The BCL2 antagonist of cell death pathway influences endometrial cancer cell sensitivity to cisplatin.

PubMed

Chon, Hye Sook; Marchion, Douglas C; Xiong, Yin; Chen, Ning; Bicaku, Elona; Stickles, Xiaomang Ba; Bou Zgheib, Nadim; Judson, Patricia L; Hakam, Ardeshir; Gonzalez-Bosquet, Jesus; Wenham, Robert M; Apte, Sachin M; Lancaster, Johnathan M

2012-01-01

To identify pathways that influence endometrial cancer (EC) cell sensitivity to cisplatin and to characterize the BCL2 antagonist of cell death (BAD) pathway as a therapeutic target to increase cisplatin sensitivity. Eight EC cell lines (Ishikawa, MFE296, RL 95-2, AN3CA, KLE, MFE280, MFE319, HEC-1-A) were subjected to Affymetrix Human U133A GeneChip expression analysis of approximately 22,000 probe sets. In parallel, endometrial cell line sensitivity to cisplatin was quantified by MTS assay, and IC(50) values were calculated. Pearson's correlation test was used to identify genes associated with response to cisplatin. Genes associated with cisplatin responsiveness were subjected to pathway analysis. The BAD pathway was identified and subjected to targeted modulation, and the effect on cisplatin sensitivity was evaluated. Pearson's correlation analysis identified 1443 genes associated with cisplatin resistance (P<0.05), which included representation of the BAD-apoptosis pathway. Small interfering RNA (siRNA) knockdown of BAD pathway protein phosphatase PP2C expression was associated with increased phosphorylated BAD (serine-155) levels and a parallel increase in cisplatin resistance in Ishikawa (P=0.004) and HEC-1-A (P=0.02) cell lines. In contrast, siRNA knockdown of protein kinase A expression increased cisplatin sensitivity in the Ishikawa (P=0.02) cell line. The BAD pathway influences EC cell sensitivity to cisplatin, likely via modulation of the phosphorylation status of the BAD protein. The BAD pathway represents an appealing therapeutic target to increase EC cell sensitivity to cisplatin. Copyright © 2011 Elsevier Inc. All rights reserved.

In vitro cytotoxicity of Indonesian stingless bee products against human cancer cell lines

PubMed Central

Kustiawan, Paula M.; Puthong, Songchan; Arung, Enos T.; Chanchao, Chanpen

2014-01-01

Objective To screen crude extracts of propolis, bee pollen and honey from four stingless bee species [Trigona incisa (T. incisa)], Timia apicalis, Trigona fusco-balteata and Trigona fuscibasis) native to East Kalimantan, Indonesia for cytotoxic activity against five human cancer cell lines (HepG2, SW620, ChaGo-I, KATO-III and BT474). Methods All samples were extracted with methanol, and then subpartitioned with n-hexane and ethyl acetate. Each crude extract was screened at 20 µg/mL for in vitro cytotoxicity against the cell lines using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. In addition, four previously shown bioactive components from propolis (apigenin, caffeic acid phenyl ester, kaempferol and naringenin) and two chemotherapeutic drugs (doxorubicin and 5-fluorouracil) were used to evaluate the sensitivity of the cell lines. Results Overall, crude extracts from propolis and honey had higher cytotoxic activities than bee pollen, but the activity was dependent upon the extraction solvent, bee species and cell line. Propolis extracts from T. incisa and Timia apicalis showed the highest and lowest cytotoxic activity, respectively. Only the HepG2 cell line was broadly sensitive to the honey extracts. For pure compounds, doxorubicin was the most cytotoxic, the four propolis compounds the least, but the ChaGo-I cell line was sensitive to kaempferol at 10 µg/mL and KATO-III was sensitive to kaempferol and apigenin at 10 µg/mL. All pure compounds were effective against the BT474 cell line. Conclusions Propolis from T. incisa and Trigona fusco-balteata contain an in vitro cytotoxic activity against human cancer cell lines. Further study is required, including the isolation and characterization of the active antiproliferative agent(s). PMID:25183275

In vitro cytotoxicity of Indonesian stingless bee products against human cancer cell lines.

PubMed

Kustiawan, Paula M; Puthong, Songchan; Arung, Enos T; Chanchao, Chanpen

2014-07-01

To screen crude extracts of propolis, bee pollen and honey from four stingless bee species [Trigona incisa (T. incisa)], Timia apicalis, Trigona fusco-balteata and Trigona fuscibasis) native to East Kalimantan, Indonesia for cytotoxic activity against five human cancer cell lines (HepG2, SW620, ChaGo-I, KATO-III and BT474). All samples were extracted with methanol, and then subpartitioned with n-hexane and ethyl acetate. Each crude extract was screened at 20 µg/mL for in vitro cytotoxicity against the cell lines using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. In addition, four previously shown bioactive components from propolis (apigenin, caffeic acid phenyl ester, kaempferol and naringenin) and two chemotherapeutic drugs (doxorubicin and 5-fluorouracil) were used to evaluate the sensitivity of the cell lines. Overall, crude extracts from propolis and honey had higher cytotoxic activities than bee pollen, but the activity was dependent upon the extraction solvent, bee species and cell line. Propolis extracts from T. incisa and Timia apicalis showed the highest and lowest cytotoxic activity, respectively. Only the HepG2 cell line was broadly sensitive to the honey extracts. For pure compounds, doxorubicin was the most cytotoxic, the four propolis compounds the least, but the ChaGo-I cell line was sensitive to kaempferol at 10 µg/mL and KATO-III was sensitive to kaempferol and apigenin at 10 µg/mL. All pure compounds were effective against the BT474 cell line. Propolis from T. incisa and Trigona fusco-balteata contain an in vitro cytotoxic activity against human cancer cell lines. Further study is required, including the isolation and characterization of the active antiproliferative agent(s).

Magnetic fluid hyperthermia enhances cytotoxicity of bortezomib in sensitive and resistant cancer cell lines.

PubMed

Alvarez-Berríos, Merlis P; Castillo, Amalchi; Rinaldi, Carlos; Torres-Lugo, Madeline

2014-01-01

The proteasome inhibitor bortezomib (BZ) has shown promising results in some types of cancer, but in others it has had minimal activity. Recent studies have reported enhanced efficacy of BZ when combined with hyperthermia. However, the use of magnetic nanoparticles to induce hyperthermia in combination with BZ has not been reported. This novel hyperthermia modality has shown better potentiation of chemotherapeutics over other types of hyperthermia. We hypothesized that inducing hyperthermia via magnetic nanoparticles (MFH) would enhance the cytotoxicity of BZ in BZ-sensitive and BZ-resistant cancer cells more effectively than hyperthermia using a hot water bath (HWH). Studies were conducted using BZ in combination with MFH in two BZ-sensitive cell lines (MDA-MB-468, Caco-2), and one BZ-resistant cell line (A2780) at two different conditions, ie, 43°C for 30 minutes and 45°C for 30 minutes. These experiments were compared with combined application of HWH and BZ. The results indicate enhanced potentiation between hyperthermic treatment and BZ. MFH combined with BZ induced cytotoxicity in sensitive and resistant cell lines to a greater extent than HWH under the same treatment conditions. The observation that MFH sensitizes BZ-resistant cell lines makes this approach a potentially effective anticancer therapy platform.

Chemo-sensitivity in a panel of B-cell precursor acute lymphoblastic leukemia cell lines, YCUB series, derived from children.

PubMed

Goto, Hiroaki; Naruto, Takuya; Tanoshima, Reo; Kato, Hiromi; Yokosuka, Tomoko; Yanagimachi, Masakatsu; Fujii, Hisaki; Yokota, Shumpei; Komine, Hiromi

2009-10-01

Sensitivity to 10 anticancer drugs was evaluated in 6 childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cell lines. Authenticity of newly established cell lines was confirmed by genomic fingerprinting. The line YCUB-5R established at relapse was more resistant to 4-hydroperoxy-cyclophosphamide, cytarabine, L-asparaginase, topotecan, fludarabine, and etoposide than YCUB-5 from the same patient at diagnosis. Of the drugs tested, etoposide and SN-38 (irinotecan) showed highest efficacy in the panel, with 50% growth inhibition at 0.22-1.8 microg/ml and 0.57-3.6 ng/ml, respectively. This cell line panel offers an in vitro model for the development of new therapies for childhood BCP-ALL.

ERK phosphorylation is predictive of resistance to IGF-1R inhibition in small cell lung cancer.

PubMed

Zinn, Rebekah L; Gardner, Eric E; Marchionni, Luigi; Murphy, Sara C; Dobromilskaya, Irina; Hann, Christine L; Rudin, Charles M

2013-06-01

New therapies are critically needed to improve the outcome for patients with small cell lung cancer (SCLC). Insulin-like growth factor 1 receptor (IGF-1R) inhibition is a potential treatment strategy for SCLC: the IGF-1R pathway is commonly upregulated in SCLC and has been associated with inhibition of apoptosis and stimulation of proliferation through downstream signaling pathways, including phosphatidylinositol-3-kinase-Akt and mitogen-activated protein kinase. To evaluate potential determinants of response to IGF-1R inhibition, we assessed the relative sensitivity of 19 SCLC cell lines to OSI-906, a small molecule inhibitor of IGF-1R, and the closely related insulin receptor. Approximately one third of these cell lines were sensitive to OSI-906, with an IC50 < 1 μmol/L. Cell line expression of IGF-1R, IR, IGF-1, IGF-2, IGFBP3, and IGFBP6 did not correlate with sensitivity to OSI-906. Interestingly, OSI-906 sensitive lines expressed significantly lower levels of baseline phospho-ERK relative to resistant lines (P = 0.006). OSI-906 treatment resulted in dose-dependent inhibition of phospho-IGF-1R and phospho-Akt in both sensitive and resistant cell lines, but induced apoptosis and cell-cycle arrest only in sensitive lines. We tested the in vivo efficacy of OSI-906 using an NCI-H187 xenograft model and two SCLC patient xenografts in mice. OSI-906 treatment resulted in 50% tumor growth inhibition in NCI-H187 and 30% inhibition in the primary patient xenograft models compared with mock-treated animals. Taken together our data support IGF-1R inhibition as a viable treatment strategy for a defined subset of SCLC and suggest that low pretreatment levels of phospho-ERK may be indicative of sensitivity to this therapeutic approach. ©2013 AACR

The Cancer Cell Line Encyclopedia enables predictive modeling of anticancer drug sensitivity

PubMed Central

Barretina, Jordi; Caponigro, Giordano; Stransky, Nicolas; Venkatesan, Kavitha; Margolin, Adam A.; Kim, Sungjoon; Wilson, Christopher J.; Lehár, Joseph; Kryukov, Gregory V.; Sonkin, Dmitriy; Reddy, Anupama; Liu, Manway; Murray, Lauren; Berger, Michael F.; Monahan, John E.; Morais, Paula; Meltzer, Jodi; Korejwa, Adam; Jané-Valbuena, Judit; Mapa, Felipa A.; Thibault, Joseph; Bric-Furlong, Eva; Raman, Pichai; Shipway, Aaron; Engels, Ingo H.; Cheng, Jill; Yu, Guoying K.; Yu, Jianjun; Aspesi, Peter; de Silva, Melanie; Jagtap, Kalpana; Jones, Michael D.; Wang, Li; Hatton, Charles; Palescandolo, Emanuele; Gupta, Supriya; Mahan, Scott; Sougnez, Carrie; Onofrio, Robert C.; Liefeld, Ted; MacConaill, Laura; Winckler, Wendy; Reich, Michael; Li, Nanxin; Mesirov, Jill P.; Gabriel, Stacey B.; Getz, Gad; Ardlie, Kristin; Chan, Vivien; Myer, Vic E.; Weber, Barbara L.; Porter, Jeff; Warmuth, Markus; Finan, Peter; Harris, Jennifer L.; Meyerson, Matthew; Golub, Todd R.; Morrissey, Michael P.; Sellers, William R.; Schlegel, Robert; Garraway, Levi A.

2012-01-01

The systematic translation of cancer genomic data into knowledge of tumor biology and therapeutic avenues remains challenging. Such efforts should be greatly aided by robust preclinical model systems that reflect the genomic diversity of human cancers and for which detailed genetic and pharmacologic annotation is available1. Here we describe the Cancer Cell Line Encyclopedia (CCLE): a compilation of gene expression, chromosomal copy number, and massively parallel sequencing data from 947 human cancer cell lines. When coupled with pharmacologic profiles for 24 anticancer drugs across 479 of the lines, this collection allowed identification of genetic, lineage, and gene expression-based predictors of drug sensitivity. In addition to known predictors, we found that plasma cell lineage correlated with sensitivity to IGF1 receptor inhibitors; AHR expression was associated with MEK inhibitor efficacy in NRAS-mutant lines; and SLFN11 expression predicted sensitivity to topoisomerase inhibitors. Altogether, our results suggest that large, annotated cell line collections may help to enable preclinical stratification schemata for anticancer agents. The generation of genetic predictions of drug response in the preclinical setting and their incorporation into cancer clinical trial design could speed the emergence of “personalized” therapeutic regimens2. PMID:22460905

Overexpression of miR-202 resensitizes imatinib resistant chronic myeloid leukemia cells through targetting Hexokinase 2

PubMed Central

Deng, Yingjun; Li, Xin; Feng, Jinxin; Zhang, Xiangliang

2018-01-01

Chronic myeloid leukemia (CML) is a myeloproliferative disease which uniquely expresses a constitutively active tyrosine kinase, BCR/ABL. As a specific inhibitor of the BCR-ABL tyrosine kinase, imatinib becomes the first choice for the treatment of CML due to its high efficacy and low toxicity. However, the development of imatinib resistance limits the long-term treatment benefits of it in CML patients. In the present study, we aimed to investigate the roles of miR-202 in the regulation of imatinib sensitivity in CML cell lines and the possible mechanisms involved in this process. We found miR-202 was down-regulated in seven CML cell lines by quantitative reverse-transcription PCR (qRT-PCR) analysis. Overexpression of miR-202 significantly suppressed proliferation rates of CML cells. By establishing imatinib resistant cell lines originating from K562 and KU812 cells, we observed expressions of miR-202 were down-regulated by imatinib treatments and imatinib resistant CML cell lines exhibited lower level of miR-202. On the contrary, imatinib resistant CML cell lines displayed up-regulated glycolysis rate than sensitive cells with the evidence that glucose uptake, lactate production, and key glycolysis enzymes were elevated in imatinib resistant cells. Importantly, the imatinib resistant CML cell lines were more sensitive to glucose starvation and glycolysis inhibitors. In addition, we identified Hexokinase 2 (HK2) as a direct target of miR-202 in CML cell lines. Overexpression of miR-202 sensitized imatinib resistant CML through the miR-202-mediated glycolysis inhibition by targetting HK2. Finally, we provided the clinical relevance that miR-202 was down-regulated in CML patients and patients with lower miR-202 expression displayed higher HK2 expression. The present study will provide new aspects on the miRNA-modulated tyrosine kinase inhibitor (TKI) sensitivity in CML, contributing to the development of new therapeutic anticancer drugs. PMID:29559564

Chandipura virus growth kinetics in vertebrate cell lines, insect cell lines & embryonated eggs.

PubMed

Jadi, R S; Sudeep, A B; Kumar, Satyendra; Arankalle, V A; Mishra, A C

2010-08-01

Since not much information on Chandipura virus is available, an attempt was made to study the growth kinetics of the virus in certain vertebrate, invertebrate cell lines and embryonated chicken eggs. Comparative study of Chandipura virus (CHPV) growth kinetics in three vertebrate cell lines [Vero E6, Rhabdo myosarcoma (RD), Porcine stable kidney (PS) cell lines], two insect cell lines [Aedes aegypti (AA) and Phlebotomus papatasi (PP-9) cell lines] and embryonated pathogen free chicken eggs was conducted, by tissue culture infective dose 50 per cent (TCID(50)) and indirect immunofluorescence assay (IFA). All the cell lines and embryonated egg supported the growth of CHPV and yielded high virus titre. The vertebrate cell lines showed distinct cytopathic effect (CPE) within 4-6 h post infection (PI), while no CPE was observed in insect cell lines. PP-9 cell line was the most sensitive system to CHPV as viral antigen could be detected at 1 h PI by IFA. Our results demonstrated that all the systems were susceptible to CHPV and achieved high yield of virus. However, the PP-9 cell line had an edge over the others due to its high sensitivity to the virus which might be useful for detection and isolation of the virus during epidemics.

Sensitivity of malignant rhabdoid tumor cell lines to PD 0332991 is inversely correlated with p16 expression

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

Katsumi, Yoshiki; Iehara, Tomoko; Miyachi, Mitsuru

Highlights: {yields} PD 0332991 (PD) could suppress four of five malignant rhabdoid tumor (MRT) cell lines. {yields} The sensitivity of the MRT cell lines to PD was inversely correlated with p16 expression (r = 0.951). {yields} p16 expression in MRT could be used to predict its sensitivity to PD. {yields} PD may be an attractive agent for patients with MRT whose tumors express low levels of p16. -- Abstract: Malignant rhabdoid tumor (MRT) is a rare and highly aggressive neoplasm of young children. MRT is characterized by inactivation of integrase interactor 1 (INI1). Cyclin-dependent kinase 4 (CDK4), which acts downstreammore » of INI1, is required for the proliferation of MRT cells. Here we investigated the effects of PD 0332991 (PD), a potent inhibitor of CDK4, against five human MRT cell lines (MP-MRT-AN, KP-MRT-RY, G401, KP-MRT-NS, KP-MRT-YM). In all of the cell lines except KP-MRT-YM, PD inhibited cell proliferation >50%, (IC{sub 50} values 0.01 to 0.6 {mu}M) by WST-8 assay, and induced G1-phase cell cycle arrest, as shown by flow cytometry and BrdU incorporation assay. The sensitivity of the MRT cell lines to PD was inversely correlated with p16 expression (r = 0.951). KP-MRT-YM cells overexpress p16 and were resistant to the growth inhibitory effect of PD. Small interfering RNA against p16 significantly increased the sensitivity of KP-MRT-YM cells to PD (p < 0.05). These results suggest that p16 expression in MRT could be used to predict its sensitivity to PD. PD may be an attractive agent for patients with MRT whose tumors express low levels of p16.« less

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.

Neuronal models for evaluation of proliferation in vitro using high content screening.

PubMed

Mundy, William R; Radio, Nicholas M; Freudenrich, Theresa M

2010-04-11

In vitro test methods can provide a rapid approach for the screening of large numbers of chemicals for their potential to produce toxicity (hazard identification). In order to identify potential developmental neurotoxicants, a battery of in vitro tests for neurodevelopmental processes such as cell proliferation, differentiation, growth, and synaptogenesis has been proposed. The development of in vitro approaches for toxicity testing will require choosing a model system that is appropriate to the endpoint of concern. This study compared several cell lines as models for neuronal proliferation. The sensitivities of neuronal cell lines derived from three species (PC12, rat; N1E-115, mouse; SH-SY5Y, human) to chemicals known to affect cell proliferation were assessed using a high content screening system. After optimizing conditions for cell growth in 96-well plates, proliferation was measured as the incorporation of 5-bromo-2'-deoxyuridine (BrdU) into replicating DNA during S phase. BrdU-labeled cells were detected by immunocytochemistry and cell counts were obtained using automated image acquisition and analysis. The three cell lines showed approximately 30-40% of the population in S phase after a 4h pulse of BrdU. Exposure to the DNA polymerase inhibitor aphidicolin for 20 h prior to the 4h pulse of BrdU significantly decreased proliferation in all three cell lines. The sensitivities of the cell lines were compared by exposure to eight chemicals known to affect proliferation (positive controls) and determination of the concentration inhibiting proliferation by 50% of control (I(50)). PC12 cells were the most sensitive to chemicals; 6 out of 8 chemicals (aphidicolin, cadmium, cytosine arabinoside, dexamethasone, 5-fluorouracil, and methylmercury) inhibited proliferation at the concentrations tested. SH-SY5Y cells were somewhat less sensitive to chemical effects, with five out of eight chemicals inhibiting proliferation; dexamethasone had no effect, and cadmium inhibited proliferation only at concentrations that decreased cell viability. Data from the N1E-115 cell line was extremely variable between experiments, and only 4 out of 8 chemicals resulted in inhibition of proliferation. Chemicals that had not been previously shown to alter proliferation (negative controls) did not affect proliferation or cell viability in any cell line. The results show that high content screening can be used to rapidly assess chemical effects on proliferation. Three neuronal cell lines exhibited differential sensitivity to the effect of chemicals on this endpoint, with PC12 cells being the most sensitive to inhibition of proliferation. Published by Elsevier Ireland Ltd.

Radiation Response in Two HPV-Infected Head-and-Neck Cancer Cell Lines in Comparison to a Non-HPV-Infected Cell Line and Relationship to Signaling Through AKT

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

Gupta, Anjali K.; Lee, John H.; Wilke, Werner W.

2009-07-01

Purpose: Human papilloma virus (HPV)-associated cancers of the head and neck (H and N) are increasing in frequency and are often treated with radiation. There are conflicting data in the literature regarding the radiation response in the presence of HPV infection, with some data suggesting they may be more sensitive to radiation. There are few studies looking at in vitro effects of HPV and further sensitization by inhibitors of specific signaling pathways. We are in the process of starting a clinical trial in H and N cancer patients using nelfinavir (NFV) (which inhibits Akt) and it would be important tomore » know the effect of HPV on radiation response {+-} NFV. Methods and Materials: Two naturally infected HPV-16 cell lines (UPCI-SCC90 and UMSCC47) and the HPV-negative SQ20B H and N squamous carcinoma cells were used. Western blots with or without 10 uM NFV were done to evaluate signaling from the PI3K-Akt pathway. Clonogenic assays were done in the three cell lines with or without NFV. Results: Both UPCI-SCC90 and UMSCC47 cells were sensitive to radiation as compared with SQ20B and the degree corresponded to Akt activation. The SQ20B cell line has an activating mutation in EGFR resulting in phosphorylation (P) of Akt; UMSCC47 has decreased P-phosphatase and TENsin (PTEN), resulting in increased P-Akt; UPCI-SCC90 had overexpression of P-PTEN and decreased P-Akt. NFV resulted in downregulation of Akt in all three cell lines, resulting in sensitization to radiation. Conclusions: HPV-infected H and N cancers are sensitive to radiation. The degree of sensitivity correlates to Akt activation and they can be further sensitized by NFV00.« less

Bcl-2/Bax protein ratio predicts 5-fluorouracil sensitivity independently of p53 status

PubMed Central

Mirjolet, J-F; Barberi-Heyob, M; Didelot, C; Peyrat, J-P; Abecassis, J; Millon, R; Merlin, J-L

2000-01-01

p53 tumour-suppressor gene is involved in cell growth control, arrest and apoptosis. Nevertheless cell cycle arrest and apoptosis induction can be observed in p53-defective cells after exposure to DNA-damaging agents such as 5-fluorouracil (5-FU) suggesting the importance of alternative pathways via p53-independent mechanisms. In order to establish relationship between p53 status, cell cycle arrest, Bcl-2/Bax regulation and 5-FU sensitivity, we examined p53 mRNA and protein expression and p53 protein functionality in wild-type (wt) and mutant (mt) p53 cell lines. p53 mRNA and p53 protein expression were determined before and after exposure to equitoxic 5-FU concentration in six human carcinoma cell lines differing in p53 status and displaying marked differences in 5-FU sensitivity, with IC 50 values ranging from 0.2–22.6 mM. 5-FU induced a rise in p53 mRNA expression in mt p53 cell lines and in human papilloma virus positive wt p53 cell line, whereas significant decrease in p53 mRNA expression was found in wt p53 cell line. Whatever p53 status, 5-FU altered p53 transcriptional and translational regulation leading to up-regulation of p53 protein. In relation with p53 functionality, but independently of p53 mutational status, after exposure to 5-FU equitoxic concentration, all cell lines were able to arrest in G1. No relationship was evidenced between G1 accumulation ability and 5-FU sensitivity. Moreover, after 5-FU exposure, Bax and Bcl-2 proteins regulation was under p53 protein control and a statistically significant relationship (r= 0.880,P= 0.0097) was observed between Bcl-2/Bax ratio and 5-FU sensitivity. In conclusion, whatever p53 status, Bcl-2 or Bax induction and Bcl-2/Bax protein ratio were correlated to 5-FU sensitivity. © 2000 Cancer Research Campaign PMID:11044365

Flow Line, Durafill VS, and Dycal toxicity to dental pulp cells: effects of growth factors

PubMed Central

Furey, Alyssa; Hjelmhaug, Julie; Lobner, Doug

2010-01-01

Introduction The objective was to determine the effects of growth factor treatment on dental pulp cell sensitivity to toxicity of two composite restoration materials, Flow Line and Durafill VS, and a calcium hydroxide pulp capping material, Dycal. Methods Toxicity of the dental materials to cultures of primary dental pulp cells was determined by the MTT metabolism assay. The ability of six different growth factors to influence the toxicity was tested. Results A 24 hour exposure to either Flow Line or Durafill VS caused approximately 40% cell death, while Dycal exposure caused approximately 80% cell death. The toxicity of Flow Line and Durafill VS was mediated by oxidative stress. Four of the growth factors tested (BMP-2, BMP-7, EGF, and TGF-β) decreased the basal MTT values while making the cells resistant to Flow Line and Durafill VS toxicity, except BMP-2 which made the cells more sensitive to Flow Line. Treatment with FGF-2 caused no change in basal MTT metabolism, prevented the toxicity of Durafill VS, but increased the toxicity of Flow Line. Treatment with IGF-I increased basal MTT metabolism and made the cells resistant to Flow Line and Durafill VS toxicity. None of the growth factors made the cells resistant to Dycal toxicity. Conclusions The results indicate that growth factors can be used to alter the sensitivity of dental pulp cells to commonly used restoration materials. The growth factors BMP-7, EGF, TGF-β, and IGF-I provided the best profile of effects, making the cells resistant to both Flow Line and Durafill VS toxicity. PMID:20630288

Effects of curcumin on stem-like cells in human esophageal squamous carcinoma cell lines.

PubMed

Almanaa, Taghreed N; Geusz, Michael E; Jamasbi, Roudabeh J

2012-10-24

Many cancers contain cell subpopulations that display characteristics of stem cells. Because these cancer stem cells (CSCs) appear to provide resistance to chemo-radiation therapy, development of therapeutic agents that target CSCs is essential. Curcumin is a phytochemical agent that is currently used in clinical trials to test its effectiveness against cancer. However, the effect of curcumin on CSCs is not well established. The current study evaluated curcumin-induced cell death in six cancer cell lines derived from human esophageal squamous cell carcinomas. Moreover, these cell lines and the ones established from cells that survived curcumin treatments were characterized. Cell loss was assayed after TE-1, TE-8, KY-5, KY-10, YES-1, and YES-2 cells were exposed to 20-80 μM curcumin for 30 hrs. Cell lines surviving 40 or 60 μM curcumin were established from these six original lines. The stem cell markers aldehyde dehydrogenase-1A1 (ALDH1A1) and CD44 as well as NF-κB were used to compare CSC-like subpopulations within and among the original lines as well as the curcumin-surviving lines. YES-2 was tested for tumorsphere-forming capabilities. Finally, the surviving lines were treated with 40 and 60 μM curcumin to determine whether their sensitivity was different from the original lines. The cell loss after curcumin treatment increased in a dose-dependent manner in all cell lines. The percentage of cells remaining after 60 μM curcumin treatment varied from 10.9% to 36.3% across the six lines. The cell lines were heterogeneous with respect to ALDH1A1, NF-κB and CD44 expression. KY-5 and YES-1 were the least sensitive and had the highest number of stem-like cells whereas TE-1 had the lowest. The curcumin-surviving lines showed a significant loss in the high staining ALDH1A1 and CD44 cell populations. Tumorspheres formed from YES-2 but were small and rare in the YES-2 surviving line. The curcumin-surviving lines showed a small but significant decrease in sensitivity to curcumin when compared with the original lines. Our results suggest that curcumin not only eliminates cancer cells but also targets CSCs. Therefore, curcumin may be an effective compound for treating esophageal and possibly other cancers in which CSCs can cause tumor recurrence.

Susceptibility of ATM-deficient pancreatic cancer cells to radiation.

PubMed

Ayars, Michael; Eshleman, James; Goggins, Michael

2017-05-19

Ataxia telangiectasia mutated (ATM) is inactivated in a significant minority of pancreatic ductal adenocarcinomas and may be predictor of treatment response. We determined if ATM deficiency renders pancreatic cancer cells more sensitive to fractionated radiation or commonly used chemotherapeutics. ATM expression was knocked down in three pancreatic cancer cell lines using ATM-targeting shRNA. Isogenic cell lines were tested for sensitivity to several chemotherapeutic agents and radiation. DNA repair kinetics were analyzed in irradiated cells using the comet assay. We find that while rendering pancreatic cancer cells ATM-deficient did not significantly change their sensitivity to several chemotherapeutics, it did render them exquisitely sensitized to radiation. Pancreatic cancer ATM status may help predict response to radiotherapy.

A systems analysis of the chemosensitivity of breast cancer cells to the polyamine analogue PG-11047

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

Kuo, Wen-Lin; Das, Debopriya; Ziyad, Safiyyah

2009-11-14

Polyamines regulate important cellular functions and polyamine dysregulation frequently occurs in cancer. The objective of this study was to use a systems approach to study the relative effects of PG-11047, a polyamine analogue, across breast cancer cells derived from different patients and to identify genetic markers associated with differential cytotoxicity. A panel of 48 breast cell lines that mirror many transcriptional and genomic features present in primary human breast tumours were used to study the antiproliferative activity of PG-11047. Sensitive cell lines were further examined for cell cycle distribution and apoptotic response. Cell line responses, quantified by the GI50 (dosemore » required for 50% relative growth inhibition) were correlated with the omic profiles of the cell lines to identify markers that predict response and cellular functions associated with drug sensitivity. The concentrations of PG-11047 needed to inhibit growth of members of the panel of breast cell lines varied over a wide range, with basal-like cell lines being inhibited at lower concentrations than the luminal cell lines. Sensitive cell lines showed a significant decrease in S phase fraction at doses that produced little apoptosis. Correlation of the GI50 values with the omic profiles of the cell lines identified genomic, transcriptional and proteomic variables associated with response. A 13-gene transcriptional marker set was developed as a predictor of response to PG-11047 that warrants clinical evaluation. Analyses of the pathways, networks and genes associated with response to PG-11047 suggest that response may be influenced by interferon signaling and differential inhibition of aspects of motility and epithelial to mesenchymal transition.« less

[Establishment and characterization of a cell line derived from human ovarian mucinous cystadenocarcinoma].

PubMed

Wan, Q; Xu, D; Li, Z

2001-07-01

To establish a cell line of human ovarian cancer, and study its characterization. The cell line was established by the cultivation of subsides walls, and kept by freezing. The morphology was observed by microscope and electromicroscope. The authors studied its growth and propagation, the agglutination test of phytohemagglutinin (PHA), the chromosome analysis, heterotransplanting, immuno-histochemistry staining, the analysis of hormone, the pollution examination and the test of sensitivity to virus etc. A new human ovarian carcinoma cell line, designated ovarian mucinous cystadenocarcinoma 685 (OMC685), was established from mucinous cystadenocarcinoma. This cell line had subcultured to 91 generations, and some had been frozen for 8 years and revived, still grew well. This cell line possessed the feature of glandular epithelium cancer cell. The cells grew exuberantly, and the agglutinating test of PHA was positive. Karyotype was subtriploid with distortion. Heterotransplantations, alcian blue periobic acid-schiff (AbPAS), mucicarmine, alcian blue stainings, estradiol (E2) and progesterone were all positive. Without being polluted, it was sensitive to polivirus-I, adenovirus 7 and measles virus. OMC685 is a distinct human ovarian tumous cell line.

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.

Quality Assurance in the Polio Laboratory. Cell Sensitivity and Cell Authentication Assays.

PubMed

Dunn, Glynis

2016-01-01

The accuracy of poliovirus surveillance is largely dependent on the quality of the cell lines used for virus isolation, which is the foundation of poliovirus diagnostic work. Many cell lines are available for the isolation of enteroviruses, whilst genetically modified L20B cells can be used as a diagnostic tool for the identification of polioviruses. To be confident that cells can consistently isolate the virus of interest, it is necessary to have a quality assurance system in place, which will ensure that the cells in use are not contaminated with other cell lines or microorganisms and that they remain sensitive to the viruses being studied.The sensitivity of cell lines can be assessed by the regular testing of a virus standard of known titer in the cell lines used for virus isolation. The titers obtained are compared to previously obtained titers in the same assay, so that any loss of sensitivity can be detected.However, the detection of cell line cross contamination is more difficult. DNA bar coding is a technique that uses a short DNA sequence from a standardized position in the genome as a molecular diagnostic assay for species-level identification. For almost all groups of higher animals, the cytochrome c oxidase subunit 1 of mitochondrial DNA (CO1) is emerging as the standard barcode region. This region is 648 nucleotide base pairs long in most phylogenetic groups and is flanked by regions of conserved sequences, making it relatively easy to isolate and analyze. DNA barcodes vary among individuals of the same species to a very minor degree (generally less than 1-2 %), and a growing number of studies have shown that the COI sequences of even closely related species differ by several per cent, making it possible to identify different species with high confidence.

Photodynamic treatment (PDT) of endometrium primary cultures serving as an in-vitro-model for endometriosis

NASA Astrophysics Data System (ADS)

Werter, Wiebke; Viereck, Volker; Keckstein, J.; Steiner, Rudolf W.; Rueck, Angelika C.

1994-05-01

As a new treatment model for endometriosis, photodynamic therapy (PDT) was applied to endometrium cultures. Endometriosis is a benign disease. Therefore primary cultures were used instead of cell lines. Endometrium is composed of epithelial and stromal cells which can also be found in primary culture. While stromal cells take a polygonal shape in culture, epithelial cells form cell colonies. PSIII (Photasan III), which is similar to hematorporphyrin derivate (HpD), meso-tetra (4-sulfonatophenyl) porphyrin (TPPS4), which posses a high fluorescence quantum yield and may be useful in fluorescence diagnosis of subtle endometriotic spots, and methylene blue (MB), a vital dye with phototoxic properties, were used as photosensitizers. Different sensitizer concentrations and incubation times were applied. The highest phototoxicity was observed for PSIII; TPPS4 and MB were less phototoxic. We compared our results with the sensitivity of cell lines described in the literature. The necessary irradiation to destroy stromal cells was relatively high but still in the same dimension as for cell lines. However they were even more sensitive than epithelial cells. This was true for all sensitizers used.

Photodynamic therapy (PDT) of endometrium primary cultures serving as an in-vitro model for endometriosis

NASA Astrophysics Data System (ADS)

Herter, Wiebke; Viereck, Volker; Keckstein, J.; Steiner, Rudolf W.; Rueck, Angelika C.

1994-05-01

As a new treatment model for endometriosis, photodynamic therapy (PDT) was applied to endometrium cultures. Endometriosis is a benign disease. Therefore primary cultures were used instead of cell lines. Endometrium is composed of epithelial and stromal cells which can also be found in primary culture. While stromal cells take a polygonal shape in culture, epithelial cells form cell colonies. PSIII (Photasan III), which is similar to hematorporphyrin derivate (HpD), meso-tetra (4-sulfonatophenyl) porphyrin (TPPS4), which posses a high fluorescence quantum yield and may be useful in fluorescence diagnosis of subtle endometriotic spots, and methylene blue (MB), a vital dye with phototoxic properties, were used as photosensitizers. Different sensitizer concentrations and incubation times were applied. The highest phototoxicity was observed for PSIII; TPPS4 and MB were less phototoxic. We compared our results with the sensitivity of cell lines described in the literature. The necessary irradiation to destroy stromal cells was relatively high but still in the same dimension as for cell lines. However they were even more sensitive than epithelial cells. This was true for all sensitizers used.

[Study on active constituents of traditional Chinese medicine reversing multidrug resistance of tumor cells in vitro].

PubMed

Zhang, H; Yang, L; Liu, S; Ren, L

2001-09-01

To screen drugs reversing multidrug resistance of tumor cells from active constituents of traditional Chinese medicine and to study the reversal action. The kill effects of the drugs on tumor cell lines in vitro were determined with MTT method. The Jin's formula was used to analyse the effect of drug combination. 5 micrograms/ml rhynchophylline, 2 micrograms/ml jatrorrhizine and 1.25 micrograms/ml indirulin could reverse multidrug resistance for vincristine on KBv200 cell line by 16.8, 5.1 and 4 fold respectively. 1.56-12.5 micrograms/ml curcumine combining with vincristine could sensitize antitumor effect both on KB and KBv200 cell lines. All rhynchophylline, jatrorrhizine and indirulin could reverse multidrug resistance for vincristine on KBv200 cell line. Curcumine combinating vincristine could sensitize antitumor effect both on kB and kBv200 cell lines.

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

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.

Differential pathway dependency discovery associated with drug response across cancer cell lines* | Office of Cancer Genomics

Cancer.gov

The effort to personalize treatment plans for cancer patients involves the identification of drug treatments that can effectively target the disease while minimizing the likelihood of adverse reactions. In this study, the gene-expression profile of 810 cancer cell lines and their response data to 368 small molecules from the Cancer Therapeutics Research Portal (CTRP) are analyzed to identify pathways with significant rewiring between genes, or differential gene dependency, between sensitive and non-sensitive cell lines.

High content screening of patient-derived cell lines highlights the potential of non-standard chemotherapeutic agents for the treatment of glioblastoma.

PubMed

Yu, Kenny Kwok-Hei; Taylor, Jessica T; Pathmanaban, Omar N; Youshani, Amir Saam; Beyit, Deniz; Dutko-Gwozdz, Joanna; Benson, Roderick; Griffiths, Gareth; Peers, Ian; Cueppens, Peter; Telfer, Brian A; Williams, Kaye J; McBain, Catherine; Kamaly-Asl, Ian D; Bigger, Brian W

2018-01-01

Glioblastoma (GBM) is the most common primary brain malignancy in adults, yet survival outcomes remain poor. First line treatment is well established, however disease invariably recurs and improving prognosis is challenging. With the aim of personalizing therapy at recurrence, we have established a high content screening (HCS) platform to analyze the sensitivity profile of seven patient-derived cancer stem cell lines to 83 FDA-approved chemotherapy drugs, with and without irradiation. Seven cancer stem cell lines were derived from patients with GBM and, along with the established cell line U87-MG, each patient-derived line was cultured in tandem in serum-free conditions as adherent monolayers and three-dimensional neurospheres. Chemotherapeutics were screened at multiple concentrations and cells double-stained to observe their effect on both cell death and proliferation. Sensitivity was classified using high-throughput algorithmic image analysis. Cell line specific drug responses were observed across the seven patient-derived cell lines. Few agents were seen to have radio-sensitizing effects, yet some drug classes showed a marked difference in efficacy between monolayers and neurospheres. In vivo validation of six drugs suggested that cell death readout in a three-dimensional culture scenario is a more physiologically relevant screening model and could be used effectively to assess the chemosensitivity of patient-derived GBM lines. The study puts forward a number of non-standard chemotherapeutics that could be useful in the treatment of recurrent GBM, namely mitoxantrone, bortezomib and actinomycin D, whilst demonstrating the potential of HCS to be used for personalized treatment based on the chemosensitivity profile of patient tumor cells.

Metabolomic Profiling of the Effects of Melittin on Cisplatin Resistant and Cisplatin Sensitive Ovarian Cancer Cells Using Mass Spectrometry and Biolog Microarray Technology

PubMed Central

Alonezi, Sanad; Tusiimire, Jonans; Wallace, Jennifer; Dufton, Mark J.; Parkinson, John A.; Young, Louise C.; Clements, Carol J.; Park, Jin Kyu; Jeon, Jong Woon; Ferro, Valerie A.; Watson, David G.

2016-01-01

In the present study, liquid chromatography-mass spectrometry (LC-MS) was employed to characterise the metabolic profiles of two human ovarian cancer cell lines A2780 (cisplatin-sensitive) and A2780CR (cisplatin-resistant) in response to their exposure to melittin, a cytotoxic peptide from bee venom. In addition, the metabolomics data were supported by application of Biolog microarray technology to examine the utilisation of carbon sources by the two cell lines. Data extraction with MZmine 2.14 and database searching were applied to provide metabolite lists. Principal component analysis (PCA) gave clear separation between the cisplatin-sensitive and resistant strains and their respective controls. The cisplatin-resistant cells were slightly more sensitive to melittin than the sensitive cells with IC50 values of 4.5 and 6.8 μg/mL respectively, although the latter cell line exhibited the greatest metabolic perturbation upon treatment. The changes induced by melittin in the cisplatin-sensitive cells led mostly to reduced levels of amino acids in the proline/glutamine/arginine pathway, as well as to decreased levels of carnitines, polyamines, adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide (NAD+). The effects on energy metabolism were supported by the data from the Biolog assays. The lipid compositions of the two cell lines were quite different with the A2780 cells having higher levels of several ether lipids than the A2780CR cells. Melittin also had some effect on the lipid composition of the cells. Overall, this study suggests that melittin might have some potential as an adjuvant therapy in cancer treatment. PMID:27754384

A Novel Inhibitor Of Topoisomerase I is Selectively Toxic For A Subset of Non-Small Cell Lung Cancer Cell Lines | Office of Cancer Genomics

Cancer.gov

SW044248, identified through a screen for chemicals that are selectively toxic for NSCLC cell lines, was found to rapidly inhibit macromolecular synthesis in sensitive, but not in insensitive cells. SW044248 killed approximately 15% of a panel of 74 NSCLC cell lines and was non-toxic to immortalized human bronchial cell lines.

Identification of biomarkers in human head and neck tumor cell lines that predict for in vitro sensitivity to gefitinib.

PubMed

Hickinson, D Mark; Marshall, Gayle B; Beran, Garry J; Varella-Garcia, Marileila; Mills, Elizabeth A; South, Marie C; Cassidy, Andrew M; Acheson, Kerry L; McWalter, Gael; McCormack, Rose M; Bunn, Paul A; French, Tim; Graham, Alex; Holloway, Brian R; Hirsch, Fred R; Speake, Georgina

2009-06-01

Potential biomarkers were identified for in vitro sensitivity to the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor gefitinib in head and neck cancer. Gefitinib sensitivity was determined in cell lines, followed by transcript profiling coupled with a novel pathway analysis approach. Eleven cell lines were highly sensitive to gefitinib (inhibitor concentration required to give 50% growth inhibition [GI(50)] < 1 microM), three had intermediate sensitivity (GI(50) 1-7 microM), and six were resistant (GI(50) > 7 microM); an exploratory principal component analysis revealed a separation between the genomic profiles of sensitive and resistant cell lines. Subsequently, a hypothesis-driven analysis of Affymetrix data (Affymetrix, Inc., Santa Clara, CA, USA) revealed higher mRNA levels for E-cadherin (CDH1); transforming growth factor, alpha (TGF-alpha); amphiregulin (AREG); FLJ22662; EGFR; p21-activated kinase 6 (PAK6); glutathione S-transferase Pi (GSTP1); and ATP-binding cassette, subfamily C, member 5 (ABCC5) in sensitive versus resistant cell lines. A hypothesis-free analysis identified 46 gene transcripts that were strongly differentiated, seven of which had a known association with EGFR and head and neck cancer (human EGF receptor 3 [HER3], TGF-alpha, CDH1, EGFR, keratin 16 [KRT16], fibroblast growth factor 2 [FGF2], and cortactin [CTTN]). Polymerase chain reaction (PCR) and enzyme-linked immunoabsorbant assay analysis confirmed Affymetrix data, and EGFR gene mutation, amplification, and genomic gain correlated strongly with gefitinib sensitivity. We identified biomarkers that predict for in vitro responsiveness to gefitinib, seven of which have known association with EGFR and head and neck cancer. These in vitro predictive biomarkers may have potential utility in the clinic and warrant further investigation.

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.

Redox Signaling and Bioenergetics Influence Lung Cancer Cell Line Sensitivity to the Isoflavone ME-344

PubMed Central

Manevich, Yefim; Reyes, Leticia; Britten, Carolyn D.; Townsend, Danyelle M.

2016-01-01

ME-344 [(3R,4S)-3,4-bis(4-hydroxyphenyl)-8-methyl-3,4-dihydro-2H-chromen-7-ol] is a second-generation derivative natural product isoflavone presently under clinical development. ME-344 effects were compared in lung cancer cell lines that are either intrinsically sensitive or resistant to the drug and in primary immortalized human lung embryonic fibroblasts (IHLEF). Cytotoxicity at low micromolar concentrations occurred only in sensitive cell lines, causing redox stress, decreased mitochondrial ATP production, and subsequent disruption of mitochondrial function. In a dose-dependent manner the drug caused instantaneous and pronounced inhibition of oxygen consumption rates (OCR) in drug-sensitive cells (quantitatively significantly less in drug-resistant cells). This was consistent with targeting of mitochondria by ME-344, with specific effects on the respiratory chain (resistance correlated with higher glycolytic indexes). OCR inhibition did not occur in primary IHLEF. ME-344 increased extracellular acidification rates in drug-resistant cells (significantly less in drug-sensitive cells), implying that ME-344 targets mitochondrial proton pumps. Only in drug-sensitive cells did ME-344 dose-dependently increase the intracellular generation of reactive oxygen species and cause oxidation of total (mainly glutathione) and protein thiols and the concomitant immediate increases in NADPH levels. We conclude that ME-344 causes complex, redox-specific, and mitochondria-targeted effects in lung cancer cells, which differ in extent from normal cells, correlate with drug sensitivity, and provide indications of a beneficial in vitro therapeutic index. PMID:27255112

Characterization of ibrutinib-sensitive and -resistant mantle lymphoma cells.

PubMed

Ma, Jiao; Lu, Pin; Guo, Ailin; Cheng, Shuhua; Zong, Hongliang; Martin, Peter; Coleman, Morton; Wang, Y Lynn

2014-09-01

Ibrutinib inhibits Bruton tyrosine kinase (BTK), a key component of early B-cell receptor (BCR) signalling pathways. A multicentre phase 2 trial of ibrutinib in patients with relapsed/refractory mantle cell lymphoma (MCL) demonstrated a remarkable response rate. However, approximately one-third of patients have primary resistance to the drug while other patients appear to lose response and develop secondary resistance. Understanding the molecular mechanisms underlying ibrutinib sensitivity is of paramount importance. In this study, we investigated cell lines and primary MCL cells that display differential sensitivity to ibrutinib. We found that the primary cells display a higher BTK activity than normal B cells and MCL cells show differential sensitivity to BTK inhibition. Genetic knockdown of BTK inhibits the growth, survival and proliferation of ibrutinib-sensitive but not resistant MCL cell lines, suggesting that ibrutinib acts through BTK to produce its anti-tumour activities. Interestingly, inhibition of ERK1/2 and AKT, but not BTK phosphorylation per se, correlates well with cellular response to BTK inhibition in cell lines as well as in primary tumours. Our study suggests that, to prevent primary resistance or to overcome secondary resistance to BTK inhibition, a combinatory strategy that targets multiple components or multiple pathways may represent the most effective approach. © 2014 John Wiley & Sons Ltd.

MHC-I modulation due to changes in tumor cell metabolism regulates tumor sensitivity to CTL and NK cells

PubMed Central

Catalán, Elena; Charni, Seyma; Jaime, Paula; Aguiló, Juan Ignacio; Enríquez, José Antonio; Naval, Javier; Pardo, Julián; Villalba, Martín; Anel, Alberto

2015-01-01

Tumor cells have a tendency to use glucose fermentation to obtain energy instead of mitochondrial oxidative phosphorylation (OXPHOS). We demonstrated that this phenotype correlated with loss of ERK5 expression and with reduced MHC class I expression. Consequently, tumor cells could evade cytotoxic T lymphocyte (CTL)-mediated immune surveillance, but also increase their sensitivity to natural killer (NK) cells. These outcomes were evaluated using two cellular models: leukemic EL4 cells and L929 transformed fibroblasts and their derived ρ° cell lines, which lack mitochondrial DNA. We have also used a L929 cell sub-line that spontaneously lost matrix attachment (L929dt), reminiscent of metastasis generation, that also downregulated MHC-I and ERK5 expression. MHC-I expression is lower in ρ° cells than in the parental cell lines, but they were equally sensitive to CTL. On the contrary, ρ° cells were more sensitive to activated NK cells than parental cells. On the other hand, L929dt cells were resistant to CTL and NK cells, showed reduced viability when forced to perform OXPHOS, and surviving cells increased MHC-I expression and became sensitive to CTL. The present results suggest that when the reduction in MHC-I levels in tumor cells due to glycolytic metabolism is partial, the increase in sensitivity to NK cells seems to predominate. However, when tumor cells completely lose MHC-I expression, the combination of treatments that increase OXPHOS with CTL-mediated immunotherapy could be a promising therapeutic approach. PMID:25949869

MHC-I modulation due to changes in tumor cell metabolism regulates tumor sensitivity to CTL and NK cells.

PubMed

Catalán, Elena; Charni, Seyma; Jaime, Paula; Aguiló, Juan Ignacio; Enríquez, José Antonio; Naval, Javier; Pardo, Julián; Villalba, Martín; Anel, Alberto

2015-01-01

Tumor cells have a tendency to use glucose fermentation to obtain energy instead of mitochondrial oxidative phosphorylation (OXPHOS). We demonstrated that this phenotype correlated with loss of ERK5 expression and with reduced MHC class I expression. Consequently, tumor cells could evade cytotoxic T lymphocyte (CTL)-mediated immune surveillance, but also increase their sensitivity to natural killer (NK) cells. These outcomes were evaluated using two cellular models: leukemic EL4 cells and L929 transformed fibroblasts and their derived ρ° cell lines, which lack mitochondrial DNA. We have also used a L929 cell sub-line that spontaneously lost matrix attachment (L929dt), reminiscent of metastasis generation, that also downregulated MHC-I and ERK5 expression. MHC-I expression is lower in ρ° cells than in the parental cell lines, but they were equally sensitive to CTL. On the contrary, ρ° cells were more sensitive to activated NK cells than parental cells. On the other hand, L929dt cells were resistant to CTL and NK cells, showed reduced viability when forced to perform OXPHOS, and surviving cells increased MHC-I expression and became sensitive to CTL. The present results suggest that when the reduction in MHC-I levels in tumor cells due to glycolytic metabolism is partial, the increase in sensitivity to NK cells seems to predominate. However, when tumor cells completely lose MHC-I expression, the combination of treatments that increase OXPHOS with CTL-mediated immunotherapy could be a promising therapeutic approach.

Effects of protein kinase C activators on phorbol ester-sensitive and -resistant EL4 thymoma cells.

PubMed

Sansbury, H M; Wisehart-Johnson, A E; Qi, C; Fulwood, S; Meier, K E

1997-09-01

Phorbol ester-sensitive EL4 murine thymoma cells respond to phorbol 12-myristate 13-acetate with activation of ERK mitogen-activated protein kinases, synthesis of interleukin-2, and death, whereas phorbol ester-resistant variants of this cell line do not exhibit these responses. Additional aspects of the resistant phenotype were examined, using a newly-established resistant cell line. Phorbol ester induced morphological changes, ERK activation, calcium-dependent activation of the c-Jun N-terminal kinase (JNK), interleukin-2 synthesis, and growth inhibition in sensitive but not resistant cells. A series of protein kinase C activators caused membrane translocation of protein kinase C's (PKCs) alpha, eta, and theta in both cell lines. While PKC eta was expressed at higher levels in sensitive than in resistant cells, overexpression of PKC eta did not restore phorbol ester-induced ERK activation to resistant cells. In sensitive cells, PKC activators had similar effects on cell viability and ERK activation, but differed in their abilities to induce JNK activation and interleukin-2 synthesis. PD 098059, an inhibitor of the mitogen activated protein (MAP)/ERK kinase kinase MEK, partially inhibited ERK activation and completely blocked phorbol ester-induced cell death in sensitive cells. Thus MEK and/or ERK activation, but not JNK activation or interleukin-2 synthesis, appears to be required for phorbol ester-induced toxicity. Alterations in phorbol ester response pathways, rather than altered expression of PKC isoforms, appear to confer phorbol ester resistance to EL4 cells.

The cytokine-dependent MUTZ-3 cell line as an in vitro model for the screening of contact sensitizers

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

Azam, Philippe; Peiffer, Jean-Luc; Chamousset, Delphine

2006-04-01

Langerhans cells (LC) are key mediators of contact allergenicity in the skin. However, no in vitro methods exist which are based on the activation process of LC to predict the sensitization potential of chemicals. In this study, we have evaluated the performances of MUTZ-3, a cytokine-dependent human monocytic cell line, in its response to sensitizers. First, we compared undifferentiated MUTZ-3 cells with several standard human cells such as THP-1, KG-1, HL-60, K-562, and U-937 in their response to the strong sensitizer DNCB and the irritant SDS by monitoring the expression levels of HLA-DR, CD54, and CD86 by flow cytometry. Onlymore » MUTZ-3 and THP-1 cells show a strong and specific response to sensitizer, while other cell lines showed very variable responses. Then, we tested MUTZ-3 cells against a wider panel of sensitizers and irritants on a broader spectrum of cell surface markers (HLA-DR, CD40, CD54, CD80, CD86, B7-H1, B7-H2, B7-DC). Of these markers, CD86 proved to be the most reliable since it detected all sensitizers, including benzocaine, a classical false negative in local lymph node assay (LLNA) but not irritants. We confirmed the MUTZ-3 response to DNCB by real-time PCR analysis. Taken together, our data suggest that undifferentiated MUTZ-3 cells may represent a valuable in vitro model for the screening of potential sensitizers.« less

Mechanistic mathematical modelling of mercaptopurine effects on cell cycle of human acute lymphoblastic leukaemia cells

PubMed Central

Panetta, J C; Evans, W E; Cheok, M H

2006-01-01

The antimetabolite mercaptopurine (MP) is widely used to treat childhood acute lymphoblastic leukaemia (ALL). To study the dynamics of MP on the cell cycle, we incubated human T-cell leukaemia cell lines (Molt-4 sensitive and resistant subline and P12 resistant) with 10 μM MP and measured total cell count, cell cycle distribution, percent viable, percent apoptotic, and percent dead cells serially over 72 h. We developed a mathematical model of the cell cycle dynamics after treatment with MP and used it to show that the Molt-4 sensitive controls had a significantly higher rate of cells entering apoptosis (2.7-fold, P<0.00001) relative to the resistant cell lines. Additionally, when treated with MP, the sensitive cell line showed a significant increase in the rate at which cells enter apoptosis compared to its controls (2.4-fold, P<0.00001). Of note, the resistant cell lines had a higher rate of antimetabolite incorporation into the DNA of viable cells (>1.4-fold, P<0.01). Lastly, in contrast to the other cell lines, the Molt-4 resistant subline continued to cycle, though at a rate slower relative to its control, rather than proceed to apoptosis. This led to a larger S-phase block in the Molt-4 resistant cell line, but not a higher rate of cell death. Gene expression of apoptosis, cell cycle, and repair genes were consistent with mechanistic dynamics described by the model. In summary, the mathematical model provides a quantitative assessment to compare the cell cycle effects of MP in cells with varying degrees of MP resistance. PMID:16333308

ATM inhibitor KU-55933 increases the TMZ responsiveness of only inherently TMZ sensitive GBM cells.

PubMed

Nadkarni, Aditi; Shrivastav, Meena; Mladek, Ann C; Schwingler, Paul M; Grogan, Patrick T; Chen, Junjie; Sarkaria, Jann N

2012-12-01

Ataxia telangiectasia mutated (ATM) kinase is critical in sensing and repairing DNA double-stranded breaks (DSBs) such as those induced by temozolomide (TMZ). ATM deficiency increases TMZ sensitivity, which suggests that ATM inhibitors may be effective TMZ sensitizing agents. In this study, the TMZ sensitizing effects of 2 ATM specific inhibitors were studied in established and xenograft-derived glioblastoma (GBM) lines that are inherently sensitive to TMZ and derivative TMZ-resistant lines. In parental U251 and U87 glioma lines, the addition of KU-55933 to TMZ significantly increased cell killing compared to TMZ alone [U251 survival: 0.004 ± 0.0015 vs. 0.08 ± 0.01 (p < 0.001), respectively, and U87 survival: 0.02 ± 0.005 vs. 0.04 ± 0.002 (p < 0.001), respectively] and also elevated the fraction of cells arrested in G2/M [U251 G2/M fraction: 61.8 ± 1.1 % vs. 35 ± 0.8 % (p < 0.001), respectively, and U87 G2/M fraction 25 ± 0.2 % vs.18.6 ± 0.4 % (p < 0.001), respectively]. In contrast, KU-55933 did not sensitize the resistant lines to TMZ, and neither TMZ alone or combined with KU-55933 induced a G2/M arrest. While KU-55933 did not enhance TMZ induced Chk1/Chk2 activation, it increased TMZ-induced residual γ-H2AX foci in the parental cells but not in the TMZ resistant cells. Similar sensitization was observed with either KU-55933 or CP-466722 combined with TMZ in GBM12 xenograft line but not in GBM12TMZ, which is resistant to TMZ due to MGMT overexpression. These findings are consistent with a model where ATM inhibition suppresses the repair of TMZ-induced DSBs in inherently TMZ-sensitive tumor lines, which suggests an ATM inhibitor potentially could be deployed with an improvement in the therapeutic window when combined with TMZ.

Low temperature caused modifications in the arrangement of cell wall pectins due to changes of osmotic potential of cells of maize leaves (Zea mays L.).

PubMed

Bilska-Kos, Anna; Solecka, Danuta; Dziewulska, Aleksandra; Ochodzki, Piotr; Jończyk, Maciej; Bilski, Henryk; Sowiński, Paweł

2017-03-01

The cell wall emerged as one of the important structures in plant stress responses. To investigate the effect of cold on the cell wall properties, the content and localization of pectins and pectin methylesterase (PME) activity, were studied in two maize inbred lines characterized by different sensitivity to cold. Low temperature (14/12 °C) caused a reduction of pectin content and PME activity in leaves of chilling-sensitive maize line, especially after prolonged treatment (28 h and 7 days). Furthermore, immunocytohistological studies, using JIM5 and JIM7 antibodies, revealed a decrease of labeling of both low- and high-methylesterified pectins in this maize line. The osmotic potential, quantified by means of incipient plasmolysis was lower in several types of cells of chilling-sensitive maize line which was correlated with the accumulation of sucrose. These studies present new finding on the effect of cold stress on the cell wall properties in conjunction with changes in the osmotic potential of maize leaf cells.

Identification of flubendazole as potential anti-neuroblastoma compound in a large cell line screen.

PubMed

Michaelis, Martin; Agha, Bishr; Rothweiler, Florian; Löschmann, Nadine; Voges, Yvonne; Mittelbronn, Michel; Starzetz, Tatjana; Harter, Patrick N; Abhari, Behnaz A; Fulda, Simone; Westermann, Frank; Riecken, Kristoffer; Spek, Silvia; Langer, Klaus; Wiese, Michael; Dirks, Wilhelm G; Zehner, Richard; Cinatl, Jaroslav; Wass, Mark N; Cinatl, Jindrich

2015-02-03

Flubendazole was shown to exert anti-leukaemia and anti-myeloma activity through inhibition of microtubule function. Here, flubendazole was tested for its effects on the viability of in total 461 cancer cell lines. Neuroblastoma was identified as highly flubendazole-sensitive cancer entity in a screen of 321 cell lines from 26 cancer entities. Flubendazole also reduced the viability of five primary neuroblastoma samples in nanomolar concentrations thought to be achievable in humans and inhibited vessel formation and neuroblastoma tumour growth in the chick chorioallantoic membrane assay. Resistance acquisition is a major problem in high-risk neuroblastoma. 119 cell lines from a panel of 140 neuroblastoma cell lines with acquired resistance to various anti-cancer drugs were sensitive to flubendazole in nanomolar concentrations. Tubulin-binding agent-resistant cell lines displayed the highest flubendazole IC50 and IC90 values but differences between drug classes did not reach statistical significance. Flubendazole induced p53-mediated apoptosis. The siRNA-mediated depletion of the p53 targets p21, BAX, or PUMA reduced the neuroblastoma cell sensitivity to flubendazole with PUMA depletion resulting in the most pronounced effects. The MDM2 inhibitor and p53 activator nutlin-3 increased flubendazole efficacy while RNAi-mediated p53-depletion reduced its activity. In conclusion, flubendazole represents a potential treatment option for neuroblastoma including therapy-refractory cells.

Identification of flubendazole as potential anti-neuroblastoma compound in a large cell line screen

PubMed Central

Michaelis, Martin; Agha, Bishr; Rothweiler, Florian; Löschmann, Nadine; Voges, Yvonne; Mittelbronn, Michel; Starzetz, Tatjana; Harter, Patrick N.; Abhari, Behnaz A.; Fulda, Simone; Westermann, Frank; Riecken, Kristoffer; Spek, Silvia; Langer, Klaus; Wiese, Michael; Dirks, Wilhelm G.; Zehner, Richard; Cinatl, Jaroslav; Wass, Mark N.; Cinatl, Jindrich

2015-01-01

Flubendazole was shown to exert anti-leukaemia and anti-myeloma activity through inhibition of microtubule function. Here, flubendazole was tested for its effects on the viability of in total 461 cancer cell lines. Neuroblastoma was identified as highly flubendazole-sensitive cancer entity in a screen of 321 cell lines from 26 cancer entities. Flubendazole also reduced the viability of five primary neuroblastoma samples in nanomolar concentrations thought to be achievable in humans and inhibited vessel formation and neuroblastoma tumour growth in the chick chorioallantoic membrane assay. Resistance acquisition is a major problem in high-risk neuroblastoma. 119 cell lines from a panel of 140 neuroblastoma cell lines with acquired resistance to various anti-cancer drugs were sensitive to flubendazole in nanomolar concentrations. Tubulin-binding agent-resistant cell lines displayed the highest flubendazole IC50 and IC90 values but differences between drug classes did not reach statistical significance. Flubendazole induced p53-mediated apoptosis. The siRNA-mediated depletion of the p53 targets p21, BAX, or PUMA reduced the neuroblastoma cell sensitivity to flubendazole with PUMA depletion resulting in the most pronounced effects. The MDM2 inhibitor and p53 activator nutlin-3 increased flubendazole efficacy while RNAi-mediated p53-depletion reduced its activity. In conclusion, flubendazole represents a potential treatment option for neuroblastoma including therapy-refractory cells. PMID:25644037

γ-radiation induces cellular sensitivity and aberrant methylation in human tumor cell lines.

PubMed

Kumar, Ashok; Rai, Padmalatha S; Upadhya, Raghavendra; Vishwanatha; Prasada, K Shama; Rao, B S Satish; Satyamoorthy, Kapettu

2011-11-01

Ionizing radiation induces cellular damage through both direct and indirect mechanisms, which may include effects from epigenetic changes. The purpose of this study was to determine the effect of ionizing radiation on DNA methylation patterns that may be associated with altered gene expression. Sixteen human tumor cell lines originating from various cancers were initially tested for radiation sensitivity by irradiating them with γ-radiation in vitro and subsequently, radiation sensitive and resistant cell lines were treated with different doses of a demethylating agent, 5-Aza-2'-Deoxycytidine (5-aza-dC) and a chromatin modifier, Trichostatin-A (TSA). Survival of these cell lines was measured using 3-(4, 5-Dimethylthiazol- 2-yl)-2, 5-diphenyltetrazolium (MTT) and clonogenic assays. The effect of radiation on global DNA methylation was measured using reverse phase high performance liquid chromatography (RP-HPLC). The transcription response of methylated gene promoters, from cyclin-dependent kinase inhibitor 2A (p16(INK4a)) and ataxia telangiectasia mutated (ATM) genes, to radiation was measured using a luciferase reporter assay. γ-radiation resistant (SiHa and MDAMB453) and sensitive (SaOS2 and WM115) tumor cell lines were examined for the relationship between radiation sensitivity and DNA methylation. Treatment of cells with 5-aza-dC and TSA prior to irradiation enhanced DNA strand breaks, G2/M phase arrest, apoptosis and cell death. Exposure to γ-radiation led to global demethylation in a time-dependent manner in tumor cells in relation to resistance and sensitivity to radiation with concomitant activation of p16(INK4a) and ATM gene promoters. These results provide important information on alterations in DNA methylation as one of the determinants of radiation effects, which may be associated with altered gene expression. Our results may help in delineating the mechanisms of radiation resistance in tumor cells, which can influence diagnosis, prognosis and eventually therapy for human cancers.

Anti-tumor activity of olaparib, a poly (ADP-ribose) polymerase (PARP) inhibitor, in cultured endometrial carcinoma cells

PubMed Central

2014-01-01

Background PTEN inactivation is the most frequent genetic aberration in endometrial cancer. One of the phosphatase-independent roles of PTEN is associated with homologous recombination (HR) in nucleus. Poly (ADP-ribose) polymerase (PARP) plays key roles in the repair of DNA single-strand breaks, and a PARP inhibitor induces synthetic lethality in cancer cells with HR deficiency. We examined the anti-tumor activity of olaparib, a PARP inhibitor, and its correlation between the sensitivity and status of PTEN in endometrial cancer cell lines. Methods The response to olaparib was evaluated using a clonogenic assay with SF50 values (concentration to inhibit cell survival to 50%) in 16 endometrial cancer cell lines. The effects of PTEN on the sensitivity to olaparib and ionizing radiation (IR) exposure were compared between parental HEC-6 (PTEN-null) and HEC-6 PTEN + (stably expressing wild-type PTEN) cells by clonogenic assay, foci formation of RAD51 and γH2AX, and induction of cleaved PARP. The effects of siRNA to PTEN were analyzed in cells with wild-type PTEN. Results The SF50 values were 100 nM or less in four (25%: sensitive) cell lines; whereas, SF50 values were 1,000 nM or more in four (25%: resistant) cell lines. PTEN mutations were not associated with sensitivity to olaparib (Mutant [n = 12]: 746 ± 838 nM; Wild-type [n = 4]: 215 ± 85 nM, p = 0.26 by Student’s t test). RAD51 expression was observed broadly and was not associated with PTEN status in the 16 cell lines. The number of colonies in the clonogenic assay, the foci formation of RAD51 and γH2AX, and the induction of apoptosis were not affected by PTEN introduction in the HEC-6 PTEN + cells. The expression level of nuclear PTEN was not elevated within 24 h following IR in the HEC-6-PTEN + cells. In addition, knocking down PTEN by siRNA did not alter the sensitivity to olaparib in 2 cell lines with wild-type PTEN. Conclusions Our results suggest that olaparib, a PARP inhibitor, is effective on certain endometrial cancer cell lines. Inactivation of PTEN might not affect the DNA repair function. Predictive biomarkers are warranted to utilize olaparib in endometrial cancer. PMID:24625059

Improvement of the antiproliferative effect of rapamycin on tumor cell lines by poly (monomethylitaconate)-based pH-sensitive, plasma stable liposomes.

PubMed

Ghanbarzadeh, Saeed; Arami, Sanam; Pourmoazzen, Zhaleh; Khorrami, Arash

2014-03-01

pH-responsive polymers produce liposomes with pH-sensitive property which can release their encapsulated drug under mild acidic conditions found inside the cellular endosomes, inflammatory tissues and cancerous cells. The aim of this study was preparing pH-sensitive and plasma stable liposomes in order to enhance the selectivity and antiproliferative effect of Rapamycin. In the present study we used PEG-poly (monomethylitaconate)-CholC6 (PEG-PMMI-CholC6) copolymer and Oleic acid (OA) to induce pH-sensitive property in Rapamycin liposomes. pH-sensitive liposomal formulations bearing copolymer PEG-PMMI-CholC6 and OA were characterized in regard to physicochemical stability, pH-responsiveness and stability in human plasma. The ability of pH-sensitive liposomes in enhancing the cytotoxicity of Rapamycin was evaluated in vitro by using colon cancer cell line (HT-29) and compared with its cytotoxicity on human umbilical vein endothelial cell (HUVEC) line. Both formulations were found to release their contents under mild acidic conditions rapidly. However, unlike OA-based liposomes, the PEG-PMMI-CholC6 bearing liposomes preserved their pH-sensitivity in plasma. Both types of pH-sensitive Rapamycin-loaded liposomes exhibited high physicochemical stability and could deliver antiproliferative agent into HT-29 cells much more efficiently in comparison with conventional liposomes. Conversely, the antiproliferative effect of pH-sensitive liposomes on HUVEC cell line was less than conventional liposomes. This study showed that both OA and PEG-PMMI-CholC6-based vesicles could submit pH-sensitive property, however, only PEG-PMMI-CholC6-based liposomes could preserve pH-sensitive property after incubation in plasma. As a result pH-sensitive PEG-PMMI-CholC6-based liposomal formulation can improve the selectivity, stability and antiproliferative effect of Rapamycin. Copyright © 2014 Elsevier B.V. All rights reserved.

Ectopic expression of protein kinase C-β sensitizes head and neck squamous cell carcinoma to diterpene esters.

PubMed

Adams, Ryan A; D'Souza, Marjorie M A; Pierce, Carly J; Korica, Natasa; Wallwork, Ben; Parsons, Peter G; Panizza, Benedict; Boyle, Glen M

2015-03-01

The objective of this study was to examine the effect of specific Protein kinase C (PKC) isoform re-expression in solid malignancies, particularly head and neck squamous cell carcinoma cell lines, and the impact this may have on treatment with known activators of PKC. The constitutive expression of PKC isoforms were determined in six head and neck squamous cell carcinoma (SCC) cell lines. Cytotoxicity of the prototypic phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA) and the novel diterpene ester PEP005 was established. Viral transduction to re-express PKCβ isoforms in two of these cell lines was performed, and its effect on the sensitivity to the compounds was quantified. Tongue and hypopharyngeal SCC cell lines were resistant to both TPA and PEP005, with the concentration required to inhibit growth by 50% (IC50) being >1,000 ng/ml. CAL-27 (tongue SCC) and FaDu (hypopharyngeal SCC) cell lines re-expressing PKCβI and -βII isoforms demonstrated IC50 of 1-5 ng/ml with TPA or PEP005. Re-expression of PKCβ in head and neck SCC cell lines leads to cells one thousand-times more sensitive to the cytotoxic effects of phorbol or diterpene esters in culture. This highlights the importance of the isoform in tumor progression and presents the potential benefit of these compounds in malignancies expressing the protein, and in combination therapy. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Combination of suberoylanilide hydroxamic acid with heavy ion therapy shows promising effects in infantile sarcoma cell lines

PubMed Central

2011-01-01

Introduction The pan-HDAC inhibitor (HDACI) suberoylanilide hydroxamic acid (SAHA) has previously shown to be a radio-sensitizer to conventional photon radiotherapy (XRT) in pediatric sarcoma cell lines. Here, we investigate its effect on the response of two sarcoma cell lines and a normal tissue cell line to heavy ion irradiation (HIT). Materials and methods Clonogenic assays after different doses of heavy ions were performed. DNA damage and repair were evaluated by measuring γH2AX via flow-cytometry. Apoptosis and cell cycle analysis were also measured via flow cytometry. Protein expression of repair proteins, p53 and p21 were measured using immunoblot analysis. Changes of nuclear architecture after treatment with SAHA and HIT were observed in one of the sarcoma cell lines via light microscopy after staining towards chromatin and γH2AX. Results Corresponding with previously reported photon data, SAHA lead to an increase of sensitivity to heavy ions along with an increase of DSB and apoptosis in the two sarcoma cell lines. In contrast, in the osteoblast cell line (hFOB 1.19), the combination of SAHA and HIT showed a significant radio-protective effect. Laser scanning microscopy revealed no significant morphologic changes after HIT compared to the combined treatment with SAHA. Immunoblot analysis revealed no significant up or down regulation of p53. However, p21 was significantly increased by SAHA and combination treatment as compared to HIT only in the two sarcoma cell lines - again in contrast to the osteoblast cell line. Changes in the repair kinetics of DSB p53-independent apoptosis with p21 involvement may be part of the underlying mechanisms for radio-sensitization by SAHA. Conclusion Our in vitro data suggest an increase of the therapeutic ratio by the combination of SAHA with HIT in infantile sarcoma cell lines. PMID:21933400

Evaluation of the skin sensitization potential of chemicals using expression of co-stimulatory molecules, CD54 and CD86, on the naive THP-1 cell line.

PubMed

Yoshida, Y; Sakaguchi, H; Ito, Y; Okuda, M; Suzuki, H

2003-04-01

It has been known that dendritic cells (DCs) including Langerhans cells (LCs) play a critical role in the skin sensitization process. Many attempts have been made to develop in vitro sensitization tests that employ DCs derived from peripheral blood mononuclear cells (PBMC-DC) or CD34+ hematopoietic progenitor cells (CD34+ HPC) purified from cord blood or bone marrow. However, the use of the DCs in in vitro methods has been difficult due to the nature of these cells such as low levels in the source and/or donor-to-donor variability. In our studies, we employed the human monocytic leukemia cell line, THP-1, in order to avoid some of these difficulties. At the start, we examined whether treatment of the cells with various cytokines could produce DCs from THP-1. Treatment of THP-1 cells with cytokines such as GM-CSF, IL-4, TNF-alpha, and/or PMA did induce some phenotypic changes in THP-1 cells that were characteristic of DCs. Subsequently, responses to a known sensitizer, dinitrochlorobenzene (DNCB), and a non-sensitizer, dimethyl sulfoxide (DMSO) or sodium lauryl sulfate (SLS), on the expression of co-stimulatory molecules, CD54 and CD86, were examined between the naive cells and the cytokine-treated cells. Interestingly, the naive THP-1 cells responded only to DNCB and the response to the sensitizer was more distinct than cytokine-treated THP-1 cells. Similar phenomena were also observed in the human myeloid leukemia cell line, KG-1. Furthermore, with treatment of DNCB, naive THP-1 cells showed augmented expression of HLA, CD80 and secretion of IL-1 beta. The response of THP-1 cells to a sensitizer was similar to that of LCs/DCs. Upon demonstrating the differentiation of monocyte cells in our system, we then evaluated a series of chemicals, including known sensitizers and non-sensitizers, for their potential to augment CD54 and CD86 expression on naive THP-1 cells. Indeed, known sensitizers such as PPD and 2-MBT significantly augmented CD54 and CD86 expression in a dose-dependent manner while non-sensitizers, such as SLS and methyl salicylate (MS), did not. To note, the metal allergens such as (NH(4))(2)[PtCl(4)], NiSO(4) and CoSO(4) augmented significantly only CD54 expression. Taking advantage of a cultured cell line, measurement of the co-stimulatory molecules, CD54 and CD86, on naive THP-1 cells following chemical exposure shows promise for the development of a simple, short-term in vitro sensitization test.

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

Raman spectroscopy differentiates between sensitive and resistant multiple myeloma cell lines

NASA Astrophysics Data System (ADS)

Franco, Domenico; Trusso, Sebastiano; Fazio, Enza; Allegra, Alessandro; Musolino, Caterina; Speciale, Antonio; Cimino, Francesco; Saija, Antonella; Neri, Fortunato; Nicolò, Marco S.; Guglielmino, Salvatore P. P.

2017-12-01

Current methods for identifying neoplastic cells and discerning them from their normal counterparts are often nonspecific and biologically perturbing. Here, we show that single-cell micro-Raman spectroscopy can be used to discriminate between resistant and sensitive multiple myeloma cell lines based on their highly reproducible biomolecular spectral signatures. In order to demonstrate robustness of the proposed approach, we used two different cell lines of multiple myeloma, namely MM.1S and U266B1, and their counterparts MM.1R and U266/BTZ-R subtypes, resistant to dexamethasone and bortezomib, respectively. Then, micro-Raman spectroscopy provides an easily accurate and noninvasive method for cancer detection for both research and clinical environments. Characteristic peaks, mostly due to different DNA/RNA ratio, nucleic acids, lipids and protein concentrations, allow for discerning the sensitive and resistant subtypes. We also explored principal component analysis (PCA) for resistant cell identification and classification. Sensitive and resistant cells form distinct clusters that can be defined using just two principal components. The identification of drug-resistant cells by confocal micro-Raman spectroscopy is thus proposed as a clinical tool to assess the development of resistance to glucocorticoids and proteasome inhibitors in myeloma cells.

Restoring E-cadherin expression increases sensitivity to epidermal growth factor receptor inhibitors in lung cancer cell lines.

PubMed

Witta, Samir E; Gemmill, Robert M; Hirsch, Fred R; Coldren, Christopher D; Hedman, Karla; Ravdel, Larisa; Helfrich, Barbara; Dziadziuszko, Rafal; Chan, Daniel C; Sugita, Michio; Chan, Zeng; Baron, Anna; Franklin, Wilbur; Drabkin, Harry A; Girard, Luc; Gazdar, Adi F; Minna, John D; Bunn, Paul A

2006-01-15

The epidermal growth factor receptor (EGFR) is overexpressed in the majority of non-small cell lung cancers (NSCLC). EGFR tyrosine kinase inhibitors, such as gefitinib and erlotinib, produce 9% to 27% response rates in NSCLC patients. E-Cadherin, a calcium-dependent adhesion molecule, plays an important role in NSCLC prognosis and progression, and interacts with EGFR. The zinc finger transcriptional repressor, ZEB1, inhibits E-cadherin expression by recruiting histone deacetylases (HDAC). We identified a significant correlation between sensitivity to gefitinib and expression of E-cadherin, and ZEB1, suggesting their predictive value for responsiveness to EGFR-tyrosine kinase inhibitors. E-Cadherin transfection into a gefitinib-resistant line increased its sensitivity to gefitinib. Pretreating resistant cell lines with the HDAC inhibitor, MS-275, induced E-cadherin along with EGFR and led to a growth-inhibitory and apoptotic effect of gefitinib similar to that in gefitinib-sensitive NSCLC cell lines including those harboring EGFR mutations. Thus, combined HDAC inhibitor and gefitinib treatment represents a novel pharmacologic strategy for overcoming resistance to EGFR inhibitors in patients with lung cancer.

The anticancer properties of iron core–gold shell nanoparticles in colorectal cancer cells

PubMed Central

Wu, Ya-Na; Wu, Ping-Ching; Yang, Li-Xing; Ratinac, Kyle R; Thordarson, Pall; Jahn, Kristina A; Chen, Dong-Hwang; Shieh, Dar-Bin; Braet, Filip

2013-01-01

Previously, iron core–gold shell nanoparticles (Fe@Au) have been shown to possess cancer-preferential cytotoxicity in oral and colorectal cancer (CRC) cells. However, CRC cell lines are less sensitive to Fe@Au treatment when compared with oral cancer cell lines. In this research, Fe@Au are found to decrease the cell viability of CRC cell lines, including Caco-2, HT-29, and SW480, through growth inhibition rather than the induction of cell death. The cytotoxicity induced by Fe@Au in CRC cells uses different subcellular pathways to the mitochondria-mediated autophagy found in Fe@Au-treated oral cancer cells, OECM1. Interestingly, the Caco-2 cell line shows a similar response to OECM1 cells and is thus more sensitive to Fe@Au treatment than the other CRC cell lines studied. We have investigated the underlying cell resistance mechanisms of Fe@Au-treated CRC cells. The resistance of CRC cells to Fe@Au does not result from the total amount of Fe@Au internalized. Instead, the different amounts of Fe and Au internalized appear to determine the different response to treatment with Fe-only nanoparticles in Fe@Au-resistant CRC cells compared with the Fe@Au-sensitive OECM1 cells. The only moderately cytotoxic effect of Fe@Au nanoparticles on CRC cells, when compared to the highly sensitive OECM1 cells, appears to arise from the CRC cells’ relative insensitivity to Fe, as is demonstrated by our Fe-only treatments. This is a surprising outcome, given that Fe has thus far been considered to be the “active” component of Fe@Au nanoparticles. Instead, we have found that the Au coatings, previously considered only as a passivating coating to protect the Fe cores from oxidation, significantly enhance the cytotoxicity of Fe@Au in certain CRC cells. Therefore, we conclude that both the Fe and Au in these core–shell nanoparticles are essential for the anticancer properties observed in CRC cells. PMID:24039416

Cytological characterization of a thermo-sensitive cytoplasmic male-sterile wheat line having K-type cytoplasm of Aegilops kotschyi.

PubMed

Meng, Liying; Liu, Zihan; Zhang, Lingli; Hu, Gan; Song, Xiyue

2016-12-01

Male sterility is an important tool for obtaining crop heterosis. A thermo-sensitive cytoplasmic male-sterile (TCMS) line was developed recently using a new method based on tiller regeneration. In the present study, we explored the critical growth stages required to maintain thermo-sensitive male sterility in TCMS lines and found that fertility is associated with abnormal tapetal and microspore development. We investigated the fertility and cytology of temperature-treated plant anthers at various developmental stages. TCMS line KTM3315A exhibited thermo-sensitive male sterility in Zadoks growth stages 41-49 and 58-59. Morphologically, the line exhibited thermo-sensitive male sterility at 3-9 days before heading and at 3-6 days before flowering, and it was partially restored in three locations during spring and summer. TCMS line KTM3315A plants exhibited premature tapetal programmed cell death (PCD) from the early uninucleate stage of microspore development until the tapetal cells degraded completely. Microspore development was then blocked and the pollen abortion type was stainable abortion. Thus, male fertility in the line KTM3315A is sensitive to temperature and premature tapetal PCD is the main cause of pollen abortion, where it determines the starting period and affects male fertility conversion in K-type TCMS lines at certain temperatures.

Social isolation increases number of newly proliferated cells in hippocampus in female flinders sensitive line rats.

PubMed

Bjørnebekk, Astrid; Mathé, Aleksander A; Gruber, Susanne H M; Brené, Stefan

2007-01-01

Genetic background influences the responsiveness to stress and plays a crucial role in the pathophysiology of depression. In an animal model of depression, Flinders Sensitive Line rats, and Sprague Dawley controls we analyzed if 7 weeks of social isolation of adult animals affect the number of newly proliferated cells in the dentate gyrus or mRNAs of Neuropeptide Y (NPY), the NPY-Y1 receptor, nociceptin, BDNF, and the serotonin 5HT1A and 5HT2A receptors, which are molecules involved in hippocampal plasticity. Since depressive illness more frequently affects women than men, and females seem to respond differently to stressful experiences than males, female rats were used in this study. Bromodeoxyuridine, which is a thymidin analogue that is incorporated into the DNA of newly formed cells, was administered during 9 days to even out the effects of hormonal fluctuations. Social isolation increased the number of newly proliferated Bromodeoxyuridine-immunoreactive cells in the Flinders Sensitive Line rats, whereas it had no impact on the number of cells in the Sprague Dawley strain. Group housed Sprague Dawley rats had a higher expression of BDNF, NPY, and the serotonin 5HT2A receptor mRNA than "depressed" Flinders Sensitive Line. Social isolation downregulated these molecules in Sprague Dawley but not in Flinders Sensitive Line rats thereby eliminating the differences between the two strains. We demonstrate strain and gender specific responses to stress induced regulation of factors important for hippocampal plasticity. (c) 2007 Wiley-Liss, Inc.

Redox Signaling and Bioenergetics Influence Lung Cancer Cell Line Sensitivity to the Isoflavone ME-344.

PubMed

Manevich, Yefim; Reyes, Leticia; Britten, Carolyn D; Townsend, Danyelle M; Tew, Kenneth D

2016-08-01

ME-344 [(3R,4S)-3,4-bis(4-hydroxyphenyl)-8-methyl-3,4-dihydro-2H-chromen-7-ol] is a second-generation derivative natural product isoflavone presently under clinical development. ME-344 effects were compared in lung cancer cell lines that are either intrinsically sensitive or resistant to the drug and in primary immortalized human lung embryonic fibroblasts (IHLEF). Cytotoxicity at low micromolar concentrations occurred only in sensitive cell lines, causing redox stress, decreased mitochondrial ATP production, and subsequent disruption of mitochondrial function. In a dose-dependent manner the drug caused instantaneous and pronounced inhibition of oxygen consumption rates (OCR) in drug-sensitive cells (quantitatively significantly less in drug-resistant cells). This was consistent with targeting of mitochondria by ME-344, with specific effects on the respiratory chain (resistance correlated with higher glycolytic indexes). OCR inhibition did not occur in primary IHLEF. ME-344 increased extracellular acidification rates in drug-resistant cells (significantly less in drug-sensitive cells), implying that ME-344 targets mitochondrial proton pumps. Only in drug-sensitive cells did ME-344 dose-dependently increase the intracellular generation of reactive oxygen species and cause oxidation of total (mainly glutathione) and protein thiols and the concomitant immediate increases in NADPH levels. We conclude that ME-344 causes complex, redox-specific, and mitochondria-targeted effects in lung cancer cells, which differ in extent from normal cells, correlate with drug sensitivity, and provide indications of a beneficial in vitro therapeutic index. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

Development of an in vitro skin sensitization test using human cell lines; human Cell Line Activation Test (h-CLAT). II. An inter-laboratory study of the h-CLAT.

PubMed

Sakaguchi, H; Ashikaga, T; Miyazawa, M; Yoshida, Y; Ito, Y; Yoneyama, K; Hirota, M; Itagaki, H; Toyoda, H; Suzuki, H

2006-08-01

Recent regulatory changes have placed a major emphasis on in vitro safety testing and alternative models. In regard to skin sensitization tests, dendritic cells (DCs) derived from human peripheral blood have been considered in the development of new in vitro alternatives. Human cell lines have been also reported recently. In our previous study, we suggested that measuring CD86 and/or CD54 expression on THP-1 cells (human monocytic leukemia cell line) could be used as an in vitro skin sensitization method. An inter-laboratory study among two laboratories was undertaken in Japan in order to further develop an in vitro skin sensitization model. In the present study, we used two human cell lines: THP-1 and U-937 (human histiocytic lymphoma cell line). First we optimized our test protocol (refer to the related paper entitled "optimization of the h-CLAT protocol" within this journal) and then we did an inter-laboratory validation with nine chemicals using the optimized protocol. We measured the expression of CD86 and CD54 on the above cells using flow cytometry after a 24h and 48h exposure to six known allergens (e.g., DNCB, pPD, NiSO(4)) and three non-allergens (e.g., SLS, tween 80). For the sample test concentration, four doses (0.1x, 0.5x, 1x, and 2x of the 50% inhibitory concentration (IC(50))) were evaluated. IC(50) was calculated using MTT assay. We found that allergens/non-allergens were better predicted using THP-1 cells compared to U-937 cells following a 24 h and a 48 h exposure. We also found that the 24h treatment time tended to have a better accuracy than the 48 h treatment time for THP-1 cells. Expression of CD86 and CD54 were good predictive markers for THP-1 cells, but for U-937 cells, expression of CD86 was a better predictor than CD54, at the 24h and the 48 h treatment time. The accuracy also improved when both markers (CD86 and CD54) were used as compared with a single marker for THP-1 cells. Both laboratories gave a good prediction of allergen/non-allergen, especially using THP-1 cells. These results suggest that our method, human Cell Line Activation Test (h-CLAT), using human cell lines THP-1 and U-937, but especially THP-1 cells at 24h treatment, may be a useful in vitro skin sensitization model to predict various contact allergens.

Pharmacodynamics of cisplatin in human head and neck cancer: correlation between platinum content, DNA adduct levels and drug sensitivity in vitro and in vivo

PubMed Central

Welters, M J P; Fichtinger-Schepman, A M J; Baan, R A; Jacobs-Bergmans, A J; Kegel, A; van der Vijgh, W J F; Braakhuis, B J M

1999-01-01

Total platinum contents and cisplatin–DNA adduct levels were determined in vivo in xenografted tumour tissues in mice and in vitro in cultured tumour cells of head and neck squamous cell carcinoma (HNSCC), and correlated with sensitivity to cisplatin. In vivo, a panel of five HNSCC tumour lines growing as xenografts in nude mice was used. In vitro, the panel consisted of five HNSCC cell lines, of which four had an in vivo equivalent. Sensitivity to cisplatin varied three- to sevenfold among cell lines and tumours respectively. However, the ranking of the sensitivities of the tumour lines (in vivo), also after reinjection of the cultured tumour cells, did not coincide with that of the corresponding cell lines, which showed that cell culture systems are not representative for the in vivo situation. Both in vitro and in vivo, however, significant correlations were found between total platinum levels, measured by atomic absorption spectrophotometry (AAS), and tumour response to cisplatin therapy at all time points tested. The levels of the two major cisplatin–DNA adduct types were determined by a recently developed and improved32P post-labelling assay at various time points after cisplatin treatment. Evidence is presented that the platinum–AG adduct, in which platinum is bound to guanine and an adjacent adenine, may be the cytotoxic lesion because a significant correlation was found between the platinum–AG levels and the sensitivities in our panel of HNSCC, in vitro as well as in vivo. This correlation with the platinum–AG levels was established at 1 h (in vitro) and 3 h (in vivo) after the start of the cisplatin treatment, which emphasizes the importance of early sampling. © 1999 Cancer Research Campaign PMID:10408697

Cytotoxicity assays with fish cells as an alternative to the acute lethality test with fish.

PubMed

Segner, Helmut

2004-10-01

In ecotoxicology, in vitro assays with fish cells are currently applied for mechanistic studies, bioanalytical purposes and toxicity screening. This paper discusses the potential of cytotoxicity assays with fish cells to reduce, refine or replace acute lethality tests using fish. Basal cytotoxicity data obtained with fish cell lines or fish primary cell cultures show a reasonable to good correlation with lethality data from acute toxicity tests, with the exception of compounds that exert a specific mode of toxic action. Basal cytotoxicity data from fish cell lines also correlate well with cytotoxicity data from mammalian cell lines. However, both the piscine and mammalian in vitro assays are clearly less sensitive than the fish test. Therefore, in vivo LC50 values (concentrations of the test compounds that are lethal to 50% of the fish in the experiment within 96 hours) currently cannot be predicted from in vitro values. This in vitro-in vivo difference in sensitivity appears to be true for both fish cell lines and mammalian cell lines. Given the good in vitro-in vivo correlation in toxicity ranking, together with the clear-cut difference in sensitivity, the role of cytotoxicity assays in a tiered alternative testing strategy could be in priority setting in relation to toxic hazard and in the toxicity classification of chemicals and environmental samples.

RasGRP1 confers the phorbol ester-sensitive phenotype to EL4 lymphoma cells.

PubMed

Han, Shujie; Knoepp, Stewart M; Hallman, Mark A; Meier, Kathryn E

2007-01-01

The murine EL4 lymphoma cell line exists in variants that are either sensitive or resistant to the tumor promoter phorbol 12-myristate 13-acetate (PMA). In sensitive EL4 cells, PMA causes robust Erk mitogen-activated protein kinase activation that results in growth arrest. In resistant cells, PMA induces minimal Erk activation, without growth arrest. PMA stimulates IL-2 production in sensitive, but not resistant, cells. The role of RasGRP1, a PMA-activated guanine nucleotide exchange factor for Ras, in EL4 phenotype was examined. Endogenous RasGRP1 protein is expressed at much higher levels in sensitive than in resistant cells. PMA-induced Ras activation is observed in sensitive cells but not in resistant cells lacking Ras-GRP1. PMA induces down-regulation of RasGRP1 protein in sensitive cells but increases RasGRP1 in resistant cells. Transfection of RasGRP1 into resistant cells enhances PMA-induced Erk activation. In the reverse experiment, introduction of small interfering RNA (siRNA) for RasGRP1 suppresses PMA-induced Ras and Erk activations in sensitive cells. Sensitive cells incubated with siRNA for RasGRP1 exhibit the PMA-resistant phenotype, in that they are able to proliferate in the presence of PMA and do not secrete IL-2 when stimulated with PMA. These studies indicate that the PMA-sensitive phenotype, as previously defined for the EL4 cell line, is conferred by endogenous expression of RasGRP1 protein.

Metabolic determinants of cancer cell sensitivity to glucose limitation and biguanides

NASA Astrophysics Data System (ADS)

Birsoy, Kıvanç; Possemato, Richard; Lorbeer, Franziska K.; Bayraktar, Erol C.; Thiru, Prathapan; Yucel, Burcu; Wang, Tim; Chen, Walter W.; Clish, Clary B.; Sabatini, David M.

2014-04-01

As the concentrations of highly consumed nutrients, particularly glucose, are generally lower in tumours than in normal tissues, cancer cells must adapt their metabolism to the tumour microenvironment. A better understanding of these adaptations might reveal cancer cell liabilities that can be exploited for therapeutic benefit. Here we developed a continuous-flow culture apparatus (Nutrostat) for maintaining proliferating cells in low-nutrient media for long periods of time, and used it to undertake competitive proliferation assays on a pooled collection of barcoded cancer cell lines cultured in low-glucose conditions. Sensitivity to low glucose varies amongst cell lines, and an RNA interference (RNAi) screen pinpointed mitochondrial oxidative phosphorylation (OXPHOS) as the major pathway required for optimal proliferation in low glucose. We found that cell lines most sensitive to low glucose are defective in the OXPHOS upregulation that is normally caused by glucose limitation as a result of either mitochondrial DNA (mtDNA) mutations in complex I genes or impaired glucose utilization. These defects predict sensitivity to biguanides, antidiabetic drugs that inhibit OXPHOS, when cancer cells are grown in low glucose or as tumour xenografts. Notably, the biguanide sensitivity of cancer cells with mtDNA mutations was reversed by ectopic expression of yeast NDI1, a ubiquinone oxidoreductase that allows bypass of complex I function. Thus, we conclude that mtDNA mutations and impaired glucose utilization are potential biomarkers for identifying tumours with increased sensitivity to OXPHOS inhibitors.

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.

Induction of arginosuccinate synthetase (ASS) expression affects the antiproliferative activity of arginine deiminase (ADI) in melanoma cells.

PubMed

Manca, Antonella; Sini, Maria Cristina; Izzo, Francesco; Ascierto, Paolo A; Tatangelo, Fabiana; Botti, Gerardo; Gentilcore, Giusy; Capone, Marilena; Mozzillo, Nicola; Rozzo, Carla; Cossu, Antonio; Tanda, Francesco; Palmieri, Giuseppe

2011-06-01

Arginine deiminase (ADI), an arginine-degrading enzyme, has been used in the treatment of tumours sensitive to arginine deprivation, such as malignant melanoma (MM) and hepatocellular carcinoma (HCC). Endogenous production of arginine is mainly dependent on activity of ornithine transcarbamylase (OTC) and argininosuccinate synthetase (ASS) enzymes. We evaluated the effect of ADI treatment on OTC and ASS expression in a series of melanoma cell lines. Twenty-five primary melanoma cell lines and normal fibroblasts as controls underwent cell proliferation assays and Western blot analyses in the presence or absence of ADI. Tissue sections from primary MMs (N = 20) and HCCs (N = 20) were investigated by immunohistochemistry for ASS expression. Overall, 21/25 (84%) MM cell lines presented a cell growth inhibition by ADI treatment; none of them presented constitutive detectable levels of the ASS protein. However, 7/21 (33%) ADI-sensitive melanoma cell lines presented markedly increased expression levels of the ASS protein following ADI treatment, with a significantly higher IC50 median value. Growth was not inhibited and the IC50 was not reached among the remaining 4/25 (16%) MM cell lines; all of them showed constitutive ASS expression. The OTC protein was found expressed in all melanoma cell lines before and after the ADI treatment. Lack of ASS immunostaining was observed in all analyzed in vivo specimens. Our findings suggest that response to ADI treatment in melanoma is significantly correlated with the ability of cells to express ASS either constitutively at basal level (inducing drug resistance) or after the treatment (reducing sensitivity to ADI).

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.

The In Vivo DNA Binding Properties of Wild-Type and Mutant p53 Proteins in Mammary Cell Lines During the Course of Cell Cycle.

DTIC Science & Technology

1996-08-01

J-4030 TITLE: The In Vivo DNA Binding Properties of Wild-Type and Mutant p53 Proteins in Mammary Cell Lines During the Course of Cell Cycle PRINCIPAL...The In Vivo DNA Binding Properties of 5. FUNDING NUMBERS Wild-Type and Mutant p53 Proteins in Mammary Cell Lines DAMD17-94-J-4030 During the Course of...ABSTRACT (Maximum 200 Using a pair of murine cell lines, one lacking p53 and a derivative cell line containing temperature sensitive p53 val 135

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.

RWEN: Response-Weighted Elastic Net For Prediction of Chemosensitivity of Cancer Cell Lines. | Office of Cancer Genomics

Cancer.gov

Motivation: In recent years there have been several efforts to generate sensitivity profiles of collections of genomically characterized cell lines to panels of candidate therapeutic compounds. These data provide the basis for the development of in silico models of sensitivity based on cellular, genetic, or expression biomarkers of cancer cells. However, a remaining challenge is an efficient way to identify accurate sets of biomarkers to validate.

Hypoxia-induced resistance to doxorubicin and methotrexate in human melanoma cell lines in vitro.

PubMed

Sanna, K; Rofstad, E K

1994-07-15

Rodent cell lines can develop resistance to doxorubicin and methotrexate during hypoxic stress. This has so far not been observed in human tumor cell lines. The purpose of our communication is to show that doxorubicin and methotrexate resistance can also develop in human melanoma cells during exposure to hypoxia. Four cell lines (BEX-c, COX-c, SAX-c, WIX-c) have been studied. Cells were exposed to hypoxia (O2 concentration < 10 ppm) for 24 hr prior to reoxygenation. Doxorubicin and methotrexate cell survival curves were determined immediately after as well as 18 and 42 hr after reoxygenation. The 4 cell lines were relatively sensitive to doxorubicin without hypoxia pre-treatment, and all developed resistance during exposure to hypoxia. Hypoxic stress also induced methotrexate resistance in BEX-c and SAX-c but not in COX-c and WIX-c. BEX-c and SAX-c were sensitive to methotrexate without hypoxia pre-treatment, whereas COX-c and WIX-c were resistant initially. Hypoxia-induced drug resistance was present immediately after reoxygenation and tended to decrease with time but remained statistically significant even 42 hr after reoxygenation.

Identification of an "Exceptional Responder" Cell Line to MEK1 Inhibition: Clinical Implications for MEK-Targeted Therapy | Office of Cancer Genomics

Cancer.gov

The identification of somatic genetic alterations that confer sensitivity to pharmacologic inhibitors has led to new cancer therapies. To identify mutations that confer an exceptional dependency, shRNA-based loss-of-function data were analyzed from a dataset of numerous cell lines to reveal genes that are essential in a small subset of cancer cell lines. Once these cell lines were determined, detailed genomic characterization from these cell lines was utilized to ascertain the genomic aberrations that led to this extreme dependency.

Diphtheria toxin translocation across cellular membranes is regulated by sphingolipids

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

Spilsberg, Bjorn; Hanada, Kentaro; Sandvig, Kirsten

2005-04-08

Diphtheria toxin is translocated across cellular membranes when receptor-bound toxin is exposed to low pH. To study the role of sphingolipids for toxin translocation, both a mutant cell line lacking the first enzyme in de novo sphingolipid synthesis, serine palmitoyltransferase, and a specific inhibitor of the same enzyme, myriocin, were used. The serine palmitoyltransferase-deficient cell line (LY-B) was found to be 10-15 times more sensitive to diphtheria toxin than the genetically complemented cell line (LY-B/cLCB1) and the wild-type cell line (CHO-K1), both when toxin translocation directly across the plasma membrane was induced by exposing cells with surface-bound toxin to lowmore » pH, and when the toxin followed its normal route via acidified endosomes into the cytosol. Toxin binding was similar in these three cell lines. Furthermore, inhibition of serine palmitoyltransferase activity by addition of myriocin sensitized the two control cell lines (LY-B/cLCB1 and CHO-K1) to diphtheria toxin, whereas, as expected, no effect was observed in cells lacking serine palmitoyltransferase (LY-B). In conclusion, diphtheria toxin translocation is facilitated by depletion of membrane sphingolipids.« less

Protein disulfide isomerases: Impact of thapsigargin treatment on their expression in melanoma cell lines.

PubMed

Silva, Zélia; Veríssimo, Teresa; Videira, Paula A; Novo, Carlos

2015-08-01

Anti-cancer treatments usually elevate the content of unfolded or misfolded proteins in the endoplasmic reticulum (ER). Here we aimed to get insights into the relation between sensitivity of melanoma cell lines to the ER stress inducer thapsigargin (THG) and the genetic expression of protein disulfide isomerase family members (PDIs). The expression of PDIs was analysed by flow cytometry and real-time PCR. The results showed that SK-MEL-30, the less THG sensitive cell line, displays higher basal PDIs' expression levels and the sensitivity is increased by the PDIs inhibitor bacitracin. While SK-MEL-30 PDIs' expression is not THG dose-dependent, an increase in glucose related protein 78 (GRP78), PDIA5, PDIA6, and thioredoxin-related-transmembrane proteins' (TMX3 and TMX4) expression, in response to higher drug concentrations, was observed in MNT-1. The differences in PDIs' gene expression in MNT-1 suggest a different response to ER stress compared to the other cell lines and highlight the importance of understanding the diversity among cancer cells. Copyright © 2015 Elsevier B.V. All rights reserved.

Establishment and characterization of a penile cancer cell line, penl1, with a deleterious TP53 mutation as a paradigm of HPV-negative penile carcinogenesis.

PubMed

Chen, Jieping; Yao, Kai; Li, Zaishang; Deng, Chuangzhong; Wang, Liangjiao; Yu, Xingsu; Liang, Peili; Xie, Qiankun; Chen, Peng; Qin, Zike; Ye, Yunlin; Liu, Zhuowei; Zhou, Fangjian; Zhang, Zhenfeng; Han, Hui

2016-08-09

To establish penile cancer (PeCa) cell lines for the study of molecular mechanisms of carcinogenesis and testing therapeutic reagents. We successfully established two PeCa cell lines from fresh tumor tissues from 21 cases. One cell line named Penl1 was isolated from a lymph node metastasis (LNM) of penile squamous cell carcinoma (PeSCC), usual type and comprehensively characterized here. Our in-depth characterization analysis of the Penl1 cell line included morphology, tumorigenicity, genetic characteristics, protein expression, biology, and chemosensitivity. Penl1 was authenticated by single tandem repeat (STR) DNA typing. Comparative histomorphology, genetic characteristics, and protein expression patterns revealed essential similarities between the cell line and its corresponding LNM. In-depth characterization analysis of Penl1 cell line revealed tumorigenicity in immunodeficient mice, negative human papilloma virus (HPV) and mycoplasma infection, TP53 mutations and sensitivity to cisplatin and epirubicin. STR DNA typing did not match any cell lines within three international cell banks. The limitation of this study is that one patient cannot represent the complete heterogeneity of PeCa, especially primary tumor. We established and characterized an HPV-negative and moderately differentiated PeCa cell model with a TP53 missense mutation from a PeSCC, usual type patient. A preliminarily study of carcinogenesis and chemosensitivity suggests that this cell model carries a tumor suppressor gene mutation and is sensitive to chemotherapy drugs.

Establishment and characterization of a penile cancer cell line, penl1, with a deleterious TP53 mutation as a paradigm of HPV-negative penile carcinogenesis

PubMed Central

Li, Zaishang; Deng, Chuangzhong; Wang, Liangjiao; Yu, Xingsu; Liang, Peili; Xie, Qiankun; Chen, Peng; Qin, Zike; Ye, Yunlin; Liu, Zhuowei; Zhou, Fangjian; Zhang, Zhenfeng; Han, Hui

2016-01-01

Purpose To establish penile cancer (PeCa) cell lines for the study of molecular mechanisms of carcinogenesis and testing therapeutic reagents. Materials and Methods We successfully established two PeCa cell lines from fresh tumor tissues from 21 cases. One cell line named Penl1 was isolated from a lymph node metastasis (LNM) of penile squamous cell carcinoma (PeSCC), usual type and comprehensively characterized here. Our in-depth characterization analysis of the Penl1 cell line included morphology, tumorigenicity, genetic characteristics, protein expression, biology, and chemosensitivity. Penl1 was authenticated by single tandem repeat (STR) DNA typing. Results Comparative histomorphology, genetic characteristics, and protein expression patterns revealed essential similarities between the cell line and its corresponding LNM. In-depth characterization analysis of Penl1 cell line revealed tumorigenicity in immunodeficient mice, negative human papilloma virus (HPV) and mycoplasma infection, TP53 mutations and sensitivity to cisplatin and epirubicin. STR DNA typing did not match any cell lines within three international cell banks. The limitation of this study is that one patient cannot represent the complete heterogeneity of PeCa, especially primary tumor. Conclusions We established and characterized an HPV-negative and moderately differentiated PeCa cell model with a TP53 missense mutation from a PeSCC, usual type patient. A preliminarily study of carcinogenesis and chemosensitivity suggests that this cell model carries a tumor suppressor gene mutation and is sensitive to chemotherapy drugs. PMID:27351128

Cytological characterization of a thermo-sensitive cytoplasmic male-sterile wheat line having K-type cytoplasm of Aegilops kotschyi

PubMed Central

Meng, Liying; Liu, Zihan; Zhang, Lingli; Hu, Gan; Song, Xiyue

2016-01-01

Male sterility is an important tool for obtaining crop heterosis. A thermo-sensitive cytoplasmic male-sterile (TCMS) line was developed recently using a new method based on tiller regeneration. In the present study, we explored the critical growth stages required to maintain thermo-sensitive male sterility in TCMS lines and found that fertility is associated with abnormal tapetal and microspore development. We investigated the fertility and cytology of temperature-treated plant anthers at various developmental stages. TCMS line KTM3315A exhibited thermo-sensitive male sterility in Zadoks growth stages 41–49 and 58–59. Morphologically, the line exhibited thermo-sensitive male sterility at 3–9 days before heading and at 3–6 days before flowering, and it was partially restored in three locations during spring and summer. TCMS line KTM3315A plants exhibited premature tapetal programmed cell death (PCD) from the early uninucleate stage of microspore development until the tapetal cells degraded completely. Microspore development was then blocked and the pollen abortion type was stainable abortion. Thus, male fertility in the line KTM3315A is sensitive to temperature and premature tapetal PCD is the main cause of pollen abortion, where it determines the starting period and affects male fertility conversion in K-type TCMS lines at certain temperatures. PMID:28163591

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

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

The relationship between CD86/CD54 expression and THP-1 cell viability in an in vitro skin sensitization test--human cell line activation test (h-CLAT).

PubMed

Sakaguchi, Hitoshi; Ashikaga, Takao; Miyazawa, Masaaki; Kosaka, Nanae; Ito, Yuichi; Yoneyama, Katsurako; Sono, Sakiko; Itagaki, Hiroshi; Toyoda, Hidekazu; Suzuki, Hiroyuki

2009-04-01

Recent regulations for cosmetics in Europe prohibit animal testing for evaluating the sensitization potential of chemicals to improve animal welfare. Yet, there is not an acceptable Organization for Economic Co-operation and Development non-animal skin sensitization test method. Several in vitro skin sensitization methods that focus on the activation of Langerhans cells, including human cell lines, are being evaluated as possible alternatives. In our previous study, we optimized our human cell line activation test (h-CLAT) using THP-1 cells (monocytic leukemia cell line) and conducted an inter-laboratory study. We found that measuring CD86/CD54 expression may be useful for predicting skin sensitization. The aim of this study was to confirm the relationship between CD86/CD54 expression and THP-1 cell viability in the h-CLAT. In this study, 21 allergens (e.g., dinitrochlorobenzene, p-phenylenediamine, Ni) and 8 non-allergens (e.g., SLS, lactic acid) were evaluated. For each chemical, more than 10 concentrations that gave a predicted cell viability range of 20-95% were used. The data showed that expression patterns of CD86/CD54 differed depending on chemical. For most allergens, cytotoxicity (65-90% cell viability) was needed for enhancement of CD86/CD54 expression. The criteria of "CD86 > or = 150 or CD54 > or = 200" resulted in an accuracy of 93%, which confirms appropriate cut-off criteria for h-CLAT. Furthermore, a good correlation was observed between EC3 of local lymph node assay and EC150(CD86) or EC200(CD54) of h-CLAT (12 or 16 chemicals, respectively), which would provide a useful estimate of allergic potency. These findings suggest that h-CLAT would be a good robust in vitro skin sensitization test.

The effect of Nullomer-derived peptides 9R, 9S1R and 124R on the NCI-60 panel and normal cell lines.

PubMed

Alileche, Abdelkrim; Hampikian, Greg

2017-08-09

Nullomer peptides are the smallest sequences absent from databases of natural proteins. We first began compiling a list of absent 5-amino acid strings in 2006 (1). We report here the effects of Nullomer-derived peptides 9R, 9S1R and 124R on the NCI-60 panel, derived from human cancers of 9 organs (kidney, ovary, skin melanoma, lung, brain, lung, colon, prostate and the hematopoietic system), and four normal cell lines (endothelial HUVEC, skin fibroblasts BJ, colon epithelial FHC and normal prostate RWPE-1). NCI-60 cancer cell panel and four normal cell lines were cultured in vitro in RPMI1640 supplemented with 10% Hyclone fetal bovine serum and exposed for 48 h to 5 μM, 25 μM and 50 μM of peptides 9R, 9S1R and 124R. Viability was assessed by CCK-8 assay. For peptide ATP depletion effects, one cell line representing each organ in the NCI-60 panel, and four normal cell lines were exposed to 50 μM of peptides 9R, 9S1R and 124R for 3 h. The ATP content was assessed in whole cells, and their supernatants. Peptides 9S1R and 9R are respectively lethal to 95 and 81.6% of the 60 cancer cell lines tested. Control peptide 124R has no effect on the growth of these cells. Especially interesting the fact that peptides 9R and 9S1R are capable of killing drug-resistant and hormone-resistant cell lines, and even cancer stem cells. Peptides 9R and 9S1R have a broader activity spectrum than many cancer drugs in current use, can completely deplete cellular ATP within 3 h, and are less toxic to 3 of the 4 normal cell lines tested than they are to several cancers. Nullomer peptides 9R and 9S1R have a large broad lethal effect on cancer cell lines derived from nine organs represented in the NCI-60 panel. This broad activity crosses many of the categorical divisions used in the general classification of cancers: solid vs liquid cancers, drug sensitive vs drug resistant, hormone sensitive vs hormone resistant, cytokine sensitive vs cytokine non sensitive, slow growing vs rapid growing, differentiated vs dedifferentiated cancers. Furthermore peptides 9R and 9S1R are lethal to cancer stem cells and breast canrcinosarcoma.

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.

Effect of the anti-neoplastic drug doxorubicin on XPD-mutated DNA repair-deficient human cells.

PubMed

Saffi, Jenifer; Agnoletto, Mateus H; Guecheva, Temenouga N; Batista, Luís F Z; Carvalho, Helotonio; Henriques, João A P; Stary, Anne; Menck, Carlos F M; Sarasin, Alain

2010-01-02

Doxorubicin (DOX), a member of the anthracycline group, is a widely used drug in cancer therapy. The mechanisms of DOX action include topoisomerase II-poisoning, free radical release, DNA adducts and interstrand cross-link (ICL) formation. Nucleotide excision repair (NER) is involved in the removal of helix-distorting lesions and chemical adducts, however, little is known about the response of NER-deficient cell lines to anti-tumoral drugs like DOX. Wild type and XPD-mutated cells, harbouring mutations in different regions of this gene and leading to XP-D, XP/CS or TTD diseases, were treated with this drug and analyzed for cell cycle arrest and DNA damage by comet assay. The formation of DSBs was also investigated by determination of gammaH2AX foci. Our results indicate that all three NER-deficient cell lines tested are more sensitive to DOX treatment, when compared to wild type cells or XP cells complemented by the wild type XPD cDNA, suggesting that NER is involved in the removal of DOX-induced lesions. The cell cycle analysis showed the characteristic G2 arrest in repair-proficient MRC5 cell line after DOX treatment, whereas the repair-deficient cell lines presented significant increase in sub-G1 fraction. The NER-deficient cell lines do not show different patterns of DNA damage formation as assayed by comet assay and phosphorylated H2AX foci formation. Knock-down of topoisomerase IIalpha with siRNA leads to increased survival in both MRC5 and XP cells, however, XP cell line still remained significantly more sensitive to the treatment by DOX. Our study suggests that the enhanced sensitivity is due to DOX-induced DNA damage that is subject to NER, as we observed decreased unscheduled DNA synthesis in XP-deficient cells upon DOX treatment. Furthermore, the complementation of the XPD-function abolished the observed sensitivity at lower DOX concentrations, suggesting that the XPD helicase activity is involved in the repair of DOX-induced lesions. Copyright (c) 2009 Elsevier B.V. All rights reserved.

Co-inhibition of Pol η and ATR sensitizes cisplatin-resistant non-small cell lung cancer cells to cisplatin by impeding DNA damage repair.

PubMed

Li, Xiao-Qin; Ren, Jin; Chen, Ping; Chen, Yu-Jiao; Wu, Min; Wu, Yan; Chen, Kang; Li, Jian

2018-05-31

For the majority of patients with advanced non-small cell lung cancer (NSCLC), the standard of care remains platinum-based chemotherapy. However, cisplatin resistance is a big obstacle to the treatment, and elucidation of its mechanism is warranted. In this study, we showed that there was no difference in intracellular uptake of cisplatin or the removal of platinum-DNA adducts between a cisplatin-resistant NSCLC cell line (A549/DR) and a cisplatin-sensitive NSCLC cell line (A549). However, the capacity to repair DNA interstrand crosslinks (ICLs) and double-strand breaks (DSBs) was significantly enhanced in the A549/DR cell line compared to 3 cisplatin-sensitive cell lines. We found that the protein and mRNA expression levels of Pol η, a Y-family translesion synthesis (TLS) polymerase, were markedly increased upon cisplatin exposure in A549/DR cells compared with A549 cells. Furthermore, intracellular co-localization of Pol η and proliferation cell nuclear antigen (PCNA) induced by cisplatin or cisplatin plus gemcitabine treatment was inhibited by depleting ataxia telangiectasia mutated and Rad-3-related (ATR). Pol η depletion by siRNA sensitized A549/DR cells to cisplatin; co-depletion of Pol η and ATR further increased A549/DR cell death induced by cisplatin or cisplatin plus gemcitabine compared to depletion of Pol η or ATR alone, concomitant with inhibition of DNA ICL and DSB repair and accumulation of DNA damage. No additional sensitization effect of co-depleting Pol η and ATR was observed in A549 cells. These results demonstrate that co-inhibition of Pol η and ATR reverses the drug resistance of cisplatin-resistant NSCLC cells by blocking the repair of DNA ICLs and DSBs induced by cisplatin or cisplatin plus gemcitabine.

Poly ADP-ribose polymerase-1 as a potential therapeutic target in Merkel cell carcinoma.

PubMed

Ferrarotto, Renata; Cardnell, Robert; Su, Shirley; Diao, Lixia; Eterovic, A Karina; Prieto, Victor; Morrisson, William H; Wang, Jing; Kies, Merrill S; Glisson, Bonnie S; Byers, Lauren Averett; Bell, Diana

2018-03-23

Patients with metastatic Merkel cell carcinoma are treated similarly to small cell lung cancer (SCLC). Poly ADP-ribose polymerase-1 (PARP1) is overexpressed in SCLC and response to PARP inhibitors have been reported in patients with SCLC. Our study explores PARP as a therapeutic target in Merkel cell carcinoma. We evaluated PARP1 expression and Merkel cell polyomavirus (MCPyV) in 19 patients with Merkel cell carcinoma. Target exome-sequencing was performed in 14 samples. Sensitivity to olaparib was tested in 4 Merkel cell carcinoma cell lines. Most Merkel cell carcinomas (74%) express PARP1 at high levels. Mutations in DNA-damage repair genes were identified in 9 samples (64%), occurred exclusively in head neck primaries, and correlated with TP53/RB1 mutations. The TP53/RB1 mutations were more frequent in MCPyV-negative tumors. Sensitivity to olaparib was seen in the Merkel cell carcinoma line with highest PARP1 expression. Based on PARP1 overexpression, DNA-damage repair gene mutations, platinum sensitivity, and activity of olaparib in a Merkel cell carcinoma line, clinical trials with PARP inhibitors are warranted in Merkel cell carcinoma. © 2018 Wiley Periodicals, Inc.

Utilization of the human cell line HL-60 for chemiluminescence based detection of microorganisms and related substances.

PubMed

Timm, Michael; Hansen, Erik W; Moesby, Lise; Christensen, Jens D

2006-02-01

In this paper we describe a new pyrogen assay using the human leukemia cell line HL-60. The cell line is differentiated using all-trans retinoic acid (ATRA) to generate a cell population that resembles mature granulocytes. The differentiated HL-60 cell is capable of generating reactive oxygen species (ROS) when challenged with pyrogenic substances. In a luminol enhanced chemilumimetric assay the responsiveness of differentiated HL-60 cells is tested towards Salmonella typhimurium, Bacillus subtilis, Saccharomyces cerevisiae, Candida albicans, lipopolysaccharide (LPS) and lipoteichoic acid (LTA). The results show a poor sensitivity to S. typhimurium but displays good sensitivity towards B. subtilis, LTA and LPS. Furthermore, the sensitivity towards the yeasts C. albicans and S. cerevisiae is considerably better than obtained in other in vitro cell systems. Overall these results indicate that the HL-60 cell assay possibly could be evolved to a supplementary assay for the known pyrogenic detection assays. Furthermore, the utilization of the assay for pyrogenic examination of recombinant drugs derived from yeast expression systems would be relevant to examine.

Characterization of LHY-821, a novel moderately differentiated endometrial carcinoma cell line.

PubMed

Hu, Qian; Yu, Li; Chen, Rui; Zhang, Yan; Xie, Ya; Liao, Qinping

2012-08-01

Endometrial cancer is a major problem for women but only a small number of comprehensively characterized cell models are available for studies. Here, we established a new cell line derived from a Stage IIIc(1) Grade 2 endometrial adenocarcinoma. The cell line, designated LHY-821, was characterized using growth curve, karyotyping, immunohistochemical staining, immunoblotting, drug sensitivity assay, invasion assay, and xenografting in nude mice. LHY-821 has a doubling time of about 46 h and a colony-forming efficiency of approximately 71 %. These cells expresse high levels of progesterone receptor but not estrogen receptor and are sensitive to medroxyprogesterone acetate (MPA). LHY-821 also expresses pan-cytokeratin, PTEN, p53, β-catenin, IGF-1, and IGF-2. In addition, karyotype analysis revealed that LHY-821 possessed a near diploid karyotype including 6q-, 10p-, Xq-, 13q+, 17p+, and Triplo-12. LHY-821 showed highly tumorigenicity in nude mice (100 %) and weak invasiveness. Chemosensitivity tests showed that LHY-821 was sensitive to both carboplatin and paclitaxel. LHY-821 is an immortalized cell line which had survived more than 80 serial passages; it may provide a novel tool to study the molecular mechanism and potential treatment for endometrial cancer.

Increased sensitivity to thiopurines in methylthioadenosine phosphorylase-deleted cancers

PubMed Central

Coulthard, Sally A.; Redfern, Christopher P.F.; Vikingsson, Svante; Appell, Malin Lindqvist; Skoglund, Karin; Falk, Ingrid Jakobsen; Hall, Andrew G.; Taylor, Gordon A.; Hogarth, Linda A.

2011-01-01

The thiopurines, 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) are used in the treatment of leukaemia. Incorporation of deoxythioguanosine nucleotides (dGs) into the DNA of thiopurine-treated cells causes cell death but there is also evidence that thiopurine metabolites, particularly the 6-MP metabolite methylthioinosine monophosphate (MeTIMP), inhibit de novo purine synthesis (DNPS). The toxicity of DNPS inhibitors is influenced by methylthioadenosine phosphorylase (MTAP), a gene frequently deleted in cancers. Since the growth of MTAP-deleted tumour cells is dependent on DNPS or hypoxanthine salvage, we would predict such cells to show differential sensitivity to 6-MP and 6-TG. To test this hypothesis, sensitivity to 6-MP and 6-TG was compared in relation to MTAP status using cytotoxicity assays in two MTAP-deficient cell lines transfected to express MTAP: the T-cell acute lymphoblastic leukaemic cell line, Jurkat, transfected with MTAP cDNA under the control of a tetracycline-inducible promoter, and a lung cancer cell line (A549-MTAP−ve) transfected to express MTAP constitutively (A549-MTAP+ve). Sensitivity to 6-MP or methyl mercaptopurine riboside, which is converted intra-cellularly to MeTIMP, was markedly higher in both cell lines under MTAP−ve conditions. Measurement of thiopurine metabolites support the hypothesis that DNPS inhibition is a major cause of cell death with 6-MP, whereas dGs incorporation is the main cause of cytotoxicity with 6-TG. These data suggest that thiopurines, particularly 6-MP, may be more effective in patients with deleted MTAP. PMID:21282358

Induction of DNA-strand breaks after X-irradiation in murine bone cells of various differentiation capacities

NASA Astrophysics Data System (ADS)

Lau, Patrick; Hellweg, Christine E.; Kirchner, Simone; Baumstark-Khan, Christa

During longterm space missions, astronauts suffer from the loss of minerals especially from weightbearing bones due to prolonged sojourn under microgravity. In addition to weightlessness, exposure to cosmic ionization radiation is another space related factor endangering health and productivity of astronauts. In order to elucidate changes in bone cell metabolism induced by ionizing radiation, ground-based bone cell models have been developed. The differentiation level of the bone cells may influence their radiation sensitivity. Therefore, our cell model comprises a collection of immortalized murine pre-osteoblast, osteoblast and osteocyte cell lines representing discrete stages of differentiation: the subclones 4 and 24 of the osteoblast cell line MC3T3-E1, the osteoblast cell line OCT-1 and the osteocyte cell line MLO-Y4 display varying potential to produce mineralized bone matrix upon incubation with ascorbic acid and β-glycerophosphate (osteogenic medium). The MLO-Y4 cells showed the highest and subclone 24 the lowest proliferation rate. The most intense von Kossa reaction after culture in osteogenic medium was observed in subclone 4, indicating mineralized bone matrix. The bone cell markers alkaline phosphatase and osteocalcin were determined to further characterize the differentiation stage. All cell lines expressed osteocalcin, as determined by reverse transcriptase polymerase chain reaction. The activity of alkaline phosphatase was highest in the cell line OCT-1 and very low in MLO-Y4 and S4. The peculiarity of the markers suggests a characterization of OCT-1 and S24 as preosteoblast, S4 as (mature) osteoblast, and MLO-Y4 as osteocyte. Survival after exposure to X-rays was determined using the colony forming ability test. The resulting dose-effect relationships revealed normal radiation sensitivity (compared to human fibroblasts). Cell clone specific variations (subclones 4 and 24) in the radiation sensitivity may be due to the differentiation level. The survival curve of MLO-Y4 shows a broad shoulder, suggesting a high repair capacity or a high DNA damage or misrepair tolerance. The quantitative acquisition of DNA-strand breaks was performed by fluorescent analysis of DNA unwinding and revealed a high level of DNA damage immediately after X-irradiation, which increases dose dependently. In conclusion, the cell line with the highest differentiation level (MLO-Y4) displays lower radiation sensitivity, regarding the shoulder width of the dose-effect curve, compared to the less differentiated osteoblast cell lines.

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

MiR-199a-3p enhances cisplatin sensitivity of cholangiocarcinoma cells by inhibiting mTOR signaling pathway and expression of MDR1.

PubMed

Li, Qiang; Xia, Xuefeng; Ji, Jie; Ma, Jianghui; Tao, Liang; Mo, Linjun; Chen, Wei

2017-05-16

Several studies have reported reduced miRNA-199a-3p (miR-199a-3p) in different human malignancies, however, little is known about miR-199a-3p in cholangiocarcinoma cells. In this study, we demonstrate the essential role and mechanism of miR-199a-3p in regulating cisplatin sensitivity in cholangiocarcinoma cell lines. Using a CCK-8 cell counting assay we found that expression of miR-199a-3p was positively correlated with cisplatin sensitivity in cholangiocarcinoma cell lines. MiR-199a-3p overexpression could decrease the proliferation rate and increase apoptosis of cholangiocarcinoma cells in the presence of cisplatin, while miR-199a-3p inhibition had the opposite effect. Further study demonstrated that mTOR was the target gene of miR-199a-3p, and that miR-199a-3p mimics could inhibit expression of mTOR, which consequently reduced the phosphorylation of its downstream proteins 4EBP1 and p70s6k. Rescue experiments proved that miR-199a-3p could increase the cisplatin sensitivity of cholangiocarcinoma cell lines by regulating mTOR expression. Moreover, we also found that miR-199a-3p overexpression could reduce cisplatin induced MDR1 expression by decreasing the synthesis and increasing the degradation of MDR1, thus enhancing the effectiveness of cisplatin in cholangiocarcinoma. In conclusion, miR-199a-3p could increase cisplatin sensitivity of cholangiocarcinoma cell lines by inhibiting the activity of the mTOR signaling pathway and decreasing the expression of MDR1.

Lapatinib-resistant cancer cells possessing epithelial cancer stem cell properties develop sensitivity during sphere formation by activation of the ErbB/AKT/cyclin D2 pathway.

PubMed

Ohnishi, Yuichi; Yasui, Hiroki; Kakudo, Kenji; Nozaki, Masami

2016-11-01

Lapatinib, a dual inhibitor of epidermal growth factor receptor (EGFR)/ErbB2, has antiproliferative effects and is used to treat patients with ErbB2-positive metastatic breast cancer. In the present study, we examined the effects of lapatinib on growth of oral and prostate cancer cells. Oral squamous cell carcinoma (OSCC) cell lines HSC3, HSC4 and Ca9-22 were sensitive to the antiproliferative effects of lapatinib in anchorage-dependent culture, but the OSCC cell lines KB and SAS and the prostate cancer cell line DU145 were resistant to lapatinib. Phosphorylation levels of EGFR in all cell lines decreased during lapatinib treatment in anchorage‑dependent culture. Furthermore, the phosphorylation levels of ErbB2, ErbB3 and Akt and the protein levels of cyclin D1 were decreased by lapatinib treatment of HSC3, HSC4 and Ca9-22 cells. ErbB3 was not expressed and cyclin D1 protein levels were not altered by lapatinib treatment in KB, DU145 and SAS cells. The phosphorylation of ErbB2 and AKT was not affected by lapatinib in SAS cells and was not detected in KB and DU145 cells. Lapatinib-resistant cell lines exhibited sphere-forming ability, and SAS cells developed sensitivity to lapatinib during sphere formation. The phosphorylation levels of ErbB2 and AKT and protein levels of cyclin D2 increased during sphere formation of SAS cells and decreased with lapatinib treatment. In addition, sphere formation of SAS cells was inhibited by the AKT inhibitor MK2206. AKT phosphorylation and cyclin D2 levels in SAS spheres were decreased by MK2206 treatment. SAS cells expressed E-cadherin, but not vimentin and KB cells expressed vimentin, but not E-cadherin. DU145 cells expressed vimentin and E-cadherin. These results suggested that phosphorylation of EGFR and ErbB2 by cell detachment from the substratum induces the AKT pathway/cyclin D2-dependent sphere growth in SAS epithelial cancer stem-like cells, thereby rendering SAS spheres sensitive to lapatinib treatment.

Copper transporters and chaperones CTR1, CTR2, ATOX1, and CCS as determinants of cisplatin sensitivity.

PubMed

Bompiani, Kristin M; Tsai, Cheng-Yu; Achatz, Felix P; Liebig, Janika K; Howell, Stephen B

2016-09-01

The development of resistance to cisplatin (cDDP) is commonly accompanied by reduced drug uptake or increased efflux. Previous studies in yeast and murine embryonic fibroblasts have reported that the copper (Cu) transporters and chaperones participate in the uptake, efflux, and intracellular distribution of cDDP. However, there is conflicting data from studies in human cells. We used CRISPR-Cas9 genome editing to individually knock out the human copper transporters CTR1 and CTR2 and the copper chaperones ATOX1 and CCS. Isogenic knockout cell lines were generated in both human HEK-293T and ovarian carcinoma OVCAR8 cells. All knockout cell lines had slowed growth compared to parental cells, small changes in basal Cu levels, and varying sensitivities to Cu depending on the gene targeted. However, all of the knockouts demonstrated only modest 2 to 5-fold changes in cDDP sensitivity that did not differ from the range of sensitivities of 10 wild type clones grown from the same parental cell population. We conclude that, under basal conditions, loss of CTR1, CTR2, ATOX1, or CCS does not produce a change in cisplatin sensitivity that exceeds the variance found within the parental population, suggesting that they are not essential to the mechanism by which cDDP enters these cell lines and is transported to the nucleus.

Combining Heavy Ion Radiation and Artificial MicroRNAs to Target the Homologous Recombination Repair Gene Efficiently Kills Human Tumor Cells

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

Zheng Zhiming; Department of Radiation Oncology, School of Medicine, Winship Cancer Institute, Emory University, Atlanta, Georgia; Wang Ping

2013-02-01

Purpose: Previously, we demonstrated that heavy ions kill more cells at the same dose than X-rays because DNA-clustered lesions produced by heavy ions affect nonhomologous end-joining (NHEJ) repair but not homologous recombination repair (HRR). We have also shown that our designed artificial microRNAs (amiRs) could efficiently target XRCC4 (an essential factor for NHEJ) or XRCC2 (an essential factor for HRR) and sensitize human tumor cells to X-rays. Based on these data, we were interested in testing the hypothesis that combining heavy ions and amiRs to target HRR but not NHEJ should more efficiently kill human tumor cells. Methods and Materials:more » Human tumor cell lines (U87MG, a brain tumor cell line, and A549, a lung cancer cell line) and their counterparts, overexpressed with amiR to target XRCC2, XRCC4 or both, were used in this study. Survival sensitivities were examined using a clonogenic assay after these cells were exposed to X-rays or heavy ions. In addition, these cell lines were subcutaneously injected into nude mice to form xenografts and the tumor size was compared after the tumor areas were exposed to X-rays or heavy ions. Results: Although targeting either XRCC4 (NHEJ factor) or XRCC2 (HRR factor) sensitized the human tumor cells to X-rays, in vitro and the xenograft animal model, targeting only XRCC2 but not XRCC4 sensitized the human tumor cells to heavy ions in vitro and in the xenograft animal model. Conclusions: Combining heavy ions with targeting the HRR pathway, but not the NHEJ pathway, could significantly improve the efficiency of tumor cell death.« less

Combining heavy ion radiation and artificial microRNAs to target the homologous recombination repair gene efficiently kills human tumor cells.

PubMed

Zheng, Zhiming; Wang, Ping; Wang, Hongyan; Zhang, Xiangming; Wang, Minli; Cucinotta, Francis A; Wang, Ya

2013-02-01

Previously, we demonstrated that heavy ions kill more cells at the same dose than X-rays because DNA-clustered lesions produced by heavy ions affect nonhomologous end-joining (NHEJ) repair but not homologous recombination repair (HRR). We have also shown that our designed artificial microRNAs (amiRs) could efficiently target XRCC4 (an essential factor for NHEJ) or XRCC2 (an essential factor for HRR) and sensitize human tumor cells to X-rays. Based on these data, we were interested in testing the hypothesis that combining heavy ions and amiRs to target HRR but not NHEJ should more efficiently kill human tumor cells. Human tumor cell lines (U87MG, a brain tumor cell line, and A549, a lung cancer cell line) and their counterparts, overexpressed with amiR to target XRCC2, XRCC4 or both, were used in this study. Survival sensitivities were examined using a clonogenic assay after these cells were exposed to X-rays or heavy ions. In addition, these cell lines were subcutaneously injected into nude mice to form xenografts and the tumor size was compared after the tumor areas were exposed to X-rays or heavy ions. Although targeting either XRCC4 (NHEJ factor) or XRCC2 (HRR factor) sensitized the human tumor cells to X-rays, in vitro and the xenograft animal model, targeting only XRCC2 but not XRCC4 sensitized the human tumor cells to heavy ions in vitro and in the xenograft animal model. Combining heavy ions with targeting the HRR pathway, but not the NHEJ pathway, could significantly improve the efficiency of tumor cell death. Copyright © 2013 Elsevier Inc. All rights reserved.

MiR-33a Decreases High-Density Lipoprotein-Induced Radiation Sensitivity in Breast Cancer

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

Wolfe, Adam R.; Bambhroliya, Arvind; Morgan Welch Inflammatory Breast Cancer Research Program and Clinic, The University of Texas MD Anderson Cancer Center, Houston, Texas

Purpose: We previously showed that high-density lipoprotein (HDL) radiosensitizes inflammatory breast cancer (IBC) cells in vitro and is associated with better local control after radiation therapy in IBC patients. The microRNA miR-33 family negatively regulates the adenosine triphosphate binding cassette transporter subfamily A member 1. We hypothesized that variations in miR-33a expression in IBC cancer cells versus non-IBC cells would correlate with radiation sensitivity following exposure to HDL in vitro. Methods and Materials: MiR-33a expression was analyzed by reverse transcriptase–polymerase chain reaction in 4 cell lines representing common clinical breast cancer subtypes. Overexpression and knockdown of miR-33a was demonstrated via transfection of anmore » miR-33a mimic or an anti-miR-33a construct in high- and low-expressing miR-33a cell lines. Clonogenic survival in vitro in these cells was quantified at baseline and following HDL treatment. MiR-33a expression on distant relapse-free survival (DRFS) of 210 cases downloaded from the Oxford breast cancer dataset was determined. Results: Expression levels of miR-33a were lower in IBC cell lines and IBC tumor samples than in non-IBC cell lines and normal breast tissue. Cholesterol concentrations in the cell membranes were higher in IBC cells than in non-IBC cells. Clonogenic survival following 24 hours of HDL treatment was decreased in response to irradiation in the low-miR-33a–expressing cell lines SUM149 and KPL4, but survival following HDL treatment decreased in the high-miR-33a–expressing cell lines MDA-MB-231 and SUM159. In the high-miR-33a–expressing cell lines, anti-miR-33a transfection decreased radiation resistance in clonogenic assays. Conversely, in the low-miR-33a–expressing cell lines, the miR-33a mimic reversed the HDL-induced radiation sensitization. Breast cancer patients in the top quartile based on miR-33a expression had markedly lower rates of DRFS than the bottom quartile (P=.0228, log-rank test). For breast cancer patients treated with radiation, high miR-33a expression predicted worse overall survival (P=.06). Conclusions: Our results reveal miR-33a negatively regulates HDL-induced radiation sensitivity in breast cancer.« less

Improved phase sensitivity in spectral domain phase microscopy using line-field illumination and self phase-referencing

PubMed Central

Yaqoob, Zahid; Choi, Wonshik; Oh, Seungeun; Lue, Niyom; Park, Yongkeun; Fang-Yen, Christopher; Dasari, Ramachandra R.; Badizadegan, Kamran; Feld, Michael S.

2010-01-01

We report a quantitative phase microscope based on spectral domain optical coherence tomography and line-field illumination. The line illumination allows self phase-referencing method to reject common-mode phase noise. The quantitative phase microscope also features a separate reference arm, permitting the use of high numerical aperture (NA > 1) microscope objectives for high resolution phase measurement at multiple points along the line of illumination. We demonstrate that the path-length sensitivity of the instrument can be as good as 41 pm/Hz, which makes it suitable for nanometer scale study of cell motility. We present the detection of natural motions of cell surface and two-dimensional surface profiling of a HeLa cell. PMID:19550464

Radio-sensitizing Effects of Novel Histone De-Acetylase Inhibitors in Prostate Cancer

DTIC Science & Technology

2007-03-01

were investigated in PC-3, LN -3 and DU-145 cells. (S)-HDAC-42 and SAHA could sensitize PC-3 and DU-145 cells to radiation. Aim 2: Effects of VAD- 18 ...combined effects of HDAC inhibitors and ionizing radiation on prostate cancer cell lines (PC-3, LN -3, LnCAP, DU-145 and 22Rv1). Aim 2. To understand the...cancer cell lines. Aim 3. To determine the combined effects of HDAC inhibitors plus ionizing radiation on the regression of (i) prostate cancer xenografts

The Fanconi anemia (FA) pathway confers glioma resistance to DNA alkylating agents.

PubMed

Chen, Clark C; Taniguchi, Toshiyasu; D'Andrea, Alan

2007-05-01

DNA alkylating agents including temozolomide (TMZ) and 1,3-bis[2-chloroethyl]-1-nitroso-urea (BCNU) are the most common form of chemotherapy in the treatment of gliomas. Despite their frequent use, the therapeutic efficacy of these agents is limited by the development of resistance. Previous studies suggest that the mechanism of this resistance is complex and involves multiple DNA repair pathways. To better define the pathways contributing to the mechanisms underlying glioma resistance, we tested the contribution of the Fanconi anemia (FA) DNA repair pathway. TMZ and BCNU treatment of FA-proficient cell lines led to a dose- and time-dependent increase in FANCD2 mono-ubiquitination and FANCD2 nuclear foci formation, both hallmarks of FA pathway activation. The FA-deficient cells were more sensitive to TMZ/BCNU relative to their corrected, isogenic counterparts. To test whether these observations were pertinent to glioma biology, we screened a panel of glioma cell lines and identified one (HT16) that was deficient in the FA repair pathway. This cell line exhibited increased sensitivity to TMZ and BCNU relative to the FA-proficient glioma cell lines. Moreover, inhibition of FA pathway activation by a small molecule inhibitor (curcumin) or by small interference RNA suppression caused increased sensitivity to TMZ/BCNU in the U87 glioma cell line. The BCNU sensitizing effect of FA inhibition appeared additive to that of methyl-guanine methyl transferase inhibition. The results presented in this paper underscore the complexity of cellular resistance to DNA alkylating agents and implicate the FA repair pathway as a determinant of this resistance.

Transdifferentiation between Luminal- and Basal-Type Cancer Cells

DTIC Science & Technology

2013-04-01

cancer patients have few choices of therapeutic agents. The MB231 cell line is a basal type cell line and is resistant to EGFR inhibitor erlotinib...mitotic inhibitor paclitaxel and DNA damaging agent cisplatin. However, over- expression of PKD1 makes the cell line sensitive to erlotinib and...approach will help to understand how PKD1 acts in basal-type cancer cells; (3) using existing small molecule inhibitors for PKD1 to treat breast cancer

HER2/neu gene amplification determines the sensitivity of uterine serous carcinoma cell lines to AZD8055, a novel dual mTORC1/2 inhibitor.

PubMed

English, Diana P; Roque, Dana M; Carrara, Luisa; Lopez, Salvatore; Bellone, Stefania; Cocco, Emiliano; Bortolomai, Ileana; Schwartz, Peter E; Rutherford, Thomas; Santin, Alessandro D

2013-12-01

To evaluate c-erbB2 gene amplification in a series of primary uterine serous carcinoma (USC) cell lines. To assess the efficacy of AZD8055, a novel dual mTORC1/2 inhibitor against primary HER2/neu amplified vs HER2/neu not amplified USC cell lines. Twenty-two primary USC cell lines were evaluated for c-erbB2 oncogene amplification by FISH assays. In vitro sensitivity to AZD8055 was evaluated by flow-cytometry-based viability and proliferation assays. Cell cycle profile and downstream cellular responses to AZD8055 were assessed by measuring the DNA content of cells and by phosphorylation of the S6 protein by flow-cytometry. Nine of 22 (40.9%) USC cell lines demonstrated c-erbB2 gene amplification by FISH. AZD8055 caused a strong differential growth inhibition in USC cell lines, with high HER-2/neu-expressors demonstrating significantly higher sensitivity when compared to low HER-2/neu-expressors (AZD-8055 IC50 mean±SEM=0.27±0.05μM in c-erbB2 amplified versus 1.67±0.68μM in c-erbB2 not amplified tumors, P=0.03). AZD8055 growth-inhibition was associated with a significant and dose-dependent increase in the percentage of cells blocked in the G0/G1 cell cycle phase and a dose-dependent decline in pS6 levels in both c-erbB2 amplified vs c-erbB2 not amplified USC cell lines. AZD8055 may represent a novel targeted therapeutic agent in patients harboring advanced/recurrent/refractory USC. c-erbB2 gene amplification may represent a biomarker to identify USC patients who may benefit most from the use of AZD8055. © 2013.

Overcoming CRPC Treatment Resistance via Novel Dual AKR1C3 Targeting

DTIC Science & Technology

2017-10-01

We therefore characterized another drug resistant line from C4-2B cells, C4-2B AbiR cells. C4-2B AbiR cells were resistant to Abi acetate in a...Testosterone level in C4-2B AbiR cell was 12 pg/50 million cells, similar to that in C4-2B MDVR or LNCaP-AKR1C3 cells. With the single drug resistant...cell lines on hand, we tested for their cross- resistance to Enza and Abi. While the parental line was sensitive to both drugs , the resistant lines

DNA excision repair in cell extracts from human cell lines exhibiting hypersensitivity to DNA-damaging agents

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

Hansson, J.; Keyse, S.M.; Lindahl, T.

Whole cell extracts from human lymphoid cell lines can perform in vitro DNA repair synthesis in plasmids damaged by agents including UV or cis-diamminedichloroplatinum(II) (cis-DDP). Extracts from xeroderma pigmentosum (XP) cells are defective in repair synthesis. We have now studied in vitro DNA repair synthesis using extracts from lymphoblastoid cell lines representing four human hereditary syndromes with increased sensitivity to DNA-damaging agents. Extracts of cell lines from individuals with the sunlight-sensitive disorders dysplastic nevus syndrome or Cockayne's syndrome (complementation groups A and B) showed normal DNA repair synthesis in plasmids with UV photoproducts. This is consistent with in vivo measurementsmore » of the overall DNA repair capacity in such cell lines. A number of extracts were prepared from two cell lines representing the variant form of XP (XP-V). Half of the extracts prepared showed normal levels of in vitro DNA repair synthesis in plasmids containing UV lesions, but the remainder of the extracts from the same cell lines showed deficient repair synthesis, suggesting the possibility of an unusually labile excision repair protein in XP-V. Fanconi's anemia (FA) cells show cellular hypersensitivity to cross-linking agents including cis-DDP. Extracts from cell lines belonging to two different complementation groups of FA showed normal DNA repair synthesis in plasmids containing cis-DDP or UV adducts. Thus, there does not appear to be an overall excision repair defect in FA, but the data do not exclude a defect in the repair of interstrand DNA cross-links.« less

Effect of sulphur mustard on human skin cell lines with differential agent sensitivity.

PubMed

Simpson, Rachel; Lindsay, Christopher D

2005-01-01

The ability of sulphur mustard (HD) to induce DNA damage places limits on the efficacy of approaches aimed at protecting human cells from the cytotoxic effects of HD using a variety of protective agents such as thiol-containing esters and protease inhibitors. In the present study, potential alternative strategies were investigated by examining the differential effects of HD on G361, SVK14, HaCaT and NCTC 2544 human skin cells. The G361 cell line was more resistant to the cytotoxic effects of HD than the NCTC, HaCaT and SVK14 cell lines at HD doses of >3 and <100 microM HD as determined by the MTT assay. At 72 h after exposure to 60 microM HD there was up to an 8.8-fold difference (P < 0.0001) between G361 and SVK14 cell culture viability. Buthionine sulphoximine (BSO) pretreatment increased the sensitivity of all four cell lines to HD. A substantial proportion of the resistance of G361 cells to HD was attributable to BSO-mediated effects on antioxidant-mediated metabolism, although G361 cultures still retained a high degree of viability at 30 microM HD following BSO pretreatment. Cell cycle analysis confirmed that SVK14 cells were relatively more sensitive to HD, as shown by the 2.1-fold reduction (P < 0.0001) in the percentage of cells in G0/G1 phase 24 h after HD exposure compared with control cultures. This compared well with a 1.2-fold increase (P < 0.05) in the percentage of G361 cells in G0/G1 phase following HD exposure, suggesting the existence of a more efficient G0/G1 checkpoint control mechanism in this cell line. Manipulation of the cell cycle using various modulating agents did not increase the resistance of cell lines to the cytotoxic effects of HD. Crown copyright 2005

Relevance of MET activation and genetic alterations of KRAS and E-cadherin for cetuximab sensitivity of gastric cancer cell lines.

PubMed

Heindl, Stefan; Eggenstein, Evelyn; Keller, Simone; Kneissl, Julia; Keller, Gisela; Mutze, Kathrin; Rauser, Sandra; Gasteiger, Georg; Drexler, Ingo; Hapfelmeier, Alexander; Höfler, Heinz; Luber, Birgit

2012-05-01

The therapeutic activity of the epidermal growth factor receptor (EGFR)-directed monoclonal antibody cetuximab in gastric cancer is currently being investigated. Reliable biomarkers for the identification of patients who are likely to benefit from the treatment are not available. The aim of the study was to examine the drug sensitivity of five gastric cancer cell lines towards cetuximab as a single agent and to establish predictive markers for chemosensitivity in this cell culture model. The effect of a combination of cetuximab with chemotherapy was compared between a sensitive and a nonsensitive cell line. EGFR expression, activation and localisation, the presence and subcellular localisation of the cell adhesion molecule E-cadherin as well as MET activation were examined by Western blot analysis, flow cytometry and immunofluorescence staining. Cells were treated with varying concentrations of cetuximab and cisplatin and 5-fluorouracil in tumour-relevant concentrations. The biological endpoint was cell viability, which was measured by XTT cell proliferation assay. Response to treatment was evaluated using statistical methods. We assessed the activity of cetuximab in five gastric cancer cell lines (AGS, KATOIII, MKN1, MKN28 and MKN45). The viability of two cell lines, MKN1 and MKN28, was significantly reduced by cetuximab treatment. High EGFR expression and low levels of receptor activation were associated with cetuximab responsiveness. MET activation as well as mutations of KRAS and CDH1 (gene encoding E-cadherin) was associated with cetuximab resistance. These data indicate that our examinations may be clinically relevant, and the candidate markers should therefore be tested in clinical studies.

l-Type Amino Acid Transporter-1 Overexpression and Melphalan Sensitivity in Barrett's Adenocarcinoma1

PubMed Central

Lin, Jules; Raoof, Duna A; Thomas, Dafydd G; Greenson, Joel K; Giordano, Thomas J; Robinson, Gregory S; Bourner, Maureen J; Bauer, Christopher T; Orringer, Mark B; Beer, David G

2004-01-01

Abstract The L-type amino acid transporter-1 (LAT-1) has been associated with tumor growth. Using cDNA microarrays, overexpression of LAT-1 was found in 87.5% (7/8) of esophageal adenocarcinomas relative to 12 Barrett's samples (33% metaplasia and 66% dysplasia) and was confirmed in 100% (28/28) of Barrett's adenocarcinomas by quantitative reverse transcription polymerase chain reaction. Immunohistochemistry revealed LAT-1 staining in 37.5% (24/64) of esophageal adenocarcinomas on tissue microarray. LAT-1 also transports the amino acid-related chemotherapeutic agent, melphalan. Two esophageal adenocarcinoma and one esophageal squamous cell line, expressing LAT-1 on Western blot analysis, were sensitive to therapeutic doses of melphalan (P < .001). Simultaneous treatment with the competitive inhibitor, BCH [2-aminobicyclo-(2,1,1)-heptane-2-carboxylic acid], decreased sensitivity to melphalan (P < .05). In addition, confluent esophageal squamous cultures were less sensitive to melphalan (P < .001) and had a decrease in LAT-1 protein expression. Tumors from two esophageal adenocarcinoma cell lines grown in nude mice retained LAT-1 mRNA expression. These results demonstrate that LAT-1 is highly expressed in a subset of esophageal adenocarcinomas and that Barrett's adenocarcinoma cell lines expressing LAT-1 are sensitive to melphalan. LAT-1 expression is also retained in cell lines grown in nude mice providing a model to evaluate melphalan as a chemotherapeutic agent against esophageal adenocarcinomas expressing LAT-1. PMID:15068672

A blood biomarker for monitoring response to anti-EGFR therapy.

PubMed

Hughes, Nicholas P; Xu, Lingyun; Nielsen, Carsten H; Chang, Edwin; Hori, Sharon S; Natarajan, Arutselvan; Lee, Samantha; Kjær, Andreas; Kani, Kian; Wang, Shan X; Mallick, Parag; Gambhir, Sanjiv Sam

2018-04-13

To monitor therapies targeted to epidermal growth factor receptors (EGFR) in non-small cell lung cancer (NSCLC), we investigated Peroxiredoxin 6 (PRDX6) as a biomarker of response to anti-EGFR agents. We studied cells that are sensitive (H3255, HCC827) or resistant (H1975, H460) to gefitinib. PRDX6 was examined with either gefitinib or vehicle treatment using enzyme-linked immunosorbent assays. We created xenograft models from one sensitive (HCC827) and one resistant cell line (H1975) and monitored serum PRDX6 levels during treatment. PRDX6 levels in cell media from sensitive cell lines increased significantly after gefitinib treatment vs. vehicle, whereas there was no significant difference for resistant lines. PRDX6 accumulation over time correlated positively with gefitinib sensitivity. Serum PRDX6 levels in gefitinib-sensitive xenograft models increased markedly during the first 24 hours of treatment and then decreased dramatically during the following 48 hours. Differences in serum PRDX6 levels between vehicle and gefitinib-treated animals could not be explained by differences in tumor burden. Our results show that changes in serum PRDX6 during the course of gefitinib treatment of xenograft models provide insight into tumor response and such an approach offers several advantages over imaging-based strategies for monitoring response to anti-EGFR agents.

Low ATM protein expression and depletion of p53 correlates with olaparib sensitivity in gastric cancer cell lines

PubMed Central

Kubota, Eiji; Williamson, Christopher T; Ye, Ruiqiong; Elegbede, Anifat; Peterson, Lars; Lees-Miller, Susan P; Bebb, D Gwyn

2014-01-01

Small-molecule inhibitors of poly (ADP-ribose) polymerase (PARP) have shown considerable promise in the treatment of homologous recombination (HR)-defective tumors, such as BRCA1- and BRCA2-deficient breast and ovarian cancers. We previously reported that mantle cell lymphoma cells with deficiency in ataxia telangiectasia mutated (ATM) are sensitive to PARP-1 inhibitors in vitro and in vivo. Here, we report that PARP inhibitors can potentially target ATM deficiency arising in a solid malignancy. We show that ATM protein expression varies between gastric cancer cell lines, with NUGC4 having significantly reduced protein levels. Significant correlation was found between ATM protein expression and sensitivity to the PARP inhibitor olaparib, with NUGC4 being the most sensitive. Moreover, reducing ATM kinase activity using a small-molecule inhibitor (KU55933) or shRNA-mediated depletion of ATM protein enhanced olaparib sensitivity in gastric cancer cell lines with depletion or inactivation of p53. Our results demonstrate that ATM is a potential predictive biomarker for PARP-1 inhibitor activity in gastric cancer harboring disruption of p53, and that combined inhibition of ATM and PARP-1 is a rational strategy for expanding the utility of PARP-1 inhibitors to gastric cancer with p53 disruption. PMID:24841718

Overcoming adaptive resistance in mucoepidermoid carcinoma through inhibition of the IKK-β/IκBα/NFκB axis

PubMed Central

Wagner, Vivian P.; Martins, Marco A.T.; Martins, Manoela D.; Warner, Kristy A.; Webber, Liana P.; Squarize, Cristiane H.; Nör, Jacques E.; Castilho, Rogerio M.

2016-01-01

Patients with mucoepidermoid carcinoma (MEC) experience low survival rates and high morbidity following treatment, yet the intrinsic resistance of MEC cells to ionizing radiation (IR) and the mechanisms underlying acquired resistance remain unexplored. Herein, we demonstrated that low doses of IR intrinsically activated NFκB in resistant MEC cell lines. Moreover, resistance was significantly enhanced in IR-sensitive cell lines when NFκB pathway was stimulated. Pharmacological inhibition of the IKK-β/IκBα/NFκB axis, using a single dose of FDA-approved Emetine, led to a striking sensitization of MEC cells to IR and a reduction in cancer stem cells. We achieved a major step towards better understanding the basic mechanisms involved in IR-adaptive resistance in MEC cell lines and how to efficiently overcome this critical problem. PMID:27682876

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.

Comparative proteomics as a tool for identifying specific alterations within interferon response pathways in human glioblastoma multiforme cells

PubMed Central

Lobas, Anna A; Solovyeva, Elizaveta M; Sidorenko, Alena S; Gorshkov, Vladimir; Kjeldsen, Frank; Bubis, Julia A; Ivanov, Mark V; Ilina, Irina Y; Moshkovskii, Sergei A; Chumakov, Peter M; Gorshkov, Mikhail V

2018-01-01

An acquisition of increased sensitivity of cancer cells to viruses is a common outcome of malignant progression that justifies the development of oncolytic viruses as anticancer therapeutics. Studying molecular changes that underlie the sensitivity to viruses would help to identify cases where oncolytic virus therapy would be most effective. We quantified changes in protein abundances in two glioblastoma multiforme (GBM) cell lines that differ in the ability to induce resistance to vesicular stomatitis virus (VSV) infection in response to type I interferon (IFN) treatment. In IFN-treated samples we observed an up-regulation of protein products of some IFN-regulated genes (IRGs). In total, the proteome analysis revealed up to 20% more proteins encoded by IRGs in the glioblastoma cell line, which develops resistance to VSV infection after pre-treatment with IFN. In both cell lines protein-protein interaction and signaling pathway analyses have revealed a significant stimulation of processes related to type I IFN signaling and defense responses to viruses. However, we observed a deficiency in STAT2 protein in the VSV-sensitive cell line that suggests a de-regulation of the JAK/STAT/IRF9 signaling. The study has shown that the up-regulation of IRG proteins induced by the IFNα treatment of GBM cells can be detected at the proteome level. Similar analyses could be applied for revealing functional alterations within the antiviral mechanisms in glioblastoma samples, accompanying by acquisition of sensitivity to oncolytic viruses. The approach can be useful for discovering the biomarkers that predict a potential sensitivity of individual glioblastoma tumors to oncolytic virus therapy. PMID:29416731

A cell clone strain from Mythimna separata (Lepidoptera: Noctuidae) highly susceptible to Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and M. separata NPV (MsNPV).

PubMed

Meng, Xiang-Qian; Zheng, Gui-Ling; Zhao, Chuan-De; Wan, Fang-Hao; Li, Chang-You

2017-08-01

In this study, we describe a cell line, Ms-10C, cloned from the line QAU-Ms-E-10 (simplified Ms-10), an embryonic line from Mythimna separata. The cloned cell line was significantly more sensitive to nucleopolyhedrovirus (NPV). Ms-10C cells were mainly spherical with a diameter of 14.42 ± 2.23 μm. DNA amplification fingerprinting (DAF) confirmed the profile of PCR-amplified bands of the cloned cell line was consistent with those of the parental cell line, Ms-10. The sequencing result of the mitochondrial cytochrome c oxidase I (mtCO I) fragment confirmed that the amplified 636-bps mtCOI fragment was 100% identical to that of M. separata. Its chromosomes exhibited the typical characters of lepidopteran cell lines. Its population doubling time was 42.2 h at 27°C. Ms-10C was more sensitive than Ms-10 to both Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and M. separata nucleopolyhedrovirus (MsNPV). At 4 d post infection, the infection rates of two viruses reached 94.2 and 92.3%, respectively. The availability of this cell clone strain will provide a useful tool for the basic research on nucleopolyhedrovirus and for potential application in expression of recombinant proteins with baculovirus expression vector system.

Efficacy and safety of cytotoxic drug chemotherapy after first-line EGFR-TKI treatment in elderly patients with non-small-cell lung cancer harboring sensitive EGFR mutations.

PubMed

Imai, Hisao; Minemura, Hiroyuki; Sugiyama, Tomohide; Yamada, Yutaka; Kaira, Kyoichi; Kanazawa, Kenya; Kasai, Takashi; Kaburagi, Takayuki; Minato, Koichi

2018-05-08

Epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) is effective as first-line chemotherapy for patients with advanced non-small-cell lung cancer (NSCLC) harboring sensitive EGFR mutations. However, whether the efficacy of second-line cytotoxic drug chemotherapy after first-line EGFR-TKI treatment is similar to that of first-line cytotoxic drug chemotherapy in elderly patients aged ≥ 75 years harboring sensitive EGFR mutations is unclear. Therefore, we aimed to investigate the efficacy and safety of cytotoxic drug chemotherapy after first-line EGFR-TKI treatment in elderly patients with NSCLC harboring sensitive EGFR mutations. We retrospectively evaluated the clinical effects and safety profiles of second-line cytotoxic drug chemotherapy after first-line EGFR-TKI treatment in elderly patients with NSCLC harboring sensitive EGFR mutations (exon 19 deletion/exon 21 L858R mutation). Between April 2008 and December 2015, 78 elderly patients with advanced NSCLC harboring sensitive EGFR mutations received first-line EGFR-TKI at four Japanese institutions. Baseline characteristics, regimens, responses to first- and second-line treatments, whether or not patients received subsequent treatment, and if not, the reasons for non-administration were recorded. Overall, 20 patients with a median age of 79.5 years (range 75-85 years) were included in our analysis. The overall response, disease control, median progression-free survival, and overall survival rates were 15.0, 60.0%, 2.4, and 13.2 months, respectively. Common adverse events included leukopenia, neutropenia, anemia, thrombocytopenia, malaise, and anorexia. Major grade 3 or 4 toxicities included leukopenia (25.0%) and neutropenia (45.0%). No treatment-related deaths were noted. Second-line cytotoxic drug chemotherapy after first-line EGFR-TKI treatment among elderly patients with NSCLC harboring sensitive EGFR mutations was effective and safe and showed equivalent outcomes to first-line cytotoxic drug chemotherapy.

Human T-Cell Leukemia Virus I Tax Protein Sensitizes p53-Mutant Cells to DNA Damage

PubMed Central

Mihaylova, Valia T.; Green, Allison M.; Khurgel, Moshe; Semmes, Oliver J.; Kupfer, Gary M.

2018-01-01

Mutations in p53 are a common cause of resistance of cancers to standard chemotherapy and, thus, treatment failure. Reports have shown that Tax, a human T-cell leukemia virus type I encoded protein that has been associated with genomic instability and perturbation of transcription and cell cycle, sensitizes HeLa cells to UV treatment. The extent to which Tax can sensitize cells and the mechanism by which it exerts its effect are unknown. In this study, we show that Tax sensitizes p53-mutant cells to a broad range of DNA-damaging agents, including mitomycin C, a bifunctional alkylator, etoposide, a topoisomerase II drug, and UV light, but not ionizing radiation, a double-strand break agent, or vinblastine, a tubulin poison. Tax caused hypersensitivity in all p53-deleted cell lines and several, but not all, mutant-expressed p53–containing cell lines, while unexpectedly being protective in p53 wild-type (wt) cells. The effect observed in p53-deleted lines could be reversed for this by transfection of wt p53. We also show that Tax activates a p53-independent proapoptotic program through decreased expression of the retinoblastoma protein and subsequent increased E2F1 expression. The expression of several proapoptotic proteins was also induced by Tax, including Puma and Noxa, culminating in a substantial increase in Bax dimerization. Our results show that Tax can sensitize p53-mutant cells to DNA damage while protecting p53 wt cells, a side benefit that might result in reduced toxicity in normal cells. Such studies hold the promise of a novel adjunctive therapy that could make cancer chemotherapy more effective. PMID:18559532

Dasatinib and Doxorubicin Treatment of Sarcoma Initiating Cells: A Possible New Treatment Strategy.

PubMed

Aggerholm-Pedersen, Ninna; Demuth, Christina; Safwat, Akmal; Meldgaard, Peter; Kassem, Moustapha; Sandahl Sorensen, Boe

2016-01-01

Background. One of the major challenges affecting sarcoma treatment outcome, particularly that of metastatic disease, is resistance to chemotherapy. Cancer-initiating cells are considered a major contributor to this resistance. Methods. An immortalised nontransformed human stromal (mesenchymal) stem cell line hMSC-TERT4 and a transformed cell line hMSC-TERT20-CE8, known to form sarcoma-like tumours when implanted in immune-deficient mice, were used as models. Receptor tyrosine kinase (RTK) activation was analysed by RTK arrays and cellular viability after tyrosine kinases inhibitor (TKI) treatment with or without doxorubicin was assessed by MTS assay. Results. Initial results showed that the hMSC-TERT4 was more doxorubicin-sensitive while hMSC-TERT20-CE8 was less doxorubicin-sensitive evidenced by monitoring cell viability in the presence of doxorubicin at different doses. The epidermal growth factor receptor (EGFR) was activated in both cell lines. However hMSC-TERT20-CE8 exhibited significantly higher expression of the EGFR ligands. EGFR inhibitors such as erlotinib and afatinib alone or in combination with doxorubicin failed to further decrease cell viability of hMSC-TERT20-CE8. However, inhibition with the TKI dasatinib in combination with doxorubicin decreased cell viability of the hMSC-TERT20-CE8 cell line. Conclusion. Our results demonstrate that dasatinib, but not EGFR-directed treatment, can decrease cell viability of stromal cancer stem cells less sensitive to doxorubicin.

High-throughput identification of genotype-specific cancer vulnerabilities in mixtures of barcoded tumor cell lines

PubMed Central

Yu, Channing; Mannan, Aristotle M.; Yvone, Griselda Metta; Ross, Kenneth N.; Zhang, Yan-Ling; Marton, Melissa A.; Taylor, Bradley R.; Crenshaw, Andrew; Gould, Joshua Z.; Tamayo, Pablo; Weir, Barbara A.; Tsherniak, Aviad; Wong, Bang; Garraway, Levi A.; Shamji, Alykhan F.; Palmer, Michelle A.; Foley, Michael A.; Winckler, Wendy; Schreiber, Stuart L.; Kung, Andrew L.; Golub, Todd R.

2016-01-01

Hundreds of genetically characterized cell lines are available for the discovery of genotype-specific cancer vulnerabilities. However, screening large numbers of compounds against large numbers of cell lines is currently impractical, and such experiments are often difficult to control1-4. Here, we report a method called PRISM that allows pooled screening of mixtures of cancer cell lines by labeling each cell line with 24-nucleotide barcodes. PRISM displayed the expected patterns of cell killing seen in conventional (unpooled) assays. In a screen of 102 cell lines across 8,400 compounds, PRISM led to the identification of BRD-7880 as a potent and highly specific inhibitor of aurora kinases B and C. Cell line pools also efficiently formed tumors as xenografts, and PRISM recapitulated the expected pattern of erlotinib sensitivity in vivo. PMID:26928769

Imaging of bioluminescent LNCaP-luc-M6 tumors: a new animal model for the study of metastatic human prostate cancer.

PubMed

Scatena, Caroline D; Hepner, Mischa A; Oei, Yoko A; Dusich, Joan M; Yu, Shang-Fan; Purchio, Tony; Contag, Pamela R; Jenkins, Darlene E

2004-05-15

Animal experiments examining hormone-sensitive metastatic prostate cancer using the human LNCaP cell line have been limited to endpoint analyses. To permit longitudinal studies, we generated a luciferase-expressing cell line and used bioluminescent imaging (BLI) to non-invasively monitor the in vivo growth of primary LNCaP tumors and metastasis. LNCaP.FGC cells were transfected to constitutively express firefly luciferase. LNCaP-luc-M6 cells were tested for bioluminescent signal intensity and hormone responsiveness in vitro. The cells were implanted in subcutaneous and orthotopic sites in SCID-bg mice and imaged over time. The LNCaP-luc-M6 cells formed subcutaneous and orthotopic tumors in SCID-bg mice, and nearly all tumor-bearing animals developed pulmonary metastases. Early detection and temporal growth of primary tumors and metastatic lesions was successfully monitored by BLI. The LNCaP-luc-M6 cell line is a bioluminescent, hormone-sensitive prostate cancer cell line applicable for BLI studies to non-invasively monitor subcutaneous and orthotopic prostate tumor growth and metastasis in vivo. Copyright 2004 Wiley-Liss, Inc.

Dendritic polymer-based nanodevices for targeted drug delivery applications

NASA Astrophysics Data System (ADS)

Kannan, R. M.; Kolhe, Parag; Gurdag, Sezen; Khandare, Jayant; Lieh-Lai, Mary

2004-03-01

Dendrimers and hyperbranched polymers are unimolecular micellar nanostructures, characterized by globular shape ( ˜ 20 nm) and large density of functional groups at periphery. The tailorable end groups make them ideal for conjugation with drugs, ligands, and imagining agents, making them an attractive molecular nanodevices for drug delivery. Compared to linear polymers and nanoparticles, these nanodevices enter cells rapidly, carrying drugs and delivering them inside cells. Performance of nanodevices prepared for asthma and cancer drug delivery will be discussed. Our conjugation procedure produced very high drug payloads. Dendritic polymer-drug conjugates were very effective in transporting methotrexate (a chemotherapy drug) into both sensitive (CCRF-CEM cell line) and resistant cell line (CEM-MTX). The conjugate nanodevice was 3 times more effective than free drug in the sensitive line, and 9 times more effective in the resistant cell line (based on IC50). The physics of cell entry and drug release from these nanodevices are being investigated. The conjugates appear to enter cells through endocytosis, with the rate of entry dependent on end-group, molecular weight, the pH of the medium, and the cancerous nature of the cells.

DeSigN: connecting gene expression with therapeutics for drug repurposing and development.

PubMed

Lee, Bernard Kok Bang; Tiong, Kai Hung; Chang, Jit Kang; Liew, Chee Sun; Abdul Rahman, Zainal Ariff; Tan, Aik Choon; Khang, Tsung Fei; Cheong, Sok Ching

2017-01-25

The drug discovery and development pipeline is a long and arduous process that inevitably hampers rapid drug development. Therefore, strategies to improve the efficiency of drug development are urgently needed to enable effective drugs to enter the clinic. Precision medicine has demonstrated that genetic features of cancer cells can be used for predicting drug response, and emerging evidence suggest that gene-drug connections could be predicted more accurately by exploring the cumulative effects of many genes simultaneously. We developed DeSigN, a web-based tool for predicting drug efficacy against cancer cell lines using gene expression patterns. The algorithm correlates phenotype-specific gene signatures derived from differentially expressed genes with pre-defined gene expression profiles associated with drug response data (IC 50 ) from 140 drugs. DeSigN successfully predicted the right drug sensitivity outcome in four published GEO studies. Additionally, it predicted bosutinib, a Src/Abl kinase inhibitor, as a sensitive inhibitor for oral squamous cell carcinoma (OSCC) cell lines. In vitro validation of bosutinib in OSCC cell lines demonstrated that indeed, these cell lines were sensitive to bosutinib with IC 50 of 0.8-1.2 μM. As further confirmation, we demonstrated experimentally that bosutinib has anti-proliferative activity in OSCC cell lines, demonstrating that DeSigN was able to robustly predict drug that could be beneficial for tumour control. DeSigN is a robust method that is useful for the identification of candidate drugs using an input gene signature obtained from gene expression analysis. This user-friendly platform could be used to identify drugs with unanticipated efficacy against cancer cell lines of interest, and therefore could be used for the repurposing of drugs, thus improving the efficiency of drug development.

Chilling-induced physiological, anatomical and biochemical responses in the leaves of Miscanthus × giganteus and maize (Zea mays L.).

PubMed

Bilska-Kos, Anna; Panek, Piotr; Szulc-Głaz, Anna; Ochodzki, Piotr; Cisło, Aneta; Zebrowski, Jacek

2018-06-08

Miscanthus × giganteus and Zea mays, closely-related C 4 grasses, originated from warm climates react differently to low temperature. To investigate the response to cold (12-14 °C) in these species, the photosynthetic and anatomical parameters as well as biochemical properties of the cell wall were studied. The research was performed using M. giganteus (MG) and two Z. mays lines differentiated for chilling-sensitivity: chilling-tolerant (Zm-T) and chilling-sensitive (Zm-S). The chilled plants of Zm-S line demonstrated strong inhibition of net CO 2 assimilation and a clear decrease in F' v /F' m , F v /F m and ɸ PSII, while in MG and Zm-T plants these parameters were almost unchanged. The anatomical studies revealed that MG plants had thinner leaves, epidermis and mesophyll cell layer as well as thicker cell walls in the comparison to both maize lines. Cold led to an increase in leaf thickness and mesophyll cell layer thickness in the Zm-T maize line, while the opposite response was observed in Zm-S. In turn, in chilled plants of MG and Zm-T lines, some anatomical parameters associated with bundle sheath cells were higher. In addition, Zm-S line showed the strong increase in the cell wall thickness at cold for mesophyll and bundle sheath cells. Chilling-treatment induced the changes in the cell wall biochemistry of tested species, mainly in the content of glucuronoarabinoxylan, uronic acid, β-glucan and phenolic compounds. This work presents a new approach in searching of mechanism(s) of tolerance/sensitivity to low temperature in two thermophilic plants: Miscanthus and maize. Copyright © 2018 Elsevier GmbH. All rights reserved.

GS-Nitroxide (JP4-039)-Mediated Radioprotection of Human Fanconi Anemia Cell Lines

PubMed Central

Bernard, Mark E.; Kim, Hyun; Berhane, Hebist; Epperly, Michael W.; Franicola, Darcy; Zhang, Xichen; Houghton, Frank; Shields, Donna; Wang, Hong; Bakkenist, Christopher J.; Frantz, Marie-Celine; Forbeck, Erin M.; Goff, Julie P.; Wipf, Peter; Greenberger, Joel S.

2011-01-01

Fanconi anemia (FA) is an inherited disorder characterized by defective DNA repair and cellular sensitivity to DNA crosslinking agents. Clinically, FA is associated with high risk for marrow failure, leukemia and head and neck squamous cell carcinoma (HNSCC). Radiosensitivity in FA patients compromises the use of total-body irradiation for hematopoietic stem cell transplantation and radiation therapy for HNSCC. A radioprotector for the surrounding tissue would therefore be very valuable during radiotherapy for HNSCC. Clonogenic radiation survival curves were determined for pre- or postirradiation treatment with the parent nitroxide Tempol or JP4-039 in cells of four FA patient-derived cell lines and two transgene-corrected subclonal lines. FancG–/– (PD326) and FancD2–/– (PD20F) patient lines were more sensitive to the DNA crosslinking agent mitomycin C (MMC) than their transgene-restored subclonal cell lines (both P < 0.0001). FancD2–/– cells were more radiosensitive than the transgene restored subclonal cell line (ñ = 2.0 ± 0.7 and 4.7 ± 2.2, respectively, P = 0.03). In contrast, FancG–/– cells were radioresistant relative to the transgene-restored subclonal cell line (ñ = 9.4 ± 1.5 and 2.2 ± 05, respectively, P = 0.001). DNA strand breaks measured by the comet assay correlated with radiosensitivity. Cell lines from a Fanc-C and Fanc-A patients showed radiosensitivity similar to that of Fanc-D2–/– cells. A fluorophore-tagged JP4-039 (BODIPY-FL) analog targeted the mitochondria of the cell lines. Preirradiation or postirradiation treatment with JP4-039 at a lower concentration than Tempol significantly increased the radioresistance and stabilized the antioxidant stores of all cell lines. Tempol increased the toxicity of MMC in FancD2–/– cells. These data provide support for the potential clinical use of JP4-039 for normal tissue radioprotection during chemoradiotherapy in FA patients. PMID:21939290

GS-nitroxide (JP4-039)-mediated radioprotection of human Fanconi anemia cell lines.

PubMed

Bernard, Mark E; Kim, Hyun; Berhane, Hebist; Epperly, Michael W; Franicola, Darcy; Zhang, Xichen; Houghton, Frank; Shields, Donna; Wang, Hong; Bakkenist, Christopher J; Frantz, Marie-Celine; Forbeck, Erin M; Goff, Julie P; Wipf, Peter; Greenberger, Joel S

2011-11-01

Fanconi anemia (FA) is an inherited disorder characterized by defective DNA repair and cellular sensitivity to DNA crosslinking agents. Clinically, FA is associated with high risk for marrow failure, leukemia and head and neck squamous cell carcinoma (HNSCC). Radiosensitivity in FA patients compromises the use of total-body irradiation for hematopoietic stem cell transplantation and radiation therapy for HNSCC. A radioprotector for the surrounding tissue would therefore be very valuable during radiotherapy for HNSCC. Clonogenic radiation survival curves were determined for pre- or postirradiation treatment with the parent nitroxide Tempol or JP4-039 in cells of four FA patient-derived cell lines and two transgene-corrected subclonal lines. FancG(-/-) (PD326) and FancD2(-/-) (PD20F) patient lines were more sensitive to the DNA crosslinking agent mitomycin C (MMC) than their transgene-restored subclonal cell lines (both P < 0.0001). FancD2(-/-) cells were more radiosensitive than the transgene restored subclonal cell line (ñ = 2.0 ± 0.7 and 4.7 ± 2.2, respectively, P = 0.03). In contrast, FancG(-/-) cells were radioresistant relative to the transgene-restored subclonal cell line (ñ = 9.4 ± 1.5 and 2.2 ± 05, respectively, P = 0.001). DNA strand breaks measured by the comet assay correlated with radiosensitivity. Cell lines from a Fanc-C and Fanc-A patients showed radiosensitivity similar to that of Fanc-D2(-/-) cells. A fluorophore-tagged JP4-039 (BODIPY-FL) analog targeted the mitochondria of the cell lines. Preirradiation or postirradiation treatment with JP4-039 at a lower concentration than Tempol significantly increased the radioresistance and stabilized the antioxidant stores of all cell lines. Tempol increased the toxicity of MMC in FancD2(-/-) cells. These data provide support for the potential clinical use of JP4-039 for normal tissue radioprotection during chemoradiotherapy in FA patients.

Microvesicle removal of anticancer drugs contributes to drug resistance in human pancreatic cancer cells

PubMed Central

Muralidharan-Chari, Vandhana; Kohan, Hamed Gilzad; Asimakopoulos, Alexandros G.; Sudha, Thangirala; Sell, Stewart; Kannan, Kurunthachalam; Boroujerdi, Mehdi; Davis, Paul J.; Mousa, Shaker A.

2016-01-01

High mortality in pancreatic cancer patients is partly due to resistance to chemotherapy. We describe that human pancreatic cancer cells acquire drug resistance by a novel mechanism in which they expel and remove chemotherapeutic drugs from the microenvironment via microvesicles (MVs). Using human pancreatic cancer cells that exhibit varied sensitivity to gemcitabine (GEM), we show that GEM exposure triggers the cancer cells to release MVs in an amount that correlates with that cell line's sensitivity to GEM. The importance of MV-release in gaining drug resistance in GEM-resistant pancreatic cancer cells was confirmed when the inhibition of MV-release sensitized the cells to GEM treatment, both in vitro and in vivo. Mechanistically, MVs remove drugs that are internalized into the cells and that are in the microenvironment. The differences between the drug-resistant and drug-sensitive pancreatic cancer cell lines tested here are explained based on the variable content of influx/efflux proteins present on MVs, which directly dictates the ability of MVs either to trap GEM or to allow GEM to flow back to the microenvironment. PMID:27391262

Cytotoxicity of the indole alkaloid reserpine from Rauwolfia serpentina against drug-resistant tumor cells.

PubMed

Abdelfatah, Sara A A; Efferth, Thomas

2015-02-15

The antihypertensive reserpine is an indole alkaloid from Rauwolfia serpentina and exerts also profound activity against cancer cells in vitro and in vivo. The present investigation was undertaken to investigate possible modes of action to explain its activity toward drug-resistant tumor cells. Sensitive and drug-resistant tumor cell lines overexpressing P-glycoprotein (ABCB1/MDR1), breast cancer resistance protein (ABCG2/BCRP), mutation-activated epidermal growth factor receptor (EGFR), wild-type and p53-knockout cells as well as the NCI panel of cell lines from different tumor origin were analyzed. Reserpine's cytotoxicity was investigated by resazurin and sulforhodamine assays, flow cytometry, and COMPARE and hierarchical cluster analyses of transcriptome-wide microarray-based RNA expressions. P-glycoprotein- or BCRP overexpressing tumor cells did not reveal cross-resistance to reserpine. EGFR-overexpressing cells were collateral sensitive and p53- Knockout cells cross-resistant to this drug compared to their wild-type parental cell lines. Reserpine increased the uptake of doxorubicin in P-glycoprotein-overexpressing cells, indicating that reserpine inhibited the efflux function of P-glycoprotein. Using molecular docking, we found that reserpine bound with even higher binding energy to P-glycoprotein and EGFR than the control drugs verapamil (P-glycoprotein inhibitor) and erlotinib (EGFR inhibitor). COMPARE and cluster analyses of microarray data showed that the mRNA expression of a panel of genes predicted the sensitivity or resistance of the NCI tumor cell line panel with statistical significance. The genes belonged to diverse pathways and biological functions, e.g. cell survival and apoptosis, EGFR activation, regulation of angiogenesis, cell mobility, cell adhesion, immunological functions, mTOR signaling, and Wnt signaling. The lack of cross-resistance to most resistance mechanisms and the collateral sensitivity in EGFR-transfectants compared to wild-type cells speak for a promising role of reserpine in cancer chemotherapy. Reserpine deserves further consideration for cancer therapy in the clinical setting. Copyright © 2015 Elsevier GmbH. All rights reserved.

A facile, branched DNA assay to quantitatively measure glucocorticoid receptor auto-regulation in T-cell acute lymphoblastic leukemia

PubMed Central

Schwartz, Jason R.; Sarvaiya, Purvaba J.; Leiva, Lily E.; Velez, Maria C.; Singleton, Tammuella C.; Yu, Lolie C.; Vedeckis, Wayne V.

2012-01-01

Glucocorticoid (GC) steroid hormones are used to treat acute lymphoblastic leukemia (ALL) because of their pro-apoptotic effects in hematopoietic cells. However, not all leukemia cells are sensitive to GC, and no assay to stratify patients is available. In the GC-sensitive T-cell ALL cell line CEM-C7, auto-up-regulation of RNA transcripts for the glucocorticoid receptor (GR) correlates with increased apoptotic response. This study aimed to determine if a facile assay of GR transcript levels might be promising for stratifying ALL patients into hormone-sensitive and hormone-resistant populations. The GR transcript profiles of various lymphoid cell lines and 4 bone marrow samples from patients with T-cell ALL were analyzed using both an optimized branched DNA (bDNA) assay and a real-time quantitative reverse transcription-polymerase chain reaction assay. There were significant correlations between both assay platforms when measuring total GR (exon 5/6) transcripts in various cell lines and patient samples, but not for a probe set that detects a specific, low abundance GR transcript (exon 1A3). Our results suggest that the bDNA platform is reproducible and precise when measuring total GR transcripts and, with further development, may ultimately offer a simple clinical assay to aid in the prediction of GC-sensitivity in ALL patients. PMID:22739263

Effect of Berry Extracts and Bioactive Compounds on Fulvestrant (ICI 182,780) Sensitive and Resistant Cell Lines.

PubMed

Woode, Denzel R; Aiyer, Harini S; Sie, Nicole; Zwart, Alan L; Li, Liya; Seeram, Navindra P; Clarke, Robert

2012-01-01

Fulvestrant (ICI 182,780; ICI) is approved for the treatment of advanced metastatic breast cancer that is unresponsive to other endocrine therapies. Berries are frequently consumed for their antioxidant, anti-inflammatory, and anticancer potential. In this study, we tested the efficacy of two berry extracts (Jamun-EJAE and red raspberry-RRE) and their bioactive compounds (Delphinidin-Del and Ellagic acid-EA) to inhibit cell proliferation with or without a sublethal dose of ICI in various breast cancer cell lines. ICI-sensitive (LCC1, ZR75-1, and BT474) and -resistant (LCC9, ZR75-1R) cells were subjected to treatment with berry extracts alone (0.1-100 μg/mL) or with a sub-lethal dose of ICI ( 1). EA, in doses tested, did not have any significant effects on any of the cell lines. Finally, we found that the extracts were more effective at lower, physiologically relevant concentrations than at higher experimental doses.

Effect of Berry Extracts and Bioactive Compounds on Fulvestrant (ICI 182,780) Sensitive and Resistant Cell Lines

PubMed Central

Woode, Denzel R.; Aiyer, Harini S.; Sie, Nicole; Zwart, Alan L.; Li, Liya; Seeram, Navindra P.; Clarke, Robert

2012-01-01

Fulvestrant (ICI 182,780; ICI) is approved for the treatment of advanced metastatic breast cancer that is unresponsive to other endocrine therapies. Berries are frequently consumed for their antioxidant, anti-inflammatory, and anticancer potential. In this study, we tested the efficacy of two berry extracts (Jamun-EJAE and red raspberry-RRE) and their bioactive compounds (Delphinidin-Del and Ellagic acid-EA) to inhibit cell proliferation with or without a sublethal dose of ICI in various breast cancer cell lines. ICI-sensitive (LCC1, ZR75-1, and BT474) and -resistant (LCC9, ZR75-1R) cells were subjected to treatment with berry extracts alone (0.1–100 μg/mL) or with a sub-lethal dose of ICI ( 1). EA, in doses tested, did not have any significant effects on any of the cell lines. Finally, we found that the extracts were more effective at lower, physiologically relevant concentrations than at higher experimental doses. PMID:23346406

Disruption of BASIGIN decreases lactic acid export and sensitizes non-small cell lung cancer to biguanides independently of the LKB1 status.

PubMed

Granja, Sara; Marchiq, Ibtissam; Le Floch, Renaud; Moura, Conceição Souto; Baltazar, Fátima; Pouysségur, Jacques

2015-03-30

Most cancers rely on aerobic glycolysis to generate energy and metabolic intermediates. To maintain a high glycolytic rate, cells must efficiently export lactic acid through the proton-coupled monocarboxylate transporters (MCT1/4). These transporters require a chaperone, CD147/BASIGIN (BSG) for trafficking to the plasma membrane and function.To validate the key role of these transporters in lung cancer, we first analysed the expression of MCT1/4 and BSG in 50 non-small lung cancer (NSCLC) cases. These proteins were specifically upregulated in tumour tissues. We then disrupted BSG in three NSCLC cell lines (A549, H1975 and H292) via 'Zinc-Finger Nucleases'. The three homozygous BSG-/- cell lines displayed a low MCT activity (10- to 5-fold reduction, for MCT1 and MCT4, respectively) compared to wild-type cells. Consequently, the rate of glycolysis, compared to the wild-type counterpart, was reduced by 2.0- to 3.5-fold, whereas the rate of respiration was stimulated in BSG-/- cell lines. Both wild-type and BSG-null cells were extremely sensitive to the mitochondria inhibitor metformin/phenformin in normoxia. However, only BSG-null cells, independently of their LKB1 status, remained sensitive to biguanides in hypoxia in vitro and tumour growth in nude mice. Our results demonstrate that inhibiting glycolysis by targeting lactic acid export sensitizes NSCLC to phenformin.

Radiosensitivity and effect of hypoxia in HPV positive head and neck cancer cells.

PubMed

Sørensen, Brita Singers; Busk, Morten; Olthof, Nadine; Speel, Ernst-Jan; Horsman, Michael R; Alsner, Jan; Overgaard, Jens

2013-09-01

HPV associated Head and Neck Squamous Cell Carcinoma (HNSCC) represents a distinct subgroup of HNSCC characterized by a favorable prognosis and a distinct molecular biology. Previous data from the randomized DAHANCA 5 trial indicated that HPV positive tumors did not benefit from hypoxic modifications by Nimorazole during radiotherapy, whereas a significant benefit was observed in the HPV negative tumors. However, more studies have demonstrated equal frequencies of hypoxic tumors among HPV-positive and HPV-negative tumors. The aim of the present study was to determine radiosensitivity, the impact of hypoxia and the effect of Nimorazole in HPV positive and HPV negative cell lines. The used cell lines were: UDSCC2, UMSCC47 and UPCISCC90 (HPV positive) and FaDuDD, UTSCC33 and UTSCC5 (HPV negative). Cells were cultured under normoxic or hypoxic conditions, and gene expression levels of previously established hypoxia induced genes were assessed by qPCR. Cells were irradiated with various doses under normoxia, hypoxia or hypoxia +1mM Nimorazole, and the clonogenic survival was determined. The HPV positive and HPV negative cell lines exhibited similar patterns of upregulation of hypoxia induced genes in response to hypoxia. The HPV positive cell lines were up to 2.4 times more radiation sensitive than HPV negative cell lines. However, all HPV positive cells displayed the same response to hypoxia in radiosensitivity, with an OER in the range 2.3-2.9, and a sensitizer effect of Nimorazole of 1.13-1.29, similar to HPV negative cells. Although HPV positive cells had a markedly higher radiosensitivity compared to HPV negative cells, they displayed the same relative radioresistance under hypoxia and the same relative sensitizer effect of Nimorazole. The clinical observation that HPV positive patients do not seem to benefit from Nimorazole treatment is not due to inherent differences in hypoxia sensitivity or response to Nimorazole, but can be accounted for by the overall higher radiosensitivity of HPV positive cells. Copyright © 2013. Published by Elsevier Ireland Ltd.

Response of Head and Neck Squamous Cell Carcinoma Cells Carrying PIK3CA Mutations to Select Targeted Therapies

PubMed Central

Wirtz, Eric D; Hoshino, Daisuke; Maldonado, Anthony T; Tyson, Darren R; Weaver, Alissa M

2015-01-01

Importance The PIK3CA mutation is one of the most common mutations in Head and Neck Squamous Cell Carcinoma (HNSCC). Through this research we attempt to elicit the role of oncogene dependence and effects of targeted therapy on this PIK3CA mutation. Objectives 1) To determine the role of oncogene dependence on one of the more common and targetable oncogenes in HNSCC – PIK3CA; 2) To evaluate the consequence of this oncogene on the effectiveness of newly developed targeted therapies. Study Design In vitro study. Setting Academic research laboratory. Participants Cell culture based study assessing the viability of PIK3CA mutated head and neck cell lines when treated with targeted therapy. Exposures PIK3CA mutated head and neck cell lines were treated with 17-AAG, GDC-0941, trametinib, and BEZ-235. Main Outcome and Measures Assessment of cell viability of HNSCC cell lines characterized for PIK3CA mutations or SCC25 cells engineered to express the PIK3CA hotspot mutations E545K or H1047R Results Surprisingly, in engineered cell lines, the hotspot E545K and H1047R mutations conferred decreased, rather than increased, sensitivity as measured by IC50 when treated with the respective HSP90, PI3K, and MEK inhibitors, 17-AAG, GDC-0941, and trametinib, compared to the SCC25 control cell lines. When treated with BEZ-235, H1047R-expressing cell lines showed increased sensitivity to inhibition compared to control while those expressing E545K showed slightly increased sensitivity of unclear significance. Conclusions and Relevance 1) The PIK3CA mutations within our engineered cell model did not lead to enhanced oncogene-dependent cell death when treated with direct inhibition of the PI3K enzyme yet did show increased sensitivity compared to control with dual PI3K/mTOR inhibition. 2) Oncogene addiction to PIK3CA hot spot mutations, if it occurs, is likely to evolve in vivo molecular changes that remain to be identified. Additional study is required to develop new model systems and approaches to determine the role of targeted therapy in the treatment of PI3K-overactive HNSCC tumors. PMID:25855885

High-sensitivity supercontinuum-based parallel line-field optical coherence tomography with 1 million A-lines/s (Conference Presentation)

NASA Astrophysics Data System (ADS)

Barrick, Jessica; Doblas, Ana; Sears, Patrick R.; Ostrowski, Lawrence E.; Oldenburg, Amy L.

2017-02-01

While traditional, flying-spot, spectral domain OCT systems can achieve MHz linerates, they are limited by the need for mechanical scanning to produce a B-mode image. Line-field OCT (LF OCT) removes the need for mechanical scanning by simultaneously recording all A-lines on a 2D CMOS sensor. Our LF OCT system operates at the highest A-line rate of any spectral domain (SD) LF OCT system reported to date (1,024,000 A-lines/s). This is comparable with the fastest flying-spot SDOCT system reported. Additionally, all OCT systems face a tradeoff between imaging speed and sensitivity. Long exposure times improve sensitivity but can lead to undesirable motion artifacts. LF OCT has the potential to relax this tradeoff between sensitivity and imaging speed because all A-lines are exposed during the entire frame acquisition time. However, this advantage has not yet been realized due to the loss of power-per-A-line by spreading the illumination light across all A-lines on the sample. Here we use a supercontinuum source to illuminate the sample with 500mW of light in the 605-950 nm wavelength band, effectively providing 480 µW of power-per-A-line, with axial and lateral resolutions of 1.8 µm and 14 µm, respectively. With this system we achieve the highest reported sensitivity (113 dB) of any LF OCT system. We then demonstrate the capability of this system by capturing the rapidly beating cilia of human bronchial-epithelial cells in vitro. The combination of high speed and high sensitivity offered by supercontinuum-based LF SD OCT offers new opportunities for studying cell and tissue dynamics.

Genotype-Dependent Efficacy of a Dual PI3K/mTOR Inhibitor, NVP-BEZ235, and an mTOR Inhibitor, RAD001, in Endometrial Carcinomas

PubMed Central

Shoji, Keiko; Oda, Katsutoshi; Kashiyama, Tomoko; Ikeda, Yuji; Nakagawa, Shunsuke; Sone, Kenbun; Miyamoto, Yuichiro; Hiraike, Haruko; Tanikawa, Michihiro; Miyasaka, Aki; Koso, Takahiro; Matsumoto, Yoko; Wada-Hiraike, Osamu; Kawana, Kei; Kuramoto, Hiroyuki; McCormick, Frank; Aburatani, Hiroyuki; Yano, Tetsu; Kozuma, Shiro; Taketani, Yuji

2012-01-01

The PI3K (phosphatidylinositol-3-kinase)/mTOR (mammalian target of rapamycin) pathway is frequently activated in endometrial cancer through various PI3K/AKT-activating genetic alterations. We examined the antitumor effect of NVP-BEZ235—a dual PI3K/mTOR inhibitor—and RAD001—an mTOR inhibitor—in 13 endometrial cancer cell lines, all of which possess one or more alterations in PTEN, PIK3CA, and K-Ras. We also combined these compounds with a MAPK pathway inhibitor (PD98059 or UO126) in cell lines with K-Ras alterations (mutations or amplification). PTEN mutant cell lines without K-Ras alterations (n = 9) were more sensitive to both RAD001 and NVP-BEZ235 than were cell lines with K-Ras alterations (n = 4). Dose-dependent growth suppression was more drastically induced by NVP-BEZ235 than by RAD001 in the sensitive cell lines. G1 arrest was induced by NVP-BEZ235 in a dose-dependent manner. We observed in vivo antitumor activity of both RAD001 and NVP-BEZ235 in nude mice. The presence of a MEK inhibitor, PD98059 or UO126, sensitized the K-Ras mutant cells to NVP-BEZ235. Robust growth suppression by NVP-BEZ235 suggests that a dual PI3K/mTOR inhibitor is a promising therapeutic for endometrial carcinomas. Our data suggest that mutational statuses of PTEN and K-Ras might be useful predictors of sensitivity to NVP-BEZ235 in certain endometrial carcinomas. PMID:22662154

A randomized and blinded comparison of qPCR and NGS-based detection of aneuploidy in a cell line mixture model of blastocyst biopsy mosaicism.

PubMed

Goodrich, David; Tao, Xin; Bohrer, Chelsea; Lonczak, Agnieszka; Xing, Tongji; Zimmerman, Rebekah; Zhan, Yiping; Scott, Richard T; Treff, Nathan R

2016-11-01

A subset of preimplantation stage embryos may possess mosaicism of chromosomal constitution, representing a possible limitation to the clinical predictive value of comprehensive chromosome screening (CCS) from a single biopsy. However, contemporary methods of CCS may be capable of predicting mosaicism in the blastocyst by detecting intermediate levels of aneuploidy within a trophectoderm biopsy. This study evaluates the sensitivity and specificity of aneuploidy detection by two CCS platforms using a cell line mixture model of a mosaic trophectoderm biopsy. Four cell lines with known karyotypes were obtained and mixed together at specific ratios of six total cells (0:6, 1:5, 2:4, 3:3, 4:2, 5:1, and 6:0). A female euploid and a male trisomy 18 cell line were used for one set, and a male trisomy 13 and a male trisomy 15 cell line were used for another. Replicates of each mixture were prepared, randomized, and blinded for analysis by one of two CCS platforms (quantitative polymerase chain reaction (qPCR) or VeriSeq next-generation sequencing (NGS)). Sensitivity and specificity of aneuploidy detection at each level of mosaicism was determined and compared between platforms. With the default settings for each platform, the sensitivity of qPCR and NGS were not statistically different, and 100 % specificity was observed (no false positives) at all levels of mosaicism. However, the use of previously published custom criteria for NGS increased sensitivity but also significantly decreased specificity (33 % false-positive prediction of aneuploidy). By demonstrating increased false-positive diagnoses when reducing the stringency of predicting an abnormality, these data illustrate the importance of preclinical evaluation of new testing paradigms before clinical implementation.

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.

α-Hispanolol sensitizes hepatocellular carcinoma cells to TRAIL-induced apoptosis via death receptor up-regulation

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

Mota, Alba, E-mail: amota@iib.uam.es; Jiménez-Garcia, Lidia, E-mail: ljimenez@isciii.es; Herránz, Sandra, E-mail: sherranz@isciii.es

Hispanolone derivatives have been previously described as anti-inflammatory and antitumoral agents. However, their effects on overcoming Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) resistance remain to be elucidated. In this study, we analyzed the cytotoxic effects of the synthetic hispanolone derivative α-hispanolol (α-H) in several tumor cell lines, and we evaluated the induction of apoptosis, as well as the TRAIL-sensitizing potential of α-H in the hepatocellular carcinoma cell line HepG2. Our data show that α-H decreased cell viability in a dose-dependent manner in HeLa, MDA-MB231, U87 and HepG2 cell lines, with a more prominent effect in HepG2 cells. Interestingly, α-H hadmore » no effect on non-tumoral cells. α-H induced activation of caspase-8 and caspase-9 and also increased levels of the proapoptotic protein Bax, decreasing antiapoptotic proteins (Bcl-2, X-IAP and IAP-1) in HepG2 cells. Specific inhibition of caspase-8 abrogated the cascade of caspase activation, suggesting that the extrinsic pathway has a critical role in the apoptotic events induced by α-H. Furthermore, combined treatment of α-H with TRAIL enhanced apoptosis in HepG2 cells, activating caspase-8 and caspase-9. This correlated with up-regulation of both the TRAIL death receptor DR4 and DR5. DR4 or DR5 neutralizing antibodies abolished the effect of α-H on TRAIL-induced apoptosis, suggesting that sensitization was mediated through the death receptor pathway. Our results demonstrate that α-H induced apoptosis in the human hepatocellular carcinoma cell line HepG2 through activation of caspases and induction of the death receptor pathway. In addition, we describe a novel function of α-H as a sensitizer on TRAIL-induced apoptotic cell death in HepG2 cells. - Highlights: • α-Hispanolol induced apoptosis in the human hepatocellular carcinoma cell line HepG2. • α-Hispanolol induced activation of caspases and the death receptor pathway. • α-Hispanolol enhanced TRAIL-induced apoptosis through upregulation of death receptors.« less

Integrating heterogeneous drug sensitivity data from cancer pharmacogenomic studies.

PubMed

Pozdeyev, Nikita; Yoo, Minjae; Mackie, Ryan; Schweppe, Rebecca E; Tan, Aik Choon; Haugen, Bryan R

2016-08-09

The consistency of in vitro drug sensitivity data is of key importance for cancer pharmacogenomics. Previous attempts to correlate drug sensitivities from the large pharmacogenomics databases, such as the Cancer Cell Line Encyclopedia (CCLE) and the Genomics of Drug Sensitivity in Cancer (GDSC), have produced discordant results. We developed a new drug sensitivity metric, the area under the dose response curve adjusted for the range of tested drug concentrations, which allows integration of heterogeneous drug sensitivity data from the CCLE, the GDSC, and the Cancer Therapeutics Response Portal (CTRP). We show that there is moderate to good agreement of drug sensitivity data for many targeted therapies, particularly kinase inhibitors. The results of this largest cancer cell line drug sensitivity data analysis to date are accessible through the online portal, which serves as a platform for high power pharmacogenomics analysis.

Paclitaxel sensitivity in relation to ABCB1 expression, efflux and single nucleotide polymorphisms in ovarian cancer.

PubMed

Gao, Bo; Russell, Amanda; Beesley, Jonathan; Chen, Xiao Qing; Healey, Sue; Henderson, Michelle; Wong, Mark; Emmanuel, Catherine; Galletta, Laura; Johnatty, Sharon E; Bowtell, David; Haber, Michelle; Norris, Murray; Harnett, Paul; Chenevix-Trench, Georgia; Balleine, Rosemary L; deFazio, Anna

2014-05-09

ABCB1 (adenosine triphosphate-binding cassette transporter B1) mediates cellular elimination of many chemotherapeutic agents including paclitaxel, which is commonly used to treat ovarian cancer. A significant association between common single nucleotide polymorphisms (SNPs) in ABCB1 and progression-free survival has been reported in patients with ovarian cancer. Variable paclitaxel clearance due to genotype specific differences in ABCB1 activity in cancer cells and/or normal tissues may underlie the association. Using cell-based models, we evaluated the correlations between ABCB1 expression, polymorphisms, transporter activity and paclitaxel sensitivity in ovarian cancer (n = 10) and lymphoblastoid (n = 19) cell lines. Close associations between ABCB1 expression, transporter function and paclitaxel sensitivity were found in lymphoblastoid cell lines, although we could not demonstrate an association with common SNPs. In ovarian cancer cell lines, ABCB1 expression was low and the association between expression and function was lost. These results suggest that ABCB1 related survival difference in ovarian cancer patients is more likely to be due to differential whole body paclitaxel clearance mediated by normal cells rather than a direct effect on cancer cells.

Cardiomyocyte H9c2 cells present a valuable alternative to fish lethal testing for azoxystrobin.

PubMed

Rodrigues, Elsa T; Pardal, Miguel Â; Laizé, Vincent; Cancela, M Leonor; Oliveira, Paulo J; Serafim, Teresa L

2015-11-01

The present study aims at identifying, among six mammalian and fish cell lines, a sensitive cell line whose in vitro median inhibitory concentration (IC50) better matches the in vivo short-term Sparus aurata median lethal concentration (LC50). IC50s and LC50 were assessed after exposure to the widely used fungicide azoxystrobin (AZX). Statistical results were relevant for most cell lines after 48 h of AZX exposure, being H9c2 the most sensitive cells, as well as the ones which provided the best prediction of fish toxicity, with a LC50,96h/IC50,48h = 0.581. H9c2 cell proliferation upon 72 h of AZX exposure revealed a LC50,96h/IC50,72h = 0.998. Therefore, identical absolute sensitivities were attained for both in vitro and in vivo assays. To conclude, the H9c2 cell-based assay is reliable and represents a suitable ethical alternative to conventional fish assays for AZX, and could be used to get valuable insights into the toxic effects of other pesticides. Copyright © 2015 Elsevier Ltd. All rights reserved.

Revisiting inconsistency in large pharmacogenomic studies

PubMed Central

Safikhani, Zhaleh; Smirnov, Petr; Freeman, Mark; El-Hachem, Nehme; She, Adrian; Rene, Quevedo; Goldenberg, Anna; Birkbak, Nicolai J.; Hatzis, Christos; Shi, Leming; Beck, Andrew H.; Aerts, Hugo J.W.L.; Quackenbush, John; Haibe-Kains, Benjamin

2017-01-01

In 2013, we published a comparative analysis of mutation and gene expression profiles and drug sensitivity measurements for 15 drugs characterized in the 471 cancer cell lines screened in the Genomics of Drug Sensitivity in Cancer (GDSC) and Cancer Cell Line Encyclopedia (CCLE). While we found good concordance in gene expression profiles, there was substantial inconsistency in the drug responses reported by the GDSC and CCLE projects. We received extensive feedback on the comparisons that we performed. This feedback, along with the release of new data, prompted us to revisit our initial analysis. We present a new analysis using these expanded data, where we address the most significant suggestions for improvements on our published analysis — that targeted therapies and broad cytotoxic drugs should have been treated differently in assessing consistency, that consistency of both molecular profiles and drug sensitivity measurements should be compared across cell lines, and that the software analysis tools provided should have been easier to run, particularly as the GDSC and CCLE released additional data. Our re-analysis supports our previous finding that gene expression data are significantly more consistent than drug sensitivity measurements. Using new statistics to assess data consistency allowed identification of two broad effect drugs and three targeted drugs with moderate to good consistency in drug sensitivity data between GDSC and CCLE. For three other targeted drugs, there were not enough sensitive cell lines to assess the consistency of the pharmacological profiles. We found evidence of inconsistencies in pharmacological phenotypes for the remaining eight drugs. Overall, our findings suggest that the drug sensitivity data in GDSC and CCLE continue to present challenges for robust biomarker discovery. This re-analysis provides additional support for the argument that experimental standardization and validation of pharmacogenomic response will be necessary to advance the broad use of large pharmacogenomic screens. PMID:28928933

Loss of the deubiquitylase BAP1 alters class I histone deacetylase expression and sensitivity of mesothelioma cells to HDAC inhibitors

PubMed Central

Sacco, Joseph J.; Kenyani, Jenna; Butt, Zohra; Carter, Rachel; Chew, Hui Yi; Cheeseman, Liam P.; Darling, Sarah; Denny, Michael; Urbé, Sylvie; Clague, Michael J.; Coulson, Judy M.

2015-01-01

Histone deacetylases are important targets for cancer therapeutics, but their regulation is poorly understood. Our data show coordinated transcription of HDAC1 and HDAC2 in lung cancer cell lines, but suggest HDAC2 protein expression is cell-context specific. Through an unbiased siRNA screen we found that BRCA1-associated protein 1 (BAP1) regulates their expression, with HDAC2 reduced and HDAC1 increased in BAP1 depleted cells. BAP1 loss-of-function is increasingly reported in cancers including thoracic malignancies, with frequent mutation in malignant pleural mesothelioma. Endogenous HDAC2 directly correlates with BAP1 across a panel of lung cancer cell lines, and is downregulated in mesothelioma cell lines with genetic BAP1 inactivation. We find that BAP1 regulates HDAC2 by increasing transcript abundance, rather than opposing its ubiquitylation. Importantly, although total cellular HDAC activity is unaffected by transient depletion of HDAC2 or of BAP1 due to HDAC1 compensation, this isoenzyme imbalance sensitizes MSTO-211H cells to HDAC inhibitors. However, other established mesothelioma cell lines with low endogenous HDAC2 have adapted to become more resistant to HDAC inhibition. Our work establishes a mechanism by which BAP1 loss alters sensitivity of cancer cells to HDAC inhibitors. Assessment of BAP1 and HDAC expression may ultimately help identify patients likely to respond to HDAC inhibitors. PMID:25970771

PIG7 promotes leukemia cell chemosensitivity via lysosomal membrane permeabilization

PubMed Central

Niu, Ting; Wu, Yu; Li, Jianjun; Wang, Fangfang; Zheng, Yuhuan; Liu, Ting

2016-01-01

PIG7 localizes to lysosomal membrane in leukemia cells. Our previous work has shown that transduction of pig7 into a series of leukemia cell lines did not result in either apoptosis or differentiation of most tested cell lines. Interestingly, it did significantly sensitize these cell lines to chemotherapeutic drugs. Here, we further investigated the mechanism underlying pig7-induced improved sensitivity of acute leukemia cells to chemotherapy. Our results demonstrated that the sensitization effect driven by exogenous pig7 was more effective in drug-resistant leukemia cell lines which had lower endogenous pig7 expression. Overexpression of pig7 did not directly activate the caspase apoptotic pathway, but decreased the lysosomal stability. The expression of pig7 resulted in lysosomal membrane permeabilization (LMP) and lysosomal protease (e.g. cathepsin B, D, L) release. Moreover, we also observed increased reactive oxygen species (ROS) and decreased mitochondrial membrane potential (ΔΨm) induced by pig7. Some autophagy markers such as LC3I/II, ATG5 and Beclin-1, and necroptosis maker MLKL were also stimulated. However, intrinsic antagonism such as serine/cysteine protease inhibitors Spi2A and Cystatin C prevented downstream effectors from triggering leukemia cells, which were only on the “verge of apoptosis”. When combined with chemotherapy, LMP increased and more proteases were released. Once this process was beyond the limit of intrinsic antagonism, it induced programmed cell death cooperatively via caspase-independent and caspase-dependent pathways. PMID:26716897

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.

ATM-Deficient Colorectal Cancer Cells Are Sensitive to the PARP Inhibitor Olaparib.

PubMed

Wang, Chen; Jette, Nicholas; Moussienko, Daniel; Bebb, D Gwyn; Lees-Miller, Susan P

2017-04-01

The ataxia telangiectasia mutated (ATM) protein kinase plays a central role in the cellular response to DNA damage. Loss or inactivation of both copies of the ATM gene (ATM) leads to ataxia telangiectasia, a devastating childhood condition characterized by neurodegeneration, immune deficiencies, and cancer predisposition. ATM is also absent in approximately 40% of mantle cell lymphomas (MCLs), and we previously showed that MCL cell lines with loss of ATM are sensitive to poly-ADP ribose polymerase (PARP) inhibitors. Next-generation sequencing of patient tumors has revealed that ATM is altered in many human cancers including colorectal, lung, prostate, and breast. Here, we show that the colorectal cancer cell line SK-CO-1 lacks detectable ATM protein expression and is sensitive to the PARP inhibitor olaparib. Similarly, HCT116 colorectal cancer cells with shRNA depletion of ATM are sensitive to olaparib, and depletion of p53 enhances this sensitivity. Moreover, HCT116 cells are sensitive to olaparib in combination with the ATM inhibitor KU55933, and sensitivity is enhanced by deletion of p53. Together our studies suggest that PARP inhibitors may have potential for treating colorectal cancer with ATM dysfunction and/or colorectal cancer with mutation of p53 when combined with an ATM kinase inhibitor. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Wee1 Kinase Inhibitor AZD1775 Radiosensitizes Hepatocellular Carcinoma Regardless of TP53 Mutational Status Through Induction of Replication Stress

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

Cuneo, Kyle C., E-mail: kcuneo@umich.edu; Morgan, Meredith A.; Davis, Mary A.

2016-06-01

Purpose: Wee1 kinase inhibitors are effective radiosensitizers in cells lacking a G{sub 1} checkpoint. In this study we examined the potential effect of Wee1 kinase inhibition on inducing replication stress in hepatocellular carcinoma (HCC). Methods and Materials: Five independent datasets from the Oncomine database comparing gene expression in HCC compared to normal tissue were combined and specific markers associated with Wee1 sensitivity were analyzed. We then performed a series of in vitro experiments to study the effect of Wee1 inhibition on irradiated HCC cell lines with varying p53 mutational status. Clonogenic survival assays and flow cytometry using anti-γH2AX and phospho-histone H3more » antibodies with propidium iodide were performed to study the effect of AZD1775 on survival, cell cycle, and DNA repair. Additionally, nucleoside enriched medium was used to examine the effect of altering nucleotide pools on Wee1 targeted radiation sensitization. Results: Our analysis of the Oncomine database found high levels of CDK1 and other cell cycle regulators indicative of Wee1 sensitivity in HCC. In our in vitro experiments, treatment with AZD1775 radiosensitized and chemosensitized Hep3B, Huh7, and HepG2 cell lines and was associated with delayed resolution of γH2AX foci and the induction of pan-nuclear γH2AX staining. Wee1 inhibition attenuated radiation-induced G{sub 2} arrest in the Hep3B (TP53 null) and Huh7 (TP53 mutant) cell lines but not in the TP53 wild-type cell line HepG2. Supplementation with nucleosides reversed the radiation-sensitizing effect of AZD1775 and reduced the amount of cells with pan-nuclear γH2AX staining after radiation. Conclusions: Radiation sensitization with Wee1 inhibition occurs in cells regardless of their p53 mutational status. In this study we show for the first time that replication stress via the overconsumption of nucleotides plays an important role in AZD1775-induced radiation sensitization.« less

Subcellular localization patterns and their relationship to photodynamic activity of pyropheophorbide-a derivatives.

PubMed

MacDonald, I J; Morgan, J; Bellnier, D A; Paszkiewicz, G M; Whitaker, J E; Litchfield, D J; Dougherty, T J

1999-11-01

To determine if subcellular localization is important to photodynamic therapy (PDT) efficacy, an in vitro fluorescence microscopy study was conducted with a congeneric series of pyropheophorbide-a derivatives in human pharyngeal squamous cell carcinoma (FaDu) cells and murine radiation-induced fibrosarcoma (RIF) mutant cells. In the FaDu cells the octyl, decyl and dodecyl ether derivatives localized to the lysosomes at extracellular concentrations less than needed to produce a 50% cell kill (LD50). At extracellular concentrations equal or greater than the LD50 the compounds localized mainly to mitochondria. The propyl, pentyl, hexyl and heptyl ether derivatives localized mainly to the mitochondria at all concentrations studied. This suggested that mitochondria are a sensitive PDT target for these derivatives. Similar experiments were performed with two Photofrin-PDT resistant RIF cell lines, one of which was found to be resistant to hexyl ether derivative (C6) mediated-PDT and the other sensitive to C6-PDT relative to the parent line. At extracellular concentrations of C6 below the LD50 of each cell line, the mutants exhibited lysosomal localization. At concentrations above these values the patterns shifted to a mainly mitochondrial pattern. In these cell lines mitochondrial localization also correlated with PDT sensitivity. Localization to mitochondria or lysosomes appeared to be affected by the aggregation state of the congeners, all of which are highly aggregated in aqueous medium. Monomers apparently were the active fraction of these compounds because equalizing the extracellular monomer concentrations produced equivalent intracellular concentrations, photoxicity and localization patterns. Compounds that were mainly aggregates localized to the lysosomes where they were rendered less active. Mitochondria appear to be a sensitive target for pyropheophorbide-a-mediated photodamage, and the degree of aggregation seems to be a determinant of the localization site.

Loss of MLH1 sensitizes colon cancer cells to DNA-PKcs inhibitor KU60648.

PubMed

Hinrichsen, Inga; Ackermann, Anne; Düding, Tonja; Graband, Annika; Filmann, Natalie; Plotz, Guido; Zeuzem, Stefan; Brieger, Angela

2017-07-01

Germline mutations of MLH1 are responsible for tumor generation in nearly 50% of patients with Lynch Syndrome, and around 15% of sporadic colorectal cancers show MLH1-deficiency due to promotor hypermethylation. Although these tumors are of lower aggressiveness the benefit for these patients from standard chemotherapy is still under discussion. Recently, it was shown that the sensitivity to the DNA-PKcs inhibitor KU60648 is linked to loss of the MMR protein MSH3. However, loss of MSH3 is rather secondary, as a consequence of MMR-deficiency, and frequently detectable in MLH1-deficient tumors. Therefore, we examined the expression of MLH1, MSH2, MSH6, and MSH3 in different MMR-deficient and proficient cell lines and determined their sensitivity to KU60648 by analyzing cell viability and survival. MLH1-dependent ability of double strand break (DSB) repair was monitored after irradiation via γH2AX detection. A panel of 12 colon cancer cell lines, two pairs of cells, where MLH1 knock down was compared to controls with the same genetic background, and one MLH1-deficient cell line where MLH1 was overexpressed, were included. In summary, we found that MLH1 and/or MSH3-deficient cells exhibited a significantly higher sensitivity to KU60648 than MMR-proficient cells and that overexpression of MLH1 in MLH1-deficient cells resulted in a decrease of cell sensitivity. KU60648 efficiency seems to be associated with reduced DSB repair capacity. Since the molecular testing of colon tumors for MLH1 expression is a clinical standard we believe that MLH1 is a much better marker and a greater number of patients would benefit from KU60648 treatment. © 2017 Wiley Periodicals, Inc.

Using genome-wide CRISPR library screening with library resistant DCK to find new sources of Ara-C drug resistance in AML.

PubMed

Kurata, Morito; Rathe, Susan K; Bailey, Natashay J; Aumann, Natalie K; Jones, Justine M; Veldhuijzen, G Willemijn; Moriarity, Branden S; Largaespada, David A

2016-11-03

Acute myeloid leukemia (AML) can display de novo or acquired resistance to cytosine arabinoside (Ara-C), a primary component of induction chemotherapy. To identify genes capable of independently imposing Ara-C resistance, we applied a genome-wide CRISPR library to human U937 cells and exposed to them to Ara-C. Interestingly, all drug resistant clones contained guide RNAs for DCK. To avoid DCK gene modification, gRNA resistant DCK cDNA was created by the introduction of silent mutations. The CRISPR screening was repeated using the gRNA resistant DCK, and loss of SLC29A was identified as also being capable of conveying Ara-C drug resistance. To determine if loss of Dck results in increased sensitivity to other drugs, we conducted a screen of 446 FDA approved drugs using two Dck-defective BXH-2 derived murine AML cell lines and their Ara-C sensitive parental lines. Both cell lines showed an increase in sensitivity to prednisolone. Guide RNA resistant cDNA rescue was a legitimate strategy and multiple DCK or SLC29A deficient human cell clones were established with one clone becoming prednisolone sensitive. Dck-defective leukemic cells may become prednisolone sensitive indicating prednisolone may be an effective adjuvant therapy in some cases of DCK-negative AML.

Inhibition of activated receptor tyrosine kinases by Sunitinib induces growth arrest and sensitizes melanoma cells to Bortezomib by blocking Akt pathway.

PubMed

Yeramian, Andree; Sorolla, Anabel; Velasco, Ana; Santacana, Maria; Dolcet, Xavier; Valls, Joan; Abal, Leandre; Moreno, Sara; Egido, Ramón; Casanova, Josep M; Puig, Susana; Vilella, Ramón; Llombart-Cussac, Antonio; Matias-Guiu, Xavier; Martí, Rosa M

2012-02-15

Despite the use of multiple therapeutic strategies, metastatic melanoma remains a challenge for oncologists. Thus, new approaches using combinational treatment may be used to try to improve the prognosis of this disease. In this report, we have analyzed the expression of receptor tyrosine kinases (RTKs) in melanoma specimens and in four metastatic melanoma cell lines. Both melanoma specimens and cell lines expressed RTKs, suggesting that they may represent eventual targets for multitargeted tyrosine kinase inhibitor, Suntinib. Sunitinib reduced the proliferation of two melanoma cell lines (M16 and M17) and increased apoptosis in one of them (M16). Moreover, the two metastatic melanoma cell lines harbored an activated receptor (PDGFRα and VEGFR, respectively), and Sunitinib suppressed the phosphorylation of the RTKs and their downstream targets Akt and ribosomal protein S6, in these two cell lines. Similar results were obtained when either PDGFRα or VEGFR2 expression was silenced by lentiviral-mediated short-hairpin RNA delivery in M16 and M17, respectively. To evaluate the interaction between Sunitinib and Bortezomib, median dose effect analysis using MTT assay was performed, and combination index was calculated. Bortezomib synergistically enhanced the Sunitinib-induced growth arrest in Sunitinib-sensitive cells (combination index < 1). Moreover, LY294002, a PI3K inhibitor, sensitized melanoma cells to Bortezomib treatment, suggesting that downregulation of phospho-Akt by Sunitinib mediates the synergy obtained by Bortezomib + Sunitinib cotreatment. Altogether, our results suggest that melanoma cells harboring an activated RTK may be clinically responsive to pharmacologic RTK inhibition by Sunitinib, and a strategy combining Sunitinib and Bortezomib, may provide therapeutic benefit. Copyright © 2011 UICC.

Fibulin-3 negatively regulates ALDH1 via c-MET suppression and increases γ-radiation-induced sensitivity in some pancreatic cancer cell lines

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

Kim, In-Gyu, E-mail: igkim@kaeri.re.kr; Department of Radiation Biotechnology and Applied Radioisotope, Korea University of Science and Technology; Lee, Jae-Ha

2014-11-21

Highlights: • FBLN-3 gene was poorly expressed in some pancreatic cancer lines. • FBLN-3 promoter region was highly methylated in some pancreatic cancer cell lines. • FBLN-3 inhibited c-MET activation and expression and reduced cellular level of ALDH1. • FBLN-3/c-Met/ALDH1 axis modulates stemness and EMT in pancreatic cancer cells. - Abstract: Fibulin-3 (FBLN-3) has been postulated to be either a tumor suppressor or promoter depending on the cell type, and hypermethylation of the FBLN-3 promoter is often associated with human disease, especially cancer. We report that the promoter region of the FBLN-3 was significantly methylated (>95%) in some pancreatic cancermore » cell lines and thus FBLN-3 was poorly expressed in pancreatic cancer cell lines such as AsPC-1 and MiaPaCa-2. FBLN-3 overexpression significantly down-regulated the cellular level of c-MET and inhibited hepatocyte growth factor-induced c-MET activation, which were closely associated with γ-radiation resistance of cancer cells. Moreover, we also showed that c-MET suppression or inactivation decreased the cellular level of ALDH1 isozymes (ALDH1A1 or ALDH1A3), which serve as cancer stem cell markers, and subsequently induced inhibition of cell growth in pancreatic cancer cells. Therefore, forced overexpression of FBLN-3 sensitized cells to cytotoxic agents such as γ-radiation and strongly inhibited the stemness and epithelial to mesenchymal transition (EMT) property of pancreatic cancer cells. On the other hand, if FBLN3 was suppressed in FBLN-3-expressing BxPC3 cells, the results were opposite. This study provides the first demonstration that the FBLN-3/c-MET/ALDH1 axis in pancreatic cancer cells partially modulates stemness and EMT as well as sensitization of cells to the detrimental effects of γ-radiation.« less

RNA interference of argininosuccinate synthetase restores sensitivity to recombinant arginine deiminase (rADI) in resistant cancer cells

PubMed Central

2011-01-01

Background Sensitivity of cancer cells to recombinant arginine deiminase (rADI) depends on expression of argininosuccinate synthetase (AS), a rate-limiting enzyme in synthesis of arginine from citrulline. To understand the efficiency of RNA interfering of AS in sensitizing the resistant cancer cells to rADI, the down regulation of AS transiently and permanently were performed in vitro, respectively. Methods We studied the use of down-regulation of this enzyme by RNA interference in three human cancer cell lines (A375, HeLa, and MCF-7) as a way to restore sensitivity to rADI in resistant cells. The expression of AS at levels of mRNA and protein was determined to understand the effect of RNA interference. Cell viability, cell cycle, and possible mechanism of the restore sensitivity of AS RNA interference in rADI treated cancer cells were evaluated. Results AS DNA was present in all cancer cell lines studied, however, the expression of this enzyme at the mRNA and protein level was different. In two rADI-resistant cell lines, one with endogenous AS expression (MCF-7 cells) and one with induced AS expression (HeLa cells), AS small interference RNA (siRNA) inhibited 37-46% of the expression of AS in MCF-7 cells. ASsiRNA did not affect cell viability in MCF-7 which may be due to the certain amount of residual AS protein. In contrast, ASsiRNA down-regulated almost all AS expression in HeLa cells and caused cell death after rADI treatment. Permanently down-regulated AS expression by short hairpin RNA (shRNA) made MCF-7 cells become sensitive to rADI via the inhibition of 4E-BP1-regulated mTOR signaling pathway. Conclusions Our results demonstrate that rADI-resistance can be altered via AS RNA interference. Although transient enzyme down-regulation (siRNA) did not affect cell viability in MCF-7 cells, permanent down-regulation (shRNA) overcame the problem of rADI-resistance due to the more efficiency in AS silencing. PMID:21453546

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

A sensitive ELISA for measuring the adhesion of leukocytic cells to human endothelial cells.

PubMed

Krakauer, T

1994-12-28

A new, sensitive ELISA using monoclonal antibodies reactive with surface molecules specific for various leukocytes was devised to measure the attachment of these cells to cultured monolayers of human umbilical vein endothelial cells. Preparations of peripheral blood mononuclear cells, a human monocytic cell line (THP-1) and a human lymphoblastic T cell line (MOLT-4) were used to test the sensitivity of this method and compare it with the conventional 51Cr-radiolabeled cell assay. The extent of adhesion to endothelial cells was assayed by measuring the optical density produced by a complex of peroxidase-labeled streptavidin, biotin-conjugated F(ab')2 anti-mouse Ig and monoclonal antibody on fixed leukocytic cells that had adhered to endothelial cells. This method is fast and sensitive, eliminates the use of radioisotopes, and, because the detection uses a specific marker on the cell of interest, can be used in preparations of unseparated mixtures of cells. As this is a microassay, using relatively small number of cells and reagents, the methodology can be applied to screen a large number of therapeutic agents that may regulate adhesion. Using this method, the anti-inflammatory corticosteroid, dexamethasone, was found to inhibit the adhesion of THP-1 and MOLT-4 cells to cytokine-activated endothelial cells.

Inhibition of Oncogenic Transcription Factor REL by the Natural Product Derivative Calafianin Monomer 101 Induces Proliferation Arrest and Apoptosis in Human B-Lymphoma Cell Lines.

PubMed

Yeo, Alan T; Chennamadhavuni, Spandan; Whitty, Adrian; Porco, John A; Gilmore, Thomas D

2015-04-23

Increased activity of transcription factor NF-κB has been implicated in many B-cell lymphomas. We investigated effects of synthetic compound calafianin monomer (CM101) on biochemical and biological properties of NF-κB. In human 293 cells, CM101 selectively inhibited DNA binding by overexpressed NF-κB subunits REL (human c-Rel) and p65 as compared to NF-κB p50, and inhibition of REL and p65 DNA binding by CM101 required a conserved cysteine residue. CM101 also inhibited DNA binding by REL in human B-lymphoma cell lines, and the sensitivity of several B-lymphoma cell lines to CM101-induced proliferation arrest and apoptosis correlated with levels of cellular and nuclear REL. CM101 treatment induced both phosphorylation and decreased expression of anti-apoptotic protein Bcl-XL, a REL target gene product, in sensitive B-lymphoma cell lines. Ectopic expression of Bcl-XL protected SUDHL-2 B-lymphoma cells against CM101-induced apoptosis, and overexpression of a transforming mutant of REL decreased the sensitivity of BJAB B-lymphoma cells to CM101-induced apoptosis. Lipopolysaccharide-induced activation of NF-κB signaling upstream components occurred in RAW264.7 macrophages at CM101 concentrations that blocked NF-κB DNA binding. Direct inhibitors of REL may be useful for treating B-cell lymphomas in which REL is active, and may inhibit B-lymphoma cell growth at doses that do not affect some immune-related responses in normal cells.

Combined use of anti-ErbB monoclonal antibodies and erlotinib enhances antibody-dependent cellular cytotoxicity of wild-type erlotinib-sensitive NSCLC cell lines

PubMed Central

2012-01-01

Background The epidermal growth factor receptor (EGFR) is an established target for anti-cancer treatment in different tumour types. Two different strategies have been explored to inhibit this pivotal molecule in epithelial cancer development: small molecules TKIs and monoclonal antibodies. ErbB/HER-targeting by monoclonal antibodies such as cetuximab and trastuzumab or tyrosine-kinase inhibitors as gefitinib or erlotinib has been proven effective in the treatment of advanced NSCLC. Results In this study we explored the potential of combining either erlotinib with cetuximab or trastuzumab to improve the efficacy of EGFR targeted therapy in EGFR wild-type NSCLC cell lines. Erlotinib treatment was observed to increase EGFR and/or HER2 expression at the plasma membrane level only in NSCLC cell lines sensitive to the drug inducing protein stabilization. The combined treatment had marginal effect on cell proliferation but markedly increased antibody-dependent, NK mediated, cytotoxicity in vitro. Moreover, in the Calu-3 xenograft model, the combination significantly inhibited tumour growth when compared with erlotinib and cetuximab alone. Conclusion Our results indicate that erlotinib increases surface expression of EGFR and/or HER2 only in EGFR-TKI sensitive NSCLC cell lines and, in turns, leads to increased susceptibility to ADCC both in vitro and in a xenograft models. The combination of erlotinib with monoclonal antibodies represents a potential strategy to improve the treatment of wild-type EGFR NSCLC patients sensitive to erlotinib. PMID:23234355

Potentiation by Tumor Necrosis Factor of Mitoxantrone Cytotoxicity to Human Ovarian Cancer Cell Lines

PubMed Central

Parodi, Silvio; Billi, Giovanna; Oliva, Cristina; Venturing, Marco; Noviello, Elvira; Conte, PierFranco

1992-01-01

The cytotoxic activity of human recombinant tumor necrosis factor (rHuTNF) (from 0.01 to 10000 U/ml) was assayed on six human ovarian cancer cell lines and one human cervical carcinoma cell line using a crystal violet assay. rHuTNF was cytotoxic to four cell lines (A2780, A2774, SW626, PAD, while 3 cell lines (IGROV1, SKOV3, Mel80) were marginally sensitive to its activity. However, under the same experimental conditions rHuTNF markedly enhanced the cytotoxicity of mitoxantrone, a chemotherapeutic drug targeted at DNA topoisomerase II, in six cell lines. The potentiation of mitoxantrone cytotoxicity was not caused by increased drug accumulation after rHuTNF treatment. No significant increase in cytotoxicity to Me180 cell line was seen when rHuTNF was added to mitoxantrone. PMID:1517145

Growth regulation of simian and human AIDS-related non-Hodgkin's lymphoma cell lines by TGF-β1 and IL-6

PubMed Central

Ruff, Kristin R; Puetter, Adriane; Levy, Laura S

2007-01-01

Background AIDS-related non-Hodgkin's lymphoma (AIDS-NHL) is the second most frequent cancer associated with AIDS, and is a frequent cause of death in HIV-infected individuals. Experimental analysis of AIDS-NHL has been facilitated by the availability of an excellent animal model, i.e., simian Acquired Immunodeficiency Syndrome (SAIDS) in the rhesus macaque consequent to infection with simian immunodeficiency virus. A recent study of SAIDS-NHL demonstrated a lymphoma-derived cell line to be sensitive to the growth inhibitory effects of the ubiquitous cytokine, transforming growth factor-beta (TGF-beta). The authors concluded that TGF-beta acts as a negative growth regulator of the lymphoma-derived cell line and, potentially, as an inhibitory factor in the regulatory network of AIDS-related lymphomagenesis. The present study was conducted to assess whether other SAIDS-NHL and AIDS-NHL cell lines are similarly sensitive to the growth inhibitory effects of TGF-beta, and to test the hypothesis that interleukin-6 (IL-6) may represent a counteracting positive influence in their growth regulation. Methods Growth stimulation or inhibition in response to cytokine treatment was quantified using trypan blue exclusion or colorimetric MTT assay. Intracellular flow cytometry was used to analyze the activation of signaling pathways and to examine the expression of anti-apoptotic proteins and distinguishing hallmarks of AIDS-NHL subclass. Apoptosis was quantified by flow cytometric analysis of cell populations with sub-G1 DNA content and by measuring activated caspase-3. Results Results confirmed the sensitivity of LCL8664, an immunoblastic SAIDS-NHL cell line, to TGF-beta1-mediated growth inhibition, and further demonstrated the partial rescue by simultaneous treatment with IL-6. IL-6 was shown to activate STAT3, even in the presence of TGF-beta1, and thereby to activate proliferative and anti-apoptotic pathways. By comparison, human AIDS-NHL cell lines differed in their responsiveness to TGF-beta1 and IL-6. Analysis of a recently derived AIDS-NHL cell line, UMCL01-101, indicated that it represents immunoblastic AIDS-DLCBL. Like LCL-8664, UMCL01-101 was sensitive to TGF-beta1-mediated inhibition, rescued partially by IL-6, and demonstrated rapid STAT3 activation following IL-6 treatment even in the presence of TGF-beta1. Conclusion These studies indicate that the sensitivity of immunoblastic AIDS- or SAIDS-DLBCL to TGF-beta1-mediated growth inhibition may be overcome through the stimulation of proliferative and anti-apoptotic signals by IL-6, particularly through the rapid activation of STAT3. PMID:17324269

Mutations of the LIM protein AJUBA mediate sensitivity of head and neck squamous cell carcinoma to treatment with cell-cycle inhibitors.

PubMed

Zhang, Ming; Singh, Ratnakar; Peng, Shaohua; Mazumdar, Tuhina; Sambandam, Vaishnavi; Shen, Li; Tong, Pan; Li, Lerong; Kalu, Nene N; Pickering, Curtis R; Frederick, Mitchell; Myers, Jeffrey N; Wang, Jing; Johnson, Faye M

2017-04-28

The genomic alterations identified in head and neck squamous cell carcinoma (HNSCC) tumors have not resulted in any changes in clinical care, making the development of biomarker-driven targeted therapy for HNSCC a major translational gap in knowledge. To fill this gap, we used 59 molecularly characterized HNSCC cell lines and found that mutations of AJUBA, SMAD4 and RAS predicted sensitivity and resistance to treatment with inhibitors of polo-like kinase 1 (PLK1), checkpoint kinases 1 and 2, and WEE1. Inhibition or knockdown of PLK1 led to cell-cycle arrest at the G 2 /M transition and apoptosis in sensitive cell lines and decreased tumor growth in an orthotopic AJUBA-mutant HNSCC mouse model. AJUBA protein expression was undetectable in most AJUBA-mutant HNSCC cell lines, and total PLK1 and Bora protein expression were decreased. Exogenous expression of wild-type AJUBA in an AJUBA-mutant cell line partially rescued the phenotype of PLK1 inhibitor-induced apoptosis and decreased PLK1 substrate inhibition, suggesting a threshold effect in which higher drug doses are required to affect PLK1 substrate inhibition. PLK1 inhibition was an effective therapy for HNSCC in vitro and in vivo. However, biomarkers to guide such therapy are lacking. We identified AJUBA, SMAD4 and RAS mutations as potential candidate biomarkers of response of HNSCC to treatment with these mitotic inhibitors. Copyright © 2017 Elsevier B.V. All rights reserved.

Quantification of sensitivity and resistance of breast cancer cell lines to anti-cancer drugs using GR metrics

PubMed Central

Hafner, Marc; Heiser, Laura M.; Williams, Elizabeth H.; Niepel, Mario; Wang, Nicholas J.; Korkola, James E.; Gray, Joe W.; Sorger, Peter K.

2017-01-01

Traditional means for scoring the effects of anti-cancer drugs on the growth and survival of cell lines is based on relative cell number in drug-treated and control samples and is seriously confounded by unequal division rates arising from natural biological variation and differences in culture conditions. This problem can be overcome by computing drug sensitivity on a per-division basis. The normalized growth rate inhibition (GR) approach yields per-division metrics for drug potency (GR50) and efficacy (GRmax) that are analogous to the more familiar IC50 and Emax values. In this work, we report GR-based, proliferation-corrected, drug sensitivity metrics for ~4,700 pairs of breast cancer cell lines and perturbagens. Such data are broadly useful in understanding the molecular basis of therapeutic response and resistance. Here, we use them to investigate the relationship between different measures of drug sensitivity and conclude that drug potency and efficacy exhibit high variation that is only weakly correlated. To facilitate further use of these data, computed GR curves and metrics can be browsed interactively at http://www.GRbrowser.org/. PMID:29112189

Epigenetic modification of α-N-acetylgalactosaminidase enhances cisplatin resistance in ovarian cancer

PubMed Central

Ha, Ye-Na; Sung, Hye Youn; Yang, San-Duk; Chae, Yun Ju

2018-01-01

Although cisplatin is one of the most effective antitumor drugs for ovarian cancer, the emergence of chemoresistance to cisplatin in over 80% of initially responsive patients is a major barrier to successful therapy. The precise mechanisms underlying the development of cisplatin resistance are not fully understood, but alteration of DNA methylation associated with aberrant gene silencing may play a role. To identify epigenetically regulated genes directly associated with ovarian cancer cisplatin resistance, we compared the expression and methylation profiles of cisplatin-sensitive and -resistant human ovarian cancer cell lines. We identified α-Nacetylgalactosaminidase (NAGA) as one of the key candidate genes for cisplatin drug response. Interestingly, in cisplatin-resistant cell lines, NAGA was significantly downregulated and hypermethylated at a promoter CpG site at position +251 relative to the transcriptional start site. Low NAGA expression in cisplatin-resistant cell lines was restored by treatment with a DNA demethylation agent, indicating transcriptional silencing by hyper-DNA methylation. Furthermore, overexpression of NAGA in cisplatin-resistant lines induced cytotoxicity in response to cisplatin, whereas depletion of NAGA expression increased cisplatin chemoresistance, suggesting an essential role of NAGA in sensitizing ovarian cells to cisplatin. These findings indicate that NAGA acts as a cisplatin sensitizer and its gene silencing by hypermethylation confers resistance to cisplatin in ovarian cancer. Therefore, we suggest NAGA may be a promising potential therapeutic target for improvement of sensitivity to cisplatin in ovarian cancer. PMID:29302211

Epigenetic modification of α-N-acetylgalactosaminidase enhances cisplatin resistance in ovarian cancer.

PubMed

Ha, Ye-Na; Sung, Hye Youn; Yang, San-Duk; Chae, Yun Ju; Ju, Woong; Ahn, Jung-Hyuck

2018-01-01

Although cisplatin is one of the most effective antitumor drugs for ovarian cancer, the emergence of chemoresistance to cisplatin in over 80% of initially responsive patients is a major barrier to successful therapy. The precise mechanisms underlying the development of cisplatin resistance are not fully understood, but alteration of DNA methylation associated with aberrant gene silencing may play a role. To identify epigenetically regulated genes directly associated with ovarian cancer cisplatin resistance, we compared the expression and methylation profiles of cisplatin-sensitive and -resistant human ovarian cancer cell lines. We identified α- N acetylgalactosaminidase ( NAGA ) as one of the key candidate genes for cisplatin drug response. Interestingly, in cisplatin-resistant cell lines, NAGA was significantly downregulated and hypermethylated at a promoter CpG site at position +251 relative to the transcriptional start site. Low NAGA expression in cisplatin-resistant cell lines was restored by treatment with a DNA demethylation agent, indicating transcriptional silencing by hyper-DNA methylation. Furthermore, overexpression of NAGA in cisplatin-resistant lines induced cytotoxicity in response to cisplatin, whereas depletion of NAGA expression increased cisplatin chemoresistance, suggesting an essential role of NAGA in sensitizing ovarian cells to cisplatin. These findings indicate that NAGA acts as a cisplatin sensitizer and its gene silencing by hypermethylation confers resistance to cisplatin in ovarian cancer. Therefore, we suggest NAGA may be a promising potential therapeutic target for improvement of sensitivity to cisplatin in ovarian cancer.

Variation in cisplatinum sensitivity is not associated with Fanconi Anemia/BRCA pathway inactivation in head and neck squamous cell carcinoma cell lines.

PubMed

Snyder, Eric R; Ricker, Justin L; Chen, Zhong; Waes, Carter Van

2007-01-08

Fanconi Anemia has recently been associated with a high risk of head and neck squamous cell carcinoma (HNSCC). Inactivation of the Fanconi Anemia (FANC-BRCA) pathway via promoter methylation of the FANCF gene has been proposed to be responsible for variation in cisplatinum (CDDP) sensitivity seen in ovarian and HNSCCs. Promoter methylation of the FANCF gene has been observed in 15% of HNSCC specimens, but the relationship to FANC pathway activation and CDDP sensitivity has not been reported. In the present study, 10 HNSCC cell lines were examined for expression of nine genes involved in the FANC-BRCA pathway by RT-PCR: FANCA, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCL, BRCA1 and BRCA2. FANC pathway function was evaluated by western blotting for FANCD2 mono-ubiquitination. All of the cell lines were also analyzed for variation in CDDP cytotoxicity. While significant differences were found in CDDP cytotoxicity, Fanconi pathway defects are an infrequent cause, as no evidence of transcriptional down-regulation of FANCF or other FANC mRNAs, or functional FANC-BRCA pathway defects were observed. These findings suggest that the variation in CDDP sensitivity of many HNSCCs is most frequently due to factors other than FANC-BRCA pathway inactivation.

mTOR ATP-competitive inhibitor INK128 inhibits neuroblastoma growth via blocking mTORC signaling

PubMed Central

Zhang, Huiyuan; Dou, Jun; Yu, Yang; Zhao, Yanling; Fan, Yihui; Cheng, Jin; Xu, Xin; Liu, Wei; Guan, Shan; Chen, Zhenghu; shi, Yan; Patel, Roma; Vasudevan, Sanjeev A; Zage, Peter E; Zhang, Hong; Nuchtern, Jed G; Kim, Eugene S; Fu, Songbin; Yang, Jianhua

2015-01-01

High-risk neuroblastoma often develops resistance to high-dose chemotherapy. The mTOR signaling cascade is frequently deregulated in human cancers and targeting mTOR signaling sensitizes many cancer types to chemotherapy. Here, using a panel of neuroblastoma cell lines, we found that the mTOR inhibitor INK128 showed inhibitory effects on both anchorage-dependent and independent growth of neuroblastoma cells and significantly enhanced the cytotoxic effects of doxorubicin (Dox) on these cell lines. Treatment of neuroblastoma cells with INK128 blocked the activation of downstream mTOR signaling and enhanced Dox-induced apoptosis. Moreover, INK128 was able to overcome the established chemoresistance in the LA-N-6 cell line. Using an orthotopic neuroblastoma mouse model, we found that INK128 significantly inhibited tumor growth in vivo. In conclusion, we have shown that INK128-mediated mTOR inhibition possessed substantial antitumor activity and could significantly increase the sensitivity of neuroblastoma cells to Doxorubicin therapy. Taken together, our results indicate that using INK128 can provide additional efficacy to current chemotherapeutic regimens and represent a new paradigm in restoring drug sensitivity in neuroblastoma. PMID:25425103

Cancer biomarker discovery is improved by accounting for variability in general levels of drug sensitivity in pre-clinical models.

PubMed

Geeleher, Paul; Cox, Nancy J; Huang, R Stephanie

2016-09-21

We show that variability in general levels of drug sensitivity in pre-clinical cancer models confounds biomarker discovery. However, using a very large panel of cell lines, each treated with many drugs, we could estimate a general level of sensitivity to all drugs in each cell line. By conditioning on this variable, biomarkers were identified that were more likely to be effective in clinical trials than those identified using a conventional uncorrected approach. We find that differences in general levels of drug sensitivity are driven by biologically relevant processes. We developed a gene expression based method that can be used to correct for this confounder in future studies.

HIF-2α dictates the susceptibility of pancreatic cancer cells to TRAIL by regulating survivin expression

PubMed Central

Harashima, Nanae; Takenaga, Keizo; Akimoto, Miho; Harada, Mamoru

2017-01-01

Cancer cells develop resistance to therapy by adapting to hypoxic microenvironments, and hypoxia-inducible factors (HIFs) play crucial roles in this process. We investigated the roles of HIF-1α and HIF-2α in cancer cell death induced by tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) using human pancreatic cancer cell lines. siRNA-mediated knockdown of HIF-2α, but not HIF-1α, increased susceptibility of two pancreatic cancer cell lines, Panc-1 and AsPC-1, to TRAIL in vitro under normoxic and hypoxic conditions. The enhanced sensitivity to TRAIL was also observed in vivo. This in vitro increased TRAIL sensitivity was observed in other three pancreatic cancer cell lines. An array assay of apoptosis-related proteins showed that knockdown of HIF-2α decreased survivin expression. Additionally, survivin promoter activity was decreased in HIF-2α knockdown Panc-1 cells and HIF-2α bound to the hypoxia-responsive element in the survivin promoter region. Conversely, forced expression of the survivin gene in HIF-2α shRNA-expressing Panc-1 cells increased resistance to TRAIL. In a xenograft mouse model, the survivin suppressant YM155 sensitized Panc-1 cells to TRAIL. Collectively, our results indicate that HIF-2α dictates the susceptibility of human pancreatic cancer cell lines, Panc-1 and AsPC-1, to TRAIL by regulating survivin expression transcriptionally, and that survivin could be a promising target to augment the therapeutic efficacy of death receptor-targeting anti-cancer therapy. PMID:28476028

Disruption of BASIGIN decreases lactic acid export and sensitizes non-small cell lung cancer to biguanides independently of the LKB1 status

PubMed Central

Floch, Renaud Le; Moura, Conceição Souto

2015-01-01

Most cancers rely on aerobic glycolysis to generate energy and metabolic intermediates. To maintain a high glycolytic rate, cells must efficiently export lactic acid through the proton-coupled monocarboxylate transporters (MCT1/4). These transporters require a chaperone, CD147/BASIGIN (BSG) for trafficking to the plasma membrane and function. To validate the key role of these transporters in lung cancer, we first analysed the expression of MCT1/4 and BSG in 50 non-small lung cancer (NSCLC) cases. These proteins were specifically upregulated in tumour tissues. We then disrupted BSG in three NSCLC cell lines (A549, H1975 and H292) via ‘Zinc-Finger Nucleases’. The three homozygous BSG−/− cell lines displayed a low MCT activity (10- to 5-fold reduction, for MCT1 and MCT4, respectively) compared to wild-type cells. Consequently, the rate of glycolysis, compared to the wild-type counterpart, was reduced by 2.0- to 3.5-fold, whereas the rate of respiration was stimulated in BSG−/− cell lines. Both wild-type and BSG-null cells were extremely sensitive to the mitochondria inhibitor metformin/phenformin in normoxia. However, only BSG-null cells, independently of their LKB1 status, remained sensitive to biguanides in hypoxia in vitro and tumour growth in nude mice. Our results demonstrate that inhibiting glycolysis by targeting lactic acid export sensitizes NSCLC to phenformin. PMID:25894929

Response of head and neck squamous cell carcinoma cells carrying PIK3CA mutations to selected targeted therapies.

PubMed

Wirtz, Eric D; Hoshino, Daisuke; Maldonado, Anthony T; Tyson, Darren R; Weaver, Alissa M

2015-06-01

The PIK3CA mutation is one of the most common mutations in head and neck squamous cell carcinoma (HNSCC). Through this research we attempt to elicit the role of oncogene dependence and effects of targeted therapy on this PIK3CA mutation. (1) To determine the role of oncogene dependence on PIK3CA-one of the more common and targetable oncogenes in HNSCC, and (2) to evaluate the consequence of this oncogene on the effectiveness of newly developed targeted therapies. This was a cell culture-based, in vitro study performed at an academic research laboratory assessing the viability of PIK3CA-mutated head and neck cell lines when treated with targeted therapy. PIK3CA-mutated head and neck cell lines were treated with 17-AAG, GDC-0941, trametinib, and BEZ-235. Assessment of cell viability of HNSCC cell lines characterized for PIK3CA mutations or SCC25 cells engineered to express the PIK3CA hotspot mutations E545K or H1047R. Surprisingly, in engineered cell lines, the hotspot E545K and H1047R mutations conferred increased, rather than reduced, IC50 assay measurements when treated with the respective HSP90, PI3K, and MEK inhibitors, 17-AAG, GDC-0941, and trametinib, compared with the SCC25 control cell lines. When treated with BEZ-235, H1047R-expressing cell lines showed increased sensitivity to inhibition compared with control, whereas those expressing E545K showed slightly increased sensitivity of unclear significance. (1) The PIK3CA mutations within our engineered cell model did not lead to enhanced oncogene-dependent cell death when treated with direct inhibition of the PI3K enzyme yet did show increased sensitivity compared with control with dual PI3K/mTOR inhibition. (2) Oncogene addiction to PIK3CA hotspot mutations, if it occurs, is likely to evolve in vivo in the context of additional molecular changes that remain to be identified. Additional study is required to develop new model systems and approaches to determine the role of targeted therapy in the treatment of PI3K-overactive HNSCC tumors.

EMT is associated with, but does not drive resistance to ALK inhibitors among EML4-ALK non-small cell lung cancer.

PubMed

Gower, Arjan; Hsu, Wei-Hsun; Hsu, Shuo-Tse; Wang, Yisong; Giaccone, Giuseppe

2016-04-01

ALK gene fusion occurs in approximately 3-7% of non-small cell lung cancer (NSCLC). For patients with ALK positive NCSLC, crizotinib and ceritinib are FDA approved ALK inhibitors, however, patients inevitably acquire resistance to such therapies typically within one to two years. Interrogation of in vitro ALK-positive NSCLC cell line models of acquired resistance to first and second-generation ALK inhibitors revealed acquired epithelial-to-mesenchymal transition (EMT) mechanisms. Here we demonstrated that knockdown of upregulated mesenchymal markers in acquired resistant lines decreased the invasive and migratory capabilities of the cells, however, it did not restore sensitivity to ALK inhibitors. Removing drug for 5 weeks from H3122 cell line that acquired resistance to ceritinib restored its sensitivity to ceritinib. In addition, HSP90 inhibitors ganetespib and 17-AAG were potent in inducing cell death in cell lines resistant to crizotinib and ceritinib. Taken together, EMT does not drive resistance to ALK inhibitors and HSP90 inhibition demonstrates more efficacy when further ALK inhibition may not. This study warrants more exploration of HSP90 inhibitors for ALK-positive patients who progress on 1st and 2nd line ALK inhibitor therapy. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

A Promising IFN-Deficient System to Manufacture IFN-Sensitive Influenza Vaccine Virus.

PubMed

Chen, Can; Fan, Wenhui; Li, Jing; Zheng, Weinan; Zhang, Shuang; Yang, Limin; Liu, Di; Liu, Wenjun; Sun, Lei

2018-01-01

Interferon (IFN)-sensitive and replication-incompetent influenza viruses are likely to be the alternatives to inactivated and attenuated virus vaccines. Some IFN-sensitive influenza vaccine candidates with modified non-structural protein 1 (NS1) are highly attenuated in IFN-competent hosts but induce robust antiviral immune responses. However, little research has been done on the manufacturability of these IFN-sensitive vaccine viruses. Here, RIG-I-knockout 293T cells were used to package the IFN-sensitive influenza A/WSN/33 (H1N1) virus expressing the mutant NS1 R38A/K41A. We found that the packaging efficiency of the NS1 R38A/K41A virus in RIG-I-knockout 293T cells was much higher than that in 293T cells. Moreover, the NS1 R38A/K41A virus almost lost its IFN antagonist activity and could no longer replicate in A549, MDCK, and Vero cells after 3-6 passages. This indicated that the replication of NS1 R38A/K41A virus is limited in conventional cells. Therefore, we further established a stable Vero cell line expressing the wild-type (WT) NS1 of the WSN virus, based on the Tet-On 3G system. The NS1 R38A/K41A virus was able to steadily propagate in this IFN-deficient cell line for at least 20 passages. In a mouse model, the NS1 R38A/K41A virus showed more than a 4-log reduction in lung virus titers compared to the WT virus at 3 and 5 days post infection. Furthermore, we observed that the NS1 R38A/K41A virus triggered high-level of IFN-α/β production in lung tissues and was eliminated from the host in a relatively short period of time. Additionally, this virus induced high-titer neutralizing antibodies against the WT WSN, A/Puerto Rico/8/1934 (PR8), or A/California/04/2009 (CA04) viruses and provided 100% protection against the WT WSN virus. Thus, we found that the replication of the NS1 R38A/K41A virus was limited in IFN-competent cells and mice. We also presented a promising IFN-deficient system, involving a RIG-I-knockout 293T cell line to package the IFN-sensitive vaccine virus and a stable Vero cell line expressing NS1 to propagate the IFN-sensitive vaccine virus. The IFN-deficient system is applicable for the manufacture of IFN-sensitive vaccine virus.

Predicting skin sensitization potential and inter-laboratory reproducibility of a human Cell Line Activation Test (h-CLAT) in the European Cosmetics Association (COLIPA) ring trials.

PubMed

Sakaguchi, Hitoshi; Ryan, Cindy; Ovigne, Jean-Marc; Schroeder, Klaus R; Ashikaga, Takao

2010-09-01

Regulatory policies in Europe prohibited the testing of cosmetic ingredients in animals for a number of toxicological endpoints. Currently no validated non-animal test methods exist for skin sensitization. Evaluation of changes in cell surface marker expression in dendritic cell (DC)-surrogate cell lines represents one non-animal approach. The human Cell Line Activation Test (h-CLAT) examines the level of CD86 and CD54 expression on the surface of THP-1 cells, a human monocytic leukemia cell line, following 24h of chemical exposure. To examine protocol transferability, between-lab reproducibility, and predictive capacity, the h-CLAT has been evaluated by five independent laboratories in several ring trials (RTs) coordinated by the European Cosmetics Association (COLIPA). The results of the first and second RTs demonstrated that the protocol was transferable and basically had good between-lab reproducibility and predictivity, but there were some false negative data. To improve performance, protocol and prediction model were modified. Using the modified prediction model in the first and second RT, accuracy was improved. However, about 15% of the outcomes were not correctly identified, which exposes some of the limitations of the assay. For the chemicals evaluated, the limitation may due to chemical being a weak allergen or having low solubility (ex. alpha-hexylcinnamaldehyde). The third RT evaluated the modified prediction model and satisfactory results were obtained. From the RT data, the feasibility of utilizing cell lines as surrogate DC in development of in vitro skin sensitization methods shows promise. The data also support initiating formal pre-validation of the h-CLAT in order to fully understand the capabilities and limitations of the assay. Copyright 2010 Elsevier Ltd. All rights reserved.

Neratinib overcomes trastuzumab resistance in HER2 amplified breast cancer

PubMed Central

Mullooly, Maeve; Bennett, Ruth; Bouguern, Noujoude; Pedersen, Kasper; O'Brien, Neil A; Roxanis, Ioannis; Li, Ji-Liang; Bridge, Esther; Finn, Richard; Slamon, Dennis; McGowan, Patricia; Duffy, Michael J.

2013-01-01

Trastuzumab has been shown to improve the survival outcomes of HER2 positive breast cancer patients. However, a significant proportion of HER2-positive patients are either inherently resistant or develop resistance to trastuzumab. We assessed the effects of neratinib, an irreversible panHER inhibitor, in a panel of 36 breast cancer cell lines. We further assessed its effects with or without trastuzumab in several sensitive and resistant breast cancer cells as well as a BT474 xenograft model. We confirmed that neratinib was significantly more active in HER2-amplified than HER2 non-amplified cell lines. Neratinib decreased the activation of the 4 HER receptors and inhibited downstream pathways. However, HER3 and Akt were reactivated at 24 hours, which was prevented by the combination of trastuzumab and neratinib. Neratinib also decreased pHER2 and pHER3 in acquired trastuzumab resistant cells. Neratinib in combination with trastuzumab had a greater growth inhibitory effect than either drug alone in 4 HER2 positive cell lines. Furthermore, trastuzumab in combination with neratinib was growth inhibitory in SKBR3 and BT474 cells which had acquired resistance to trastuzumab as well as in a BT474 xenograft model. Innately trastuzumab resistant cell lines showed sensitivity to neratinib, but the combination did not enhance response compared to neratinib alone. Levels of HER2 and phospho-HER2 showed a direct correlation with sensitivity to neratinib. Our data indicate that neratinib is an effective anti-HER2 therapy and counteracted both innate and acquired trastuzumab resistance in HER2 positive breast cancer. Our results suggest that combined treatment with trastuzumab and neratinib is likely to be more effective than either treatment alone for both trastuzumab-sensitive breast cancer as well as HER2-positive tumors with acquired resistance to trastuzumab. PMID:24009064

Neratinib overcomes trastuzumab resistance in HER2 amplified breast cancer.

PubMed

Canonici, Alexandra; Gijsen, Merel; Mullooly, Maeve; Bennett, Ruth; Bouguern, Noujoude; Pedersen, Kasper; O'Brien, Neil A; Roxanis, Ioannis; Li, Ji-Liang; Bridge, Esther; Finn, Richard; Siamon, Dennis; McGowan, Patricia; Duffy, Michael J; O'Donovan, Norma; Crown, John; Kong, Anthony

2013-10-01

Trastuzumab has been shown to improve the survival outcomes of HER2 positive breast cancer patients. However, a significant proportion of HER2-positive patients are either inherently resistant or develop resistance to trastuzumab. We assessed the effects of neratinib, an irreversible panHER inhibitor, in a panel of 36 breast cancer cell lines. We further assessed its effects with or without trastuzumab in several sensitive and resistant breast cancer cells as well as a BT474 xenograft model. We confirmed that neratinib was significantly more active in HER2-amplified than HER2 non-amplified cell lines. Neratinib decreased the activation of the 4 HER receptors and inhibited downstream pathways. However, HER3 and Akt were reactivated at 24 hours, which was prevented by the combination of trastuzumab and neratinib. Neratinib also decreased pHER2 and pHER3 in acquired trastuzumab resistant cells. Neratinib in combination with trastuzumab had a greater growth inhibitory effect than either drug alone in 4 HER2 positive cell lines. Furthermore, trastuzumab in combination with neratinib was growth inhibitory in SKBR3 and BT474 cells which had acquired resistance to trastuzumab as well as in a BT474 xenograft model. Innately trastuzumab resistant cell lines showed sensitivity to neratinib, but the combination did not enhance response compared to neratinib alone. Levels of HER2 and phospho-HER2 showed a direct correlation with sensitivity to neratinib. Our data indicate that neratinib is an effective anti-HER2 therapy and counteracted both innate and acquired trastuzumab resistance in HER2 positive breast cancer. Our results suggest that combined treatment with trastuzumab and neratinib is likely to be more effective than either treatment alone for both trastuzumab-sensitive breast cancer as well as HER2-positive tumors with acquired resistance to trastuzumab.

Integrated modulation of phorbol ester-induced Raf activation in EL4 lymphoma cells.

PubMed

Han, Shujie; Meier, Kathryn E

2009-05-01

The EL4 murine lymphoma cell line exists in variant phenotypes that differ with respect to responses to the tumor promoter phorbol 12-myristate 13-acetate (PMA1). Previous work showed that "PMA-sensitive" cells, characterized by a high magnitude of PMA-induced Erk activation, express RasGRP, a phorbol ester receptor that directly activates Ras. In "PMA-resistant" and "intermediate" EL4 cell lines, PMA induces Erk activation to lesser extents, but with a greater response in intermediate cells. In the current study, these cell lines were used to examine mechanisms of Raf-1 modulation. Phospho-specific antibodies were utilized to define patterns and kinetics of Raf-1 phosphorylation on several sites. Further studies showed that Akt is constitutively activated to a greater extent in PMA-resistant than in PMA-sensitive cells, and also to a greater extent in resistant than intermediate cells. Akt negatively regulates Raf-1 activation (Ser259), partially explaining the difference between resistant and intermediate cells. Erk activation exerts negative feedback on Raf-1 (Ser289/296/301), thus resulting in earlier termination of the signal in cells with a higher level of Erk activation. RKIP, a Raf inhibitory protein, is expressed at higher levels in resistant cells than in sensitive or intermediate cells. Knockdown of RKIP increases Erk activation and also negative feedback. In conclusion, this study delineates Raf-1 phosphorylation events occurring in response to PMA in cell lines with different extents of Erk activation. Variations in the levels of expression and activation of multiple signaling proteins work in an integrated fashion to modulate the extent and duration of Erk activation.

Challenging the roles of CD44 and lipolysis stimulated lipoprotein receptor in conveying Clostridium perfringens iota toxin cytotoxicity in breast cancer

PubMed Central

2014-01-01

Background Translational exploration of bacterial toxins has come to the forefront of research given their potential as a chemotherapeutic tool. Studies in select tissues have demonstrated that Clostridium perfringens iota toxin binds to CD44 and lipolysis stimulated lipoprotein receptor (LSR) cell-surface proteins. We recently demonstrated that LSR expression correlates with estrogen receptor positive breast cancers and that LSR signaling directs aggressive, tumor-initiating cell behaviors. Herein, we identify the mechanisms of iota toxin cytotoxicity in a tissue-specific, breast cancer model with the ultimate goal of laying the foundation for using iota toxin as a targeted breast cancer therapy. Methods In vitro model systems were used to determine the cytotoxic effect of iota toxin on breast cancer intrinsic subtypes. The use of overexpression and knockdown technologies confirmed the roles of LSR and CD44 in regulating iota toxin endocytosis and induction of cell death. Lastly, cytotoxicity assays were used to demonstrate the effect of iota toxin on a validated set of tamoxifen resistant breast cancer cell lines. Results Treatment of 14 breast cancer cell lines revealed that LSR+/CD44- lines were highly sensitive, LSR+/CD44+ lines were slightly sensitive, and LSR-/CD44+ lines were resistant to iota cytotoxicity. Reduction in LSR expression resulted in a significant decrease in toxin sensitivity; however, overexpression of CD44 conveyed toxin resistance. CD44 overexpression was correlated with decreased toxin-stimulated lysosome formation and decreased cytosolic levels of iota toxin. These findings indicated that expression of CD44 drives iota toxin resistance through inhibition of endocytosis in breast cancer cells, a role not previously defined for CD44. Moreover, tamoxifen-resistant breast cancer cells exhibited robust expression of LSR and were highly sensitive to iota-induced cytotoxicity. Conclusions Collectively, these data are the first to show that iota toxin has the potential to be an effective, targeted therapy for breast cancer. PMID:24990559

Challenging the roles of CD44 and lipolysis stimulated lipoprotein receptor in conveying Clostridium perfringens iota toxin cytotoxicity in breast cancer.

PubMed

Fagan-Solis, Katerina D; Reaves, Denise K; Rangel, M Cristina; Popoff, Michel R; Stiles, Bradley G; Fleming, Jodie M

2014-07-02

Translational exploration of bacterial toxins has come to the forefront of research given their potential as a chemotherapeutic tool. Studies in select tissues have demonstrated that Clostridium perfringens iota toxin binds to CD44 and lipolysis stimulated lipoprotein receptor (LSR) cell-surface proteins. We recently demonstrated that LSR expression correlates with estrogen receptor positive breast cancers and that LSR signaling directs aggressive, tumor-initiating cell behaviors. Herein, we identify the mechanisms of iota toxin cytotoxicity in a tissue-specific, breast cancer model with the ultimate goal of laying the foundation for using iota toxin as a targeted breast cancer therapy. In vitro model systems were used to determine the cytotoxic effect of iota toxin on breast cancer intrinsic subtypes. The use of overexpression and knockdown technologies confirmed the roles of LSR and CD44 in regulating iota toxin endocytosis and induction of cell death. Lastly, cytotoxicity assays were used to demonstrate the effect of iota toxin on a validated set of tamoxifen resistant breast cancer cell lines. Treatment of 14 breast cancer cell lines revealed that LSR+/CD44- lines were highly sensitive, LSR+/CD44+ lines were slightly sensitive, and LSR-/CD44+ lines were resistant to iota cytotoxicity. Reduction in LSR expression resulted in a significant decrease in toxin sensitivity; however, overexpression of CD44 conveyed toxin resistance. CD44 overexpression was correlated with decreased toxin-stimulated lysosome formation and decreased cytosolic levels of iota toxin. These findings indicated that expression of CD44 drives iota toxin resistance through inhibition of endocytosis in breast cancer cells, a role not previously defined for CD44. Moreover, tamoxifen-resistant breast cancer cells exhibited robust expression of LSR and were highly sensitive to iota-induced cytotoxicity. Collectively, these data are the first to show that iota toxin has the potential to be an effective, targeted therapy for breast cancer.

ABCB1 as predominant resistance mechanism in cells with acquired SNS-032 resistance

PubMed Central

Rothweiler, Florian; Voges, Yvonne; Balónová, Barbora; Blight, Barry A.; Cinatl, Jindrich

2016-01-01

The CDK inhibitor SNS-032 had previously exerted promising anti-neuroblastoma activity via CDK7 and 9 inhibition. ABCB1 expression was identified as major determinant of SNS-032 resistance. Here, we investigated the role of ABCB1 in acquired SNS-032 resistance. In contrast to ABCB1-expressing UKF-NB-3 sub-lines resistant to other ABCB1 substrates, SNS-032-adapted UKF-NB-3 (UKF-NB-3rSNS- 032300nM) cells remained sensitive to the non-ABCB1 substrate cisplatin and were completely re-sensitized to cytotoxic ABCB1 substrates by ABCB1 inhibition. Moreover, UKF-NB-3rSNS-032300nM cells remained similarly sensitive to CDK7 and 9 inhibition as UKF-NB-3 cells. In contrast, SHEPrSNS-0322000nM, the SNS-032-resistant sub-line of the neuroblastoma cell line SHEP, displayed low level SNS-032 resistance also when ABCB1 was inhibited. This discrepancy may be explained by the higher SNS-032 concentrations that were used to establish SHEPrSNS-0322000nM cells, since SHEP cells intrinsically express ABCB1 and are less sensitive to SNS-032 (IC50 912 nM) than UKF-NB-3 cells (IC50 153 nM). In conclusion, we show that ABCB1 expression represents the primary (sometimes exclusive) resistance mechanism in neuroblastoma cells with acquired resistance to SNS-032. Thus, ABCB1 inhibitors may increase the SNS-032 efficacy in ABCB1-expressing cells and prolong or avoid resistance formation. PMID:27517323

Sensitive detection of EML4-ALK fusion oncoprotein of lung cancer by in situ proximity ligation assay.

PubMed

Rho, Jin Kyung; Lee, Hyangsin; Park, Chan-Sik; Choi, Chang-Min; Lee, Jae Cheol

2013-09-01

EML4-ALK fusion oncogene has emerged as a novel molecular target in non-small cell lung cancer (NSCLC). Although break-apart fluorescent in situ hybridization (FISH) is the standard method for diagnosis, it is expensive, not readily available and sometimes difficult to interpret. In addition, ALK immunohistochemistry (IHC) may miss the diagnosis because of relatively low level of ALK transcription. In situ proximity ligation assay (PLA) originally developed for precise detection and quantification of proteins by dual recognition and amplification process was used for sensitive detection of EML4-ALK fusion oncoprotein in NSCLC cell lines (ALK negative cell: PC-9 and H460, ALK positive cell: H3122 and H2228). EML4-ALK oncogene and protein in lung cancer cells were confirmed by multiplex RT-PCR and Western blots. We detected 117 kDa variant 1 of EML4-ALK in H3122 and 90 kDa variant 3 of EML4-ALK in H2228. These cells were more sensitive to crizotinib, an ALK inhibitor compared with PC-9 and H460 cells without EML4-ALK rearrangement. After fixing on glass slides by cytospin centrifuge, in situ PLA test was performed. Among four cell lines, distinct, tiny spots were visible only in H3122 and H2228 cell lines with ALK rearrangement. The same results were also obtained when paraffin-embedded cell blocks were used. Highly specific and sensitive detection of EML4-ALK fusion oncoprotein is possible by in situ PLA method suggesting its clinical application.

Modulating cell-to-cell variability and sensitivity to death ligands by co-drugging

NASA Astrophysics Data System (ADS)

Flusberg, Deborah A.; Sorger, Peter K.

2013-06-01

TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) holds promise as an anti-cancer therapeutic but efficiently induces apoptosis in only a subset of tumor cell lines. Moreover, even in clonal populations of responsive lines, only a fraction of cells dies in response to TRAIL and individual cells exhibit cell-to-cell variability in the timing of cell death. Fractional killing in these cell populations appears to arise not from genetic differences among cells but rather from differences in gene expression states, fluctuations in protein levels and the extent to which TRAIL-induced death or survival pathways become activated. In this study, we ask how cell-to-cell variability manifests in cell types with different sensitivities to TRAIL, as well as how it changes when cells are exposed to combinations of drugs. We show that individual cells that survive treatment with TRAIL can regenerate the sensitivity and death-time distribution of the parental population, demonstrating that fractional killing is a stable property of cell populations. We also show that cell-to-cell variability in the timing and probability of apoptosis in response to treatment can be tuned using combinations of drugs that together increase apoptotic sensitivity compared to treatment with one drug alone. In the case of TRAIL, modulation of cell-to-cell variability by co-drugging appears to involve a reduction in the threshold for mitochondrial outer membrane permeabilization.

Retrovirus-mediated conditional immortalization and analysis of established cell lines of osteoclast precursor cells

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

Kawata, Shigehisa; Suzuki, Jun; Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita, Osaka 565-0871

2006-11-10

Osteoclast precursor cells (OPCs) have previously been established from bone marrow cells of SV40 temperature-sensitive T antigen-expressing transgenic mice. Here, we use retrovirus-mediated gene transfer to conditionally immortalize OPCs by expressing temperature-sensitive large T antigen (tsLT) from wild type bone marrow cells. The immortalized OPCs proliferated at the permissive temperature of 33.5 deg. C, but stopped growing at the non-permissive temperature of 39 deg. C. In the presence of receptor activator of NF{kappa}B ligand (RANKL), the OPCs differentiated into tartrate-resistant acid phosphatase (TRAP)-positive cells and formed multinucleate osteoclasts at 33.5 deg. C. From these OPCs, we cloned two types ofmore » cell lines. Both differentiated into TRAP-positive cells, but one formed multinucleate osteoclasts while the other remained unfused in the presence of RANKL. These results indicate that the established cell lines are useful for analyzing mechanisms of differentiation, particularly multinucleate osteoclast formation. Retrovirus-mediated conditional immortalization should be a useful method to immortalize OPCs from primary bone marrow cells.« less

Combining targeted drugs to overcome and prevent resistance of solid cancers with some stem-like cell features

PubMed Central

Koivunen, Peppi; Koivunen, Jussi P.

2014-01-01

Treatment resistance significantly inhibits the efficiency of targeted cancer therapies in drug-sensitive genotypes. In the current work, we studied mechanisms for rapidly occurring, adaptive resistance in targeted therapy-sensitive lung, breast, and melanoma cancer cell lines. The results show that in ALK translocated lung cancer lines H3122 and H2228, cells with cancer stem-like cell features characterized by high expression of cancer stem cell markers and/or in vivo tumorigenesis can mediate adaptive resistance to oncogene ablative therapy. When pharmacological ablation of ALK oncogene was accompanied with PI3K inhibitor or salinomycin therapy, cancer stem-like cell features were reversed which was accompanied with decreased colony formation. Furthermore, co-targeting was able to block the formation of acquired resistance in H3122 line. The results suggest that cells with cancer stem-like cell features can mediate adaptive resistance to targeted therapies. Since these cells follow the stochastic model, concurrent therapy with an oncogene ablating agent and a stem-like cell-targeting drug is needed for maximal therapeutic efficiency. PMID:25238228

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.

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

HMOX1 and NQO1 genes are upregulated in response to contact sensitizers in dendritic cells and THP-1 cell line: role of the Keap1/Nrf2 pathway.

PubMed

Ade, Nadège; Leon, Fanny; Pallardy, Marc; Peiffer, Jean-Luc; Kerdine-Romer, Saadia; Tissier, Marie-Hélène; Bonnet, Pierre-Antoine; Fabre, Isabelle; Ourlin, Jean-Claude

2009-02-01

Electrophilicity is one of the most common features of skin contact sensitizers and is necessary for protein haptenation. The Keap1 (Kelch-like ECH-associated protein 1)/Nrf2 -signaling pathway is dedicated to the detection of electrophilic stress in cells leading to the upregulation of genes involved in protection or neutralization of chemical reactive species. Signals provided by chemical stress could play an important role in dendritic cell activation and the aim of this work was to test whether contact sensitizers were specific activators of the Keap1/Nrf2 pathway. CD34-derived dendritic cells (CD34-DC) and the THP-1 myeloid cell line were treated by a panel of sensitizers (Ni, 1-chloro 2,4-dinitrobenzene, cinnamaldehyde, 7-hydroxycitronellal, 1,4-dihydroquinone, alpha-methyl-trans-cinnamaldehyde, 2-4-tert-(butylbenzyl)propionaldehyde or Lilial, and 1,4-phenylenediamine), irritants (sodium dodecyl sulfate, benzalkonium chloride), and a nonsensitizer molecule (chlorobenzene). Three well-known Nrf2 activators (tert-butylhydroquinone, lipoic acid, sulforaphane) were also tested. Expression of hmox1 and nqo1 was measured using real-time PCR and cellular accumulation of Nrf2 was assessed by Western blot. Our results showed an increased expression at early time points of hmox1 and nqo1 mRNAs in response to sensitizers but not to irritants. Accumulation of the Nrf2 protein was also observed only with chemical sensitizers. A significant inhibition of the expression of hmox1 and nqo1 mRNAs and CD86 expression was found in 1-chloro 2,4-dinitrobenzene-treated THP-1 cells preincubated with N-acetyl cysteine, a glutathione precursor. Altogether, these data suggested that the Keap1/Nrf2-signaling pathway was activated by electrophilic molecules including sensitizers in dendritic cells and in the THP-1 cell line. Monitoring of this pathway may provide new biomarkers (e.g., Nrf2, hmox1) for the detection of the sensitization potential of chemicals.

Absence of death receptor translocation into lipid rafts in acquired TRAIL-resistant NSCLC cells.

PubMed

Ouyang, Wen; Yang, Chunxu; Zhang, Simin; Liu, Yu; Yang, Bo; Zhang, Junhong; Zhou, Fuxiang; Zhou, Yunfeng; Xie, Conghua

2013-02-01

Resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a major limitation for its clinical use. The mechanisms of TRAIL resistance have been mostly studied in the context of cell lines that are intrinsically resistant to TRAIL. However, little is known about the molecular alterations that contribute to the development of acquired resistance during treatment with TRAIL. In this study, we established H460R, an isogenic cell line with acquired TRAIL resistance, from the TRAIL‑sensitive human lung cancer cell line H460 to investigate the mechanisms of acquired resistance. The acquired TRAIL‑resistant H460R cells remained sensitive to cisplatin. The mRNA and protein expression levels of death receptor 4 (DR4) and death receptor 5 (DR5) were not altered in either of the TRAIL-treated cell lines. Nevertheless, tests in which the DR4 or DR5 gene was overexpressed or silenced suggest that death receptor expression is necessary but not sufficient for TRAIL‑induced apoptosis. Compared with parental TRAIL-sensitive H460 cells, H460R cells showed a decreased TRAIL-induced translocation of DR4/DR5 into lipid rafts. Further studies showed that nystatin partially prevented lipid raft aggregation and DR4 and DR5 clustering and reduced apoptosis in H460 cells again. Analysis of apoptotic molecules showed that more pro-caspase-8, FADD, caspase-3 and Bid, but less cFLIP in H460 cells than in H460R cells. Our findings suggest that the lack of death receptor redistribution negatively impacts DISC assembly in lipid rafts, which at least partially leads to the development of acquired resistance to TRAIL in H460R cells.

Reactivating p53 and Inducing Tumor Apoptosis (RITA) Enhances the Response of RITA-Sensitive Colorectal Cancer Cells to Chemotherapeutic Agents 5-Fluorouracil and Oxaliplatin.

PubMed

Wiegering, Armin; Matthes, Niels; Mühling, Bettina; Koospal, Monika; Quenzer, Anne; Peter, Stephanie; Germer, Christoph-Thomas; Linnebacher, Michael; Otto, Christoph

2017-04-01

Colorectal carcinoma (CRC) is the most common cancer of the gastrointestinal tract with frequently dysregulated intracellular signaling pathways, including p53 signaling. The mainstay of chemotherapy treatment of CRC is 5-fluorouracil (5FU) and oxaliplatin. The two anticancer drugs mediate their therapeutic effect via DNA damage-triggered signaling. The small molecule reactivating p53 and inducing tumor apoptosis (RITA) is described as an activator of wild-type and reactivator of mutant p53 function, resulting in elevated levels of p53 protein, cell growth arrest, and cell death. Additionally, it has been shown that RITA can induce DNA damage signaling. It is expected that the therapeutic benefits of 5FU and oxaliplatin can be increased by enhancing DNA damage signaling pathways. Therefore, we highlighted the antiproliferative response of RITA alone and in combination with 5FU or oxaliplatin in human CRC cells. A panel of long-term established CRC cell lines (n=9) including p53 wild-type, p53 mutant, and p53 null and primary patient-derived, low-passage cell lines (n=5) with different p53 protein status were used for this study. A substantial number of CRC cells with pronounced sensitivity to RITA (IC 50 <3.0 μmol/l) were identified within established (4/9) and primary patient-derived (2/5) CRC cell lines harboring wild-type or mutant p53 protein. Sensitivity to RITA appeared independent of p53 status and was associated with an increase in antiproliferative response to 5FU and oxaliplatin, a transcriptional increase of p53 targets p21 and NOXA, and a decrease in MYC mRNA. The effect of RITA as an inducer of DNA damage was shown by a strong elevation of phosphorylated histone variant H2A.X, which was restricted to RITA-sensitive cells. Our data underline the primary effect of RITA, inducing DNA damage, and demonstrate the differential antiproliferative effect of RITA to CRC cells independent of p53 protein status. We found a substantial number of RITA-sensitive CRC cells within both panels of established CRC cell lines and primary patient-derived CRC cell lines (6/14) that provide a rationale for combining RITA with 5FU or oxaliplatin to enhance the antiproliferative response to both chemotherapeutic agents. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Transcription Factor Activities Enhance Markers of Drug Sensitivity in Cancer.

PubMed

Garcia-Alonso, Luz; Iorio, Francesco; Matchan, Angela; Fonseca, Nuno; Jaaks, Patricia; Peat, Gareth; Pignatelli, Miguel; Falcone, Fiammetta; Benes, Cyril H; Dunham, Ian; Bignell, Graham; McDade, Simon S; Garnett, Mathew J; Saez-Rodriguez, Julio

2018-02-01

Transcriptional dysregulation induced by aberrant transcription factors (TF) is a key feature of cancer, but its global influence on drug sensitivity has not been examined. Here, we infer the transcriptional activity of 127 TFs through analysis of RNA-seq gene expression data newly generated for 448 cancer cell lines, combined with publicly available datasets to survey a total of 1,056 cancer cell lines and 9,250 primary tumors. Predicted TF activities are supported by their agreement with independent shRNA essentiality profiles and homozygous gene deletions, and recapitulate mutant-specific mechanisms of transcriptional dysregulation in cancer. By analyzing cell line responses to 265 compounds, we uncovered numerous TFs whose activity interacts with anticancer drugs. Importantly, combining existing pharmacogenomic markers with TF activities often improves the stratification of cell lines in response to drug treatment. Our results, which can be queried freely at dorothea.opentargets.io, offer a broad foundation for discovering opportunities to refine personalized cancer therapies. Significance: Systematic analysis of transcriptional dysregulation in cancer cell lines and patient tumor specimens offers a publicly searchable foundation to discover new opportunities to refine personalized cancer therapies. Cancer Res; 78(3); 769-80. ©2017 AACR . ©2017 American Association for Cancer Research.

Comparison of primary human fibroblasts and keratinocytes with immortalized cell lines regarding their sensitivity to sodium dodecyl sulfate in a neutral red uptake cytotoxicity assay.

PubMed

Olschläger, Veronika; Schrader, Andreas; Hockertz, Stefan

2009-01-01

Cell lines present a valuable tool for in vitro assessment of skin damage caused by application of cosmeticals or pharmaceuticals. They form a reproducible test system under controllable test conditions and, in many cases, can be used as alternatives to animal testing in order to assess the compatibility of drugs or cosmetics and human skin. Yet, it can not necessarily be assumed that the behavior of cultured cells, when treated with different substances, is exactly consistent with the behavior of cells being part of a live organism. Becoming immortal, cells exhibit changes in genotype and/or phenotype, possibly resulting in modified reactions to external influences. Therefore, to obtain results close to in vivo studies, it seems apparent to use primary cells for testing that have not yet undergone any modifications. To compare the properties of primary fibroblasts (Normal Human Dermal Fibroblasts, NHDF) and primary keratinocytes (Normal Human Epidermal Keratinocytes, NHEK) with those of immortal cell lines (3T3 (ACC 173) Swiss albino mouse fibroblasts and HaCaT (human, adult, low calcium, high temperature, human adult skin keratinocytes) cells), their sensitivities in cytotoxicity assays have been assessed. While both fibroblast cell cultures showed similar sensitivities towards sodium dodecyl sulfate (SDS), primary keratinocytes died at SDS concentrations about three times lower than the immortal HaCaT cells.

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

Integrated analysis of breast cancer cell lines reveals unique signaling pathways

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

Heiser, Laura M.; Wang, Nicholas J.; Talcott, Carolyn L.

Cancer is a heterogeneous disease resulting from the accumulation of genetic defects that negatively impact control of cell division, motility, adhesion and apoptosis. Deregulation in signaling along the EGFR-MAPK pathway is common in breast cancer, though the manner in which deregulation occurs varies between both individuals and cancer subtypes. We were interested in identifying subnetworks within the EGFR-MAPK pathway that are similarly deregulated across subsets of breast cancers. To that end, we mapped genomic, transcriptional and proteomic profiles for 30 breast cancer cell lines onto a curated Pathway Logic symbolic systems model of EGFR-MEK signaling. This model was comprised ofmore » 539 molecular states and 396 rules governing signaling between active states. We analyzed these models and identified several subtype specific subnetworks, including one that suggested PAK1 is particularly important in regulating the MAPK cascade when it is over-expressed. We hypothesized that PAK1 overexpressing cell lines would have increased sensitivity to MEK inhibitors. We tested this experimentally by measuring quantitative responses of 20 breast cancer cell lines to three MEK inhibitors. We found that PAK1 over-expressing luminal breast cancer cell lines are significantly more sensitive to MEK inhibition as compared to those that express PAK1 at low levels. This indicates that PAK1 over-expression may be a useful clinical marker to identify patient populations that may be sensitive to MEK inhibitors. All together, our results support the utility of symbolic system biology models for identification of therapeutic approaches that will be effective against breast cancer subsets.« less

Integrated analysis of breast cancer cell lines reveals unique signaling pathways.

PubMed

Heiser, Laura M; Wang, Nicholas J; Talcott, Carolyn L; Laderoute, Keith R; Knapp, Merrill; Guan, Yinghui; Hu, Zhi; Ziyad, Safiyyah; Weber, Barbara L; Laquerre, Sylvie; Jackson, Jeffrey R; Wooster, Richard F; Kuo, Wen Lin; Gray, Joe W; Spellman, Paul T

2009-01-01

Cancer is a heterogeneous disease resulting from the accumulation of genetic defects that negatively impact control of cell division, motility, adhesion and apoptosis. Deregulation in signaling along the EgfR-MAPK pathway is common in breast cancer, though the manner in which deregulation occurs varies between both individuals and cancer subtypes. We were interested in identifying subnetworks within the EgfR-MAPK pathway that are similarly deregulated across subsets of breast cancers. To that end, we mapped genomic, transcriptional and proteomic profiles for 30 breast cancer cell lines onto a curated Pathway Logic symbolic systems model of EgfR-MAPK signaling. This model was composed of 539 molecular states and 396 rules governing signaling between active states. We analyzed these models and identified several subtype-specific subnetworks, including one that suggested Pak1 is particularly important in regulating the MAPK cascade when it is over-expressed. We hypothesized that Pak1 over-expressing cell lines would have increased sensitivity to Mek inhibitors. We tested this experimentally by measuring quantitative responses of 20 breast cancer cell lines to three Mek inhibitors. We found that Pak1 over-expressing luminal breast cancer cell lines are significantly more sensitive to Mek inhibition compared to those that express Pak1 at low levels. This indicates that Pak1 over-expression may be a useful clinical marker to identify patient populations that may be sensitive to Mek inhibitors. All together, our results support the utility of symbolic system biology models for identification of therapeutic approaches that will be effective against breast cancer subsets.

Inhibition of JNK Sensitizes Hypoxic Colon Cancer Cells to DNA Damaging Agents

PubMed Central

Vasilevskaya, Irina A.; Selvakumaran, Muthu; Hierro, Lucia Cabal; Goldstein, Sara R.; Winkler, Jeffrey D.; O'Dwyer, Peter J.

2015-01-01

Purpose We showed previously that in HT29 colon cancer cells, modulation of hypoxia-induced stress signaling affects oxaliplatin cytotoxicity. To further study the significance of hypoxia-induced signaling through JNK, we set out to investigate how modulation of kinase activities influences cellular responses of hypoxic colon cancer cells to cytotoxic drugs. Experimental design In a panel of cell lines we investigated effects of pharmacological and molecular inhibition of JNK on sensitivity to oxaliplatin, SN-38 and 5-FU. Combination studies for the drugs and JNK inhibitor CC-401 were carried out in vitro and in vivo. Results Hypoxia-induced JNK activation was associated with resistance to oxaliplatin. CC-401 in combination with chemotherapy demonstrates synergism in colon cancer cell lines, though synergy is not always hypoxia-specific. A more detailed analysis focused on HT29 and SW620 (responsive), and HCT116 (non-responsive) lines. In HT29 and SW620 cells CC-401 treatment results in greater DNA damage in the sensitive cells. In vivo, potentiation of bevacizumab, oxaliplatin, and the combination by JNK inhibition was confirmed in HT29-derived mouse xenografts, where tumor growth delay was greater in the presence of CC-401. Finally, stable introduction of a dominant negative JNK1, but not JNK2, construct into HT29 cells rendered them more sensitive to oxaliplatin under hypoxia, suggesting differing input of JNK isoforms in cellular responses to chemotherapy. Conclusions These findings demonstrate that signaling through JNK is a determinant of response to therapy in colon cancer models, and support the testing of JNK inhibition to sensitize colon tumors in the clinic. PMID:26023085

Preclinical evaluation of the PARP inhibitor BMN-673 for the treatment of ovarian clear cell cancer

PubMed Central

Samartzis, Eleftherios Pierre; Dedes, Ioannis; Lambros, Maryou B; Natrajan, Rachael; Gauthier, Arnaud; Piscuoglio, Salvatore; Töpfer, Chantal; Vukovic, Vesna; Daley, Frances; Weigelt, Britta; Reis-Filho, Jorge S

2017-01-01

Purpose To determine if models of ovarian clear cell carcinomas (OCCCs) harbouring defects in homologous recombination (HR) DNA repair of double strand breaks (DSBs) are sensitive to cisplatin and/or PARP inhibition. Experimental Design The HR status of 12 OCCC cell lines was determined using RAD51/γH2AX foci formation assays. Sensitivity to cisplatin and the PARP inhibitor BMN-673 was correlated with HR status. BRCA1, BRCA2, MRE11 and PTEN loss of expression was investigated as a potential determinant of BMN-673 sensitivity. A tissue microarray containing 50 consecutive primary OCCC was assessed for PTEN expression using immunohistochemistry. Results A subset of OCCC cells displayed reduced RAD51 foci formation in the presence of DNA DSBs, suggestive of HR defects. HR-defective OCCC cells, with the exception of KOC-7c, had higher sensitivity to cisplatin/ BMN-673 than HR-competent OCCC cell lines (Log10 SF50 –9.4 (SD +/− 0.29) vs –8.1 (SD +/− 0.35), mean difference 1.3, p < 0.01). Of the cell lines studied, two, TOV-21G and KOC-7c, showed loss of PTEN expression. In primary OCCCs, loss of PTEN expression was observed in 10% (5/49) of cases. Conclusions A subset of OCCC cells are sensitive to PARP inhibition in vitro, which can be predicted by HR defects as defined by γH2AX/RAD51 foci formation. These results provide a rationale for the testing of HR deficiency and PARP inhibitors as a targeted therapy in a subset of OCCCs. PMID:28002809

Comparative DNA microarray analysis of human monocyte derived dendritic cells and MUTZ-3 cells exposed to the moderate skin sensitizer cinnamaldehyde

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

Python, Francois; Goebel, Carsten; Aeby, Pierre

2009-09-15

The number of studies involved in the development of in vitro skin sensitization tests has increased since the adoption of the EU 7th amendment to the cosmetics directive proposing to ban animal testing for cosmetic ingredients by 2013. Several studies have recently demonstrated that sensitizers induce a relevant up-regulation of activation markers such as CD86, CD54, IL-8 or IL-1{beta} in human myeloid cell lines (e.g., U937, MUTZ-3, THP-1) or in human peripheral blood monocyte-derived dendritic cells (PBMDCs). The present study aimed at the identification of new dendritic cell activation markers in order to further improve the in vitro evaluation ofmore » the sensitizing potential of chemicals. We have compared the gene expression profiles of PBMDCs and the human cell line MUTZ-3 after a 24-h exposure to the moderate sensitizer cinnamaldehyde. A list of 80 genes modulated in both cell types was obtained and a set of candidate marker genes was selected for further analysis. Cells were exposed to selected sensitizers and non-sensitizers for 24 h and gene expression was analyzed by quantitative real-time reverse transcriptase-polymerase chain reaction. Results indicated that PIR, TRIM16 and two Nrf2-regulated genes, CES1 and NQO1, are modulated by most sensitizers. Up-regulation of these genes could also be observed in our recently published DC-activation test with U937 cells. Due to their role in DC activation, these new genes may help to further refine the in vitro approaches for the screening of the sensitizing properties of a chemical.« less

MicroRNA-133b targets glutathione S-transferase π expression to increase ovarian cancer cell sensitivity to chemotherapy drugs.

PubMed

Chen, Shuo; Jiao, Jin-Wen; Sun, Kai-Xuan; Zong, Zhi-Hong; Zhao, Yang

2015-01-01

Accumulating studies reveal that aberrant microRNA (miRNA) expression can affect the development of chemotherapy drug resistance by modulating the expression of relevant target proteins. The aim of this study was to investigate the role of miR-133b in the development of drug resistance in ovarian cancer cells. We examined the levels of miR-133b expression in ovarian carcinoma tissues and the human ovarian carcinoma cell lines (A2780, A2780/DDP and A2780/Taxol, respectively). We determined the cell viability of these cell lines treated with cisplatin or paclitaxel in the presence or absence of miR-133b or anti-miR-133b transfection using the MTT assay. Reverse transcription polymerase chain reaction and Western blotting were used to assess the mRNA and protein expression levels of two drug-resistance-related genes: glutathione S-transferase (GST)-π and multidrug resistance protein 1 (MDR1). The dual-luciferase reporter assay was used to detect the promoter activity of GST-π in the presence and absence of miR-133b. The expression of miR-133b was significantly lower in primary resistant ovarian carcinomas than in the chemotherapy-sensitive carcinomas (P<0.05), and the same results were found in primary resistant ovarian cell lines (A2780/Taxol and A2780/DDP) compared to the chemotherapy-sensitive cell line (A2780; P<0.05). Following miR-133b transfection, four cell lines showed increased sensitivity to paclitaxel and cisplatin, while anti-miR-133b transfection reduced cell sensitivity to paclitaxel and cisplatin. Dual-luciferase reporter assay showed that miR-133b interacted with the 3'-untranslated region of GST-π. Compared to controls, the mRNA and protein levels of MDR1 and GST-π were downregulated after miR-133b transfection and upregulated after anti-miR-133b transfection. MicroRNA-133b may reduce ovarian cancer drug resistance by silencing the expression of the drug-resistance-related proteins, GST-π and MDR1. In future, the combination of miR-133b with chemotherapy agents may prevent the development of drug resistance in ovarian cancers.

Induction of NKCF-like activity in mixed lymphocyte-tumor cell culture: direct involvement of mycoplasma infection of tumor cells.

PubMed

Wayner, E A; Brooks, C G

1984-04-01

Co-culture of CBA/J spleen cells and certain lines of YAC-1 stimulators resulted in the appearance of NKCF-like activity in 24- to 48-hr supernatants. Numerous other in vitro cell lines were effective stimulators of this splenic cytotoxic factor (SCF). The cells participating in SCF production were absent from normal thymocytes and were present in BALB/c nu/nu spleen, were nonadherent, asialo GM1+, and bore low levels of Thy-1.2. SCF could mediate lysis of certain NK-sensitive tumor targets in an 18-hr 51Cr-release assay. However, the induction of SCF was not correlated with the ability of a particular cell line to be lysed by NK cells, but showed an absolute correlation with the presence of mycoplasma contamination in cultured tumor cell lines. Mycoplasma negative cell lines, including an uninfected but NK-sensitive subline of YAC-1, were unable to induce SCF. Decontamination of mycoplasma-infected lines with antibiotics or by passage through syngeneic mice abrogated the ability of infected tumor cells to stimulate SCF. The ability to induce SCF could be restored by reinfection with mycoplasma. Tumor cell-free supernatants from contaminated cultures were mitogenic for CBA spleen cells and could themselves induce SCF activity in spleen cell supernatants. SCF production and the agent responsible could be removed by passing such supernatants through 0.1-micron filters. The organism apparently responsible for SCF induction from CBA spleen cells was typed and found to be Mycoplasma orale, a nonfermentative, arginine-dependent, common tissue culture contaminant. About 50 to 60% of SCF activity could be removed by 0.1-micron filters, suggesting that SCF is composed of two components: mycoplasma organisms themselves and a soluble cytotoxic factor produced in response to mycoplasma.

MicroRNA-9 enhances sensitivity to cetuximab in epithelial phenotype hepatocellular carcinoma cells through regulation of the eukaryotic translation initiation factor 5A-2.

PubMed

Xue, Fei; Liang, Yuntian; Li, Zhenrong; Liu, Yanhui; Zhang, Hongwei; Wen, Yu; Yan, Lei; Tang, Qiang; Xiao, Erhui; Zhang, Dongyi

2018-01-01

Hepatocellular carcinoma (HCC) is one of the most widespread malignant human tumors worldwide. Treatment options include radiotherapy, surgical intervention and chemotherapy; however, drug resistance is an ongoing treatment concern. In the present study, the effects of a microRNA (miR/miRNA), miR-9, on the sensitivity of HCC cell lines to the epidermal growth factor receptor inhibitor, cetuximab, were examined. miR-9 has been proposed to serve a role in tumorigenesis and tumor progression. In the present study, bioinformatics analyses identified the eukaryotic translation initiation factor 5A2 (eIF-5A-2) as a target of miR-9. The expression levels of miR-9 and eIF-5A-2 were examined by reverse transcription-quantitative polymerase chain reaction and HCC cell lines were transfected with miR-9 mimics and inhibitors to determine the effects of the miRNA on cell proliferation and viability. The miR-9 mimic was revealed to significantly increase the sensitivity of epithelial phenotype HCC cells (Hep3B and Huh7) to cetuximab, while the miR-9 inhibitor triggered the opposite effect. There were no significant differences in sensitivity to cetuximab observed in mesenchymal phenotype HCC cells (SNU387 and SNU449). Cells lines displaying high expression levels of eIF-5A-2 were more resistant to cetuximab. Transfection of cells with a miR-9 mimic resulted in downregulation of the expression of eIF-5A-2 mRNA, while an miR-9 inhibitor increased expression. When expression of eIF-5A-2 was knocked down with siRNA, the effects of miR-9 on cetuximab sensitivity were no longer observed. Taken together, these data support a role for miR-9 in enhancing the sensitivity of epithelial phenotype HCC cells to cetuximab through regulation of eIF-5A-2.

Identifying Determinants of PARP Inhibitor Sensitivity in Ovarian Cancer

DTIC Science & Technology

2016-10-01

inhibitors. Ovarian cancer patients that harbored germ- line BRCA1 mutations treated with PARP inhibitors exhibited meaningful responses in early phase...hypothesized that a range of common ovarian cancer predisposing germ- line BRCA1 gene mutations produce semi-functional proteins that are capable of...we have started our work examining exome sequences and gene expression in PARPi sensitive and resistance cancer cell lines . I attended and presented

Effectiveness of imatinib mesylate over etoposide in the treatment of sensitive and resistant chronic myeloid leukaemia cells in vitro.

PubMed

Husaini, Roslina; Ahmad, Munirah; Zakaria, Zubaidah

2017-06-01

Chronic myeloid leukaemia (CML) is a form of leukaemia derived from the myeloid cell lineage. Imatinib mesylate, the breakpoint cluster region-abelson murine leukeamia kinase inhibitor, is a specific reagent used in the clinical treatment of CML. The DNA topoisomerase II inhibitor, etoposide, is also employed as a therapeutic, though it is used to a lesser extent. The present study aims to evaluate the effects of CML-targeted therapy, utilising imatinib mesylate and etoposide in the in vitro treatment of parental sensitive and adriamycin-resistant CML in the K562 and K562/ADM cell lines, respectively. Preliminary work involved the screening of multidrug resistant (MDR) gene expression, including MDR1, MRP1 and B-cell lymphoma 2 (BCL-2) at the mRNA levels. The sensitive and resistant CML cell lines expressed the MRP1 gene, though the sensitive K562 cells expressed low, almost undetectable levels of MDR1 and BCL-2 genes relative to the K562/ADM cells. Following treatment with imatinib mesylate or etoposide, the IC50 for imatinib mesylate did not differ between the sensitive and resistant cell lines (0.492±0.024 and 0.378±0.029, respectively), indicating that imatinib mesylate is effective in the treatment of CML regardless of cell chemosensitivity. However, the IC50 for etoposide in sensitive K562 cells was markedly lower than that of K562/ADM cells (50.6±16.5 and 194±8.46 µM, respectively), suggesting that the higher expression levels of MDR1 and/or BCL-2 mRNA in resistant cells may be partially responsible for this effect. This is supported by terminal deoxynucleotidyl transferase dUTP nick-end labeling data, whereby a higher percentage of apoptotic cells were found in the sensitive and resistant K562 cells treated with imatinib mesylate (29.3±0.2 and 31.9±16.7%, respectively), whereas etoposide caused significant apoptosis of sensitive K562 cells (18.3±8.35%) relative to K562/ADM cells (5.17±3.3%). In addition, the MDR genes in K562/ADM cells were knocked down by short interfering RNAs. The percentage knockdowns were 15.4% for MRP1, 17.8% for MDR and 30.7% for BCL-2, which resulted in a non-significant difference in the half maximal inhibitory concentration value of K562/ADM cells relative to K562 cells upon treatment with etoposide.

Evaluation of selected biomarkers for the detection of chemical sensitization in human skin: a comparative study applying THP-1, MUTZ-3 and primary dendritic cells in culture.

PubMed

Hitzler, Manuel; Bergert, Antje; Luch, Andreas; Peiser, Matthias

2013-09-01

Dendritic cells (DCs) exhibit the unique capacity to induce T cell differentiation and proliferation, two processes that are crucially involved in allergic reactions. By combining the exclusive potential of DCs as the only professional antigen-presenting cells of the human body with the well known handling advantages of cell lines, cell-based alternative methods aimed at detecting chemical sensitization in vitro commonly apply DC-like cells derived from myeloid cell lines. Here, we present the new biomarkers programmed death-ligand 1 (PD-L1), DC immunoreceptor (DCIR), IL-16, and neutrophil-activating protein-2 (NAP-2), all of which have been detectable in primary human DCs upon exposure to chemical contact allergens. To evaluate the applicability of DC-like cells in the prediction of a chemical's sensitization potential, the expression of cell surface PD-L1 and DCIR was analyzed. In contrast to primary DCs, only minor subpopulations of MUTZ-3 and THP-1 cells presented PD-L1 or DCIR at their surface. After exposure to increasing concentrations of nickel and cinnamic aldehyde, the expression level of PD-L1 and DCIR revealed much stronger affected on monocyte-derived DCs (MoDCs) or Langerhans cells (MoLCs) when compared to THP-1 and MUTZ-3 cells. Applying protein profiler arrays we further identified the soluble factors NAP-2, IL-16, IL-8 and MIP-1α as sensitive biomarkers showing the capacity to discriminate sensitizing from non-sensitizing chemicals or irritants. An allergen-specific release of IL-8 and MIP-1α could be detected in the supernatants of MoDCs and MoLCs and also in MUTZ-3 and THP-1 cells, though at much lower levels. On the protein and transcriptional level, NAP-2 and IL-16 indicated sensitizers most sensitively and specifically in MoDCs. Altogether, we have proven the reciprocal regulated surface molecules PD-L1 and DCIR and the soluble factors MIP-1α, NAP-2 and IL-16 as reliable biomarkers for chemical sensitization. We further show that primary DCs are significantly different in their phenotype and function compared to DC-like cell lines. Since they demonstrated higher absolute values and a broader range in biomarker expression, we propose that MoDCs represent an optimal and robust sensor test system well suited to identify and classify chemicals with an allergic potential. Copyright © 2013 Elsevier Ltd. All rights reserved.

Characterization of a hydroxyurea-resistant human KB cell line with supersensitivity to 6-thioguanine.

PubMed

Yen, Y; Grill, S P; Dutschman, G E; Chang, C N; Zhou, B S; Cheng, Y C

1994-07-15

Hydroxyurea (HU) is currently used in the clinic for the treatment of chronic myelogenous leukemia, head and neck carcinoma, and sarcoma. One of its drawbacks, however, is the development of HU resistance. To study this problem, we developed a HU-resistant human KB cell line which exhibits a 15-fold resistance to HU. The characterization of this HU-resistant phenotype revealed a gene amplification of the M2 subunit of ribonucleotide reductase (RR), increased levels of M2 mRNA and protein, and a 3-fold increase of RR activity. This HU-resistant cell line also expressed a "collateral sensitivity" to 6-thioguanine (6-TG), with a 10-fold decrease in the dose inhibiting cell growth by 50% as compared to the KB parental line. The mechanism responsible for this supersensitivity to 6-TG is believed to be related to an increasingly efficient conversion of 6-TG to its triphosphate form, which is subsequently incorporated into DNA. After passage of the resistant cells in the absence of HU, the cell line reverts. The revertant cells lose their resistance to HU and concomitantly their sensitivity to 6-TG. This phenomenon is due to the return of RR to levels comparable to that of the KB parental cell line. These observations and their relevance to cancer chemotherapy will be discussed in this paper. Our results suggest that a clinical protocol could be designed which would allow for a lower dose of 6-TG to be used by taking advantage of the increased RR activity in HU-refractory cancer patients. Two drugs which display collateral sensitivity are known as a "Ying-Yang" pair. Alternate treatment with two different Ying-Yang pairs is the rationale for the "Ying-Yang Ping-Pong" theory in cancer treatment. This rationale allows for effective cancer chemotherapy with reduced toxicity.

The dual PI3K/mTOR inhibitor PKI-587 enhances sensitivity to cetuximab in EGFR-resistant human head and neck cancer models

PubMed Central

D'Amato, V; Rosa, R; D'Amato, C; Formisano, L; Marciano, R; Nappi, L; Raimondo, L; Di Mauro, C; Servetto, A; Fusciello, C; Veneziani, B M; De Placido, S; Bianco, R

2014-01-01

Background: Cetuximab is the only targeted agent approved for the treatment of head and neck squamous cell carcinomas (HNSCC), but low response rates and disease progression are frequently reported. As the phosphoinositide 3-kinase (PI3K) and the mammalian target of rapamycin (mTOR) pathways have an important role in the pathogenesis of HNSCC, we investigated their involvement in cetuximab resistance. Methods: Different human squamous cancer cell lines sensitive or resistant to cetuximab were tested for the dual PI3K/mTOR inhibitor PF-05212384 (PKI-587), alone and in combination, both in vitro and in vivo. Results: Treatment with PKI-587 enhances sensitivity to cetuximab in vitro, even in the condition of epidermal growth factor receptor (EGFR) resistance. The combination of the two drugs inhibits cells survival, impairs the activation of signalling pathways and induces apoptosis. Interestingly, although significant inhibition of proliferation is observed in all cell lines treated with PKI-587 in combination with cetuximab, activation of apoptosis is evident in sensitive but not in resistant cell lines, in which autophagy is pre-eminent. In nude mice xenografted with resistant Kyse30 cells, the combined treatment significantly reduces tumour growth and prolongs mice survival. Conclusions: Phosphoinositide 3-kinase/mammalian target of rapamycin inhibition has an important role in the rescue of cetuximab resistance. Different mechanisms of cell death are induced by combined treatment depending on basal anti-EGFR responsiveness. PMID:24823695

The dual PI3K/mTOR inhibitor PKI-587 enhances sensitivity to cetuximab in EGFR-resistant human head and neck cancer models.

PubMed

D'Amato, V; Rosa, R; D'Amato, C; Formisano, L; Marciano, R; Nappi, L; Raimondo, L; Di Mauro, C; Servetto, A; Fusciello, C; Veneziani, B M; De Placido, S; Bianco, R

2014-06-10

Cetuximab is the only targeted agent approved for the treatment of head and neck squamous cell carcinomas (HNSCC), but low response rates and disease progression are frequently reported. As the phosphoinositide 3-kinase (PI3K) and the mammalian target of rapamycin (mTOR) pathways have an important role in the pathogenesis of HNSCC, we investigated their involvement in cetuximab resistance. Different human squamous cancer cell lines sensitive or resistant to cetuximab were tested for the dual PI3K/mTOR inhibitor PF-05212384 (PKI-587), alone and in combination, both in vitro and in vivo. Treatment with PKI-587 enhances sensitivity to cetuximab in vitro, even in the condition of epidermal growth factor receptor (EGFR) resistance. The combination of the two drugs inhibits cells survival, impairs the activation of signalling pathways and induces apoptosis. Interestingly, although significant inhibition of proliferation is observed in all cell lines treated with PKI-587 in combination with cetuximab, activation of apoptosis is evident in sensitive but not in resistant cell lines, in which autophagy is pre-eminent. In nude mice xenografted with resistant Kyse30 cells, the combined treatment significantly reduces tumour growth and prolongs mice survival. Phosphoinositide 3-kinase/mammalian target of rapamycin inhibition has an important role in the rescue of cetuximab resistance. Different mechanisms of cell death are induced by combined treatment depending on basal anti-EGFR responsiveness.

Ferrocene-cinchona hybrids with triazolyl-chalcone linkers act as pro-oxidants and sensitize human cancer cell lines to paclitaxel.

PubMed

Podolski-Renić, Ana; Bősze, Szilvia; Dinić, Jelena; Kocsis, László; Hudecz, Ferenc; Csámpai, Antal; Pešić, Milica

2017-08-16

Recently, we demonstrated that ferrocene-containing compounds with a cinchona moiety displayed marked anticancer activity. Here we report on the effects of the most promising isomers encompassing quinine- (compounds 4 and 5) and quinidine-epimers (compounds 6 and 7) - synthesized using improved methods providing controlled diastereoselectivity - in three different human multidrug resistant (MDR) cancer cell lines and their sensitive counterparts (non-small cell lung carcinoma NCI-H460/R/NCI-H460, colorectal carcinoma DLD1-TxR/DLD1 and glioblastoma U87-TxR/U87). We observed that the presence of the MDR phenotype did not diminish the activity of the compounds suggesting that ferrocene quinine- and quinidine-epimers are not substrates for P-glycoprotein, which has been indicated as a major mechanism of MDR in the cell lines used. Considering that metal-based anticancer agents mainly act by increasing ROS production, we investigated the potential of ferrocene-quinidine epimers to generate ROS. We found that 6 and 7 more readily increased ROS production and induced mitochondrial damage in MDR cancer cells. According to cell death analysis, 6 and 7 were more active against MDR cancer cells showing collateral sensitivity. In addition, our data suggest that these compounds could act as inhibitors of autophagy. Importantly, simultaneous treatments of 6 and 7 with paclitaxel (PTX) increased the sensitivity of MDR cancer cells to PTX. In conclusion, the ferrocene-quinidine epimers, besides being selective towards MDR cancer cells, could also possess potential to overcome PTX resistance.

Chromatin status of apoptosis genes correlates with sensitivity to chemo-, immune- and radiation therapy in colorectal cancer cell lines.

PubMed

Benard, Anne; Janssen, Connie M; van den Elsen, Peter J; van Eggermond, Marja C J A; Hoon, Dave S B; van de Velde, Cornelis J H; Kuppen, Peter J K

2014-12-01

The apoptosis pathway of programmed cell death is frequently deregulated in cancer. An intact apoptosis pathway is required for proper response to anti-cancer treatment. We investigated the chromatin status of key apoptosis genes in the apoptosis pathway in colorectal cancer cell lines in relation to apoptosis induced by chemo-, immune- or radiation therapy. Using chromatin immunoprecipitation (ChIP), we measured the presence of transcription-activating histone modifications H3Ac and H3K4me3 and silencing modifications H3K9me3 and H3K27me3 at the gene promoter regions of key apoptosis genes Bax, Bcl2, Caspase-9, Fas (CD95) and p53. Cell lines DLD1, SW620, Colo320, Caco2, Lovo and HT29 were treated with cisplatin, anti-Fas or radiation. The apoptotic response was measured by flow cytometry using propidium iodide and annexin V-FITC. The chromatin status of the apoptosis genes reflected the activation status of the intrinsic (Bax, Bcl2, Caspase-9 and p53) and extrinsic (Fas) pathways. An active intrinsic apoptotic pathway corresponded to sensitivity to cisplatin and radiation treatment of cell lines DLD1, SW620 and Colo320. An active Fas promoter corresponded to an active extrinsic apoptotic pathway in cell line DLD1. mRNA expression data correlated with the chromatin status of the apoptosis genes as measured by ChIP. In conclusion, the results presented in this study indicate that the balance between activating and silencing histone modifications, reflecting the chromatin status of apoptosis genes, can be used to predict the response of tumor cells to different anti-cancer therapies and could provide a novel target to sensitize tumors to obtain adequate treatment responses.

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.

Poly(ADP-ribose) polymerase inhibitors sensitize cancer cells to death receptor-mediated apoptosis by enhancing death receptor expression.

PubMed

Meng, X Wei; Koh, Brian D; Zhang, Jin-San; Flatten, Karen S; Schneider, Paula A; Billadeau, Daniel D; Hess, Allan D; Smith, B Douglas; Karp, Judith E; Kaufmann, Scott H

2014-07-25

Recombinant human tumor necrosis factor-α-related apoptosis inducing ligand (TRAIL), agonistic monoclonal antibodies to TRAIL receptors, and small molecule TRAIL receptor agonists are in various stages of preclinical and early phase clinical testing as potential anticancer drugs. Accordingly, there is substantial interest in understanding factors that affect sensitivity to these agents. In the present study we observed that the poly(ADP-ribose) polymerase (PARP) inhibitors olaparib and veliparib sensitize the myeloid leukemia cell lines ML-1 and K562, the ovarian cancer line PEO1, non-small cell lung cancer line A549, and a majority of clinical AML isolates, but not normal marrow, to TRAIL. Further analysis demonstrated that PARP inhibitor treatment results in activation of the FAS and TNFRSF10B (death receptor 5 (DR5)) promoters, increased Fas and DR5 mRNA, and elevated cell surface expression of these receptors in sensitized cells. Chromatin immunoprecipitation demonstrated enhanced binding of the transcription factor Sp1 to the TNFRSF10B promoter in the presence of PARP inhibitor. Knockdown of PARP1 or PARP2 (but not PARP3 and PARP4) not only increased expression of Fas and DR5 at the mRNA and protein level, but also recapitulated the sensitizing effects of the PARP inhibition. Conversely, Sp1 knockdown diminished the PARP inhibitor effects. In view of the fact that TRAIL is part of the armamentarium of natural killer cells, these observations identify a new facet of PARP inhibitor action while simultaneously providing the mechanistic underpinnings of a novel therapeutic combination that warrants further investigation.

Analysing intracellular deformation of polymer capsules using structured illumination microscopy

NASA Astrophysics Data System (ADS)

Chen, Xi; Cui, Jiwei; Sun, Huanli; Müllner, Markus; Yan, Yan; Noi, Ka Fung; Ping, Yuan; Caruso, Frank

2016-06-01

Understanding the behaviour of therapeutic carriers is important in elucidating their mechanism of action and how they are processed inside cells. Herein we examine the intracellular deformation of layer-by-layer assembled polymer capsules using super-resolution structured illumination microscopy (SIM). Spherical- and cylindrical-shaped capsules were studied in three different cell lines, namely HeLa (human epithelial cell line), RAW264.7 (mouse macrophage cell line) and differentiated THP-1 (human monocyte-derived macrophage cell line). We observed that the deformation of capsules was dependent on cell line, but independent of capsule shape. This suggests that the mechanical forces, which induce capsule deformation during cell uptake, vary between cell lines, indicating that the capsules are exposed to higher mechanical forces in HeLa cells, followed by RAW264.7 and then differentiated THP-1 cells. Our study demonstrates the use of super-resolution SIM in analysing intracellular capsule deformation, offering important insights into the cellular processing of drug carriers in cells and providing fundamental knowledge of intracellular mechanobiology. Furthermore, this study may aid in the design of novel drug carriers that are sensitive to deformation for enhanced drug release properties.Understanding the behaviour of therapeutic carriers is important in elucidating their mechanism of action and how they are processed inside cells. Herein we examine the intracellular deformation of layer-by-layer assembled polymer capsules using super-resolution structured illumination microscopy (SIM). Spherical- and cylindrical-shaped capsules were studied in three different cell lines, namely HeLa (human epithelial cell line), RAW264.7 (mouse macrophage cell line) and differentiated THP-1 (human monocyte-derived macrophage cell line). We observed that the deformation of capsules was dependent on cell line, but independent of capsule shape. This suggests that the mechanical forces, which induce capsule deformation during cell uptake, vary between cell lines, indicating that the capsules are exposed to higher mechanical forces in HeLa cells, followed by RAW264.7 and then differentiated THP-1 cells. Our study demonstrates the use of super-resolution SIM in analysing intracellular capsule deformation, offering important insights into the cellular processing of drug carriers in cells and providing fundamental knowledge of intracellular mechanobiology. Furthermore, this study may aid in the design of novel drug carriers that are sensitive to deformation for enhanced drug release properties. Electronic supplementary information (ESI) available: Additional figures. See DOI: 10.1039/c6nr02151d

MRP- and BCL-2-mediated drug resistance in human SCLC: effects of apoptotic sphingolipids in vitro.

PubMed

Khodadadian, M; Leroux, M E; Auzenne, E; Ghosh, S C; Farquhar, D; Evans, R; Spohn, W; Zou, Y; Klostergaard, J

2009-10-01

Multidrug-resistance-associated protein (MRP) and BCL-2 contribute to drug resistance expressed in SCLC. To establish whether MRP-mediated drug resistance affects sphingolipid (SL)-induced apoptosis in SCLC, we first examined the human SCLC cell line, UMCC-1, and its MRP over-expressing, drug-resistant subline, UMCC-1/VP. Despite significantly decreased sensitivity to doxorubicin (Dox) and to the etoposide, VP-16, the drug-selected line was essentially equally as sensitive to treatment with exogenous ceramide (Cer), sphingosine (Sp) or dimethyl-sphingosine (DMSP) as the parental line. Next, we observed that high BCL-2-expressing human H69 SCLC cells, that were approximately 160-fold more sensitive to Dox than their combined BCL-2 and MRP-over-expressing (H69AR) counterparts, were only approximately 5-fold more resistant to DMSP. Time-lapse fluorescence microscopy of either UMCC cell line treated with DMSP-Coumarin revealed comparable extents and kinetics of SL uptake, further ruling out MRP-mediated effects on drug uptake. DMSP potentiated the cytotoxic activity of VP-16 and Taxol, but not Dox, in drug-resistant UMCC-1/VP cells. However, this sensitization did not appear to involve DMSP-mediated effects on the function of MRP in drug export; nor did DMSP strongly shift the balance of pro-apoptotic Sps and anti-apoptotic Sp-1-Ps in these cells. We conclude that SL-induced apoptosis markedly overcomes or bypasses MRP-mediated drug resistance relevant to SCLC and may suggest a novel therapeutic approach to chemotherapy for these tumors.

Investigating a signature of temozolomide resistance in GBM cell lines using metabolomics.

PubMed

St-Coeur, Patrick-Denis; Poitras, Julie J; Cuperlovic-Culf, Miroslava; Touaibia, Mohamed; Morin, Pier

2015-10-01

Glioblastoma multiforme (GBM) is the most common form of malignant glioma. Current therapeutic approach to treat this malignancy involves a combination of surgery, radiotherapy and chemotherapy with temozolomide. Numerous mechanisms contributing to inherent and acquired resistance to this chemotherapeutic agent have been identified and can lead to treatment failure. This study undertook a metabolomics-based approach to characterize the metabolic profiles observed in temozolomide-sensitive and temozolomide-resistant GBM cell lines as well as in a small sub-set of primary GBM tumors. This approach was also utilized to explore the metabolic changes modulated upon cell treatment with temozolomide and lomeguatrib, an MGMT inhibitor with temozolomide-sensitizing potential. Metabolites previously explored for their potential role in chemoresistance including glucose, citrate and isocitrate demonstrated elevated levels in temozolomide-resistant GBM cells. In addition, a signature of metabolites comprising alanine, choline, creatine and phosphorylcholine was identified as up-regulated in sensitive GBM cell line across different treatments. These results present the metabolic profiles associated with temozolomide response in selected GBM models and propose interesting leads that could be leveraged for the development of therapeutic or diagnostic tools to impact temozolomide response in GBMs.

Enhanced expression of IL-8 in normal human keratinocytes and human keratinocyte cell line HaCaT in vitro after stimulation with contact sensitizers, tolerogens and irritants.

PubMed

Mohamadzadeh, M; Müller, M; Hultsch, T; Enk, A; Saloga, J; Knop, J

1994-12-01

To investigate the interleukin-8 production of keratinocytes after stimulation in vitro we have used various agents: (i) contact sensitizer (2,4-dinitrofluorobenzene, 3-n-pentadecylcatechol); (ii) tolerogen (5-methyl-3-n-pentadecylcatechol); (iii) irritant (sodium lauryl sulfate). Interleukin-8 gene expression was assessed by northern blot hybridization of the total cytoplasmic RNA extracted from subconfluent normal human keratinocyte cultures and the keratinocyte cell line HaCaT using a radiolabeled DNA probe specific for human interleukin-8. Interleukin-8 gene expression was markedly increased upon in vitro stimulation after 1-6 h with contact sensitizers, tolerogen and the irritant. In contrast, interleukin-8 production was not detectable in unstimulated normal human keratinocytes or the HaCaT keratinocyte cell line. These results suggest that the induction and production of interleukin-8 is a response to nonspecific stimuli and may play a critical role in the early response to immunogenic or inflammatory signals in man.

The anti-proliferative and apoptotic effects of crocin on chemosensitive and chemoresistant cervical cancer cells.

PubMed

Mollaei, Homa; Safaralizadeh, Reza; Babaei, Esmaeil; Abedini, Mohamad Reza; Hoshyar, Reyhane

2017-10-01

Cervical cancer is the fourth cause of cancer-related mortality among females worldwide. Although current therapies reduce disease symptoms, resistance of tumor cells to chemotherapy agents after a while is a serious problem. Therefore, utilization of novel adjuvant agents to increase efficiency of chemotherapy is essential. In the last two decades, botanicals with effective anticancer activities have been studied. Among them, the anticancer properties of crocin have been more attended. In this study, the molecular mechanism of crocin action was investigated in sensitive human cervical cancer cell line (OV2008) in comparison with the resistant one (C13). A 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay showed that crocin inhibits proliferation of sensitive cells (OV2008) at a time- and dose-dependent manner at 48 and 72h. Also, this inhibitory effect has been shown on resistant cells (C13) at 72h. Hoechst staining and flow cytometry assay also confirmed these results and revealed that antiproliferative effect of crocin might be due to the induction of apoptosis. Moreover, the genetic mechanism of crocin-induced apoptosis was accomplished by studying the relative expressions of P53, Bax, Bcl2 and miR-365, an upstream regulator of the last two ones. Real-time PCR analysis indicated that 1.5 and 3mg/ml crocin led to up-regulation of Bax and P53 and down-regulation of Bcl2 and miR-365 at all time intervals in both two cell lines. However, OV2008 cell line was more sensitive to crocin, and alternation of gene expretion was more obvious in this cell line. In this regard, the present study demonstrated the anti-proliferative and apoptotic activities of crocin against both sensitive and resistant cervical cancer cells that may benefit cervical cancer treatment as an adjuvant agent to decrease chemoresistance and increase the efficiency of therapy. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Isolation and characterization of circulating tumor cells from human gastric cancer patients.

PubMed

Yuan, Dandan; Chen, Liang; Li, Mingxing; Xia, Hongwei; Zhang, Yuchen; Chen, Tie; Xia, Rui; Tang, Qiulin; Gao, Fabao; Mo, Xianming; Liu, Ming; Bi, Feng

2015-04-01

Circulating tumor cells (CTCs) have been proved to be responsible for tumor metastasis and resistant to anticancer therapies. This study aims to isolate and characterize circulating tumor cells from human gastric cancer patients, and investigate characteristic differences between gastric CTCs and gastric cancer cell lines. We analyzed 31 cases of gastric cancer patients using anti-CD45 antibody-conjugated magnetic microbeads negative separation, combined with fluorescence activated cell sorter CD44 positive screening. Abilities of tumor formation, metastasis, invasion, migration, irradiation and drug sensitivity of CTCs and gastric cancer cell lines were detected and compared. Of all the 31 patients, CD44(+)/CD45(-)CTCs were isolated in 14 patients, of which 3 cases were stage IIA, 2 cases stage IIB, 2 cases stage IIIC and 7 cases stage IV. The malignant behavior was demonstrated by both clonogenetic assay and tumor xenograft in nude mice. Compared with human gastric cancer cell lines, the migration and invasion abilities of CTCs increased to 3.21-12.6-fold and 2.3-6.7-fold, respectively (all p values <0.05). In addition, the metastatic potential of CTCs is much higher in vivo than that of the control. Furthermore, CTCs were found to be relatively sensitive to FU, cisplatin and paclitaxel, but relatively resistant to irradiation, oxaliplatin, cetuximab and trastuzumab. CD44(+)/CD45(-) gastric CTCs were isolated and found to exhibit stronger malignant behavior when compared with human gastric cancer cell lines. Furthermore, CTCs cultured in vitro have potential implications in drug sensitivity screening for the future anticancer treatments.

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.

Establishment and proteomic characterization of NCC-LMS1-C1, a novel cell line of primary leiomyosarcoma of the bone.

PubMed

Sakumoto, Marimu; Takahashi, Mami; Oyama, Rieko; Takai, Yoko; Kito, Fusako; Shiozawa, Kumiko; Qiao, Zhiwei; Yoshida, Akihiko; Endo, Makoto; Kawai, Akira; Kondo, Tadashi

2017-10-01

Leiomyosarcoma (LMS) is one of most aggressive mesenchymal malignancies that differentiate towards smooth muscle. The clinical outcome of LMS patients is poor; as such, there is an urgent need for novel therapeutic approaches. Experimental models such as patient-derived cell lines are invaluable tools for pre-clinical studies. In the present study, we established a stable cell line from the tumor tissue of a patient with a primary LMS of the bone. Despite the urgent need for novel therapeutic strategies in LMS, there are only a few LMS cell lines available in public cell banks, none of which are primary to the bone. Bone primary LMS tumor tissues were sampled to establish cell lines. Morphological and proteomic analyses were performed and sensitivity to pazopanib was evaluated. NCC-LMS1-C1 cells were maintained for over 100 passages. The cells exhibited a spindle shape and aggressive growth; they also expressed smooth muscle actin, reflecting the original LMS tissue (i.e. smooth muscle cells). The cells also showed tumor characteristics such as colony formation on soft agar and sensitivity to pazopanib, doxorubicin and cisplatin, with half-maximal inhibitory concentrations of 4.5, 0.11 and 20 μM, respectively. Proteomic analyses by mass spectrometry and antibody array revealed some differences in the protein expression profiles of these cells as compared to the original tumor tissue. Our results indicate that the NCC-LMS1-C1 cell lines will be useful for LMS research. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

HRD1 sensitizes breast cancer cells to Tamoxifen by promoting S100A8 degradation

PubMed Central

Liang, XiuBin; Li, Min; Shi, Ming; Li, Yan; Jenkins, Gareth; Lin, XiaWen; Wei, XueFei; Jia, ZhiJun; Feng, XueFeng; Su, DongMing; Guo, WanHua

2017-01-01

Estrogen receptor alpha positive (ER+) of breast cancer could develop resistance to antiestrogens including Tamoxifen. Our previous study showed that the E3 ubiquitin ligase HRD1 played an important role in anti-breast cancer. However, its role in chemotherapy resistance hasn't been reported. In this study, we found that HRD1 expression was downregulated in Tamoxifen-resistant breast cancer cell line MCF7/Tam compared to the Tamoxifen sensitive cell line MCF7. Moreover, S100A8 is the direct target of HRD1 by proteome analysis. Our data showed that HRD1 decreased the protein level of S100A8 through ubiquitination while HRD1 was regulated by acetylation of histone. More importantly, HRD1 knockdown significantly increased the cell survival of MCF7 cells to the Tamoxifen treatment. HRD1 overexpression sensitized MCF7/Tam cells to the Tamoxifen treatment in vitro and in vivo. In conclusion, the decrease of HRD1 expression contributed to Tamoxifen resistance in breast cancer. PMID:28423597

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

Paris Saponin I Sensitizes Gastric Cancer Cell Lines to Cisplatin via Cell Cycle Arrest and Apoptosis.

PubMed

Song, Shuichuan; Du, Leiwen; Jiang, Hao; Zhu, Xinhai; Li, Jinhui; Xu, Ji

2016-10-18

BACKGROUND Dose-related toxicity is the major restriction of cisplatin and cisplatin-combination chemotherapy, and is a challenge for advanced gastric cancer treatment. We explored the possibility of using Paris saponin I as an agent to sensitize gastric cancer cells to cisplatin, and examined the underlying mechanism. MATERIAL AND METHODS Growth inhibition was detected by MTT assay. The cell cycle and apoptosis were detected using flow cytometry and Annexin V/PI staining. The P21waf1/cip1, Bcl-2, Bax, and caspase-3 protein expression were detected using Western blot analysis. RESULTS The results revealed that PSI sensitized gastric cancer cells to cisplatin, with low toxicity. The IC50 value of cisplatin in SGC-7901 cell lines was decreased when combined with PSI. PSI promoted cisplatin-induced G2/M phase arrest and apoptosis in a cisplatin concentration-dependent manner. Bcl-2 protein expression decreased, but Bax, caspase-3, and P21waf1/cip1 protein expression increased with PSI treatment. CONCLUSIONS The underlying mechanism of Paris saponin I may be related to targeting the apoptosis pathway and cell cycle blocking, which suggests that PSI is a potential therapeutic sensitizer for cisplatin in treating gastric cancer.

Differential cytotoxicity of MEX: a component of Neem oil whose action is exerted at the cell membrane level.

PubMed

Ricci, Francesca; Berardi, Valerio; Risuleo, Gianfranco

2008-12-31

Neem oil is obtained from the seeds of the tree Azadirachta indica. Its chemical composition is very complex, being rich in terpenoids and limonoids, as well as volatile sulphur modified compounds. This work focused on the evaluation of a component of the whole Neem oil obtained by methanolic extraction and defined as MEX. Cytotoxicity was assessed on two different cell populations: a stabilized murine fibroblast line (3T6) and a tumor cell line (HeLa). The data presented here suggest a differential sensitivity of these two populations, the tumor line exhibiting a significantly higher sensitivity to MEX. The data strongly suggest that its toxic target is the cell membrane. In addition the results presented here imply that MEX may contain one or more agents that could find a potential use in anti-proliferative therapy.

Doxorubicin-anti-carcinoembryonic antigen immunoconjugate activity in vitro.

PubMed

Richardson, V J; Ford, C H; Tsaltas, G; Gallant, M E

1989-04-01

An in vitro model consisting of a series of 11 human cancer cell lines with varying density of expression of membrane carcinoembryonic antigen (CEA) has been used to evaluate conjugates of doxorubicin (Adriamycin) covalently linked by a carbodiimide method to goat polyclonal antibodies and mouse monoclonal antibodies to CEA. Conjugates were produced which retained both antigen binding and drug cytotoxicity. IC50 values were determined for free drug, free drug mixed with unconjugated antibodies and for the immunoconjugates. Cell lines that were very sensitive to free drug (IC50 less than 100 ng/ml) were also found to be highly sensitive to conjugated drug and similarly cell lines resistant to drug (IC50 greater than 1,000 ng/ml) were also resistant to conjugated drug. Although there was no correlation between CEA expression and conjugates efficacy, competitive inhibition studies using autologous antibody to block conjugate binding to cells indicated immunoconjugates specificity for the CEA target.

Real-time PCR assay is superior to other methods for the detection of mycoplasma contamination in the cell lines of the National Cell Bank of Iran.

PubMed

Molla Kazemiha, Vahid; Bonakdar, Shahin; Amanzadeh, Amir; Azari, Shahram; Memarnejadian, Arash; Shahbazi, Shirin; Shokrgozar, Mohammad Ali; Mahdian, Reza

2016-08-01

Mycoplasmas are the most important contaminants of cell cultures throughout the world. They are considered as a major problem in biological studies and biopharmaceutical economic issues. In this study, our aim was to find the best standard technique as a rapid method with high sensitivity, specificity and accuracy for the detection of mycoplasma contamination in the cell lines of the National Cell Bank of Iran. Thirty cell lines suspected to mycoplasma contamination were evaluated by five different techniques including microbial culture, indirect DNA DAPI staining, enzymatic mycoalert(®) assay, conventional PCR and real-time PCR. Five mycoplasma-contaminated cell lines were assigned as positive controls and five mycoplasma-free cell lines as negative controls. The enzymatic method was performed using the mycoalert(®) mycoplasma detection kit. Real-time PCR technique was conducted by PromoKine diagnostic kits. In the conventional PCR method, mycoplasma genus-specific primers were designed to analyze the sequences based on a fixed and common region on 16S ribosomal RNA with PCR product size of 425 bp. Mycoplasma contamination was observed in 60, 56.66, 53.33, 46.66 and 33.33 % of 30 different cell cultures by real-time PCR, PCR, enzymatic mycoalert(®), indirect DNA DAPI staining and microbial culture methods, respectively. The analysis of the results of the different methods showed that the real-time PCR assay was superior the other methods with the sensitivity, specificity, accuracy, predictive value of positive and negative results of 100 %. These values were 94.44, 100, 96.77, 100 and 92.85 % for the conventional PCR method, respectively. Therefore, this study showed that real-time PCR and PCR assays based on the common sequences in the 16S ribosomal RNA are reliable methods with high sensitivity, specificity and accuracy for detection of mycoplasma contamination in cell cultures and other biological products.

Proliferation inhibitory effect of human alpha interferon on primary explants of Burkitt lymphoma: inverse relationship to patient survival.

PubMed

Ernberg, I; Einhorn, S; Strander, H; Klein, G

1981-11-01

Eleven biopsies from 9 patients with Burkitt's lymphoma were tested for their sensitivity to the cell multiplication inhibitory activity of interferon. Three were resistant to interferon while 8 were sensitive to various degrees. Different biopsies from the same patient did not differ in interferon sensitivity. These results indicate that Burkitt's lymphoma cells might be resistant to interferon already in vivo as previously shown for some derived cell lines tested in vitro. The results imply an inverse relationship between patient survival and interferon sensitivity of the tumor cells.

Intratumoral genetic heterogeneity in metastatic melanoma is accompanied by variation in malignant behaviors.

PubMed

Anaka, Matthew; Hudson, Christopher; Lo, Pu-Han; Do, Hongdo; Caballero, Otavia L; Davis, Ian D; Dobrovic, Alexander; Cebon, Jonathan; Behren, Andreas

2013-10-11

Intratumoral heterogeneity is a major obstacle for the treatment of cancer, as the presence of even minor populations that are insensitive to therapy can lead to disease relapse. Increased clonal diversity has been correlated with a poor prognosis for cancer patients, and we therefore examined genetic, transcriptional, and functional diversity in metastatic melanoma. Amplicon sequencing and SNP microarrays were used to profile somatic mutations and DNA copy number changes in multiple regions from metastatic lesions. Clonal genetic and transcriptional heterogeneity was also assessed in single cell clones from early passage cell lines, which were then subjected to clonogenicity and drug sensitivity assays. MAPK pathway and tumor suppressor mutations were identified in all regions of the melanoma metastases analyzed. In contrast, we identified copy number abnormalities present in only some regions in addition to homogeneously present changes, suggesting ongoing genetic evolution following metastatic spread. Copy number heterogeneity from a tumor was represented in matched cell line clones, which also varied in their clonogenicity and drug sensitivity. Minor clones were identified based on dissimilarity to the parental cell line, and these clones were the most clonogenic and least sensitive to drugs. Finally, treatment of a polyclonal cell line with paclitaxel to enrich for drug-resistant cells resulted in the adoption of a gene expression profile with features of one of the minor clones, supporting the idea that these populations can mediate disease relapse. Our results support the hypothesis that minor clones might have major consequences for patient outcomes in melanoma.

Development and characterization of a cell line from Pacific herring, Clupea harengus pallasi, sensitive to both naphthalene cytotoxicity and infection by viral hemorrhagic septicemia virus.

PubMed

Ganassin, R C; Sanders, S M; Kennedy, C J; Joyce, E M; Bols, N C

1999-01-01

A cell line, PHL, has been successfully established from newly hatched herring larvae. The cells are maintained in growth medium consisting of Leibovitz's L-15 supplemented with 15% fetal bovine serum (FBS), and have been cryopreserved and maintain viability after thawing. These cells retain a diploid karotype after 65 population doublings. PHL are susceptible to infection by the North American strain of viral hemorrhagic septicemia (VHS) virus, and are sensitive to the cytotoxic effects of naphthalene, a common environmental contaminant. Naphthalene is a component of crude and refined oil, and may be found in the marine environment following acute events such as oil spills. In addition, chronic sources of naphthalene contamination include offshore drilling and petroleum contamination from areas such as docks and marinas that have creosote-treated docks and pilings and also receive constant small inputs of petroleum products. This cell line should be useful for investigations of the toxicity of naphthalene and other petroleum components to juvenile herring. In addition, studies of the VHS virus will be facilitated by the availability of a susceptible cell line from an alternative species.

Radiosensitization by PARP Inhibition in DNA Repair Proficient and Deficient Tumor Cells: Proliferative Recovery in Senescent Cells

PubMed Central

Alotaibi, Moureq; Sharma, Khushboo; Saleh, Tareq; Povirk, Lawrence F.; Hendrickson, Eric A.; Gewirtz, David A.

2016-01-01

Radiotherapy continues to be a primary modality in the treatment of cancer. DNA damage induced by radiation can promote apoptosis as well as both autophagy and senescence, where autophagy and senescence can theoretically function to prolong tumor survival. A primary aim of this work was to investigate the hypothesis that autophagy and/or senescence could be permissive for DNA repair, thereby facilitating tumor cell recovery from radiation-induced growth arrest and/or cell death. In addition, studies were designed to elucidate the involvement of autophagy and senescence in radiation sensitization by PARP inhibitors and the re-emergence of a proliferating tumor cell population. In the context of this work, the relationship between radiation-induced autophagy and senescence was also determined. Studies were performed using DNA repair proficient HCT116 colon carcinoma cells and a repair deficient Ligase IV (−/−) isogenic cell line. Irradiation promoted a parallel induction of autophagy and senescence that was strongly correlated with the extent of persistent H2AX phosphorylation in both cell lines; however inhibition of autophagy failed to suppress senescence, indicating that the two responses were dissociable. Irradiation resulted in a transient arrest in the HCT116 cells while arrest was prolonged in the Ligase IV (−/−) cells; however, both cell lines ultimately recovered proliferative function, which may reflect maintenance of DNA repair capacity. The PARP inhibitors (Olaparib) and (Niraparib) increased the extent of persistent DNA damage induced by radiation as well as the extent of both autophagy and senescence; neither cell line underwent significant apoptosis by radiation alone or in the presence of the PARP inhibitors. Inhibition of autophagy failed to attenuate radiation sensitization, indicating that autophagy was not involved in the action of the PARP inhibitors. As with radiation alone, despite sensitization by PARP inhibition, proliferative recovery was evident within a period of 10–20 days. While inhibition of DNA repair via PARP inhibition may initially sensitize tumor cells to radiation via the promotion of senescence, this strategy does not appear to interfere with proliferative recovery, which could ultimately contribute to disease recurrence. PMID:26934368

Impact of BRAF kinase inhibitors on the miRNomes and transcriptomes of melanoma cells.

PubMed

Kozar, Ines; Cesi, Giulia; Margue, Christiane; Philippidou, Demetra; Kreis, Stephanie

2017-11-01

Melanoma is an aggressive skin cancer with increasing incidence worldwide. The development of BRAF kinase inhibitors as targeted treatments for patients with BRAF-mutant tumours contributed profoundly to an improved overall survival of patients with metastatic melanoma. Despite these promising results, the emergence of rapid resistance to targeted therapy remains a serious clinical issue. To investigate the impact of BRAF inhibitors on miRNomes and transcriptomes, we used in vitro melanoma models consisting of BRAF inhibitor-sensitive and -resistant cell lines generated in our laboratory. Subsequently, microarray analyses were performed followed by RT-qPCR validations. Regarding miRNome and transcriptome changes, the long-term effects of BRAF inhibition differed in a cell line-specific manner with the two different BRAF inhibitors inducing comparable responses in three melanoma cell lines. Despite this heterogeneity, several miRNAs (e.g. miR-92a-1-5p, miR-708-5p) and genes (e.g. DOK5, PCSK2) were distinctly differentially expressed in drug-resistant versus -sensitive cell lines. Analyses of coexpressed miRNAs, as well as inversely correlated miRNA-mRNA pairs, revealed a low MITF/AXL ratio in two drug-resistant cell lines that might be regulated by miRNAs. Several genes and miRNAs were differentially regulated in the drug-resistant and -sensitive cell lines and might be considered as prognostic and/or diagnostic resistance biomarkers in melanoma drug resistance. Thus far, only little information is available on the significance and role of miRNAs with respect to kinase inhibitor treatments and emergence of drug resistance. In this study, promising miRNAs and genes were identified and associated to BRAF inhibitor-mediated resistance in melanoma. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue. Copyright © 2017 Elsevier B.V. All rights reserved.

High early growth response 1 (EGR1) expression correlates with resistance to anti-EGFR treatment in vitro and with poorer outcome in metastatic colorectal cancer patients treated with cetuximab.

PubMed

Kumar, S S; Tomita, Y; Wrin, J; Bruhn, M; Swalling, A; Mohammed, M; Price, T J; Hardingham, J E

2017-06-01

Biomarkers, such as mutant RAS, predict resistance to anti-EGFR therapy in only a proportion of patients, and hence, other predictive biomarkers are needed. The aims were to identify candidate genes upregulated in colorectal cancer cell lines resistant to anti-EGFR monoclonal antibody treatment, to knockdown (KD) these genes in the resistant cell lines to determine if sensitivity to anti-EGFR antibody was restored, and finally to perform a pilot correlative study of EGR1 expression and outcomes in a cohort of metastatic colorectal cancer (mCRC) patients given cetuximab therapy. Comparative expression array analysis of resistant cell lines (SW48, COLO-320DM, and SNU-C1) vs sensitive cell lines (LIM1215, CaCo2, and SW948) was performed. The highest up-regulated gene in each resistant cell line was knocked down (KD) using RNA interference, and effect on proliferation was assessed with and without anti-EGFR treatment. Expression of the candidate genes in patients' tumours treated with cetuximab was assessed by immunohistochemistry; survival analyses were performed comparing high vs low expression. Genes significantly upregulated in resistant cell lines were EGR1 (early growth response protein 1), HBEGF (heparin-binding epidermal growth factor-like growth factor), and AKT3 (AKT serine/threonine kinase 3). KD of each gene resulted in the respective cells being more sensitive to anti-EGFR treatment, suggesting that the resistant phenotype was reversed. In the pilot study of mCRC patients treated with cetuximab, both median PFS (1.38 months vs 6.79 months; HR 2.77 95% CI 1.2-19.4) and median OS (2.59 months vs 9.82 months; HR 3.0 95% CI 1.3-23.2) were significantly worse for those patients with high EGR1 expression. High EGR1 expression may be a candidate biomarker of resistance to anti-EGFR therapy.

Susceptibility to cytotoxic T cell lysis of cancer stem cells derived from cervical and head and neck tumor cell lines.

PubMed

Liao, Tian; Kaufmann, Andreas M; Qian, Xu; Sangvatanakul, Voramon; Chen, Chao; Kube, Tina; Zhang, Guoyou; Albers, Andreas E

2013-01-01

To explore cancer stem cell susceptibility to a host's cytotoxic T lymphocyte (CTL)-mediated immune response. We compared the susceptibility of putative CSC generated from cancer cell lines to immunologic recognition and killing by alloantigen-specific CD8(+) CTL. CSC-enriched spheroid culture-derived cells (SDC) exhibited higher expression of ALDH, ICAM1 and of stem/progenitor cell markers on all 3 tumor cell lines investigated and lower MHC class I on the cervical cancer cell line as compared to their monolayer-derived cells (MDC). The expression of ICAM1 and MHCI was upregulated by IFN-γ treatment. CSC populations were less sensitive to MHC class I-restricted alloantigen-specific CD8(+) CTL lysis as compared to matched MDC. IFN-γ pretreatment resulted in over-proportionally enhanced lysis of SDC. Finally, the subset of ALDH(high) expressing SDC presented more sensitivity toward CD8(+) CTL killing than the ALDH(low) SDC. Tumor therapy resistance has been attributed to cancer stem cells (CSC). We show in vitro susceptibility of CSC to CTL-mediated lysis. Immunotherapy targeting of ALDH(+) CSC may therefore be a promising approach. Our results and method may be helpful for the development and optimization of adjuvants, as here exemplified for INF-γ, for CSC-targeted vaccines, independent of the availability of CSC-specific antigens.

Anti-proliferative effect of 20-hydroxyecdysone in a lepidopteran cell line.

PubMed

Auzoux-Bordenave, Stéphanie; Hatt, Philippe-Jacques; Porcheron, Patrick

2002-02-01

Ecdysteroids are steroid hormones involved in the epidermal growth of arthropods, controlling cell proliferation and further differentiation of target cells. The epidermal cell line IAL-PID2, established from imaginal discs of the Indian meal moth Plodia interpunctella kept its sensitivity to ecdysteroids in vitro, cells being able to respond to them by cytological and biochemical changes. When added to the culture medium, 20-hydroxyecdysone (20E) stopped cell proliferation and induced formation of epithelial-like aggregates. In order to better understand the cellular sequence of ecdysteroids signalling in epidermal cells we used the IAL-PID2 cell line for in vitro investigations of cytological events induced by the moulting hormone. After a 40 h serum deprivation, formazan assay (XTT) was routinely used to evaluate anti-proliferative effects of 20E during cell cycle. We established a more precise timing of the period of cell sensitivity to the hormone during the cell cycle, by the use of the mitotic index and the BrdU incorporation test. These in vitro assays were performed in parallel with the description of some hormone dependant cytological events, using immunofluorescent labelling with anti-beta tubulin/FITC antibodies and DNA staining.

Krüppel-Like Factor 4 Enhances Sensitivity of Cisplatin to Esophageal Squamous Cell Carcinoma (ESCC) Cells

PubMed Central

Chen, Chuangui; Ma, Zhao; Zhang, Hongdian; Liu, Xiaoqiong; Yu, Zhentao

2017-01-01

Background The aim of this study was to elucidate the role of Krüppel-Like factor 4 (KLF4) in cisplatin resistance in esophageal squamous cell carcinoma (ESCC) cells, which may eventually help to improve the treatment efficacy. Material/Methods Human esophageal squamous cell carcinoma (ESCC) cell line CaEs-17, TE-1, EC109, KYSE510, KYSE140, KYSE70, and KYSE30 were selected to detect their sensitivity to cisplatin. 5-Azacytidine-2′-deoxycytidine (5′-Aza-CdR) treatment and methylation-specific PCR (MS-PCR) were used to detect the methylation status for KLF4. Cell viability, apoptosis, and cell cycle were measured using methyl thiazolyl tetrazolium (MTT) assay, Annexin V affinity assay, and flow cytometry, respectively. Results The sensitivity to cisplatin was different in the seven ESCC cell lines, with TE-1 having the lowest sensitivity and KYSE140 having the highest sensitivity. Interestingly, the level of KLF4 was relatively low in TE-1 cells; while it was high in KYSE140 cells. These results suggested that KLF4 may be involved in cisplatin resistance. The promoter region was mostly unmethylated in KYSE140 cells; while it was hypermethylated in TE-1 cells. After treatment with demethylation reagent 5-Aza-CdR, cisplatin sensitivities were significantly increased after upregulation of KLF4, as the IC50 values were significantly decreased in the TE-1 cell treated with 5-Aza-CdR. Furthermore, upregulation of KLF4 induced cell apoptosis and cell cycle arrest at S phase. Conclusions KLF4 enhances the sensitivity of cisplatin to ESCC cells through apoptosis induction and cell cycle arrest. Our data provided a novel insight to the mechanism of cisplatin resistance; overexpression of KLF4 may be a potential therapeutic strategy for cisplatin resistance in human ESCC. PMID:28694421

Krüppel-Like Factor 4 Enhances Sensitivity of Cisplatin to Esophageal Squamous Cell Carcinoma (ESCC) Cells.

PubMed

Chen, Chuangui; Ma, Zhao; Zhang, Hongdian; Liu, Xiaoqiong; Yu, Zhentao

2017-07-11

BACKGROUND The aim of this study was to elucidate the role of Krüppel-Like factor 4 (KLF4) in cisplatin resistance in esophageal squamous cell carcinoma (ESCC) cells, which may eventually help to improve the treatment efficacy. MATERIAL AND METHODS Human esophageal squamous cell carcinoma (ESCC) cell line CaEs-17, TE-1, EC109, KYSE510, KYSE140, KYSE70, and KYSE30 were selected to detect their sensitivity to cisplatin. 5-Azacytidine-2'-deoxycytidine (5'-Aza-CdR) treatment and methylation-specific PCR (MS-PCR) were used to detect the methylation status for KLF4. Cell viability, apoptosis, and cell cycle were measured using methyl thiazolyl tetrazolium (MTT) assay, Annexin V affinity assay, and flow cytometry, respectively. RESULTS The sensitivity to cisplatin was different in the seven ESCC cell lines, with TE-1 having the lowest sensitivity and KYSE140 having the highest sensitivity. Interestingly, the level of KLF4 was relatively low in TE-1 cells; while it was high in KYSE140 cells. These results suggested that KLF4 may be involved in cisplatin resistance. The promoter region was mostly unmethylated in KYSE140 cells; while it was hypermethylated in TE-1 cells. After treatment with demethylation reagent 5-Aza-CdR, cisplatin sensitivities were significantly increased after upregulation of KLF4, as the IC50 values were significantly decreased in the TE-1 cell treated with 5-Aza-CdR. Furthermore, upregulation of KLF4 induced cell apoptosis and cell cycle arrest at S phase. CONCLUSIONS KLF4 enhances the sensitivity of cisplatin to ESCC cells through apoptosis induction and cell cycle arrest. Our data provided a novel insight to the mechanism of cisplatin resistance; overexpression of KLF4 may be a potential therapeutic strategy for cisplatin resistance in human ESCC.

Flavopiridol sensitivity of cancer cells isolated from ascites and pleural fluids.

PubMed

Richard, Christina; Matthews, Donald; Duivenvoorden, Wilhelmina; Yau, Jonathan; Wright, Paul S; Th'ng, John P H

2005-05-01

We examined the efficacy of flavopiridol, a cyclin-dependent kinase inhibitor that is undergoing clinical trials, on primary cancer cells isolated from the ascites or pleural fluids of patients with metastatic cancers. Metastasized cancer cells were isolated from the pleural fluids (n = 20) or ascites (n = 15) of patients, most of whom were refractory to chemotherapy. These primary cancer cells were used within 2 weeks of isolation without selecting for proliferative capacities. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide viability assay was used to characterize the response of these cancer cells to commonly used chemotherapeutic agents, and their response to flavopiridol was compared with rapidly dividing cultured cell lines. The primary cancer cells displayed phenotypes that were different from established cell lines; they had very low replication rates, dividing every 1 to 2 weeks, and underwent replicative senescence within five passages. These primary tumor cells retained their resistance to chemotherapeutic drugs exhibited by the respective patients but did not show cross-resistance to other agents. However, these cancer cells showed sensitivity to flavopiridol with an average LD50 of 50 nmol/L (range, 21.5-69 nmol/L), similar to the LD50 in established cell lines. Because senescent cells also showed similar sensitivity to flavopiridol, it suggests that the mechanism of action is not dependent on the activity of cyclin-dependent kinases that regulate the progression of the cell cycle. Using cancer cells isolated from the ascites or pleural fluids, this study shows the potential of flavopiridol against cancer cells that have developed resistance to conventional chemotherapeutic agents.

Improved method increases sensitivity for circulating hepatocellular carcinoma cells

PubMed Central

Liu, Hui-Ying; Qian, Hai-Hua; Zhang, Xiao-Feng; Li, Jun; Yang, Xia; Sun, Bin; Ma, Jun-Yong; Chen, Lei; Yin, Zheng-Feng

2015-01-01

AIM: To improve an asialoglycoprotein receptor (ASGPR)-based enrichment method for detection of circulating tumor cells (CTCs) of hepatocellular carcinoma (HCC). METHODS: Peripheral blood samples were collected from healthy subjects, patients with HCC or various other cancers, and patients with hepatic lesions or hepatitis. CTCs were enriched from whole blood by extracting CD45-expressing leukocytes with monoclonal antibody coated-beads following density gradient centrifugation. The remaining cells were cytocentrifuged on polylysine-coated slides. Isolated cells were treated by triple immunofluorescence staining with CD45 antibody and a combination of antibodies against ASGPR and carbamoyl phosphate synthetase 1 (CPS1), used as liver-specific markers, and costained with DAPI. The cell slide was imaged and stained tumor cells that met preset criteria were counted. Recovery, sensitivity and specificity of the detection methods were determined and compared by spiking experiments with various types of cultured human tumor cell lines. Expression of ASGPR and CPS1 in cultured tumor cells and tumor tissue specimens was analyzed by flow cytometry and triple immunofluorescence staining, respectively. RESULTS: CD45 depletion of leukocytes resulted in a significantly greater recovery of multiple amounts of spiked HCC cells than the ASGPR+ selection (Ps < 0.05). The expression rates of either ASGPR or CPS1 were different in various liver cancer cell lines, ranging between 18% and 99% for ASGPR and between 9% and 98% for CPS1. In both human HCC tissues and liver cancer cell lines, there were a few HCC cells that did not stain positive for ASGPR or CPS1. The mixture of monoclonal antibodies against ASGPR and CPS1 identified more HCC cells than either antibody alone. However, these antibodies did not detect any tumor cells in blood samples spiked with the human breast cancer cell line MCF-7 and the human renal cancer cell line A498. ASGPR+ or/and CPS1+ CTCs were detected in 29/32 (91%) patients with HCC, but not in patients with any other kind of cancer or any of the other test subjects. Furthermore, the improved method detected a higher CTC count in all patients examined than did the previous method (P = 0.001), and consistently achieved 12%-21% higher sensitivity of CTC detection in all seven HCC patients with more than 40 CTCs. CONCLUSION: Negative depletion enrichment combined with identification using a mixture of antibodies against ASGPR and CPS1 improves sensitivity and specificity for detecting circulating HCC cells. PMID:25780289

Evidence of drug-response heterogeneity rapidly generated from a single cancer cell.

PubMed

Wang, Rong; Jin, Chengmeng; Hu, Xun

2017-06-20

One cancer cell line is believed to be composed of numerous clones with different drug sensitivity. We sought to investigate the difference of drug-response pattern in clones from a cell line or from a single cell. We showed that 22 clones derived from 4T1 cells were drastically different from each other with respect to drug-response pattern against 11 anticancer drugs and expression profile of 19 genes associated with drug resistance or sensitivity. Similar results were obtained using daughter clones derived from a single 4T1 cell. Each daughter clone showed distinct drug-response pattern and gene expression profile. Similar results were also obtained using Bcap37 cells. We conclude that a single cancer cell can rapidly produce a population of cells with high heterogeneity of drug response and the acquisition of drug-response heterogeneity is random.

Sensitizing effects of gallium citrate on hyperthermic cell killing in vitro.

PubMed

Miyazaki, N; Nakano, H; Kawakami, N; Kugotani, M; Nishihara, K; Aoki, Y; Shinohara, K

2000-01-01

The lethal effects of gallium citrate in combination with heat were studied using four cell lines, L5178Y, FM3A, P388 and HeLa. Cells were incubated with different concentrations (0.2 2 mM) of gallium citrate at 37 degrees C for 24 h and heated at a range of temperatures from 40-44 degrees C for various time periods up to 6 h in the absence of gallium citrate. Survival and cell viability were determined by clonogenic assay and the dye-exclusion test, respectively. All of the cell lines tested were insensitive to heat below 41 degrees C, but were very sensitive to heat above 43 degrees C. Gallium citrate was cytotoxic to these cell lines at different levels: P388 and HeLa were far more sensitive than L5178Y and FM3A. The killing effects of heat at 41 degrees C were greatly enhanced by gallium citrate in L5178Y and P388 cells. The Arrhenius analysis for the lethal effect of heat, determined by clonogenic assay, in L5178Y cells showed that the transition temperature was remarkably decreased for the gallium-treated cells from approximately 43 degrees C to 41 degrees C. The mechanism for this decrease in the transition temperature may be attributable to the additional effects of gallium citrate on energy metabolism. Preincubation with 0.05 mM gallium citrate at 37 degrees C for 7 days also enhanced heat sensitization at 41 degrees C in L5178Y. This preincubation condition may correspond to the condition for the continuous infusion of gallium that is clinically used for cancer treatment. In contrast, treatment with gallium did not greatly enhance the sensitivity of FM3A or HeLa cells to heat at 41 degrees C, but the effects of gallium were significant.

Chrysin enhances doxorubicin-induced cytotoxicity in human lung epithelial cancer cell lines: The role of glutathione

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

Brechbuhl, Heather M.; Kachadourian, Remy; Min, Elysia

We hypothesized that flavonoid-induced glutathione (GSH) efflux through multi-drug resistance proteins (MRPs) and subsequent intracellular GSH depletion is a viable mechanism to sensitize cancer cells to chemotherapies. This concept was demonstrated using chrysin (5–25 μM) induced GSH efflux in human non-small cell lung cancer lines exposed to the chemotherapeutic agent, doxorubicin (DOX). Treatment with chrysin resulted in significant and sustained intracellular GSH depletion and the GSH enzyme network in the four cancer cell types was predictive of the severity of chrysin induced intracellular GSH depletion. Gene expression data indicated a positive correlation between basal MRP1, MRP3 and MRP5 expression andmore » total GSH efflux before and after chrysin exposure. Co-treating the cells for 72 h with chrysin (5–30 μM) and DOX (0.025–3.0 μM) significantly enhanced the sensitivity of the cells to DOX as compared to 72-hour DOX alone treatment in all four cell lines. The maximum decrease in the IC{sub 50} values of cells treated with DOX alone compared to co-treatment with chrysin and DOX was 43% in A549 cells, 47% in H157 and H1975 cells and 78% in H460 cells. Chrysin worked synergistically with DOX to induce cancer cell death. This approach could allow for use of lower concentrations and/or sensitize cancer cells to drugs that are typically resistant to therapy. -- Graphical abstract: Possible mechanisms by which chrysin enhances doxorubicin-induced toxicity in cancer cells. Highlights: ► Chyrsin sustains a significant depletion of GSH levels in lung cancer cells. ► Chyrsin synergistically potentiates doxorubicin-induced cancer cell cytotoxicity. ► Cancer cell sensitivity correlated with GSH and MRP gene network expression. ► This approach could allow for lower side effects and targeting resistant tumors.« less

Copper (II) complexes of bidentate ligands exhibit potent anti-cancer activity regardless of platinum sensitivity status.

PubMed

Wehbe, Mohamed; Lo, Cody; Leung, Ada W Y; Dragowska, Wieslawa H; Ryan, Gemma M; Bally, Marcel B

2017-12-01

Insensitivity to platinum, either through inherent or acquired resistance, is a major clinical problem in the treatment of many solid tumors. Here, we explored the therapeutic potential of diethyldithiocarbamate (DDC), pyrithione (Pyr), plumbagin (Plum), 8-hydroxyquinoline (8-HQ), clioquinol (CQ) copper complexes in a panel of cancer cell lines that differ in their sensitivity to platins (cisplatin/carboplatin) using a high-content imaging system. Our data suggest that the copper complexes were effective against both platinum sensitive (IC 50  ~ 1 μM platinum) and insensitive (IC 50  > 5 μM platinum) cell lines. Furthermore, copper complexes of DDC, Pyr and 8-HQ had greater therapeutic activity compared to the copper-free ligands in all cell lines; whereas the copper-dependent activities of Plum and CQ were cell-line specific. Four of the copper complexes (Cu(DDC) 2 , Cu(Pyr) 2 , Cu(Plum) 2 and Cu(8-HQ) 2 ) showed IC 50 values less than that of cisplatin in all tested cell lines. The complex copper DDC (Cu(DDC) 2 ) was selected for in vivo evaluation due to its low nano-molar range activity in vitro and the availability of an injectable liposomal formulation. Liposomal (Cu(DDC) 2 ) was tested in a fast-growing platinum-resistant A2780-CP ovarian xenograft model and was found to achieve a statistically significant reduction (50%; p < 0.05) in tumour size. This work supports the potential use of copper-based therapeutics to treat cancers that are insensitive to platinum drugs.

Belinostat and vincristine demonstrate mutually synergistic cytotoxicity associated with mitotic arrest and inhibition of polyploidy in a preclinical model of aggressive diffuse large B cell lymphoma.

PubMed

Havas, Aaron P; Rodrigues, Kameron B; Bhakta, Anvi; Demirjian, Joseph A; Hahn, Seongmin; Tran, Jack; Scavello, Margarethakay; Tula-Sanchez, Ana A; Zeng, Yi; Schmelz, Monika; Smith, Catharine L

2016-12-01

Diffuse Large B-cell lymphoma (DLBCL) is an aggressive malignancy that has a 60 percent 5-year survival rate, highlighting a need for new therapeutic approaches. Histone deacetylase inhibitors (HDACi) are novel therapeutics being clinically-evaluated in combination with a variety of other drugs. However, rational selection of companion therapeutics for HDACi is difficult due to their poorly-understood, cell-type specific mechanisms of action. To address this, we developed a pre-clinical model system of sensitivity and resistance to the HDACi belinostat using DLBCL cell lines. In the current study, we demonstrate that cell lines sensitive to the cytotoxic effects of HDACi undergo early mitotic arrest prior to apoptosis. In contrast, HDACi-resistant cell lines complete mitosis after a short delay and arrest in G1. To force mitotic arrest in HDACi-resistant cell lines, we used low dose vincristine or paclitaxel in combination with belinostat and observed synergistic cytotoxicity. Belinostat curtails vincristine-induced mitotic arrest and triggers a strong apoptotic response associated with downregulated MCL-1 expression and upregulated BIM expression. Resistance to microtubule targeting agents (MTAs) has been associated with their propensity to induce polyploidy and thereby increase the probability of genomic instability that enables cancer progression. Co-treatment with belinostat effectively eliminated a vincristine-induced, actively cycling polyploid cell population. Our study demonstrates that vincristine sensitizes DLBCL cells to the cytotoxic effects of belinostat and that belinostat prevents polyploidy that could cause vincristine resistance. Our findings provide a rationale for using low dose MTAs in conjunction with HDACi as a potential therapeutic strategy for treatment of aggressive DLBCL.

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

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

Silencing of high-mobility group box 2 (HMGB2) modulates cisplatin and 5-fluorouracil sensitivity in head and neck squamous cell carcinoma.

PubMed

Syed, Nazia; Chavan, Sandip; Sahasrabuddhe, Nandini A; Renuse, Santosh; Sathe, Gajanan; Nanjappa, Vishalakshi; Radhakrishnan, Aneesha; Raja, Remya; Pinto, Sneha M; Srinivasan, Anand; Prasad, T S Keshava; Srikumar, Kotteazeth; Gowda, Harsha; Santosh, Vani; Sidransky, David; Califano, Joseph A; Pandey, Akhilesh; Chatterjee, Aditi

2015-01-01

Dysregulation of protein expression is associated with most diseases including cancer. MS-based proteomic analysis is widely employed as a tool to study protein dysregulation in cancers. Proteins that are differentially expressed in head and neck squamous cell carcinoma (HNSCC) cell lines compared to the normal oral cell line could serve as biomarkers for patient stratification. To understand the proteomic complexity in HNSCC, we carried out iTRAQ-based MS analysis on a panel of HNSCC cell lines in addition to a normal oral keratinocyte cell line. LC-MS/MS analysis of total proteome of the HNSCC cell lines led to the identification of 3263 proteins, of which 185 proteins were overexpressed and 190 proteins were downregulated more than twofold in at least two of the three HNSCC cell lines studied. Among the overexpressed proteins, 23 proteins were related to DNA replication and repair. These included high-mobility group box 2 (HMGB2) protein, which was overexpressed in all three HNSCC lines studied. Overexpression of HMGB2 has been reported in various cancers, yet its role in HNSCC remains unclear. Immunohistochemical labeling of HMGB2 in a panel of HNSCC tumors using tissue microarrays revealed overexpression in 77% (54 of 70) of tumors. The HMGB proteins are known to bind to DNA structure resulting from cisplatin-DNA adducts and affect the chemosensitivity of cells. We observed that siRNA-mediated silencing of HMGB2 increased the sensitivity of the HNSCC cell lines to cisplatin and 5-FU. We hypothesize that targeting HMGB2 could enhance the efficacy of existing chemotherapeutic regimens for treatment of HNSCC. All MS data have been deposited in the ProteomeXchange with identifier PXD000737 (http://proteomecentral.proteomexchange.org/dataset/PXD000737). © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Enhancement of Radiation Response in Osteosarcoma and Rhabomyosarcoma Cell Lines by Histone Deacetylase Inhibition

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

Blattmann, Claudia, E-mail: claudia.blattmann@med.uni-heidelberg.d; Oertel, Susanne; Ehemann, Volker

2010-09-01

Purpose: Histone deacetylase inhibitors (HDACIs) can enhance the sensitivity of cells to photon radiation treatment (XRT) by altering numerous molecular pathways. We investigated the effect of pan-HDACIs such as suberoylanilide hydroxamic acid (SAHA) on radiation response in two osteosarcoma (OS) and two rhabdomyosarcoma (RMS) cell lines. Methods and Materials: Clonogenic survival, cell cycle analysis, and apoptosis were examined in OS (KHOS-24OS, SAOS2) and RMS (A-204, RD) cell lines treated with HDACI and HDACI plus XRT, respectively. Protein expression was investigated via immunoblot analysis, and cell cycle analysis and measurement of apoptosis were performed using flow cytometry. Results: SAHA induced anmore » inhibition of cell proliferation and clonogenic survival in OS and RMS cell lines and led to a significant radiosensitization of all tumor cell lines. Other HDACI such as M344 and valproate showed similar effects as investigated in one OS cell line. Furthermore, SAHA significantly increased radiation-induced apoptosis in the OS cell lines, whereas in the RMS cell lines radiation-induced apoptosis was insignificant with and without SAHA. In all investigated sarcoma cell lines, SAHA attenuated radiation-induced DNA repair protein expression (Rad51, Ku80). Conclusion: Our results show that HDACIs enhance radiation action in OS and RMS cell lines. Inhibition of DNA repair, as well as increased apoptosis induction after exposure to HDACIs, can be mechanisms of radiosensitization by HDACIs.« less

Plasma stable, pH-sensitive non-ionic surfactant vesicles simultaneously enhance antiproliferative effect and selectivity of Sirolimus.

PubMed

Ghanbarzadeh, Saeed; Khorrami, Arash; Pourmoazzen, Zhaleh; Arami, Sanam

2015-05-01

The purpose of the present investigation was to prepare a plasma stable, pH-sensitive niosomal formulation to enhance Sirolimus efficacy and selectivity. pH-sensitive niosomal formulations bearing PEG-Poly (monomethyl itaconate)-CholC6 (PEG-PMMI-CholC6) copolymers and cholesteryl hemisuccinate (CHEMS) were prepared by a modified ethanol injection method and characterized with regard to pH-responsiveness and stability in human serum. The ability of pH-sensitive niosomes to enhance the Sirolimus cytotoxicity was evaluated in vitro using human erythromyeloblastoid leukemia cell line (K562) and compared with cytotoxicity effect on human umbilical vein endothelial cells (HUVEC). This study showed that both formulations can be rendered pH-sensitive property and were found to rapidly release their contents under mildly acidic conditions. However, the CHEMS-based niosomes lost their pH-sensitivity after incubation in plasma, whereas, PEG-PMMI-CholC6 niosomes preserved their ability to respond to pH change. Sirolimus encapsulated in pH-sensitive niosomes exhibited a higher cytotoxicity than the control conventional formulation on K562 cell line. On the other hand, both pH-sensitive niosomes showed lower antiproliferative effect on HUVEC cells. Plasma stable, pH-sensitive PEG-PMMI-CholC6-based niosomes can improve the in vitro efficiency and also reduce the side effects of Sirolimus.

MicroRNA-566 activates EGFR signaling and its inhibition sensitizes glioblastoma cells to nimotuzumab.

PubMed

Zhang, Kai-Liang; Zhou, Xuan; Han, Lei; Chen, Lu-Yue; Chen, Ling-Chao; Shi, Zhen-Dong; Yang, Ming; Ren, Yu; Yang, Jing-Xuan; Frank, Thomas S; Zhang, Chuan-Bao; Zhang, Jun-Xia; Pu, Pei-Yu; Zhang, Jian-Ning; Jiang, Tao; Wagner, Eric J; Li, Min; Kang, Chun-Sheng

2014-03-20

Epidermal growth factor receptor (EGFR) is amplified in 40% of human glioblastomas. However, most glioblastoma patients respond poorly to anti-EGFR therapy. MicroRNAs can function as either oncogenes or tumor suppressor genes, and have been shown to play an important role in cancer cell proliferation, invasion and apoptosis. Whether microRNAs can impact the therapeutic effects of EGFR inhibitors in glioblastoma is unknown. miR-566 expression levels were detected in glioma cell lines, using real-time quantitative RT-PCR (qRT-PCR). Luciferase reporter assays and Western blots were used to validate VHL as a direct target gene of miR-566. Cell proliferation, invasion, cell cycle distribution and apoptosis were also examined to confirm whether miR-566 inhibition could sensitize anti-EGFR therapy. In this study, we demonstrated that miR-566 is up-regulated in human glioma cell lines and inhibition of miR-566 decreased the activity of the EGFR pathway. Lentiviral mediated inhibition of miR-566 in glioblastoma cell lines significantly inhibited cell proliferation and invasion and led to cell cycle arrest in the G0/G1 phase. In addition, we identified von Hippel-Lindau (VHL) as a novel functional target of miR-566. VHL regulates the formation of the β-catenin/hypoxia-inducible factors-1α complex under miR-566 regulation. miR-566 activated EGFR signaling and its inhibition sensitized glioblastoma cells to anti-EGFR therapy.

Cytotoxicity of Vitex agnus-castus fruit extract and its major component, casticin, correlates with differentiation status in leukemia cell lines.

PubMed

Kikuchi, Hidetomo; Yuan, Bo; Nishimura, Yoshio; Imai, Masahiko; Furutani, Ryota; Kamoi, Saki; Seno, Misako; Fukushima, Shin; Hazama, Shingo; Hirobe, Chieko; Ohyama, Kunio; Hu, Xiao-Mei; Takagi, Norio; Hirano, Toshihiko; Toyoda, Hiroo

2013-12-01

We have demonstrated that an extract from the ripe fruit of Vitex agnus-castus (Vitex) exhibits cytotoxic activities against various types of solid tumor cells, whereas its effects on leukemia cells has not been evaluated to date. In this study, the effects of Vitex and its major component, casticin, on leukemia cell lines, HL-60 and U-937, were investigated by focusing on proliferation, induction of apoptosis and differentiation. Identification and quantitation by NMR spectroscopy showed that casticin accounted for approximate 1% weight of Vitex. Dose-dependent cytotoxicity of Vitex and casticin was observed in both cell lines, and HL-60 cells were more sensitive to the cytotoxicity of Vitex/casticin compared to U-937 cells. Furthermore, compared to unstimulated HL-60 cells, phorbol 12-myristate 13-acetate (PMA)- and 1,25-dihydroxyvitamin D₃ (VD₃)-differentiated HL-60 cells acquired resistance to Vitex/casticin based on the results from cell viability and apoptosis induction analysis. Since the HL-60 cell line is more immature than the U-937 cell line, these results suggested that the levels of cytotoxicity of Vitex/casticin were largely attributed to the degree of differentiation of leukemia cells; that is, cell lines with less differentiated phenotype were more susceptible than the differentiated ones. RT-PCR analysis demonstrated that PMA upregulated the expression of intercellular adhesion molecule-1 (ICAM-1) in HL-60 cells, and that anti-ICAM-1 monoclonal antibody not only abrogated PMA-induced aggregation and adhesion of the cells but also restored its sensitivity to Vitex. These results suggested that ICAM-1 plays a crucial role in the acquired resistance in PMA-differentiated HL-60 cells by contributing to cell adhesion. These findings provide fundamental insights into the clinical application of Vitex/casticin for hematopoietic malignancy.

Effects of mycoplasma contamination on phenotypic expression of mitochondrial mutants in human cells.

PubMed

Doersen, C J; Stanbridge, E J

1981-04-01

HeLa cells sensitive to the mitochondrial protein synthesis inhibitors erythromycin (ERY) and chloramphenicol (CAP) and HeLa variants resistant to the effects of these drugs were purposefully infected with drug-sensitive and -resistant mycoplasma strains. Mycoplasma hyorhinis and the ERY-resistant strain of Mycoplasma orale, MO-ERYr, did not influence the growth of HeLa and ERY-resistant ERY2301 cells in the presence or absence of ERY. M. hyorhinis also did not affect the growth of HeLa and CAP-resistant Cap-2 cells in the presence or absence of CAP. However, both HeLa and Cap-2 cells infected with the CAP-resistant strain of M. hyorhinis, MH-CAPr, were more sensitive to the cytotoxic effect of CAP. This may be due to the glucose dependence of the cells, which was compromised by the increased utilization of glucose by MH-CAPr in these infected cell cultures. In vitro protein synthesis by isolated mitochondria was significantly altered by mycoplasma infection of the various cell lines. A substantial number of mycoplasmas copurified with the mitochondria, resulting in up to a sevenfold increase in the incorporation of [3H]leucine into the trichloroacetic acid-insoluble material. More importantly, the apparent drug sensitivity or resistance of mitochondrial preparations from mycoplasma-infected cells reflected the drug sensitivity or resistance of the contaminating mycoplasmas. These results illustrate the hazards in interpreting mitochondrial protein synthesis data derived from mycoplasma-infected cell lines, particularly putative mitochondrially encoded mutants resistant to inhibitors of mitochondrial protein synthesis.

Mutation and repair in an ultraviolet-sensitive Chinese hamster ovary cell line

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

Wood, R.D.

1981-11-01

An ultraviolet (UV) light-sensitive mutant of Chinese hamster ovary cells (CHO) has been isolated and characterized with respect to a number of post-irradiation responses. The UV-sensitive mutant, termed 43-3B, has the same growth rate and chromosome number as the wild-type CHO-9. 43-3B is hypersensitive to the lethal effects of UV light (D/sub 0/ of 0.3 J/m/sup 2/ as compared to 3.2 J/m/sup 2/ for the wild-type). A marked UV-hypermutability is observed in 43-3B as compared to the wild-type, as measured with markers for induced resistance to 6-thioguanine, ouabain, and diphtheria toxin. A factor of 38 to 65 more mutations aremore » induced per unit fluence in 43-3B than in CHO-9. The UV-sensitive mutant is also sensitive to killing by simulated solar light, although the D/sub 0/ ratio is not as great as for germicidal UV. 43-3B exhibits only about 17% of the wild-type level of UV-stimulated DNA repair synthesis, as measured by autoradiography of G/sub 1/ phase cells. A much reduced ability to recover control rates of semiconservative DNA synthesis after UV irradiation was observed in the repair-deficient 43-3B cell line. Recovery of colony-forming ability between fractionated UV exposures was observed in the wild-type CHO-9, but little recovery was seen in 43-3B. The present investigation demonstrates that a sensitive/wild-type pair of CHO cell lines can be used in comparative studies to determine the involvement of repair in a wide range of post-irradiation phenomena.« less

BAD phosphorylation determines ovarian cancer chemo-sensitivity and patient survival

PubMed Central

Marchion, Douglas C.; Cottrill, Hope M.; Xiong, Yin; Chen, Ning; Bicaku, Elona; Fulp, William J.; Bansal, Nisha; Chon, Hye Sook; Stickles, Xiaomang B.; Kamath, Siddharth G.; Hakam, Ardeshir; Li, Lihua; Su, Dan; Moreno, Carolina; Judson, Patricia L.; Berchuck, Andrew; Wenham, Robert M.; Apte, Sachin M.; Gonzalez-Bosquet, Jesus; Bloom, Gregory C.; Eschrich, Steven A.; Sebti, Said; Chen, Dung-Tsa; Lancaster, Johnathan M.

2011-01-01

Purpose Despite initial sensitivity to chemotherapy, ovarian cancers (OVCA) often develop drug-resistance, which limits patient survival. Using specimens and/or genomic data from 289 patients and a panel of cancer cell lines, we explored genome-wide expression changes that underlie the evolution of OVCA chemo-resistance and characterized the BCL2 antagonist of cell death (BAD) apoptosis pathway as a determinant of chemo-sensitivity and patient survival. Experimental Design Serial OVCA cell cisplatin treatments were performed in parallel with measurements of genome-wide expression changes. Pathway analysis was performed on genes associated with increasing cisplatin-resistance (EC50). BAD-pathway expression and BAD-protein phosphorylation were evaluated in patient samples and cell lines as determinants of chemo-sensitivity and/or clinical outcome and as therapeutic targets. Results Induced in vitro OVCA cisplatin-resistance was associated with BAD-pathway expression (P < 0.001). In OVCA cell lines and primary specimens, BAD-protein phosphorylation was associated with platinum-resistance (n = 147, P < 0.0001) and also with overall patient survival (n = 134, P = 0.0007). Targeted modulation of BAD-phosphorylation levels influenced cisplatin sensitivity. A 47-gene BAD-pathway score was associated with in vitro phosphorylated-BAD levels and with survival in 142 patients with advanced-stage (III/IV) serous OVCA. Integration of BAD-phosphorylation or BAD-pathway score with OVCA surgical cytoreductive status was significantly associated with overall survival by log-rank test (P = 0.004 and <0.0001, respectively). Conclusion The BAD apoptosis pathway influences OVCA chemo-sensitivity and overall survival, likely via modulation of BAD-phosphorylation. The pathway has clinical relevance as a biomarker of therapeutic response, patient survival, and as a promising therapeutic target. PMID:21849418

Rituximab-induced inhibition of YY1 and Bcl-xL expression in Ramos non-Hodgkin's lymphoma cell line via inhibition of NF-kappa B activity: role of YY1 and Bcl-xL in Fas resistance and chemoresistance, respectively.

PubMed

Vega, Mario I; Jazirehi, Ali R; Huerta-Yepez, Sara; Bonavida, Benjamin

2005-08-15

Rituximab treatment of B non-Hodgkin's lymphoma (NHL) cell lines inhibits the constitutive NF-kappaB activity and results in the sensitization of tumor cells to both chemotherapy and Fas-induced apoptosis. Cells expressing dominant active IkappaB or treated with NF-kappaB-specific inhibitors were sensitive to both drugs and Fas agonist mAb (CH-11)-induced apoptosis. Down-regulation of Bcl-xL expression via inhibition of NF-kappaB activity correlated with chemosensitivity. The direct role of Bcl-xL in chemoresistance was demonstrated by the use of Bcl-xL-overexpressing Ramos cells, Ramos hemagglutinin (HA)-Bcl-x, which were not sensitized by rituximab to drug-induced apoptosis. However, inhibition of Bcl-xL in Ramos HA-Bcl-x resulted in sensitization to drug-induced apoptosis. The role of Bcl-xL expression in the regulation of Fas resistance was not apparent; Ramos HA-Bcl-x cells were as sensitive as the wild type to CH-11-induced apoptosis. Several lines of evidence support the direct role of the transcription repressor yin-yang 1 (YY1) in the regulation of resistance to CH-11-induced apoptosis. Inhibition of YY1 activity by either rituximab or the NO donor DETANONOate or after transfection with YY1 small interfering RNA resulted in up-regulation of Fas expression and sensitization to CH-11-induced apoptosis. These findings suggest two mechanisms underlying the chemosensitization and immunosensitization of B-NHL cells by rituximab via inhibition of NF-kappaB. The regulation of chemoresistance by NF-kappaB is mediated via Bcl-xL expression, whereas the regulation of Fas resistance by NF-kappaB is mediated via YY1 expression and activity. The potential clinical significance of these findings is discussed.

Novel poly (ADP-ribose) polymerase inhibitor, AZD2281, enhances radiosensitivity of both normoxic and hypoxic esophageal squamous cancer cells.

PubMed

Zhan, L; Qin, Q; Lu, J; Liu, J; Zhu, H; Yang, X; Zhang, C; Xu, L; Liu, Z; Cai, J; Ma, J; Dai, S; Tao, G; Cheng, H; Sun, X

2016-04-01

Radiotherapy plays an important role in the treatment of esophageal squamous cell carcinoma (ESCC). However, the outcome of radiotherapy in ESCC remains unsatisfactory because esophageal squamous cancer cells, particularly those under hypoxic condition, exhibit radioresistance. The aim of this study was to determine whether or not AZD2281, a potent poly (ADP-ribose) polymerase (PARP) inhibitor, could enhance the radiation sensitivity of two ESCC cell lines, namely ECA109 and TE13. The radiosensitizing effect of AZD2281 was evaluated on the basis of cell death, clonogenic survival and tumor xenograft progression. AZD2281 alone was slightly toxic to ESCC cell lines. Apoptosis was increased and clonogenic survival was decreased in both cell lines when AZD2281 was combined with ionizing radiation (IR) under normoxic condition. AZD2281 enhanced IR-induced apoptosis to a more significant level under chronic hypoxic condition (0.2% O(2), 48 hour) than under normoxic condition. AZD2281 also slightly enhanced clonogenic cell death under chronic hypoxic condition compared with that under normoxic condition. This result could be associated with increased radiation-induced DNA double-strand breaks (DSB), decreased DSB repair and increased apoptosis of ESCC cells. Furthermore, homologous recombination (HR) protein Rad51 expression and focus formation were decreased in ESCC cells exposed to moderate chronic hypoxic condition (0.2% O(2), 48 hour); this result indicated that chronic hypoxic ESCC cells were HR deficient, possibly causing contextual synthetic lethality with PARP inhibitor in radiation sensitization. AZD2281 was also a radiation sensitizer in ESCC tumor xenograft models. Hence, in vitro and in vivo findings provide evidence that AZD2281 potently sensitizes ESCC cells to X-ray irradiation. The selective cell killing of HR-defective hypoxic cells contributes to radiosensitization by PARP inhibitor in ESCC cells under hypoxic condition. © 2015 International Society for Diseases of the Esophagus.

RELATIVE POTENCY VALUES FOR POLYCHLORINATED DIBENZO-P-DIOXIN, DIBENZOFURAN AND BIPHENYL CONGENERS TO INDUCE CYTOCHROME P4501A MRNA IN A ZEBRAFISH LIVER CELL LINE (ZF-L)

EPA Science Inventory

Induction of cytochrome P450 (CYPIA) mRNA by polychlorinated dibenzo-p-dioxin (PCDD), polychlorinated dibenzofuran (PCDF) and polychlorinated biphenyl (PCB) congeners was measured in a zebrafish liver cell line (ZF-L). ZF-L cells were far less sensitive to PCDD, PCDF and PCB cong...

Poly(ADP-ribose) Polymerase Inhibitors Sensitize Cancer Cells to Death Receptor-mediated Apoptosis by Enhancing Death Receptor Expression*

PubMed Central

Meng, X. Wei; Koh, Brian D.; Zhang, Jin-San; Flatten, Karen S.; Schneider, Paula A.; Billadeau, Daniel D.; Hess, Allan D.; Smith, B. Douglas; Karp, Judith E.; Kaufmann, Scott H.

2014-01-01

Recombinant human tumor necrosis factor-α-related apoptosis inducing ligand (TRAIL), agonistic monoclonal antibodies to TRAIL receptors, and small molecule TRAIL receptor agonists are in various stages of preclinical and early phase clinical testing as potential anticancer drugs. Accordingly, there is substantial interest in understanding factors that affect sensitivity to these agents. In the present study we observed that the poly(ADP-ribose) polymerase (PARP) inhibitors olaparib and veliparib sensitize the myeloid leukemia cell lines ML-1 and K562, the ovarian cancer line PEO1, non-small cell lung cancer line A549, and a majority of clinical AML isolates, but not normal marrow, to TRAIL. Further analysis demonstrated that PARP inhibitor treatment results in activation of the FAS and TNFRSF10B (death receptor 5 (DR5)) promoters, increased Fas and DR5 mRNA, and elevated cell surface expression of these receptors in sensitized cells. Chromatin immunoprecipitation demonstrated enhanced binding of the transcription factor Sp1 to the TNFRSF10B promoter in the presence of PARP inhibitor. Knockdown of PARP1 or PARP2 (but not PARP3 and PARP4) not only increased expression of Fas and DR5 at the mRNA and protein level, but also recapitulated the sensitizing effects of the PARP inhibition. Conversely, Sp1 knockdown diminished the PARP inhibitor effects. In view of the fact that TRAIL is part of the armamentarium of natural killer cells, these observations identify a new facet of PARP inhibitor action while simultaneously providing the mechanistic underpinnings of a novel therapeutic combination that warrants further investigation. PMID:24895135

A genomic biomarker signature can predict skin sensitizers using a cell-based in vitro alternative to animal tests

PubMed Central

2011-01-01

Background Allergic contact dermatitis is an inflammatory skin disease that affects a significant proportion of the population. This disease is caused by an adverse immune response towards chemical haptens, and leads to a substantial economic burden for society. Current test of sensitizing chemicals rely on animal experimentation. New legislations on the registration and use of chemicals within pharmaceutical and cosmetic industries have stimulated significant research efforts to develop alternative, human cell-based assays for the prediction of sensitization. The aim is to replace animal experiments with in vitro tests displaying a higher predictive power. Results We have developed a novel cell-based assay for the prediction of sensitizing chemicals. By analyzing the transcriptome of the human cell line MUTZ-3 after 24 h stimulation, using 20 different sensitizing chemicals, 20 non-sensitizing chemicals and vehicle controls, we have identified a biomarker signature of 200 genes with potent discriminatory ability. Using a Support Vector Machine for supervised classification, the prediction performance of the assay revealed an area under the ROC curve of 0.98. In addition, categorizing the chemicals according to the LLNA assay, this gene signature could also predict sensitizing potency. The identified markers are involved in biological pathways with immunological relevant functions, which can shed light on the process of human sensitization. Conclusions A gene signature predicting sensitization, using a human cell line in vitro, has been identified. This simple and robust cell-based assay has the potential to completely replace or drastically reduce the utilization of test systems based on experimental animals. Being based on human biology, the assay is proposed to be more accurate for predicting sensitization in humans, than the traditional animal-based tests. PMID:21824406

AML sensitivity to YM155 is modulated through AKT and Mcl-1

PubMed Central

de Necochea-Campion, Rosalia; Diaz Osterman, Carlos J.; Hsu, Heng-Wei; Fan, Junjie; Mirshahidi, Saied; Wall, Nathan R.; Chen, Chien-Shing

2015-01-01

HL60 and U937 (acute myeloid leukemia (AML) cell lines) were assessed for sensitivity to YM155, and found to have distinct sensitive and resistant phenotypes, respectively. In HL60 cells, YM155 inhibition of growth proliferation was due to apoptosis which was measured by annexin V/PI staining. YM155 induced apoptosis through activation of intrinsic and extrinsic pathways that also culminated in caspase-3 activity and PARP cleavage. YM155 sensitivity was partially associated with this compound’s ability to downregulate survivin transcription since this was more pronounced in the HL60 cell line. However, marked differences were also observed in XIAP, Bcl-2, and Mcl-1L, and Mcl-1s. Furthermore, YM155 treatment completely inhibited production of total Akt protein in HL60, but not U937 cells. Importantly, Akt activity (pAkt-Ser473) levels were maintained in YM155 treated U937 cells which may help stabilize other anti-apoptotic proteins. Combination treatments with an Akt inhibitor, MK-2206, reduced levels of pAkt-Ser473 in U937 cells and synergistically sensitized them to YM155 cytotoxicity. Collectively our results indicate that Akt signaling may be an important factor mediating YM155 response in AML, and combinatorial therapies with Akt inhibitors could improve treatment efficacy in YM155-resistant cells. PMID:26118775

Establishment and characterization of 13 cell lines from a green turtle (Chelonia mydas) with fibropapillomas

USGS Publications Warehouse

Lu, Y.; Nerurkar, V.R.; Aguirre, A.A.; Work, Thierry M.; Balazs, G.H.; Yanagihara, R.

1999-01-01

Thirteen cell lines were established and characterized from brain, kidney, lung, spleen, heart, liver, gall bladder, urinary bladder, pancreas, testis, skin, and periorbital and tumor tissues of an immature male green turtle (Chelonia mydas) with fibropapillomas. Cell lines were optimally maintained at 30A? C in RPMI 1640 medium supplemented with 10% fetal bovine serum. Propagation of the turtle cell lines was serum dependent, and plating efficiencies ranged from 13 to 37%. The cell lines, which have been subcultivated more than 20 times, had a doubling time of approximately 30 to 36 h. When tested for their sensitivity to several fish viruses, most of the cell lines were susceptible to a rhabdovirus, spring viremia carp virus, but refractory to channel catfish virus (a herpesvirus), infectious pancreatic necrosis virus (a birnavirus), and two other fish rhabdoviruses, infectious hematopoietic necrosis virus and viral hemorrhagic septicemia virus. During in vitro subcultivation, tumor-like cell aggregates appeared in cell lines derived from lungs, testis, and periorbital and tumor tissues, and small, naked intranuclear virus particles were detected by thin-section electron microscopy. These cell lines are currently being used in attempts to isolate the putative etiologic virus of green turtle fibropapilloma.

Cytotoxic human peripheral blood-derived γδT cells kill glioblastoma cell lines: implications for cell-based immunotherapy for patients with glioblastoma.

PubMed

Nakazawa, Tsutomu; Nakamura, Mitsutoshi; Park, Young Soo; Motoyama, Yasushi; Hironaka, Yasuo; Nishimura, Fumihiko; Nakagawa, Ichiro; Yamada, Shuichi; Matsuda, Ryosuke; Tamura, Kentaro; Sugimoto, Tadashi; Takeshima, Yasuhiro; Marutani, Akiko; Tsujimura, Takahiro; Ouji, Noriko; Ouji, Yukiteru; Yoshikawa, Masahide; Nakase, Hiroyuki

2014-01-01

Glioblastoma (GBM) is a highly aggressive brain tumor for which novel therapeutic approaches, such as immunotherapy, are urgently needed. Zoledronate (ZOL), an inhibitor of osteoclastic activity, is known to stimulate peripheral blood-derived γδT cells and sensitize tumors to γδT cell-mediated killing. To investigate the feasibility of γδT cell-based immunotherapy for patients with GBM, we focused on the killing of GBM cell lines by γδT cells and the molecular mechanisms involved in these cell-cell interactions. Peripheral blood mononuclear cells were expanded in ZOL and interleukin (IL)-2 for 14 days, and γδT cells were enriched in the expanded cells by the immunomagnetic depletion of αβT cells. Gliomas are resistant to NK cells but susceptible to lymphokine-activated killer cells and some cytotoxic T lymphocytes. When the γδT cell-mediated killing of three GBM cell lines (U87MG, U138MG and A172 cells) and an NK-sensitive leukemia cell line (K562 cells) were tested, 32% U87MG, 15% U138MG, 1% A172, and 50% K562 cells were killed at an effector:target ratio of 5:1. The γδT cell-mediated killing of all three GBM cell lines was significantly enhanced by ZOL and this ZOL-enhanced killing was blocked by an anti-T cell receptor (TcR) antibody. These results indicated that TcR γδ is crucial for the recognition of ZOL-treated GBM cells by γδT cells. Since the low level killing of GBM cells by the γδT cells was enhanced by ZOL, γδT cell-targeting therapy in combination with ZOL treatment could be effective for patients with GBM.

Differential Sensitivity of Bat Cells to Infection by Enveloped RNA Viruses: Coronaviruses, Paramyxoviruses, Filoviruses, and Influenza Viruses

PubMed Central

Hoffmann, Markus; Müller, Marcel Alexander; Drexler, Jan Felix; Glende, Jörg; Erdt, Meike; Gützkow, Tim; Losemann, Christoph; Binger, Tabea; Deng, Hongkui; Schwegmann-Weßels, Christel; Esser, Karl-Heinz; Drosten, Christian; Herrler, Georg

2013-01-01

Bats (Chiroptera) host major human pathogenic viruses including corona-, paramyxo, rhabdo- and filoviruses. We analyzed six different cell lines from either Yinpterochiroptera (including African flying foxes and a rhinolophid bat) or Yangochiroptera (genera Carollia and Tadarida) for susceptibility to infection by different enveloped RNA viruses. None of the cells were sensitive to infection by transmissible gastroenteritis virus (TGEV), a porcine coronavirus, or to infection mediated by the Spike (S) protein of SARS-coronavirus (SARS-CoV) incorporated into pseudotypes based on vesicular stomatitis virus (VSV). The resistance to infection was overcome if cells were transfected to express the respective cellular receptor, porcine aminopeptidase N for TGEV or angiotensin-converting enzyme 2 for SARS-CoV. VSV pseudotypes containing the S proteins of two bat SARS-related CoV (Bg08 and Rp3) were unable to infect any of the six tested bat cell lines. By contrast, viral pseudotypes containing the surface protein GP of Marburg virus from the family Filoviridae infected all six cell lines though at different efficiency. Notably, all cells were sensitive to infection by two paramyxoviruses (Sendai virus and bovine respiratory syncytial virus) and three influenza viruses from different subtypes. These results indicate that bat cells are more resistant to infection by coronaviruses than to infection by paramyxoviruses, filoviruses and influenza viruses. Furthermore, these results show a receptor-dependent restriction of the infection of bat cells by CoV. The implications for the isolation of coronaviruses from bats are discussed. PMID:24023659

PIK3CA dependence and sensitivity to therapeutic targeting in urothelial carcinoma.

PubMed

Ross, R L; McPherson, H R; Kettlewell, L; Shnyder, S D; Hurst, C D; Alder, O; Knowles, M A

2016-07-28

Many urothelial carcinomas (UC) contain activating PIK3CA mutations. In telomerase-immortalized normal urothelial cells (TERT-NHUC), ectopic expression of mutant PIK3CA induces PI3K pathway activation, cell proliferation and cell migration. However, it is not clear whether advanced UC tumors are PIK3CA-dependent and whether PI3K pathway inhibition is a good therapeutic option in such cases. We used retrovirus-mediated delivery of shRNA to knock down mutant PIK3CA in UC cell lines and assessed effects on pathway activation, cell proliferation, migration and tumorigenicity. The effect of the class I PI3K inhibitor GDC-0941 was assessed in a panel of UC cell lines with a range of known molecular alterations in the PI3K pathway. Specific knockdown of PIK3CA inhibited proliferation, migration, anchorage-independent growth and in vivo tumor growth of cells with PIK3CA mutations. Sensitivity to GDC-0941 was dependent on hotspot PIK3CA mutation status. Cells with rare PIK3CA mutations and co-occurring TSC1 or PTEN mutations were less sensitive. Furthermore, downstream PI3K pathway alterations in TSC1 or PTEN or co-occurring AKT1 and RAS gene mutations were associated with GDC-0941 resistance. Mutant PIK3CA is a potent oncogenic driver in many UC cell lines and may represent a valuable therapeutic target in advanced bladder cancer.

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.

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.

Phenylbutyrate interferes with the Fanconi anemia and BRCA pathway and sensitizes head and neck cancer cells to cisplatin

PubMed Central

Burkitt, Kyunghee; Ljungman, Mats

2008-01-01

Background Cisplatin has been widely used to treat head and neck cancer. One of the clinical limitations with this treatment, however, is that tumors that are initially responsive to cisplatin later acquire resistance. We have recently shown that a subset of head and neck cancer cell lines has a defective Fanconi anemia DNA damage response pathway and this defect correlates to cisplatin sensitivity. We have also shown that the histone deacetylase inhibitor phenylbutyrate sensitize human cells to cisplatin. In this study we explored whether phenylbutyrate may sensitize head and neck cancer cells by interfering with the Fanconi anemia pathway. Results We found that the phenylbutyrate sensitizes head and neck cancer cell lines to cisplatin. This sensitization by phenylbutyrate correlated to a significant decrease in the formation of cisplatin-induced FANCD2 nuclear foci, which is a functional read out of the Fanconi anemia and BRCA (FA/BRCA) pathway. This abrogation of the FA/BRCA pathway by phenylbutyrate was not due to loss of FANCD2 monoubiquitylation but rather correlated to a phenylbutyrate-mediated reduction in the expression of the BRCA1 protein. Furthermore, we found that cancer cells defective in the FA pathway were also sensitized to cisplatin by phenylbutyrate suggesting that phenylbutyrate targets additional pathways. Conclusion The results from this study suggest that phenylbutyrate may have therapeutic utility as a cisplatin sensitizer in head and neck cancer by inhibiting the FA/BRCA pathway through the down regulation of BRCA1 as well as by an FA/BRCA-independent mechanism. PMID:18325101

Cytokine receptor signaling is required for the survival of ALK− anaplastic large cell lymphoma, even in the presence of JAK1/STAT3 mutations

PubMed Central

Chen, Jing; Zhang, Yong; Petrus, Michael N.; Xiao, Wenming; Nicolae, Alina; Raffeld, Mark; Pittaluga, Stefania; Bamford, Richard N.; Nakagawa, Masao; Ouyang, Sunny Tianyi; Epstein, Alan L.; Kadin, Marshall E.; Del Mistro, Annarose; Woessner, Richard; Jaffe, Elaine S.; Waldmann, Thomas A.

2017-01-01

Activating Janus kinase (JAK) and signal transducer and activator of transcription (STAT) mutations have been discovered in many T-cell malignancies, including anaplastic lymphoma kinase (ALK)− anaplastic large cell lymphomas (ALCLs). However, such mutations occur in a minority of patients. To investigate the clinical application of targeting JAK for ALK− ALCL, we treated ALK− cell lines of various histological origins with JAK inhibitors. Interestingly, most exogenous cytokine-independent cell lines responded to JAK inhibition regardless of JAK mutation status. JAK inhibitor sensitivity correlated with the STAT3 phosphorylation status of tumor cells. Using retroviral shRNA knockdown, we have demonstrated that these JAK inhibitor-sensitive cells are dependent on both JAK1 and STAT3 for survival. JAK1 and STAT3 gain-of-function mutations were found in some, but not all, JAK inhibitor-sensitive cells. Moreover, the mutations alone cannot explain the JAK1/STAT3 dependency, given that wild-type JAK1 or STAT3 was sufficient to promote cell survival in the cells that had either JAK1or STAT3 mutations. To investigate whether other mechanisms were involved, we knocked down upstream receptors GP130 or IL-2Rγ. Knockdown of GP130 or IL-2Rγ induced cell death in selected JAK inhibitor-sensitive cells. High expression levels of cytokines, including IL-6, were demonstrated in cell lines as well as in primary ALK− ALCL tumors. Finally, ruxolitinib, a JAK1/2 inhibitor, was effective in vivo in a xenograft ALK− ALCL model. Our data suggest that cytokine receptor signaling is required for tumor cell survival in diverse forms of ALK− ALCL, even in the presence of JAK1/STAT3 mutations. Therefore, JAK inhibitor therapy might benefit patients with ALK− ALCL who are phosphorylated STAT3+. PMID:28356514

[Hypoxia responsive element regulated herpes simplex virus-thymidine kinase system enhances killing effect of gancyclovir on Ewing's sarcoma cell line under hypoxic condition].

PubMed

Si, Ying-jian; Guang, Li-xia; Yuan, Fa-huan; Zhang, Ke-bin

2006-08-01

To find out a possible approach to improve the effectiveness of radiotherapy and chemotherapy for Ewing's sarcoma by constructing a eukaryotic expression vector expressing herpes simplex virus-thymidine kinase (HSV-TK) regulated by hypoxia responsive element (HRE) under hypoxia and to evaluate the effects of this HRE regulated HSV-TK system on killing effect of gancyclovir (GCV) on Ewing's sarcoma cell line SK-ES under hypoxic condition. The HRE was synthesized according to the literature and cloned into the enhancer site of pIRES(2)-EGFP vector to obtain the pHRE recombinant plasmid. The HSV-TK was amplified by PCR and cloned into the multiple clone site of pIRES(2)-EGFP and pHRE to obtain pTK and pHRE-TK recombinant plasmid. The human Ewing's sarcoma cell line SK-ES was transfected by pTK or pHRE-TK recombinant plasmid with liposome and then was exposed to normoxic (21% oxygen) or hypoxic (3% oxygen) condition. The expression of enhanced green fluorescent protein (EGFP) was monitored by fluorescent microscopy. The sensitivity of human Ewing's sarcoma cell line SK-ES transfected with pTK or pHRE-TK recombinant plasmid to the anti-tumour drug GCV was determined with the method of tetrazolium (MTT) after treating with GCV for five days. (1) The result of sequencing showed that the recombinant plasmid pHRE contained HRE, and that the recombinant plasmid pTK and pHRE-TK contained HSV-TK gene in the sense direction. (2) Comparison of fluorescent optical density (FOD) showed that (1) the EGFP FOD value of pHRE and pHRE-TK group cells exposed to hypoxia was significantly higher than those exposed to normoxia (P < 0.01); (2) when the cells were exposed to hypoxia, the EGFP FOD value of pHRE and pHRE-TK group cells was significantly higher than that of pTK and empty vector group (P < 0.01); (3) there was no significant difference among the four groups of cells when they were exposed to normoxia (P > 0.05). (3) Comparison of the sensitivity of four groups of cells to GCV showed that (1) the cells in pHRE-TK and pTK groups were much more sensitive to GCV than the cells in pHRE group under hypoxia condition (P < 0.01), the higher the GCV concentration, the greater the difference; (2) the cells of pHRE-TK group were more sensitive to GCV than those in pTK group under hypoxic condition (P < 0.01), but was almost equally sensitive under normoxic condition (P > 0.05); (3) the pHRE-TK group cells had higher sensitivity to GCV under hypoxia than normoxia (P < 0.01) while the pTK group cells had almost the same sensitivity to GCV under hypoxia and normoxia (P > 0.05). (1) The eukaryotic expression vector expressing herpes simplex virus-thymidine kinase (HSV-TK) regulated by hypoxia responsive element (HRE) under hypoxia was constructed successfully. (2) HRE could up-regulate expression of EGFP by SK-ES cells under hypoxia condition. (3) HRE could enhance the killing effect of HSV-TK/GCV system on human Ewing's sarcoma cell line SK-ES under hypoxic condition.

Prioritization of anticancer drugs against a cancer using genomic features of cancer cells: A step towards personalized medicine

PubMed Central

Gupta, Sudheer; Chaudhary, Kumardeep; Kumar, Rahul; Gautam, Ankur; Nanda, Jagpreet Singh; Dhanda, Sandeep Kumar; Brahmachari, Samir Kumar; Raghava, Gajendra P. S.

2016-01-01

In this study, we investigated drug profile of 24 anticancer drugs tested against a large number of cell lines in order to understand the relation between drug resistance and altered genomic features of a cancer cell line. We detected frequent mutations, high expression and high copy number variations of certain genes in both drug resistant cell lines and sensitive cell lines. It was observed that a few drugs, like Panobinostat, are effective against almost all types of cell lines, whereas certain drugs are effective against only a limited type of cell lines. Tissue-specific preference of drugs was also seen where a drug is more effective against cell lines belonging to a specific tissue. Genomic features based models have been developed for each anticancer drug and achieved average correlation between predicted and actual growth inhibition of cell lines in the range of 0.43 to 0.78. We hope, our study will throw light in the field of personalized medicine, particularly in designing patient-specific anticancer drugs. In order to serve the scientific community, a webserver, CancerDP, has been developed for predicting priority/potency of an anticancer drug against a cancer cell line using its genomic features (http://crdd.osdd.net/raghava/cancerdp/). PMID:27030518

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.

Intracellular Doppler Signatures of Platinum Sensitivity Captured by Biodynamic Profiling in Ovarian Xenografts

NASA Astrophysics Data System (ADS)

Merrill, Daniel; An, Ran; Sun, Hao; Yakubov, Bakhtiyor; Matei, Daniela; Turek, John; Nolte, David

2016-01-01

Three-dimensional (3D) tissue cultures are replacing conventional two-dimensional (2D) cultures for applications in cancer drug development. However, direct comparisons of in vitro 3D models relative to in vivo models derived from the same cell lines have not been reported because of the lack of sensitive optical probes that can extract high-content information from deep inside living tissue. Here we report the use of biodynamic imaging (BDI) to measure response to platinum in 3D living tissue. BDI combines low-coherence digital holography with intracellular Doppler spectroscopy to study tumor drug response. Human ovarian cancer cell lines were grown either in vitro as 3D multicellular monoculture spheroids or as xenografts in nude mice. Fragments of xenografts grown in vivo in nude mice from a platinum-sensitive human ovarian cell line showed rapid and dramatic signatures of induced cell death when exposed to platinum ex vivo, while the corresponding 3D multicellular spheroids grown in vitro showed negligible response. The differences in drug response between in vivo and in vitro growth have important implications for predicting chemotherapeutic response using tumor biopsies from patients or patient-derived xenografts.

IGF1R signalling in testicular germ cell tumour cells impacts on cell survival and acquired cisplatin resistance.

PubMed

Selfe, Joanna; Goddard, Neil C; McIntyre, Alan; Taylor, Kathryn R; Renshaw, Jane; Popov, Sergey D; Thway, Khin; Summersgill, Brenda; Huddart, Robert A; Gilbert, Duncan C; Shipley, Janet M

2018-02-01

Testicular germ cell tumours (TGCTs) are the most frequent malignancy and cause of death from solid tumours in the 20- to 40-year age group. Although most cases show sensitivity to cis-platinum-based chemotherapy, this is associated with long-term toxicities and chemo-resistance. Roles for receptor tyrosine kinases other than KIT are largely unknown in TGCT. We therefore conducted a phosphoproteomic screen and identified the insulin growth factor receptor-1 (IGF1R) as both highly expressed and activated in TGCT cell lines representing the nonseminomatous subtype. IGF1R was also frequently expressed in tumour samples from patients with nonseminomas. Functional analysis of cell line models showed that long-term shRNA-mediated IGF1R silencing leads to apoptosis and complete ablation of nonseminoma cells with active IGF1R signalling. Cell lines with high levels of IGF1R activity also showed reduced AKT signalling in response to decreased IGF1R expression as well as sensitivity to the small-molecule IGF1R inhibitor NVP-AEW541. These results were in contrast to those in the seminoma cell line TCAM2 that lacked IGF1R signalling via AKT and was one of the two cell lines least sensitive to the IGF1R inhibitor. The dependence on IGF1R activity in the majority of nonseminomas parallels the known role of IGF signalling in the proliferation, migration, and survival of primordial germ cells, the putative cell of origin for TGCT. Upregulation of IGF1R expression and signalling was also found to contribute to acquired cisplatin resistance in an in vitro nonseminoma model, providing a rationale for targeting IGF1R in cisplatin-resistant disease. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

The taccalonolides: microtubule stabilizers that circumvent clinically relevant taxane resistance mechanisms.

PubMed

Risinger, April L; Jackson, Evelyn M; Polin, Lisa A; Helms, Gregory L; LeBoeuf, Desiree A; Joe, Patrick A; Hopper-Borge, Elizabeth; Ludueña, Richard F; Kruh, Gary D; Mooberry, Susan L

2008-11-01

The taccalonolides are a class of structurally and mechanistically distinct microtubule-stabilizing agents isolated from Tacca chantrieri. A crucial feature of the taxane family of microtubule stabilizers is their susceptibility to cellular resistance mechanisms including overexpression of P-glycoprotein (Pgp), multidrug resistance protein 7 (MRP7), and the betaIII isotype of tubulin. The ability of four taccalonolides, A, E, B, and N, to circumvent these multidrug resistance mechanisms was studied. Taccalonolides A, E, B, and N were effective in vitro against cell lines that overexpress Pgp and MRP7. In addition, taccalonolides A and E were highly active in vivo against a doxorubicin- and paclitaxel-resistant Pgp-expressing tumor, Mam17/ADR. An isogenic HeLa-derived cell line that expresses the betaIII isotype of tubulin was generated to evaluate the effect of betaIII-tubulin on drug sensitivity. When compared with parental HeLa cells, the betaIII-tubulin-overexpressing cell line was less sensitive to paclitaxel, docetaxel, epothilone B, and vinblastine. In striking contrast, the betaIII-tubulin-overexpressing cell line showed greater sensitivity to all four taccalonolides. These data cumulatively suggest that the taccalonolides have advantages over the taxanes in their ability to circumvent multiple drug resistance mechanisms. The ability of the taccalonolides to overcome clinically relevant mechanisms of drug resistance in vitro and in vivo confirms that the taccalonolides represent a valuable addition to the family of microtubule-stabilizing compounds with clinical potential.

[Biological behavior of hypopharyngeal carcinoma].

PubMed

Zhou, L X

1997-01-01

Hypopharyngeal squamous cell carcinomas (HPC) has an extremely poor prognosis. Characteristics of cell lines of head and neck squamous cell carcinomas including HPC were studied by various methods, e.g., chemosensitivity test and the immunohistochemistry staining method, to determine whether this poor prognosis is due to the biological behavior of this cancer. An HPC cell line was found to be resistant to anti tumor drugs, i.e., PEP, MTX and CPM and moderately sensitive to CDDP, 5-FU and ADM. Thermoresistance to hyperthermatic treatment and weak expression of ICAM-1 on the HPC cell line were observed. DNA synthesis by the HPC cell line was induced by stimulation with a low concentration of EGF and the amount of EGFR on these HPC cells was very high. In addition, cyclinD1 overexpression was found in the HPC cell line. Based on the above findings, further analysis of hypopharyngeal carcinoma cells and the development of a new treatment modality to control tumor growth and metastatic factors influencing the poor outcome are necessary to improve the prognosis of this cancer.

mTOR is a Promising Therapeutic Target Both in Cisplatin-Sensitive and Cisplatin-Resistant Clear Cell Carcinoma of the Ovary

PubMed Central

Mabuchi, Seiji; Kawase, Chiaki; Altomare, Deborah A.; Morishige, Kenichirou; Sawada, Kenjiro; Hayashi, Masami; Tsujimoto, Masahiko; Yamoto, Mareo; Klein-Szanto, Andres J.; Schilder, Russell J.; Ohmichi, Masahide; Testa, Joseph R.; Kimura, Tadashi

2009-01-01

Translational Relevance Clear cell carcinoma (CCC) of the ovary is a distinctive subtype of epithelial ovarian cancer associated with a poorer sensitivity to platinum-based chemotherapy and a worse prognosis than the more common serous adenocarcinoma (SAC). To improve survival, the development of new treatment strategies that target CCC more effectively is necessary. Our results show that mTOR is more frequently activated in CCCs than in SACs. Our data have relevance for the design of future clinical studies of first-line treatment for patients with CCC of the ovary. Moreover, the finding of increased expression of phospho-mTOR and greater sensitivity to RAD001 in cisplatin-resistant CCC cells than in cisplatin-sensitive cells suggests a novel treatment option for patients with recurrent disease after cisplatin-based first-line chemotherapy. Purpose mTOR (mammalian target of rapamycin) plays a central role in cell proliferation and is regarded as a promising target in cancer therapy including for ovarian cancer. This study aims to examine the role of mTOR as a therapeutic target in clear cell carcinoma (CCC) of the ovary which is regarded as aggressive, chemo-resistant histological subtype. Experimental Design Using tissue microarrays of 98 primary ovarian cancers (52 clear cell carcinomas and 46 serous adenocarcinomas), the expression of phospho-mTOR was assessed by immunohistochemistry. Then, the growth-inhibitory effect of mTOR inhibition by RAD001 (everolimus) was examined using 2 pairs of cisplatin-sensitive parental (RMG1 and KOC7C) and cisplatin-resistant human CCC cell lines (RMG1-CR and KOC7C-CR) both in vitro and in vivo. Results Immunohistochemical analysis demonstrated mTOR was more frequently activated in CCCs than in serous adenocarcinomas (86.6% vs 50%). Treatment with RAD001 markedly inhibited the growth of both RMG1 and KOC7C cells both in vitro and in vivo. Increased expression of phospho-mTOR was observed in cisplatin-resistant RMG1-CR and KOC7C-CR cells, compared to the respective parental cells. This increased expression of phospho-mTOR in cisplatin-resistant cells was associated with increased activation of AKT. RMG1-CR and KOC7C-CR cells showed greater sensitivity to RAD001 than parental RMG1 and KOC7C cells, respectively, in vitro and in vivo. Conclusion mTOR is frequently activated in CCC and can be a promising therapeutic target in the management of CCC. Moreover, mTOR inhibition by RAD001 may be efficacious as a second-line treatment of recurrent disease in patients previously treated with cisplatin. PMID:19690197

Prediction of cancer cell sensitivity to natural products based on genomic and chemical properties.

PubMed

Yue, Zhenyu; Zhang, Wenna; Lu, Yongming; Yang, Qiaoyue; Ding, Qiuying; Xia, Junfeng; Chen, Yan

2015-01-01

Natural products play a significant role in cancer chemotherapy. They are likely to provide many lead structures, which can be used as templates for the construction of novel drugs with enhanced antitumor activity. Traditional research approaches studied structure-activity relationship of natural products and obtained key structural properties, such as chemical bond or group, with the purpose of ascertaining their effect on a single cell line or a single tissue type. Here, for the first time, we develop a machine learning method to comprehensively predict natural products responses against a panel of cancer cell lines based on both the gene expression and the chemical properties of natural products. The results on two datasets, training set and independent test set, show that this proposed method yields significantly better prediction accuracy. In addition, we also demonstrate the predictive power of our proposed method by modeling the cancer cell sensitivity to two natural products, Curcumin and Resveratrol, which indicate that our method can effectively predict the response of cancer cell lines to these two natural products. Taken together, the method will facilitate the identification of natural products as cancer therapies and the development of precision medicine by linking the features of patient genomes to natural product sensitivity.

Genetic load makes cancer cells more sensitive to common drugs: evidence from Cancer Cell Line Encyclopedia.

PubMed

Pavel, Ana B; Korolev, Kirill S

2017-05-16

Genetic alterations initiate tumors and enable the evolution of drug resistance. The pro-cancer view of mutations is however incomplete, and several studies show that mutational load can reduce tumor fitness. Given its negative effect, genetic load should make tumors more sensitive to anticancer drugs. Here, we test this hypothesis across all major types of cancer from the Cancer Cell Line Encyclopedia, which provides genetic and expression data of 496 cell lines together with their response to 24 common anticancer drugs. We found that the efficacy of 9 out of 24 drugs showed significant association with genetic load in a pan-cancer analysis. The associations for some tissue-drug combinations were remarkably strong, with genetic load explaining up to 83% of the variance in the drug response. Overall, the role of genetic load depended on both the drug and the tissue type with 10 tissues being particularly vulnerable to genetic load. We also identified changes in gene expression associated with increased genetic load, which included cell-cycle checkpoints, DNA damage and apoptosis. Our results show that genetic load is an important component of tumor fitness and can predict drug sensitivity. Beyond being a biomarker, genetic load might be a new, unexplored vulnerability of cancer.

Reduced mtDNA copy number increases the sensitivity of tumor cells to chemotherapeutic drugs.

PubMed

Mei, H; Sun, S; Bai, Y; Chen, Y; Chai, R; Li, H

2015-04-02

Many cancer drugs are toxic to cells by activating apoptotic pathways. Previous studies have shown that mitochondria have key roles in apoptosis in mammalian cells, but the role of mitochondrial DNA (mtDNA) copy number variation in the pathogenesis of tumor cell apoptosis remains largely unknown. We used the HEp-2, HNE2, and A549 tumor cell lines to explore the relationship between mtDNA copy number variation and cell apoptosis. We first induced apoptosis in three tumor cell lines and one normal adult human skin fibroblast cell line (HSF) with cisplatin (DDP) or doxorubicin (DOX) treatment and found that the mtDNA copy number significantly increased in apoptotic tumor cells, but not in HSF cells. We then downregulated the mtDNA copy number by transfection with shRNA-TFAM plasmids or treatment with ethidium bromide and found that the sensitivity of tumor cells to DDP or DOX was significantly increased. Furthermore, we observed that levels of reactive oxygen species (ROS) increased significantly in tumor cells with lower mtDNA copy numbers, and this might be related to a low level of antioxidant gene expression. Finally, we rescued the increase of ROS in tumor cells with lipoic acid or N-acetyl-L-cysteine and found that the apoptosis rate decreased. Our studies suggest that the increase of mtDNA copy number is a self-protective mechanism of tumor cells to prevent apoptosis and that reduced mtDNA copy number increases ROS levels in tumor cells, increases the tumor cells' sensitivity to chemotherapeutic drugs, and increases the rate of apoptosis. This research provides evidence that mtDNA copy number variation might be a promising new therapeutic target for the clinical treatment of tumors.

Proteomic differential display analysis for TS-1-resistant and -sensitive pancreatic cancer cells using two-dimensional gel electrophoresis and mass spectrometry.

PubMed

Yoshida, Kanako; Kuramitsu, Yasuhiro; Murakami, Kohei; Ryozawa, Shomei; Taba, Kumiko; Kaino, Seiji; Zhang, Xiulian; Sakaida, Isao; Nakamura, Kazuyuki

2011-06-01

TS-1 is an oral anticancer agent containing two biochemical modulators for 5-fluorouracil (5-FU) and tegafur (FT), a metabolically activated prodrug of 5-FU. TS-1 has been recognized as an effective anticancer drug using standard therapies for patients with advanced pancreatic cancer along with gemcitabine. However, a high level of inherent and acquired tumor resistance to TS-1 induces difficulty in the treatment. To identify proteins linked to the TS-1-resistance of pancreatic cancer, we profiled protein expression levels in samples of TS-1-resistant and -sensitive pancreatic cancer cell lines by using two-dimensional gel electrophoresis (2-DE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The cytotoxicity of a 5-FU/5-chloro-2,4-dihydroxypyridine (CDHP) combination towards pancreatic cancer cell lines was evaluated by MTS assay. Panc-1, BxPC-3, MiaPaCa-2 and PK59 showed high sensitivity to the 5-FU/CDHP combination (TS-1-sensitive), whereas PK45p and KLM-1 were much less sensitive (TS-1-resistant). Proteomic analysis showed that eleven spots, including T-complex protein 1 subunit beta, ribonuclease inhibitor, elongation factor 1-delta, peroxiredoxin-2 and superoxide dismutase (Cu-Zn), appeared to be down-regulated, and 29 spots, including hypoxia up-regulated protein 1, lamin-A/C, endoplasmin, fascin and annexin A1, appeared to be up-regulated in TS-1-resistant cells compared with -sensitive cells. These results suggest that the identified proteins showing different expression between TS-1-sensitive and -resistant pancreatic cancer cells possibly relate to TS-1-sensitivity. These findings could be useful to overcome the TS-1-resistance of pancreatic cancer cells.

Intratumoral genetic heterogeneity in metastatic melanoma is accompanied by variation in malignant behaviors

PubMed Central

2013-01-01

Background Intratumoral heterogeneity is a major obstacle for the treatment of cancer, as the presence of even minor populations that are insensitive to therapy can lead to disease relapse. Increased clonal diversity has been correlated with a poor prognosis for cancer patients, and we therefore examined genetic, transcriptional, and functional diversity in metastatic melanoma. Methods Amplicon sequencing and SNP microarrays were used to profile somatic mutations and DNA copy number changes in multiple regions from metastatic lesions. Clonal genetic and transcriptional heterogeneity was also assessed in single cell clones from early passage cell lines, which were then subjected to clonogenicity and drug sensitivity assays. Results MAPK pathway and tumor suppressor mutations were identified in all regions of the melanoma metastases analyzed. In contrast, we identified copy number abnormalities present in only some regions in addition to homogeneously present changes, suggesting ongoing genetic evolution following metastatic spread. Copy number heterogeneity from a tumor was represented in matched cell line clones, which also varied in their clonogenicity and drug sensitivity. Minor clones were identified based on dissimilarity to the parental cell line, and these clones were the most clonogenic and least sensitive to drugs. Finally, treatment of a polyclonal cell line with paclitaxel to enrich for drug-resistant cells resulted in the adoption of a gene expression profile with features of one of the minor clones, supporting the idea that these populations can mediate disease relapse. Conclusion Our results support the hypothesis that minor clones might have major consequences for patient outcomes in melanoma. PMID:24119551

Inhibition of WEE1 kinase and cell cycle checkpoint activation sensitizes head and neck cancers to natural killer cell therapies.

PubMed

Friedman, Jay; Morisada, Megan; Sun, Lillian; Moore, Ellen C; Padget, Michelle; Hodge, James W; Schlom, Jeffrey; Gameiro, Sofia R; Allen, Clint T

2018-06-21

Natural killer (NK) cells recognize and lyse target tumor cells in an MHC-unrestricted fashion and complement antigen- and MHC-restricted killing by T-lymphocytes. NK cells and T-lymphocytes mediate early killing of targets through a common granzyme B-dependent mechanism. Tumor cell resistance to granzyme B and how this alters NK cell killing is not clearly defined. Tumor cell sensitivity to cultured murine KIL and human high affinity NK (haNK) cells in the presence or absence of AZD1775, a small molecule inhibitor of WEE1 kinase, was assessed via real time impedance analysis. Mechanisms of enhanced sensitivity to NK lysis were determined and in vivo validation via adoptive transfer of KIL cells into syngeneic mice was performed. Cultured murine KIL cells lyse murine oral cancer 2 (MOC2) cell targets more efficiently than freshly isolated peripheral murine NK cells. MOC2 sensitivity to granzyme B-dependent KIL cell lysis was enhanced by inhibition of WEE1 kinase, reversing G2/M cell cycle checkpoint activation and resulting in enhanced DNA damage and apoptosis. Treatment of MOC2 tumor-bearing wild-type C57BL/6 mice with AZD1775 and adoptively transferred KIL cells resulted in enhanced tumor growth control and survival over controls or either treatment alone. Validating these findings in human models, WEE1 kinase inhibition sensitized two human head and neck cancer cell lines to direct lysis by haNK cells. Further, WEE1 kinase inhibition sensitized these cell lines to antibody-dependent cell-mediated cytotoxicity when combined with the anti-PD-L1 IgG1 mAb Avelumab. Tumor cell resistance to granzyme B-induced cell death can be reversed through inhibition of WEE1 kinase as AZD1775 sensitized both murine and human head and neck cancer cells to NK lysis. These data provide the pre-clinical rationale for the combination of small molecules that reverse cell cycle checkpoint activation and NK cellular therapies.

Sensitivity of C6 Glioma Cells Carrying the Human Poliovirus Receptor to Oncolytic Polioviruses.

PubMed

Sosnovtseva, A O; Lipatova, A V; Grinenko, N F; Baklaushev, V P; Chumakov, P M; Chekhonin, V P

2016-10-01

A humanized line of rat C6 glioma cells expressing human poliovirus receptor was obtained and tested for the sensitivity to oncolytic effects of vaccine strains of type 1, 2, and 3 polioviruses. Presentation of the poliovirus receptor on the surface of C6 glioma cells was shown to be a necessary condition for the interaction of cells with polioviruses, but insufficient for complete poliovirus oncolysis.

Differential Expression of FAK and Pyk2 in Metastatic and Non-metastatic EL4 Lymphoma Cell Lines

PubMed Central

Zhang, Zhihong; Knoepp, Stewart M.; Ku, Hsun; Sansbury, Heather M.; Xie, Yuhuan; Chahal, Manpreet S.; Tomlinson, Stephen; Meier, Kathryn E.

2011-01-01

The murine EL4 lymphoma cell line exists in variants that are either sensitive or resistant to phorbol 12-myristate 13-acetate (PMA). In sensitive cells, PMA causes Erk MAPK activation and Erk-mediated growth arrest. In resistant cells, PMA induces a low level of Erk activation, without growth arrest. A relatively unexplored aspect of the phenotypes is that resistant cells are more adherent to culture substrate than are sensitive cells. In this study, the roles of the protein tyrosine kinases FAK and Pyk2 in EL4 phenotype were examined, with a particular emphasis on the role of these proteins in metastasis. FAK is expressed only in PMA-resistant (or intermediate phenotype) EL4 cells, correlating with enhanced cell-substrate adherence, while Pyk2 is more highly expressed in non-adherent PMA-sensitive cells. PMA treatment causes modulation of mRNA for FAK (up-regulation) and Pyk2 (down-regulation) in PMA-sensitive but not PMA-resistant EL4 cells. The increase in Pyk2 mRNA is correlated with an increase in Pyk2 protein expression. The roles of FAK in cell phenotype were further explored using transfection and knockdown experiments. The results showed that FAK does not play a major role in modulating PMA-induced Erk activation in EL4 cells. However, the knockdown studies demonstrated that FAK expression is required for proliferation and migration of PMA-resistant cells. In an experimental metastasis model using syngeneic mice, only FAK-expressing (PMA-resistant) EL4 cells form liver tumors. Taken together, these studies suggest that FAK expression promotes metastasis of EL4 lymphoma cells. PMID:21533871

Differential expression of FAK and Pyk2 in metastatic and non-metastatic EL4 lymphoma cell lines.

PubMed

Zhang, Zhihong; Knoepp, Stewart M; Ku, Hsun; Sansbury, Heather M; Xie, Yuhuan; Chahal, Manpreet S; Tomlinson, Stephen; Meier, Kathryn E

2011-08-01

The murine EL4 lymphoma cell line exists in variants that are either sensitive or resistant to phorbol 12-myristate 13-acetate (PMA). In sensitive cells, PMA causes Erk MAPK activation and Erk-mediated growth arrest. In resistant cells, PMA induces a low level of Erk activation, without growth arrest. A relatively unexplored aspect of the phenotypes is that resistant cells are more adherent to culture substrate than are sensitive cells. In this study, the roles of the protein tyrosine kinases FAK and Pyk2 in EL4 phenotype were examined, with a particular emphasis on the role of these proteins in metastasis. FAK is expressed only in PMA-resistant (or intermediate phenotype) EL4 cells, correlating with enhanced cell-substrate adherence, while Pyk2 is more highly expressed in non-adherent PMA-sensitive cells. PMA treatment causes modulation of mRNA for FAK (up-regulation) and Pyk2 (down-regulation) in PMA-sensitive but not PMA-resistant EL4 cells. The increase in Pyk2 mRNA is correlated with an increase in Pyk2 protein expression. The roles of FAK in cell phenotype were further explored using transfection and knockdown experiments. The results showed that FAK does not play a major role in modulating PMA-induced Erk activation in EL4 cells. However, the knockdown studies demonstrated that FAK expression is required for proliferation and migration of PMA-resistant cells. In an experimental metastasis model using syngeneic mice, only FAK-expressing (PMA-resistant) EL4 cells form liver tumors. Taken together, these studies suggest that FAK expression promotes metastasis of EL4 lymphoma cells.

Chromosomal Aberrations in DNA Repair Defective Cell Lines: Comparisons of Dose Rate and Radiation Quality

NASA Technical Reports Server (NTRS)

George, K. A.; Hada, M.; Patel, Z.; Huff, J.; Pluth, J. M.; Cucinotta, F. A.

2009-01-01

Chromosome aberration yields were assessed in DNA double-strand break repair (DSB) deficient cells after acute doses of gamma-rays or high-LET iron nuclei, or low dose-rate (0.018 Gy/hr) gamma-rays. We studied several cell lines including fibroblasts deficient in ATM (product of the gene that is mutated in ataxia telangiectasia patients) or NBS (product of the gene mutated in the Nijmegen breakage syndrome), and gliomablastoma cells that are proficient or lacking in DNA-dependent protein kinase, DNA-PK activity. Chromosomes were analyzed using the fluorescence in-situ hybridization (FISH) chromosome painting method in cells at the first division post-irradiation and chromosome aberrations were identified as either simple exchanges (translocations and dicentrics) or complex exchanges (involving >2 breaks in 2 or more chromosomes). Gamma radiation induced higher yields of both simple and complex exchanges in the DSB repair defective cells than in the normal cells. The quadratic dose-response terms for both chromosome exchange types were significantly higher for the ATM and NBS defective lines than for normal fibroblasts. However, the linear dose-response term was significantly higher only for simple exchanges in the NBS cells. Large increases in the quadratic dose response terms indicate the important roles of ATM and NBS in chromatin modifications that facilitate correct DSB repair and minimize aberration formation. Differences in the response of AT and NBS deficient cells at lower doses suggests important questions about the applicability of observations of radiation sensitivity at high dose to low dose exposures. For all iron nuclei irradiated cells, regression models preferred purely linear and quadratic dose responses for simple and complex exchanges, respectively. All the DNA repair defective cell lines had lower Relative biological effectiveness (RBE) values than normal cells, the lowest being for the DNA-PK-deficient cells, which was near unity. To further investigate the sensitivity differences for low and low high doses, we performed chronic low dose-rate irradiation, and have begun studies with ATM and Nibrin inhibitors and siRNA knockout of these proteins. Results support the conclusion that for the endpoint of simple chromosomal aberrations (translocation or dicentrics), the increased radiation sensitivity of AT cells found at high doses (>1 Gy) does not carry over to low doses or doserates, while NBS cells show increased sensitivity for both high and low dose exposures.

Intensified autophagy compromises the efficacy of radiotherapy against prostate cancer

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

Koukourakis, Michael I., E-mail: targ@her.forthnet.gr; Kalamida, Dimitra; Mitrakas, Achilleas

2015-05-29

Introduction: Radiotherapy is an equivalent alternative or complement to radical prostatectomy, with high therapeutic efficacy. High risk patients, however, experience high relapse rates, so that research on radio-sensitization is the most evident route to improve curability of this common disease. Materials and methods: In the current study we investigated the autophagic activity in a series of patients with localized prostate tumors treated with radical radiotherapy, using the LC3A and the LAMP2a proteins as markers of autophagosome and lysosome cellular content, respectively. The role of autophagy on prostate cancer cell line resistance to radiation was also examined. Results: Using confocal microscopymore » on tissue biopsies, we showed that prostate cancer cells have, overall, high levels of LC3A and low levels of LAMP2a compared to normal prostate glands. Tumors with a ‘highLC3A/lowLAMP2a’ phenotype, suggestive of intensified lysosomal consumption, had a significantly poorer biochemical relapse free survival. The PC3 radioresistant cell line sustained remarkably its autophagic flux ability after radiation, while the DU145 radiosensitive one experiences a prolonged blockage of the autophagic process. This was assessed with aggresome accumulation detection and LC3A/LAMP2a double immunofluorescence, as well as with sequestrosome/p62 protein detection. By silencing the LC3A or LAMP2a expression, both cell lines became more sensitive to escalated doses of radiation. Conclusions: High base line autophagy activity and cell ability to sustain functional autophagy define resistance of prostate cancer cells to radiotherapy. This can be reversed by blocking up-regulated components of the autophagy pathway, which may prove of importance in the field of clinical radiotherapy. - Highlights: • High LC3A and low LAMP2a levels is a frequent expression pattern of prostate carcinoma. • This pattern of intensified autophagic flux relates with high relapse rates after radiotherapy. • The PC3 radio-resistant cell line sustains remarkably its autophagic flux ability after radiation. • Irradiation of the DU145 radio-sensitive cell line blocks the autophagic flux. • Intense autophagy activity defines prostate cancer radio-resistance, in vivo and in vitro.« less

Neratinib shows efficacy in the treatment of HER2/neu amplified uterine serous carcinoma in vitro and in vivo.

PubMed

Schwab, Carlton L; English, Diana P; Roque, Dana M; Bellone, Stefania; Lopez, Salvatore; Cocco, Emiliano; Nicoletti, Roberta; Rutherford, Thomas J; Schwartz, Peter E; Santin, Alessandro D

2014-10-01

Uterine serous carcinoma (USC) represents an aggressive variant of endometrial cancer and accounts for a large proportion of deaths annually. HER2/neu amplification is associated with USC in approximately 30-35% of cases. The objective of this study was to determine the sensitivity of a panel of primary USC cell lines to the small tyrosine kinase inhibitor neratinib, an ErbB1 and HER2 inhibitor, both in vitro and in vivo. HER2/neu amplification was determined by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) in 24 USC cell lines. Flow cytometry was used to determine the effects of neratinib on cell viability, cell cycle distribution and signaling in vitro. Mice harboring HER2/neu amplified xenografts were treated with neratinib to assess the efficacy of the drug in vivo. HER2/neu amplification was noted in 8/24 primary cell lines. Data regarding the efficacy of neratinib was determined using 4 HER2 amplified cell lines and 4 non-amplified cell lines with similar growth rates. Data revealed that cell lines with HER2/neu amplification were exquisitely more sensitive to neratinib compared to non-amplified cell lines (mean ± SEM IC50: 0.011μM ± 0.0008 vs. 0.312μM ± 0.0456 p<0.0001). Neratinib caused arrest in the G0/G1 phase of the cell cycle and resulted in decreased autophosphorylation of HER2 and activation of S6. Neratinib treated mice harboring xenografts of HER2/neu amplified USC showed delayed tumor growth and improved overall survival compared to vehicle (p=0.0019). Neratinib may be a potential treatment option for patients harboring HER2/neu amplified USC. Clinical trials for this subset of endometrial cancer patients are warranted. Copyright © 2014 Elsevier Inc. All rights reserved.

miR-370 regulates ISG15 expression and influences IFN-α sensitivity in hepatocellular carcinoma cells.

PubMed

Liu, Zhuo; Ma, Min; Yan, Lei; Chen, Shilin; Li, Sha; Yang, Darong; Wang, Xiaohong; Xiao, Hua; Deng, Hongyu; Zhu, Haizhen; Zuo, Chaohui; Xia, Man

2018-05-05

Interferon-α (IFN-α) is an adjuvant to chemotherapy and radiotherapy for hepatocellular carcinoma (HCC), but some HCC patients do not respond to treatment with IFN-α. We performed loss-of-function and gain-of-function experiments to examine the role of ISG15 in the IFN-α sensitivity of LH86, HLCZ01, SMMC7721, and Huh7 cell lines and tumor samples. The overexpression of ISG15 reduced apoptosis in Huh7 and LH86 cells in the presence of IFN-α, whereas the shRNA-mediated knock down of ISG15 expression increased apoptosis in both Huh7 and LH86 cells. We identified a putative miR-370 target site in the 3'-UTR in the ISG15 mRNA, and the level of miR-370 expression in HCC cell lines reflected the level of IFN-α-induced apoptosis exhibited by each. Both HCC cell lines and tumor samples had significantly lower levels of miR-370 than the control cells and tissues (P< 0.05). The overexpression of miR-370 in IFN-α-treated LH86 and Huh7 cells increased apoptosis and reduced the volume of LH86- and Huh7-derived xenograft tumors in mice treated with IFN-α compared with the control tumors. Our findings suggest that miR-370 functions as an HCC tumor suppressor and regulator of IFN-α sensitivity and that miR-370 might be a useful prognostic marker for HCC patients.

New Small Molecules Targeting Apoptosis and Cell Viability in Osteosarcoma

PubMed Central

Maugg, Doris; Rothenaigner, Ina; Schorpp, Kenji; Potukuchi, Harish Kumar; Korsching, Eberhard; Baumhoer, Daniel; Hadian, Kamyar

2015-01-01

Despite the option of multimodal therapy in the treatment strategies of osteosarcoma (OS), the most common primary malignant bone tumor, the standard therapy has not changed over the last decades and still involves multidrug chemotherapy and radical surgery. Although successfully applied in many patients a large number of patients eventually develop recurrent or metastatic disease in which current therapeutic regimens often lack efficacy. Thus, new therapeutic strategies are urgently needed. In this study, we performed a phenotypic high-throughput screening campaign using a 25,000 small-molecule diversity library to identify new small molecules selectively targeting osteosarcoma cells. We could identify two new small molecules that specifically reduced cell viability in OS cell lines U2OS and HOS, but affected neither hepatocellular carcinoma cell line (HepG2) nor primary human osteoblasts (hOB). In addition, the two compounds induced caspase 3 and 7 activity in the U2OS cell line. Compared to conventional drugs generally used in OS treatment such as doxorubicin, we indeed observed a greater sensitivity of OS cell viability to the newly identified compounds compared to doxorubicin and staurosporine. The p53-negative OS cell line Saos-2 almost completely lacked sensitivity to compound treatment that could indicate a role of p53 in the drug response. Taken together, our data show potential implications for designing more efficient therapies in OS. PMID:26039064

O(6)-methylguanine DNA-methyltransferase (MGMT) overexpression in melanoma cells induces resistance to nitrosoureas and temozolomide but sensitizes to mitomycin C.

PubMed

Passagne, Isabelle; Evrard, Alexandre; Depeille, Philippe; Cuq, Pierre; Cupissol, Didier; Vian, Laurence

2006-03-01

Alkylating agents play an important role in the chemotherapy of malignant melanomas. The activity of alkylating agents depends on their capacity to form alkyl adducts with DNA, in some cases causing cross-linking of DNA strands. However, the use of these agents is limited by cellular resistance induced by the DNA repair enzyme O(6)-methylguanine DNA-methyltransferase (MGMT) which removes alkyl groups from alkylated DNA strands. To determine to what extent the expression of MGMT in melanoma cells induces resistance to alkylating agents, the human cell line CAL77 Mer- (i.e., MGMT deficient) were transfected with pcMGMT vector containing human MGMT cDNA. Several clones expressing MGMT at a high level were selected to determine their sensitivity to chemotherapeutic drugs. Melanoma-transfected cells were found to be significantly less sensitive to nitrosoureas (carmustine, fotemustine, streptozotocin) and temozolomide with an increase of IC(50) values between 3 and 14 when compared to parent cells. No difference in cell survival rates between MGMT-proficient and -deficient cells was observed for melphalan, chlorambucil, busulphan, thiotepa and cisplatin which preferentially induce N(7) guanine lesions. Surprisingly, MGMT overexpression increased the sensitivity of CAL77 cells to mitomycin C by approximately 10-fold. Treatment of clonal cell lines with buthionine-[S,R]-sulfoximine (BSO), an inhibitor of gamma-glutamylcysteine synthetase which depletes cellular glutathione, completely reversed this unexpected increase in sensitivity to mitomycin C. This observation suggests that glutathione is involved in the sensitivity of MGMT-transfected cells to mitomycin C and may act synergistically with MGMT via an unknown mechanism.

Immortalisation of a human diploid fibroblast cell strain: a DT-diaphorase paradox.

PubMed Central

Kuehl, B. L.; Brezden, C. B.; Traver, R. D.; Siegel, D.; Ross, D.; Renzing, J.; Rauth, A. M.

1996-01-01

Transfection of a normal human diploid fibroblast cell strain, GM38, with a simian virus 40 (SV40) large T antigen containing plasmid, yielded an immortal cell line, G38-8X, which had a similar sensitivity as the parental cell strain to the quinone-containing chemotherapeutic agent mitomycin C (MMC), under both aerobic and hypoxic exposure conditions. The activity level of DT-diaphorase was similar in both the parental GM38 and G38-8X cells. Although DT-diaphorase could be detected by Western blot analysis, using two mouse anti-human monoclonal antibodies, in GM38 cells, it was not detected in the G38-8X cells. G38-8X cells have a slightly increased P450R activity (2-fold), and have elevated P-glycoprotein levels compared with the parental GM38 cell strain. The immortal G38-8X cell line is 2-fold more resistant to ionising radiation than the parental GM38 cell strain (D10 approximately 5 Gy). Although these SV40 large T antigen immortalised human diploid fibroblasts behaved similarly to their parental cell strain in terms of MMC sensitivity and DT-diaphorase activity, careful characterisation revealed that these cells had enhanced P-glycoprotein activity and had a decreased sensitivity to ionising radiation. Images Figure 3 PMID:8763839

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.

Method for rapid isolation of sensitive mutants

DOEpatents

Freyer, James P.

1997-01-01

Sensitive mammalian cell mutants are rapidly isolated using flow cytometry. A first population of clonal spheroids is established to contain both normal and mutant cells. The population may be naturally occurring or may arise from mutagenized cells. The first population is then flow sorted by size to obtain a second population of clonal spheroids of a first uniform size. The second population is then exposed to a DNA-damaging agent that is being investigated. The exposed second population is placed in a growth medium to form a third population of clonal spheroids comprising spheroids of increased size from the mammalian cells that are resistant to the DNA-damaging agent and spheroids of substantially the first uniform size formed from the mammalian cells that are sensitive to the DNA-damaging agent. The third population is not flow sorted to differentiate the spheroids formed from resistant mammalian cells from spheroids formed from sensitive mammalian cells. The spheroids formed from sensitive mammalian cells are now treated to recover viable sensitive cells from which a sensitive cell line can be cloned.

Method for rapid isolation of sensitive mutants

DOEpatents

Freyer, J.P.

1997-07-29

Sensitive mammalian cell mutants are rapidly isolated using flow cytometry. A first population of clonal spheroids is established to contain both normal and mutant cells. The population may be naturally occurring or may arise from mutagenized cells. The first population is then flow sorted by size to obtain a second population of clonal spheroids of a first uniform size. The second population is then exposed to a DNA-damaging agent that is being investigated. The exposed second population is placed in a growth medium to form a third population of clonal spheroids comprising spheroids of increased size from the mammalian cells that are resistant to the DNA-damaging agent and spheroids of substantially the first uniform size formed from the mammalian cells that are sensitive to the DNA-damaging agent. The third population is not flow sorted to differentiate the spheroids formed from resistant mammalian cells from spheroids formed from sensitive mammalian cells. The spheroids formed from sensitive mammalian cells are now treated to recover viable sensitive cells from which a sensitive cell line can be cloned. 15 figs.

NK sensitivity of neuroblastoma cells determined by a highly sensitive coupled luminescent method

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

Ogbomo, Henry; Hahn, Anke; Geiler, Janina

2006-01-06

The measurement of natural killer (NK) cells toxicity against tumor or virus-infected cells especially in cases with small blood samples requires highly sensitive methods. Here, a coupled luminescent method (CLM) based on glyceraldehyde-3-phosphate dehydrogenase release from injured target cells was used to evaluate the cytotoxicity of interleukin-2 activated NK cells against neuroblastoma cell lines. In contrast to most other methods, CLM does not require the pretreatment of target cells with labeling substances which could be toxic or radioactive. The effective killing of tumor cells was achieved by low effector/target ratios ranging from 0.5:1 to 4:1. CLM provides highly sensitive, safe,more » and fast procedure for measurement of NK cell activity with small blood samples such as those obtained from pediatric patients.« less

A gene expression profile indicative of early stage HER2 targeted therapy response.

PubMed

O'Neill, Fiona; Madden, Stephen F; Clynes, Martin; Crown, John; Doolan, Padraig; Aherne, Sinéad T; O'Connor, Robert

2013-07-01

Efficacious application of HER2-targetting agents requires the identification of novel predictive biomarkers. Lapatinib, afatinib and neratinib are tyrosine kinase inhibitors (TKIs) of HER2 and EGFR growth factor receptors. A panel of breast cancer cell lines was treated with these agents, trastuzumab, gefitinib and cytotoxic therapies and the expression pattern of a specific panel of genes using RT-PCR was investigated as a potential marker of early drug response to HER2-targeting therapies. Treatment of HER2 TKI-sensitive SKBR3 and BT474 cell lines with lapatinib, afatinib and neratinib induced an increase in the expression of RB1CC1, ERBB3, FOXO3a and NR3C1. The response directly correlated with the degree of sensitivity. This expression pattern switched from up-regulated to down-regulated in the HER2 expressing, HER2-TKI insensitive cell line MDAMB453. Expression of the CCND1 gene demonstrated an inversely proportional response to drug exposure. A similar expression pattern was observed following the treatment with both neratinib and afatinib. These patterns were retained following exposure to traztuzumab and lapatinib plus capecitabine. In contrast, gefitinib, dasatinib and epirubicin treatment resulted in a completely different expression pattern change. In these HER2-expressing cell line models, lapatinib, neratinib, afatinib and trastuzumab treatment generated a characteristic and specific gene expression response, proportionate to the sensitivity of the cell lines to the HER2 inhibitor.Characterisation of the induced changes in expression levels of these genes may therefore give a valuable, very early predictor of the likely extent and specificity of tumour HER2 inhibitor response in patients, potentially guiding more specific use of these agents.

A gene expression profile indicative of early stage HER2 targeted therapy response

PubMed Central

2013-01-01

Background Efficacious application of HER2-targetting agents requires the identification of novel predictive biomarkers. Lapatinib, afatinib and neratinib are tyrosine kinase inhibitors (TKIs) of HER2 and EGFR growth factor receptors. A panel of breast cancer cell lines was treated with these agents, trastuzumab, gefitinib and cytotoxic therapies and the expression pattern of a specific panel of genes using RT-PCR was investigated as a potential marker of early drug response to HER2-targeting therapies. Results Treatment of HER2 TKI-sensitive SKBR3 and BT474 cell lines with lapatinib, afatinib and neratinib induced an increase in the expression of RB1CC1, ERBB3, FOXO3a and NR3C1. The response directly correlated with the degree of sensitivity. This expression pattern switched from up-regulated to down-regulated in the HER2 expressing, HER2-TKI insensitive cell line MDAMB453. Expression of the CCND1 gene demonstrated an inversely proportional response to drug exposure. A similar expression pattern was observed following the treatment with both neratinib and afatinib. These patterns were retained following exposure to traztuzumab and lapatinib plus capecitabine. In contrast, gefitinib, dasatinib and epirubicin treatment resulted in a completely different expression pattern change. Conclusions In these HER2-expressing cell line models, lapatinib, neratinib, afatinib and trastuzumab treatment generated a characteristic and specific gene expression response, proportionate to the sensitivity of the cell lines to the HER2 inhibitor. Characterisation of the induced changes in expression levels of these genes may therefore give a valuable, very early predictor of the likely extent and specificity of tumour HER2 inhibitor response in patients, potentially guiding more specific use of these agents. PMID:23816254

Sensitizing Triple-Negative Breast Cancer to PI3K Inhibition by Cotargeting IGF1R.

PubMed

de Lint, Klaas; Poell, Jos B; Soueidan, Hayssam; Jastrzebski, Katarzyna; Vidal Rodriguez, Jordi; Lieftink, Cor; Wessels, Lodewyk F A; Beijersbergen, Roderick L

2016-07-01

Targeted therapies have proven invaluable in the treatment of breast cancer, as exemplified by tamoxifen treatment for hormone receptor-positive tumors and trastuzumab treatment for HER2-positive tumors. In contrast, a subset of breast cancer negative for these markers, triple-negative breast cancer (TNBC), has met limited success with pathway-targeted therapies. A large fraction of TNBCs depend on the PI3K pathway for proliferation and survival, but inhibition of PI3K alone generally has limited clinical benefit. We performed an RNAi-based genetic screen in a human TNBC cell line to identify kinases whose knockdown synergizes with the PI3K inhibitor GDC-0941 (pictilisib). We discovered that knockdown of insulin-like growth factor-1 receptor (IGF1R) expression potently increased sensitivity of these cells to GDC-0941. Pharmacologic inhibition of IGF1R using OSI-906 (linsitinib) showed a strong synergy with PI3K inhibition. Furthermore, we found that the combination of GDC-0941 and OSI-906 is synergistic in 8 lines from a panel of 18 TNBC cell lines. In these cell lines, inhibition of IGF1R further decreases the activity of downstream PI3K pathway components when PI3K is inhibited. Expression analysis of the panel of TNBC cell lines indicates that the expression levels of IGF2BP3 can be used as a potential predictor for sensitivity to the PI3K/IGF1R inhibitor combination. Our data show that combination therapy consisting of PI3K and IGF1R inhibitors could be beneficial in a subset of TNBCs. Mol Cancer Ther; 15(7); 1545-56. ©2016 AACR. ©2016 American Association for Cancer Research.

DNA damage induced by boron neutron capture therapy is partially repaired by DNA ligase IV.

PubMed

Kondo, Natsuko; Sakurai, Yoshinori; Hirota, Yuki; Tanaka, Hiroki; Watanabe, Tsubasa; Nakagawa, Yosuke; Narabayashi, Masaru; Kinashi, Yuko; Miyatake, Shin-ichi; Hasegawa, Masatoshi; Suzuki, Minoru; Masunaga, Shin-ichiro; Ohnishi, Takeo; Ono, Koji

2016-03-01

Boron neutron capture therapy (BNCT) is a particle radiation therapy that involves the use of a thermal or epithermal neutron beam in combination with a boron ((10)B)-containing compound that specifically accumulates in tumor. (10)B captures neutrons and the resultant fission reaction produces an alpha ((4)He) particle and a recoiled lithium nucleus ((7)Li). These particles have the characteristics of high linear energy transfer (LET) radiation and therefore have marked biological effects. High-LET radiation is a potent inducer of DNA damage, specifically of DNA double-strand breaks (DSBs). The aim of the present study was to clarify the role of DNA ligase IV, a key player in the non-homologous end-joining repair pathway, in the repair of BNCT-induced DSBs. We analyzed the cellular sensitivity of the mouse embryonic fibroblast cell lines Lig4-/- p53-/- and Lig4+/+ p53-/- to irradiation using a thermal neutron beam in the presence or absence of (10)B-para-boronophenylalanine (BPA). The Lig4-/- p53-/- cell line had a higher sensitivity than the Lig4+/+ p53-/-cell line to irradiation with the beam alone or the beam in combination with BPA. In BNCT (with BPA), both cell lines exhibited a reduction of the 50 % survival dose (D 50) by a factor of 1.4 compared with gamma-ray and neutron mixed beam (without BPA). Although it was found that (10)B uptake was higher in the Lig4+/+ p53-/- than in the Lig4-/- p53-/- cell line, the latter showed higher sensitivity than the former, even when compared at an equivalent (10)B concentration. These results indicate that BNCT-induced DNA damage is partially repaired using DNA ligase IV.

Female mice lack adult germ-line stem cells but sustain oogenesis using stable primordial follicles.

PubMed

Lei, Lei; Spradling, Allan C

2013-05-21

Whether or not mammalian females generate new oocytes during adulthood from germ-line stem cells to sustain the ovarian follicle pool has recently generated controversy. We used a sensitive lineage-labeling system to determine whether stem cells are needed in female adult mice to compensate for follicular losses and to directly identify active germ-line stem cells. Primordial follicles generated during fetal life are highly stable, with a half-life during adulthood of 10 mo, and thus are sufficient to sustain adult oogenesis without a source of renewal. Moreover, in normal mice or following germ-cell depletion with Busulfan, only stable, single oocytes are lineage-labeled, rather than cell clusters indicative of new oocyte formation. Even one germ-line stem cell division per 2 wk would have been detected by our method, based on the kinetics of fetal follicle formation. Thus, adult female mice neither require nor contain active germ-line stem cells or produce new oocytes in vivo.

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

JNK1 Inhibition Attenuates Hypoxia-Induced Autophagy and Sensitizes to Chemotherapy.

PubMed

Vasilevskaya, Irina A; Selvakumaran, Muthu; Roberts, David; O'Dwyer, Peter J

2016-08-01

Inhibition of hypoxia-induced stress signaling through JNK potentiates the effects of oxaliplatin. The JNK pathway plays a role in both autophagy and apoptosis; therefore, it was determined how much of the effect of JNK inhibition on oxaliplatin sensitivity is dependent on its effect on autophagy. We studied the impact of JNK isoform downregulation in the HT29 colon adenocarcinoma cell line on hypoxia- and oxaliplatin-induced responses. Electron microscopic analyses demonstrated that both oxaliplatin- and hypoxia-induced formations of autophagosomes were reduced significantly in HT29 cells treated with the JNK inhibitor SP600125. The role of specific JNK isoforms was defined using HT29-derived cell lines stably expressing dominant-negative constructs for JNK1 and JNK2 (HTJ1.3 and HTJ2.2, respectively). These cell lines demonstrated that functional JNK1 is required for hypoxia-induced autophagy and that JNK2 does not substitute for it. Inhibition of autophagy in HTJ1.3 cells also coincided with enhancement of intrinsic apoptosis. Analysis of Bcl2-family proteins revealed hyperphosphorylation of Bcl-XL in the HTJ1.3 cell line, but this did not lead to the expected dissociation from Beclin 1. Consistent with this, knockdown of Bcl-XL in HT29 cells did not significantly affect the induction of autophagy, but abrogated hypoxic resistance to oxaliplatin due to the faster and more robust activation of apoptosis. These data suggest that balance between autophagy and apoptosis is shifted toward apoptosis by downregulation of JNK1, contributing to oxaliplatin sensitization. These findings further support the investigation of JNK inhibition in colorectal cancer treatment. Mol Cancer Res; 14(8); 753-63. ©2016 AACR. ©2016 American Association for Cancer Research.

Afatinib demonstrates remarkable activity against HER2-amplified uterine serous endometrial cancer in vitro and in vivo.

PubMed

Schwab, C L; Bellone, S; English, D P; Roque, D M; Lopez, S; Cocco, E; Nicoletti, R; Bortolomai, I; Bonazzoli, E; Ratner, E; Silasi, D-A; Azodi, M; Schwartz, P E; Rutherford, T J; Santin, A D

2014-10-28

Uterine serous carcinomas (USCs) are an aggressive form of uterine cancer that may rely on HER2/neu amplification as a driver of proliferation. The objective of this paper is to assess the sensitivity of USC cell lines with and without HER2/neu gene amplification to afatinib, an irreversible ErbB tyrosine kinase inhibitor, and to test the efficacy of afatinib in the treatment of HER2-amplified USC xenografts. Eight of fifteen primary USC cell lines (four with HER2 amplification and four without) demonstrating similar in vitro growth rates were treated with scalar concentrations of afatinib. Effects on cell growth, signalling and cell cycle distribution were determined by flow cytometry assays. Mice harbouring xenografts of HER2/neu-amplified USC were treated with afatinib by gavage to determine the effect on tumour growth and overall survival. Primary chemotherapy-resistant USC cell lines harbouring HER2/neu gene amplification were exquisitely sensitive to afatinib exposure (mean ± s.e.m. IC50=0.0056 ± 0.0006 μM) and significantly more sensitive than HER2/neu-non-amplified USC cell lines (mean ± s.e.m. IC50=0.563 ± 0.092 μM, P<0.0001). Afatinib exposure resulted in abrogation of cell survival, inhibition of HER2/neu autophosphorylation and S6 transcription factor phosphorylation in HER2/neu overexpressing USC and inhibited the growth of HER2-amplified tumour xenografts improving overall survival (P=0.0017). Afatinib may be highly effective against HER2/neu-amplified chemotherapy-resistant USC. The investigation of afatinib in patients harbouring HER2/neu-amplified USC is warranted.

Role of reactive oxygen species in the anticancer activity of botanicals: Comparing sensitivity profiles

PubMed Central

Cohen, Zoya; Maimon, Yair; Samuels, Noah; Berger, Raanan

2017-01-01

Numerous botanicals have been shown to exhibit in vitro and in vivo anticancer activity, some of which is the result of the induction of reactive oxygen species (ROS) in cancer cells with a high ROS content. The present study compared sensitivities to a series of botanicals among cancer cell lines, using an XTT viability test, in order to create a specific cancer-herb profile. Of the 27 botanicals screened, 10 exhibited a cytotoxic effect, 7 of which were ROS-mediated. The sensitivity profiles of the ROS-inducing botanicals in 10 cancer cell lines were similar, unlike 3 cytotoxic ROS-independent botanicals that displayed divergent botanical-specific profiles. The correlation between sensitivity profiles of ROS-inducing botanicals suggests a common mechanism of action, in contrast to the varied mechanism of ROS-independent botanicals. This implies that the investigation of the anticancer activity of botanicals should start with the examination of ROS-mediated activity. Further investigation of ROS sensitivity among various tumor types is required in order to guide research into developing evidence-based guidelines in the use of botanicals for cancer treatment. PMID:28454445

Interaction of celecoxib with different anti-cancer drugs is antagonistic in breast but not in other cancer cells

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

El-Awady, Raafat A., E-mail: relawady@sharjah.ac.ae; Department of Pharmacology and Pharmaceutics, College of Pharmacy, University of Sharjah, University City road, 27272 Sharjah; Saleh, Ekram M.

Celecoxib, an inhibitor of cyclooxygenase-2, is being investigated for enhancement of chemotherapy efficacy in cancer clinical trials. This study investigates the ability of cyclooxygenase-2 inhibitors to sensitize cells from different origins to several chemotherapeutic agents. The effect of the drug's mechanism of action and sequence of administration are also investigated. The sensitivity, cell cycle, apoptosis and DNA damage of five different cancer cell lines (HeLa, HCT116, HepG2, MCF7 and U251) to 5-FU, cisplatin, doxorubicin and etoposide {+-} celecoxib following different incubation schedules were analyzed. We found antagonism between celecoxib and the four drugs in the breast cancer cells MCF7 followingmore » all incubation schedules and between celecoxib and doxorubicin in all cell lines except for two combinations in HCT116 cells. Celecoxib with the other three drugs in the remaining four cell lines resulted in variable interactions. Mechanistic investigations revealed that celecoxib exerts different molecular effects in different cells. In some lines, it abrogates the drug-induced G2/M arrest enhancing pre-mature entry into mitosis with damaged DNA thus increasing apoptosis and resulting in synergism. In other cells, it enhances drug-induced G2/M arrest allowing time to repair drug-induced DNA damage before entry into mitosis and decreasing cell death resulting in antagonism. In some synergistic combinations, celecoxib-induced abrogation of G2/M arrest was not associated with apoptosis but permanent arrest in G1 phase. These results, if confirmed in-vivo, indicate that celecoxib is not a suitable chemosensitizer for breast cancer or with doxorubicin for other cancers. Moreover, combination of celecoxib with other drugs should be tailored to the tumor type, drug and administration schedule. - Graphical abstract: Display Omitted Highlights: > Celecoxib may enhance effects of anticancer drugs. > Its combination with four drugs was tested in five cancer cell lines. > It antagonized the effects of the four drugs in the breast cancer cell line MCF7. > Doxorubicin's cytotoxic effects were antagonized by celecoxib in four cell lines. > Cell cycle, apoptosis and DNA damage explain the different interactive effects.« less

TRAIL-induced programmed necrosis as a novel approach to eliminate tumor cells

PubMed Central

2014-01-01

Background The cytokine TRAIL represents one of the most promising candidates for the apoptotic elimination of tumor cells, either alone or in combination therapies. However, its efficacy is often limited by intrinsic or acquired resistance of tumor cells to apoptosis. Programmed necrosis is an alternative, molecularly distinct mode of programmed cell death that is elicited by TRAIL under conditions when the classical apoptosis machinery fails or is actively inhibited. The potential of TRAIL-induced programmed necrosis in tumor therapy is, however, almost completely uncharacterized. We therefore investigated its impact on a panel of tumor cell lines of wide-ranging origin. Methods Cell death/viability was measured by flow cytometry/determination of intracellular ATP levels/crystal violet staining. Cell surface expression of TRAIL receptors was detected by flow cytometry, expression of proteins by Western blot. Ceramide levels were quantified by high-performance thin layer chromatography and densitometric analysis, clonogenic survival of cells was determined by crystal violet staining or by soft agarose cloning. Results TRAIL-induced programmed necrosis killed eight out of 14 tumor cell lines. Clonogenic survival was reduced in all sensitive and even one resistant cell lines tested. TRAIL synergized with chemotherapeutics in killing tumor cell lines by programmed necrosis, enhancing their effect in eight out of 10 tested tumor cell lines and in 41 out of 80 chemotherapeutic/TRAIL combinations. Susceptibility/resistance of the investigated tumor cell lines to programmed necrosis seems to primarily depend on expression of the pro-necrotic kinase RIPK3 rather than the related kinase RIPK1 or cell surface expression of TRAIL receptors. Furthermore, interference with production of the lipid ceramide protected all tested tumor cell lines. Conclusions Our study provides evidence that TRAIL-induced programmed necrosis represents a feasible approach for the elimination of tumor cells, and that this treatment may represent a promising new option for the future development of combination therapies. Our data also suggest that RIPK3 expression may serve as a potential predictive marker for the sensitivity of tumor cells to programmed necrosis and extend the previously established role of ceramide as a key mediator of death receptor-induced programmed necrosis (and thus as a potential target for future therapies) also to the tumor cell lines examined here. PMID:24507727

The Akt-inhibitor Erufosine induces apoptotic cell death in prostate cancer cells and increases the short term effects of ionizing radiation.

PubMed

Rudner, Justine; Ruiner, Carola-Ellen; Handrick, René; Eibl, Hans-Jörg; Belka, Claus; Jendrossek, Verena

2010-11-16

The phosphatidylinositol-3-kinase (PI3K)/Akt pathway is frequently deregulated in prostate cancer and associated with neoplastic transformation, malignant progression, and enhanced resistance to classical chemotherapy and radiotherapy. Thus, it is a promising target for therapeutic intervention. In the present study, the cytotoxic action of the Akt inhibitor Erufosine (ErPC3) was analyzed in prostate cancer cells and compared to the cytotoxicity of the PI3K inhibitor LY294002. Moreover, the efficacy of combined treatment with Akt inhibitors and ionizing radiation in prostate cancer cells was examined. Prostate cancer cell lines PC3, DU145, and LNCaP were treated with ErPC3 (1-100 µM), LY294002 (25-100 µM), irradiated (0-10 Gy), or subjected to combined treatments. Cell viability was determined by the WST-1 assay. Apoptosis induction was analyzed by flow cytometry after staining with propidium iodide in a hypotonic citrate buffer, and by Western blotting using antibodies against caspase-3 and its substrate PARP. Akt activity and regulation of the expression of Bcl-2 family members and key downstream effectors involved in apoptosis regulation were examined by Western blot analysis. The Akt inhibitor ErPC3 exerted anti-neoplastic effects in prostate cancer cells, however with different potency. The anti-neoplastic action of ErPC3 was associated with reduced phosphoserine 473-Akt levels and induction of apoptosis. PC3 and LNCaP prostate cancer cells were also sensitive to treatment with the PI3K inhibitor LY294002. However, the ErPC3-sensitive PC3-cells were less susceptible to LY294002 than the ErPC3-refractory LNCaP cells. Although both cell lines were largely resistant to radiation-induced apoptosis, both cell lines showed higher levels of apoptotic cell death when ErPC3 was combined with radiotherapy. Our data suggest that constitutive Akt activation and survival are controlled by different different molecular mechanisms in the two prostate cancer cell lines - one which is sensitive to the Akt-inhibitor ErPC3 and one which is more sensitive to the PI3K-inhibitor LY294002. Our findings underline the importance for the definition of predictive biomarkers that allow the selection patients that may benefit from the treatment with a specific signal transduction modifier.

Comprehensive Evaluation of the Contribution of X Chromosome Genes to Platinum Sensitivity

PubMed Central

Gamazon, Eric R.; Im, Hae Kyung; O’Donnell, Peter H.; Ziliak, Dana; Stark, Amy L.; Cox, Nancy J.; Dolan, M. Eileen; Huang, Rong Stephanie

2011-01-01

Utilizing a genome-wide gene expression dataset generated from Affymetrix GeneChip® Human Exon 1.0ST array, we comprehensively surveyed the role of 322 X chromosome gene expression traits on cellular sensitivity to cisplatin and carboplatin. We identified 31 and 17 X chromosome genes whose expression levels are significantly correlated (after multiple testing correction) with sensitivity to carboplatin and cisplatin, respectively, in the combined HapMap CEU and YRI populations (false discovery rate, FDR<0.05). Of those, 14 overlap for both cisplatin and carboplatin. Employing an independent gene expression quantification method, the Illumina Sentrix Human-6 Expression BeadChip, measured on the same HapMap cell lines, we found that 4 and 2 of these genes are significantly associated with carboplatin and cisplatin sensitivity respectively in both analyses. Two genes, CTPS2 and DLG3, were identified by both genome-wide gene expression analyses as correlated with cellular sensitivity to both platinating agents. The expression of DLG3 gene was also found to correlate with cellular sensitivity to platinating agents in NCI60 cancer cell lines. In addition, we evaluated the role of X chromosome gene expression to the observed differences in sensitivity to the platinums between CEU and YRI derived cell lines. Of the 34 distinct genes significantly correlated with either carboplatin or cisplatin sensitivity, 14 are differentially expressed (defined as p<0.05) between CEU and YRI. Thus, sex chromosome genes play a role in cellular sensitivity to platinating agents and differences in the expression level of these genes are an important source of variation that should be included in comprehensive pharmacogenomic studies. PMID:21252287

Vinblastine 20' Amides: Synthetic Analogues That Maintain or Improve Potency and Simultaneously Overcome Pgp-Derived Efflux and Resistance.

PubMed

Lukesh, John C; Carney, Daniel W; Dong, Huijun; Cross, R Matthew; Shukla, Vyom; Duncan, Katharine K; Yang, Shouliang; Brody, Daniel M; Brütsch, Manuela M; Radakovic, Aleksandar; Boger, Dale L

2017-09-14

A series of 180 vinblastine 20' amides were prepared in three steps from commercially available starting materials, systematically exploring a typically inaccessible site in the molecule enlisting a powerful functionalization strategy. Clear structure-activity relationships and a structural model were developed in the studies which provided many such 20' amides that exhibit substantial and some even remarkable enhancements in potency, many that exhibit further improvements in activity against a Pgp overexpressing resistant cancer cell line, and an important subset of the vinblastine analogues that display little or no differential in activity against a matched pair of vinblastine sensitive and resistant (Pgp overexpressing) cell lines. The improvements in potency directly correlated with target tubulin binding affinity, and the reduction in differential functional activity against the sensitive and Pgp overexpressing resistant cell lines was found to correlate directly with an impact on Pgp-derived efflux.

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.

Comparative performance of fetal goat tongue cell line ZZ-R 127 and fetal porcine kidney cell line LFBK-αvβ6 for Foot-and-mouth disease virus isolation.

PubMed

Fukai, Katsuhiko; Morioka, Kazuki; Yamada, Manabu; Nishi, Tatsuya; Yoshida, Kazuo; Kitano, Rie; Yamazoe, Reiko; Kanno, Toru

2015-07-01

The fetal goat tongue cell line ZZ-R 127 and the fetal porcine kidney cell line LFBK-α(v)β(6) have been reported to have high sensitivity to various Foot-and-mouth disease virus (FMDV) strains. The suitability of ZZ-R 127 cells for FMDV isolation not only from epithelial suspensions but also from other clinical samples has already been confirmed in a previous study. However, to our knowledge, the suitability of LFBK-α(v)β(6) cells has not been evaluated using clinical samples other than epithelial materials. In addition, both cell lines have never been compared, in terms of use for FMDV isolation, under the same conditions. Therefore, in the current study, the virus isolation rates of both cell lines were compared using clinical samples collected from animals infected experimentally with FMDV. Viruses were successfully isolated from clinical samples other than epithelial suspensions for both cell lines. The virus isolation rates for the 2 cell lines were not significantly different. The Cohen kappa coefficients between the virus isolation results for both cell lines were significantly high. Taken together, these results confirmed the suitability of LFBK-α(v)β(6) cells for FMDV isolation from clinical samples other than epithelial suspensions. The levels of susceptibility of both cell lines to FMDV isolation were also confirmed to be almost the same. © 2015 The Author(s).

Assessment of the U937 cell line for the detection of contact allergens

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

Python, Francois; Goebel, Carsten; Aeby, Pierre

2007-04-15

The human myeloid cell line U937 was evaluated as an in vitro test system to identify contact sensitizers in order to develop alternatives to animal tests for the cosmetic industry. Specific culture conditions (i.e., presence of interleukin-4, IL-4) were applied to obtain a dendritic cell-like phenotype. In the described test protocol, these cells were exposed to test chemicals and then analyzed by flow cytometry for CD86 expression and by quantitative real-time reverse transcriptase-polymerase chain reaction for IL-1{beta} and IL-8 gene expressions. Eight sensitizers, three non-sensitizers and five oxidative hair dye precursors were examined after 24-, 48- and 72-h exposure times.more » Test item-specific modulations of the chosen activation markers (CD86, IL-1{beta} and IL-8) suggest that this U937 activation test could discriminate test items classified as contact sensitizers or non-sensitizers in the local lymph node assay in mice (LLNA). More specifically, a test item can be considered as a potential sensitizer when it significantly induced the upregulation of the expression of at least two markers. Using this approach, we could correctly evaluate the dendritic cell (DC) activation potential for 15 out of 16 tested chemicals. We conclude that the U937 activation test may represent an useful tool in a future in vitro test battery for predicting sensitizing properties of chemicals.« less

ILs-3, 6 and 11 increase, but ILs-10 and 24 decrease stemness of human prostate cancer cells in vitro.

PubMed

Yu, Dandan; Zhong, Yali; Li, Xiaoran; Li, Yaqing; Li, Xiaoli; Cao, Jing; Fan, Huijie; Yuan, Yuan; Ji, Zhenyu; Qiao, Baoping; Wen, Jian-Guo; Zhang, Mingzhi; Kvalheim, Gunnar; Nesland, Jahn M; Suo, Zhenhe

2015-12-15

Cancer stem cells (CSCs) are associated with cancer recurrence and metastasis. Prostate cancer cells often metastasize to the bone with a complex microenvironment of cytokines favoring cell survival. In this study, the cell stemness influence of a group of interleukins including IL-3, 6, 10, 11 and 24 on human prostate cancer cell lines LNCaP and PC-3 was explored in vitro. Sulforhodamine B(SRB) and 5-ethynyl-2'-deoxyuridine (EdU) assays were applied to examine the effect on cell proliferation, and wound healing and transwell assays were used for migration and invasion studies, in addition to colony formation, Western blotting and flowcytometry for the expression of stemness factors and chemotherapy sensitivity. We observed that ILs-3, 6 and 11 stimulated while ILs-10 and 24 inhibited the growth, invasion and migration of both cell lines. Interestingly, ILs-3, 6 and 11 significantly promoted colony formation and increased the expression of SOX2, CD44 and ABCG2 in both prostate cancer cell lines. However, ILs-10 and 24 showed the opposite effect on the expression of these factors. In line with the above findings, treatment with either IL-3 or IL-6 or IL-11 decreased the chemosensitivity to docetaxel while treatment with either IL-10 or IL-24 increased the sensitivity of docetaxel chemotherapy. In conclusion, our results suggest that ILs-3, 6 and 11 function as tumor promoters while ILs-10 and 24 function as tumor suppressors in the prostate cancer cell lines PC-3 and LNCaP in vitro, and such differences may attribute to their different effect on the stemness of PCa cells.

Anti-tumor activity of the ATR inhibitor AZD6738 in HER2 positive breast cancer cells.

PubMed

Kim, Hee-Jun; Min, Ahrum; Im, Seock-Ah; Jang, Hyemin; Lee, Kyung Hun; Lau, Alan; Lee, Miso; Kim, Seongyeong; Yang, Yaewon; Kim, Jungeun; Kim, Tae Yong; Oh, Do-Youn; Brown, Jeffrey; O'Connor, Mark J; Bang, Yung-Jue

2017-01-01

Ataxia telangiectasia and Rad3-related (ATR) proteins are sensors of DNA damage, which induces homologous recombination (HR)-dependent repair. ATR is a master regulator of DNA damage repair (DDR), signaling to control DNA replication, DNA repair and apoptosis. Therefore, the ATR pathway might be an attractive target for developing new drugs. This study was designed to investigate the antitumor effects of the ATR inhibitor, AZD6738 and its underlying mechanism in human breast cancer cells. Growth inhibitory effects of AZD6738 against human breast cancer cell lines were studied using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (methyl thiazolyl tetrazolium, MTT) assay. Cell cycle analysis, Western blotting, immunofluorescence and comet assays were also performed to elucidate underlying mechanisms of AZD6738 action. Anti-proliferative and DDR inhibitory effects of AZD6738 were demonstrated in human breast cancer cell lines. Among 13 cell lines, the IC 50 values of nine cell lines were less than 1 μmol/L using MTT assay. Two cell lines, SK-BR-3 and BT-474, were chosen for further evaluation focused on human epidermal growth factor receptor 2 (HER2)-positive breast cancer cells. Sensitive SK-BR-3 but not the less sensitive BT-474 breast cancer cells showed increased level of apoptosis and S phase arrest and reduced expression levels of phosphorylated check-point kinase 1 (CHK1) and other repair markers. Decreased functional CHK1 expression induced DNA damage accumulation due to HR inactivation. AZD6738 showed synergistic activity with cisplatin. Understanding the antitumor activity and mechanisms of AZD6738 in HER2-positive breast cancer cells creates the possibility for future clinical trials targeting DDR in HER2-positive breast cancer treatment. © 2016 UICC.

ILs-3, 6 and 11 increase, but ILs-10 and 24 decrease stemness of human prostate cancer cells in vitro

PubMed Central

Yu, Dandan; Zhong, Yali; Li, Xiaoran; Li, Yaqing; Li, Xiaoli; Cao, Jing; Fan, Huijie; Yuan, Yuan; Ji, Zhenyu; Qiao, Baoping; Wen, Jian-Guo; Zhang, Mingzhi; Kvalheim, Gunnar; Nesland, Jahn M.; Suo, Zhenhe

2015-01-01

Cancer stem cells (CSCs) are associated with cancer recurrence and metastasis. Prostate cancer cells often metastasize to the bone with a complex microenvironment of cytokines favoring cell survival. In this study, the cell stemness influence of a group of interleukins including IL-3, 6, 10, 11 and 24 on human prostate cancer cell lines LNCaP and PC-3 was explored in vitro. Sulforhodamine B(SRB) and 5-ethynyl-2′-deoxyuridine (EdU) assays were applied to examine the effect on cell proliferation, and wound healing and transwell assays were used for migration and invasion studies, in addition to colony formation, Western blotting and flowcytometry for the expression of stemness factors and chemotherapy sensitivity. We observed that ILs-3, 6 and 11 stimulated while ILs-10 and 24 inhibited the growth, invasion and migration of both cell lines. Interestingly, ILs-3, 6 and 11 significantly promoted colony formation and increased the expression of SOX2, CD44 and ABCG2 in both prostate cancer cell lines. However, ILs-10 and 24 showed the opposite effect on the expression of these factors. In line with the above findings, treatment with either IL-3 or IL-6 or IL-11 decreased the chemosensitivity to docetaxel while treatment with either IL-10 or IL-24 increased the sensitivity of docetaxel chemotherapy. In conclusion, our results suggest that ILs-3, 6 and 11 function as tumor promoters while ILs-10 and 24 function as tumor suppressors in the prostate cancer cell lines PC-3 and LNCaP in vitro, and such differences may attribute to their different effect on the stemness of PCa cells. PMID:26528857

Effects of mycoplasma contamination on phenotypic expression of mitochondrial mutants in human cells.

PubMed Central

Doersen, C J; Stanbridge, E J

1981-01-01

HeLa cells sensitive to the mitochondrial protein synthesis inhibitors erythromycin (ERY) and chloramphenicol (CAP) and HeLa variants resistant to the effects of these drugs were purposefully infected with drug-sensitive and -resistant mycoplasma strains. Mycoplasma hyorhinis and the ERY-resistant strain of Mycoplasma orale, MO-ERYr, did not influence the growth of HeLa and ERY-resistant ERY2301 cells in the presence or absence of ERY. M. hyorhinis also did not affect the growth of HeLa and CAP-resistant Cap-2 cells in the presence or absence of CAP. However, both HeLa and Cap-2 cells infected with the CAP-resistant strain of M. hyorhinis, MH-CAPr, were more sensitive to the cytotoxic effect of CAP. This may be due to the glucose dependence of the cells, which was compromised by the increased utilization of glucose by MH-CAPr in these infected cell cultures. In vitro protein synthesis by isolated mitochondria was significantly altered by mycoplasma infection of the various cell lines. A substantial number of mycoplasmas copurified with the mitochondria, resulting in up to a sevenfold increase in the incorporation of [3H]leucine into the trichloroacetic acid-insoluble material. More importantly, the apparent drug sensitivity or resistance of mitochondrial preparations from mycoplasma-infected cells reflected the drug sensitivity or resistance of the contaminating mycoplasmas. These results illustrate the hazards in interpreting mitochondrial protein synthesis data derived from mycoplasma-infected cell lines, particularly putative mitochondrially encoded mutants resistant to inhibitors of mitochondrial protein synthesis. PMID:6965101

Micro-RNA expression in cisplatin resistant germ cell tumor cell lines

PubMed Central

2011-01-01

Background We compared microRNA expression patterns in three cisplatin resistant sublines derived from paternal cisplatin sensitive germ cell tumor cell lines in order to improve our understanding of the mechanisms of cisplatin resistance. Methods Three cisplatin resistant sublines (NTERA-2-R, NCCIT-R, 2102EP-R) showing 2.7-11.3-fold increase in drug resistance after intermittent exposure to increasing doses of cisplatin were compared to their parental counterparts, three well established relatively cisplatin sensitive germ cell tumor cell lines (NTERA-2, NCCIT, 2102EP). Cells were cultured and total RNA was isolated from all 6 cell lines in three independent experiments. RNA was converted into cDNA and quantitative RT-PCR was run using 384 well low density arrays covering almost all (738) known microRNA species of human origin. Results Altogether 72 of 738 (9.8%) microRNAs appeared differentially expressed between sensitive and resistant cell line pairs (NTERA-2R/NTERA-2 = 43, NCCIT-R/NCCIT = 53, 2102EP-R/2102EP = 15) of which 46.7-95.3% were up-regulated (NTERA-2R/NTERA-2 = 95.3%, NCCIT-R/NCCIT = 62.3%, 2102EP-R/2102EP = 46.7%). The number of genes showing differential expression in more than one of the cell line pairs was 34 between NTERA-2R/NTERA-2 (79%) and NCCIT-R/NCCIT (64%), and 3 and 4, respectively, between these two cell lines and 2102EP-R/2102EP (about 27%). Only the has-miR-10b involved in breast cancer invasion and metastasis and has-miR-512-3p appeared to be up-regulated (2-3-fold) in all three cell lines. The hsa-miR-371-373 cluster (counteracting cellular senescence and linked with differentiation potency), as well as hsa-miR-520c/-520h (inhibiting the tumor suppressor p21) were 3.9-16.3 fold up-regulated in two of the three cisplatin resistant cell lines. Several new micro-RNA species missing an annotation towards cisplatin resistance could be identified. These were hsa-miR-512-3p/-515/-517/-518/-525 (up to 8.1-fold up-regulated) and hsa-miR-99a/-100/-145 (up to 10-fold down-regulated). Conclusion Examining almost all known human micro-RNA species confirmed the miR-371-373 cluster as a promising target for explaining cisplatin resistance, potentially by counteracting wild-type P53 induced senescence or linking it with the potency to differentiate. Moreover, we describe for the first time an association of the up-regulation of micro-RNA species such as hsa-miR-512-3p/-515/-517/-518/-525 and down-regulation of hsa-miR-99a/-100/-145 with a cisplatin resistant phenotype in human germ cell tumors. Further functional analyses are warranted to gain insight into their role in drug resistance. PMID:21575166

The regrowth kinetic of the surviving population is independent of acute and chronic responses to temozolomide in glioblastoma cell lines

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

Silva, Andrew Oliveira, E-mail: andrewbiomed@gmail.com; Dalsin, Eloisa, E-mail: dalsineloisa@gmail.com; Onzi, Giovana Ravizzoni, E-mail: gioonzi@gmail.com

Chemotherapy acts on cancer cells by producing multiple effects on a cell population including cell cycle arrest, necrosis, apoptosis and senescence. However, often a subpopulation of cells survives and the behavior of this subpopulation, which is responsible for cancer recurrence, remains obscure. Here we investigated the in vitro short- and long-term responses of six glioblastoma cell lines to clinically relevant doses of temozolomide for 5 days followed by 23 days of recovery, mimicking the standard schedule used in glioblastoma patient for this drug. These cells presented different profiles of sensitivity to temozolomide with varying levels of cell cycle arrest, autophagymore » and senescence, followed by a regrowth of the surviving cells. The initial reduction in cell number and the subsequent regrowth was analyzed with four new parameters applied to Cumulative Population Doubling (CPD) curves that describe the overall sensitivity of the population and the characteristic of the regrowth: the relative end point CPD (RendCPD); the relative Area Under Curve (rAUC); the Relative Time to Cross a Threshold (RTCT); and the Relative Proliferation Rate (RPR). Surprisingly, the kinetics of regrowth were not predicted by the mechanisms activated after treatment nor by the acute or overall sensitivity. With this study we added new parameters that describe key responses of glioblastoma cell populations to temozolomide treatment. These parameters can also be applied to other cell types and treatments and will help to understand the behavior of the surviving cancer cells after treatment and shed light on studies of cancer resistance and recurrence. - Highlights: • Little is known about the behavior of the glioma cells surviving to TMZ. • The short- and long-term response of six glioma cells lines to TMZ varies considerably. • These glioma cells lines recovered proliferation after therapeutic levels of TMZ. • The growth velocity of the surviving cells was different from the untreated cells. • The kinetic of regrowth was not predicted by any TMZ-triggered mechanism.« less

Protective Role of Hsp27 Protein Against Gamma Radiation-Induced Apoptosis and Radiosensitization Effects of Hsp27 Gene Silencing in Different Human Tumor Cells

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

Aloy, Marie-Therese; Hospices Civils de Lyon, Service de Radiotherapie, Centre Hospitalier Lyon-Sud, Pierre-Benite; Hadchity, Elie

Purpose: The ability of heat shock protein 27 (Hsp27) to protect cells from stressful stimuli and its increased levels in tumors resistant to anticancer therapeutics suggest that it may represent a target for sensitization to radiotherapy. In this study, we investigate the protective role of Hsp27 against radiation-induced apoptosis and the effect of its attenuation in highly expressing radioresistant cancer cell lines. Methods and Materials: We examined clonogenic death and the kinetics of apoptotic events in different tumor cell lines overexpressing or underexpressing Hsp27 protein irradiated with photons. The radiosensitive Jurkat cell line, which does not express Hsp27 constitutively ormore » in response to {gamma}-rays, was stably transfected with Hsp27 complementary DNA. Attenuation of Hsp27 expression was accomplished by antisense or RNAi (interfering RNA) strategies in SQ20B head-and-neck squamous carcinoma, PC3 prostate cancer, and U87 glioblastoma radioresistant cells. Results: We measured concentration-dependent protection against the cytotoxic effects of radiation in Jurkat-Hsp27 cells, which led to a 50% decrease in apoptotic cells at 48 hours in the highest expressing cells. Underlying mechanisms leading to radiation resistance involved a significant increase in glutathione levels associated with detoxification of reactive oxygen species, a delay in mitochondrial collapse, and caspase activation. Conversely, attenuation of Hsp27 in SQ20B cells, characterized by their resistance to apoptosis, sensitizes cells to irradiation. This was emphasized by increased apoptosis, decreased glutathione basal level, and clonogenic cell death. Sensitization to irradiation was confirmed in PC3 and U87 radioresistant cells. Conclusion: Hsp27 gene therapy offers a potential adjuvant to radiation-based therapy of resistant tumors.« less

Arsenic trioxide inhibits cell proliferation and human papillomavirus oncogene expression in cervical cancer cells

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

Wang, Hongtao; Gao, Peng; Zheng, Jie, E-mail: jiezheng54@126.com

Highlights: • As{sub 2}O{sub 3} inhibits growth of cervical cancer cells and expression of HPV oncogenes in these cells. • HPV-negative cervical cancer cells are more sensitive to As{sub 2}O{sub 3} than HPV-positive cervical cancer cells. • HPV-18 positive cervical cancer cells are more sensitive to As{sub 2}O{sub 3} than HPV-16 positive cancer cells. • Down-regulation of HPV oncogenes by As{sub 2}O{sub 3} is partially due to the diminished AP-1 binding. - Abstract: Arsenic trioxide (As{sub 2}O{sub 3}) has shown therapeutic effects in some leukemias and solid cancers. However, the molecular mechanisms of its anticancer efficacy have not been clearlymore » elucidated, particularly in solid cancers. Our previous data showed that As{sub 2}O{sub 3} induced apoptosis of human papillomavirus (HPV) 16 DNA-immortalized human cervical epithelial cells and cervical cancer cells and inhibited the expression of HPV oncogenes in these cells. In the present study, we systemically examined the effects of As{sub 2}O{sub 3} on five human cervical cancer cell lines and explored the possible molecular mechanisms. MTT assay showed that HPV-negative C33A cells were more sensitive to growth inhibition induced by As{sub 2}O{sub 3} than HPV-positive cervical cancer cells, and HPV 18-positive HeLa and C4-I cells were more sensitive to As{sub 2}O{sub 3} than HPV 16-positive CaSki and SiHa cells. After As{sub 2}O{sub 3} treatment, both mRNA and protein levels of HPV E6 and E7 obviously decreased in all HPV positive cell lines. In contrast, p53 and Rb protein levels increased in all tested cell lines. Transcription factor AP-1 protein expression decreased significantly in HeLa, CaSki and C33A cells with ELISA method. These results suggest that As{sub 2}O{sub 3} is a potential anticancer drug for cervical cancer.« less

Accumulation of prohibitin is a common cellular response to different stressing stimuli and protects melanoma cells from ER stress and chemotherapy-induced cell death

PubMed Central

Tortelli, Tharcisio Citrangulo; de Godoy, Lyris Martins Franco; de Souza, Gustavo Antonio; Bonatto, Diego; Otake, Andreia Hanada; de Freitas Saito, Renata; Rosa, Jose Cesar; Greene, Lewis Joel; Chammas, Roger

2017-01-01

Melanoma is responsible for most deaths among skin cancers and conventional and palliative care chemotherapy are limited due to the development of chemoresistance. We used proteomic analysis to identify cellular responses that lead to chemoresistance of human melanoma cell lines to cisplatin. A systems approach to the proteomic data indicated the participation of specific cellular processes such as oxidative phosphorylation, mitochondrial organization and homeostasis, as well as the unfolded protein response (UPR) to be required for the survival of cells treated with cisplatin. Prohibitin (PHB) was among the proteins consistently accumulated, interacting with the functional clusters associated with resistance to cisplatin. We showed PHB accumulated at different levels in melanoma cell lines under stressing stimuli, such as (i) treatment with temozolomide (TMZ), dacarbazine (DTIC) and cisplatin; (ii) serum deprivation; (iii) tunicamycin, an UPR inducer. Prohibitin accumulated in the mitochondria of melanoma cells after cisplatin and tunicamycin treatment and its de novo accumulation led to chemoresistance melanoma cell lines. In contrast, PHB knock-down sensitized melanoma cells to cisplatin and tunicamycin treatment. We conclude that PHB participates in the survival of cells exposed to different stress stimuli, and can therefore serve as a target for the sensitization of melanoma cells to chemotherapy. PMID:28562344

[Identification of occult disseminated tumor cells by recombinant herpes simplex virus expressing GFP (HSV(GFP))].

PubMed

Han, Xiang-ping; Shi, Gui-lan; Wang, Cheng-feng; Li, Jie; Zhang, Jian-wei; Zhang, Yu; Zhang, Shu-ren; Liu, Bin-lei

2012-12-01

To develop a novel rapid protocol for the detection of occult disseminated tumor cells by a recombinant herpes simplex virus expressing GFP (HSV(GFP)). Tumor cells of seven cell lines were exposed to HSV(GFP) and then examined for GFP expression by fluorescence microscopy. Various numbers of tumor cells (10, 100, 1000, 10 000) were mixed into 2 ml human whole blood, separated with lymphocytes separation medium, exposed to HSV(GFP), incubated at 37°C for 6 - 24 h and then counted for the number of green cells under the fluorescence microscope. Some clinical samples including peripheral blood, pleural effusion, ascites, spinal fluid from tumor-bearing patients were screened using this protocol in parallel with routine cytological examination. HSV(GFP) was able to infect all 7 tumor cell lines indicating that the HSV(GFP) can be used to detect different types of tumor cells. The detection sensitivity was 10 cancer cells in 2 ml whole blood. In the clinical samples, there were 4/15 positive by routine cytological examination but 11/15 positive by HSV(GFP), indicating a higher sensitivity of this new protocol. Recombinant herpes simplex virus-mediated green fluorescence is a simple and sensitive technique for the identification of occult disseminated cancer cells including circulating tumor cells (CTCs).

Influence of Mesenchymal Stem Cells Conditioned Media on Proliferation of Urinary Tract Cancer Cell Lines and Their Sensitivity to Ciprofloxacin.

PubMed

Maj, Malgorzata; Bajek, Anna; Nalejska, Ewelina; Porowinska, Dorota; Kloskowski, Tomasz; Gackowska, Lidia; Drewa, Tomasz

2017-06-01

Mesenchymal stem cells (MSCs) are known to interact with cancer cells through direct cell-to-cell contact and secretion of paracrine factors, although their exact influence on tumor progression in vivo remains unclear. To better understand how fetal and adult stem cells affect tumors, we analyzed viability of human renal (786-0) and bladder (T24) carcinoma cell lines cultured in conditioned media harvested from amniotic fluid-derived stem cells (AFSCs) and adipose-derived stem cells (ASCs). Both media reduced metabolic activity of 786-0 cells, however, decreased viability of T24 cells was noted only after incubation with conditioned medium from ASCs. To test the hypothesis that MSCs-secreted factors might be involved in chemoresistance acquisition, we further analyzed influence of mesenchymal stem cell conditioned media (MSC-CM) on cancer cells sensitivity to ciprofloxacin, that is considered as potential candidate agent for urinary tract cancers treatment. Significantly increased resistance to tested drug indicates that MSCs may protect cancer cells from chemotherapy. J. Cell. Biochem. 118: 1361-1368, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Expression of surface markers on the human monocytic leukaemia cell line, THP-1, as indicators for the sensitizing potential of chemicals.

PubMed

An, Susun; Kim, Seoyoung; Huh, Yong; Lee, Tae Ryong; Kim, Han-Kon; Park, Kui-Lea; Eun, Hee Chul

2009-04-01

Evaluation of skin sensitization potential is an important part of the safety assessment of cosmetic ingredients and topical drugs. Recently, evaluation of changes in surface marker expression induced in dendritic cells (DC) or DC surrogate cell lines following exposure to chemicals represents one approach for in vitro test methods. The study aimed to test the change of expression patterns of surface markers on THP-1 cells by chemicals as a predictive in vitro method for contact sensitization. We investigated the expression of CD54, CD86, CD83, CD80, and CD40 after a 1-day exposure to sensitizers (1-chloro-2,4-dinitrobenzene; 2,4-dinitrofluorobenzene; benzocaine; 5-chloro-2-methyl-4-isothiazolin-3-one; hexyl cinnamic aldehyde; eugenol; nickel sulfate hexahydrate; potassium dichromate; cobalt sulfate; 2-mercaptobenzothiazole; and ammonium tetrachloroplatinate) and non-sensitizers (sodium lauryl sulfate, benzalkonium chloride, lactic acid, salicylic acid, isopropanol, and dimethyl sulphoxide). The test concentrations were 0.1x, 0.5x, and 1x of the 50% inhibitory concentration, and the relative fluorescence intensity was used as an expression indicator. By evaluating the expression patterns of CD54, CD86, and CD40, we could classify the chemicals as sensitizers or non-sensitizers, but CD80 and CD83 showed non-specific patterns of expression. These data suggest that the THP-1 cells are good model for screening contact sensitizers and CD40 could be a useful marker complementary to CD54 and CD86.

Drug/Cell-line Browser: interactive canvas visualization of cancer drug/cell-line viability assay datasets.

PubMed

Duan, Qiaonan; Wang, Zichen; Fernandez, Nicolas F; Rouillard, Andrew D; Tan, Christopher M; Benes, Cyril H; Ma'ayan, Avi

2014-11-15

Recently, several high profile studies collected cell viability data from panels of cancer cell lines treated with many drugs applied at different concentrations. Such drug sensitivity data for cancer cell lines provide suggestive treatments for different types and subtypes of cancer. Visualization of these datasets can reveal patterns that may not be obvious by examining the data without such efforts. Here we introduce Drug/Cell-line Browser (DCB), an online interactive HTML5 data visualization tool for interacting with three of the recently published datasets of cancer cell lines/drug-viability studies. DCB uses clustering and canvas visualization of the drugs and the cell lines, as well as a bar graph that summarizes drug effectiveness for the tissue of origin or the cancer subtypes for single or multiple drugs. DCB can help in understanding drug response patterns and prioritizing drug/cancer cell line interactions by tissue of origin or cancer subtype. DCB is an open source Web-based tool that is freely available at: http://www.maayanlab.net/LINCS/DCB CONTACT: avi.maayan@mssm.edu Supplementary data are available at Bioinformatics online. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Interferon-alpha and interferon-gamma modulate Fas-mediated apoptosis in mitomycin-C-resistant human Tenon's fibroblasts.

PubMed

Wang, Xiao Yang; Crowston, Jonathan G; White, Andrew J R; Zoellner, Hans; Healey, Paul R

2014-08-01

The aim of the study was to investigate, using a native mitomycin-C-resistant human Tenon's fibroblast cell line, the possibility that interferon-alpha and gamma could be used with Fas agonists as an alternative anti-fibrotic strategy to mitomycin-C in trabeculectomy. A clinically resistant and in vitro verified mitomycin-C-resistant human Tenon's fibroblast cell line was pretreated with interferon-alpha and interferon-gamma for 48 h before stimulation with an agonistic Fas antibody (CH11) for 2 days to induce cell death. Cell death assays were undertaken. Changes in apoptosis-related proteins were determined by flow cytometry and Western blot. Pretreatment with interferon-alpha or interferon-gamma for 48 h increased Fas, Fas-associated protein with death domain and caspase-8 expression. Protein expression was further increased by combined exposure to interferon-alpha and gamma. Pretreatment with cytokines had no effect on Fas-L and Bcl-2. Interferon-alpha alone did not change the rate of induced cell death. A combination of interferon-alpha and gamma synergistically increased the sensitivity of mitomycin-C-resistant human Tenon's fibroblast cell line to induced cell death. An antagonistic anti-Fas antibody (ZB4) completely blocked induced cell death. Broad caspase inhibitors specific for caspases-8 and -3 reduced induced deaths in interferon pretreated mitomycin-C-resistant human Tenon's fibroblast cell line in a dose-dependent manner. Interferon-alpha and interferon-gamma render mitomycin-C-resistant human Tenon's fibroblast cell line sensitive to Fas-mediated apoptosis. The mechanism involves increased death-inducing signalling complex formation by upregulation of Fas, Fas-associated protein with death domain and caspase-8 expression. © 2013 Royal Australian and New Zealand College of Ophthalmologists.

Sigma-2 ligands and PARP inhibitors synergistically trigger cell death in breast cancer cells

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

McDonald, Elizabeth S.; Mankoff, Julia; Makvandi, Mehran

The sigma-2 receptor is overexpressed in proliferating cells compared to quiescent cells and has been used as a target for imaging solid tumors by positron emission tomography. Recent work has suggested that the sigma-2 receptor may also be an effective therapeutic target for cancer therapy. Poly (ADP-ribose) polymerase (PARP) is a family of enzymes involved in DNA damage response. In this study, we looked for potential synergy of cytotoxicity between PARP inhibitors and sigma-2 receptor ligands in breast cancer cell lines. We showed that the PARP inhibitor, YUN3-6, sensitized mouse breast cancer cell line, EMT6, to sigma-2 receptor ligand (SV119,more » WC-26, and RHM-138) induced cell death determined by cell viability assay and colony forming assay. The PARP inhibitor, olaparib, sensitized tumor cells to a different sigma-2 receptor ligand SW43-induced apoptosis and cell death in human triple negative cell line, MDA-MB-231. Olaparib inhibited PARP activity and cell proliferation, and arrested cells in G2/M phase of the cell cycle in MDA-MB-231 cells. Subsequently cells became sensitized to SW43 induced cell death. In conclusion, the combination of sigma-2 receptor ligands and PARP inhibitors appears to hold promise for synergistically triggering cell death in certain types of breast cancer cells and merits further investigation. - Highlights: • PARPi, YUN3-6 and olaparib, and σ2 ligands, SV119 and SW43, were evaluated. • Mouse and human breast cancer cells, EMT6 and MDA-MB-231 respectively, were used. • YUN3-6 and SV119 synergistically triggered cell death in EMT6 cells. • Olaparib and SW43 additively triggered cell death in MDA-MB-231 cells. • Olaparib arrested cells in G2/M in MDA-MB-231 cells.« less

The Flint Animal Cancer Center (FACC) Canine Tumour Cell Line Panel: a resource for veterinary drug discovery, comparative oncology and translational medicine.

PubMed

Fowles, J S; Dailey, D D; Gustafson, D L; Thamm, D H; Duval, D L

2017-06-01

Mammalian cell tissue culture has been a critical tool leading to our current understanding of cancer including many aspects of cellular transformation, growth and response to therapies. The current use of large panels of cell lines with associated phenotypic and genotypic information now allows for informatics approaches and in silico screens to rapidly test hypotheses based on simple as well as complex relationships. Current cell line panels with large amounts of associated drug sensitivity and genomics data are comprised of human cancer cell lines (i.e. NCI60 and GDSC). There is increased recognition of the contribution of canine cancer to comparative cancer research as a spontaneous large animal model with application in basic and translational studies. We have assembled a panel of canine cancer cell lines to facilitate studies in canine cancer and report here phenotypic and genotypic data associated with these cells. © 2016 John Wiley & Sons Ltd.

Zinc Finger Nuclease Mediated Knockout of ADP-Dependent Glucokinase in Cancer Cell Lines: Effects on Cell Survival and Mitochondrial Oxidative Metabolism

PubMed Central

Richter, Susan; Morrison, Shona; Connor, Tim; Su, Jiechuang; Print, Cristin G.; Ronimus, Ron S.; McGee, Sean L.; Wilson, William R.

2013-01-01

Zinc finger nucleases (ZFN) are powerful tools for editing genes in cells. Here we use ZFNs to interrogate the biological function of ADPGK, which encodes an ADP-dependent glucokinase (ADPGK), in human tumour cell lines. The hypothesis we tested is that ADPGK utilises ADP to phosphorylate glucose under conditions where ATP becomes limiting, such as hypoxia. We characterised two ZFN knockout clones in each of two lines (H460 and HCT116). All four clones had frameshift mutations in all alleles at the target site in exon 1 of ADPGK, and were ADPGK-null by immunoblotting. ADPGK knockout had little or no effect on cell proliferation, but compromised the ability of H460 cells to survive siRNA silencing of hexokinase-2 under oxic conditions, with clonogenic survival falling from 21±3% for the parental line to 6.4±0.8% (p = 0.002) and 4.3±0.8% (p = 0.001) for the two knockouts. A similar increased sensitivity to clonogenic cell killing was observed under anoxia. No such changes were found when ADPGK was knocked out in HCT116 cells, for which the parental line was less sensitive than H460 to anoxia and to hexokinase-2 silencing. While knockout of ADPGK in HCT116 cells caused few changes in global gene expression, knockout of ADPGK in H460 cells caused notable up-regulation of mRNAs encoding cell adhesion proteins. Surprisingly, we could discern no consistent effect on glycolysis as measured by glucose consumption or lactate formation under anoxia, or extracellular acidification rate (Seahorse XF analyser) under oxic conditions in a variety of media. However, oxygen consumption rates were generally lower in the ADPGK knockouts, in some cases markedly so. Collectively, the results demonstrate that ADPGK can contribute to tumour cell survival under conditions of high glycolytic dependence, but the phenotype resulting from knockout of ADPGK is cell line dependent and appears to be unrelated to priming of glycolysis in these lines. PMID:23799003

Radiation sensitivities of 31 human oesophageal squamous cell carcinoma cell lines

PubMed Central

Ban, Sadayuki; Michikawa, Yuichi; Ishikawa, Ken-ichi; Sagara, Masashi; Watanabe, Koji; Shimada, Yutaka; Inazawa, Johji; Imai, Takashi

2005-01-01

The purpose of this study was to determine the radiosensitivities of 31 human oesophageal squamous cell carcinoma cell lines with a colony-formation assay. A large variation in radiosensitivity existed among 31 cell lines. Such a large variation may partly explain the poor result of radiotherapy for this cancer. One cell line (KYSE190) demonstrated an unusual radiosensitivity. Ataxia-telangiectasia-mutated (ATM) gene in these cells had five missense mutations, and ATM protein was truncated or degraded. Inability to phosphorylate Chk2 in the irradiated KYSE190 cells suggests that the ATM protein in these cells had lost its function. The dysfunctional ATM protein may be a main cause of unusual radiosensitivity of KYSE190 cells. Because the donor of these cells was not diagnosed with ataxia telangiectasia, mutations in ATM gene might have occurred during the initiation and progression of cancer. Radiosensitive cancer developed in non-hereditary diseased patients must be a good target for radiotherapy. PMID:16045545

Intrinsic resistance to the lethal effects of x-irradiation in insect and arachnid cells

PubMed Central

Koval, Thomas M.

1983-01-01

Twelve cell lines representing 10 genera of three orders (Diptera, Lepidoptera, and Orthoptera) of the class Insecta and one cell line (Acarina) from the class Arachnida were examined to discern their sensitivity to the lethal effects of x-irradiation. Radiosensitivity was measured by a combination of colony formation and population growth curve techniques. Each of these arthropod cell lines is significantly more radioresistant than mammalian cells, though the degree of resistance varies greatly with order. Dipteran cells are 3 to 9 times and lepidopteran cells 52 to 104 times more radioresistant than mammalian cells. Orthopteran and acarine cells are intermediate in radiosensitivity between dipteran and lepidopteran cells. These cells, especially the lepidopteran, should be valuable in determining the molecular nature of repair mechanisms that result in resistance to ionizing radiation. PMID:16593348

Zidovudine, abacavir and lamivudine increase the radiosensitivity of human esophageal squamous cancer cell lines.

PubMed

Chen, Xuan; Wang, Cong; Guan, Shanghui; Liu, Yuan; Han, Lihui; Cheng, Yufeng

2016-07-01

Telomerase is a type of reverse transcriptase that is overexpressed in almost all human tumor cells, but not in normal tissues, which provides an opportunity for radiosensitization targeting telomerase. Zidovudine, abacavir and lamivudine are reverse transcriptase inhibitors that have been applied in clinical practice for several years. We sought to explore the radiosensitization effect of these three drugs on human esophageal cancer cell lines. Eca109 and Eca9706 cells were treated with zidovudine, abacavir and lamivudine for 48 h before irradiation was administered. Samples were collected 1 h after irradiation. Clonal efficiency assay was used to evaluate the effect of the combination of these drugs with radiation doses of 2, 4, 6 and 8 Gy. DNA damage was measured by comet assay. Telomerase activity (TA) and relative telomere length (TL) were detected and evaluated by real-time PCR. Apoptosis rates were assessed by flow cytometric analysis. The results showed that all the drugs tested sensitized the esophageal squamous cell carcinoma (ESCC) cell lines to radiation through an increase in radiation-induced DNA damage and cell apoptosis, deregulation of TA and decreasing the shortened TL caused by radiation. Each of the drugs investigated (zidovudine, abacavir and lamivudine) could be used for sensitizing human esophageal cancer cell lines to radiation. Consequently, the present study supports the potential of these three drugs as therapeutic agents for the radiosensitization of esophageal squamous cell cancer.

Genistein enhances the effect of epidermal growth factor receptor tyrosine kinase inhibitors and inhibits nuclear factor kappa B in nonsmall cell lung cancer cell lines.

PubMed

Gadgeel, Shirish M; Ali, Shadan; Philip, Philip A; Wozniak, Antoinette; Sarkar, Fazlul H

2009-05-15

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) have shown modest clinical benefit in patients with relapsed nonsmall cell lung cancer (NSCLC). Down-regulation of Akt appears to correlate with the antitumor activity of EGFR-TKIs. Akt activates nuclear factor kappa B (NF-kappaB), which transcribes genes important for cell survival, invasion, and metastasis. The authors hypothesized that genistein, through the inhibition of NF-kappaB, could enhance the activity of EGFR-TKIs in NSCLCs. Three NSCLC cell lines with various EGFR mutation status and sensitivities to EGFR-TKIs were selected: H3255 (L858R), H1650 (del E746-A750), and H1781 (wild-type EGFR). Cells were treated with erlotinib, gefitinib, genistein, or the combination of each of the EGFR-TKIs with genistein. Cell survival and apoptosis were assessed, and expression levels of EGFR, pAkt, cyclooxygenase-2 (COX-2), E-cadherin, prostaglandin E(2) (PGE(2)), and NF-kappaB were measured. Both EGFR-TKIs demonstrated growth inhibition and apoptosis in each of the cell lines, but H1650 and H1781 were much less sensitive. Genistein demonstrated some antitumor activity in all cell lines, but enhanced growth inhibition and apoptosis when combined with the EGFR-TKIs in each of the cell lines. Both combinations down-regulated NF-kappaB significantly more than either agent alone in H3255. In addition, the combinations reduced the expression of EGFR, pAkt, COX-2, and PGE(2,) consistent with inactivation of NF-kappaB. The authors concluded that genistein enhances the antitumor effects of EGFR-TKIs in 3 separate NSCLC cell lines. This enhanced activity is in part because of greater reduction in the DNA-binding activity of NF-kappaB when EGFR-TKIs were combined with genistein.

Isolation and characterization of an IGROV-1 human ovarian cancer cell line made resistant to Ecteinascidin-743 (ET-743)

PubMed Central

Erba, E; Bergamaschi, D; Bassano, L; Ronzoni, S; Liberti, G Di; Muradore, I; Vignati, S; Faircloth, G; Jimeno, J; D'Incalci, M

2000-01-01

By exposing Igrov-1 human ovarian cancer cells to increasing concentrations of Ecteinascidin-743 (ET-743), either for a short or prolonged time, we obtained sublines resistant to ET-743 which overexpress Pgp. The most resistant clone (Igrov-1/25 ET) was evaluated for biological and pharmacological characterizations. The increased Pgp levels of Igrov-1/25 ET were not due to amplification of the mdr-1 gene but to increased mRNA levels. No increase in other multidrug resistance-related proteins such as MRP or LRP was observed in Igrov-1/25 ET. The IC50values of ET-743 against Igrov-1/25 ET was approximately 50 times higher than the parental cell line. Resistance was not reversed while maintaining the cell line in drug-free medium for at least 24 months. Igrov-1/25 ET was cross-resistant to Doxorubicin and VP16 while it was equally sensitive to L-PAM, MNNG, CPT and only marginally less sensitive to Cis-DDP and Oxaliplatin compared to the parental cell line. Igrov-1/25 ET exposed to Doxorubicin retained this drug much less, mainly because of a more efficient drug efflux. The cyclosporine analogue SDZ PSC-833 reversed the resistance of Igrov-1/25 ET to ET-743, without any enhancement of the drug activity against the parental Igrov-1 cell line. Igrov-1/25 ET exhibits typical features of cell lines overexpressing the mdr-1 gene and can be a potentially useful tool in selecting ET-743 non-cross-resistant analogues as well as to investigate methods to counteract resistance to this drug. © 2000 Cancer Research Campaign PMID:10817511

Preclinical evaluation of dasatinib alone and in combination with cabozantinib for the treatment of diffuse intrinsic pontine glioma

PubMed Central

Philippe, Cathy; Paulsson, Janna; Andreiuolo, Felipe; Guerrini-Rousseau, Léa; Cornilleau, Gaétan; Le Dret, Ludivine; Richon, Catherine; Lacroix, Ludovic; Puget, Stéphanie; Geoerger, Birgit; Vassal, Gilles; Östman, Arne; Grill, Jacques

2015-01-01

Abstract Background Platelet-derived growth factor receptor A is altered by amplification and/or mutation in diffuse intrinsic pontine glioma (DIPG). We explored in vitro on new DIPG models the efficacy of dasatinib, a multi-tyrosine kinase inhibitor targeting this receptor. Methods Gene expression profiles were generated from 41 DIPGs biopsied at diagnosis and compared with the signature associated with sensitivity/resistance to dasatinib. A panel of 12 new DIPG cell lines were established from biopsy at diagnosis, serially passaged, and characterized by gene expression analyses. Effects of dasatinib (1–10 μM) on proliferation, invasion, and cytotoxicity were determined on 4 of these cell lines using live-cell imaging and flow cytometry assays. Downstream signaling and receptor tyrosine kinases (RTKs) were assessed by western blot and phospho-RTK array. The effect of the combination with the c-Met inhibitor cabozantinib was studied on cellular growth and invasion analyzed by the Chou–Talaly method. Results DIPG primary tumors and cell lines exhibited the gene expression signature of sensitivity to dasatinib. Dasatinib reduced proliferation (half-maximal inhibitory concentration = 10–100 nM) and invasion (30%–60% reduction) at 100 nM in 4/4 cultures and induced apoptosis in 1 of 4 DIPG cell lines. Activity of downstream effectors of dasatinib targets including activin receptor 1 was strongly reduced. Since multiple RTKs were activated simultaneously in DIPG cell lines, including c-Met, which can be also amplified in DIPG, the benefit of the combination of dasatinib with cabozantinib was explored for its synergistic effects on proliferation and migration/invasion in these cell lines. Conclusion Dasatinib exhibits antitumor effects in vitro that could be increased by the combination with another RTK inhibitor targeting c-Met. PMID:25534822

Using the MCF10A/MCF10CA1a Breast Cancer Progression Cell Line Model to Investigate the Effect of Active, Mutant Forms of EGFR in Breast Cancer Development and Treatment Using Gefitinib

PubMed Central

Bessette, Darrell C.; Tilch, Erik; Seidens, Tatjana; Quinn, Michael C. J.; Wiegmans, Adrian P.; Shi, Wei; Cocciardi, Sibylle; McCart-Reed, Amy; Saunus, Jodi M.; Simpson, Peter T.; Grimmond, Sean M.; Lakhani, Sunil R.; Khanna, Kum Kum; Waddell, Nic; Al-Ejeh, Fares; Chenevix-Trench, Georgia

2015-01-01

Background Basal-like and triple negative breast cancer (TNBC) share common molecular features, poor prognosis and a propensity for metastasis to the brain. Amplification of epidermal growth factor receptor (EGFR) occurs in ~50% of basal-like breast cancer, and mutations in the epidermal growth factor receptor (EGFR) have been reported in up to ~ 10% of Asian TNBC patients. In non-small cell lung cancer several different mutations in the EGFR tyrosine kinase domain confer sensitivity to receptor tyrosine kinase inhibitors, but the tumourigenic potential of EGFR mutations in breast cells and their potential for targeted therapy is unknown. Materials and Methods Constructs containing wild type, G719S or E746-A750 deletion mutant forms of EGFR were transfected into the MCF10A breast cells and their tumorigenic derivative, MCF10CA1a. The effects of EGFR over-expression and mutation on proliferation, migration, invasion, response to gefitinib, and tumour formation in vivo was investigated. Copy number analysis and whole exome sequencing of the MCF10A and MCF10CA1a cell lines were also performed. Results Mutant EGFR increased MCF10A and MCF10CA1a proliferation and MCF10A gefitinib sensitivity. The EGFR-E746-A750 deletion increased MCF10CA1a cell migration and invasion, and greatly increased MCF10CA1a xenograft tumour formation and growth. Compared to MCF10A cells, MCF10CA1a cells exhibited large regions of gain on chromosomes 3 and 9, deletion on chromosome 7, and mutations in many genes implicated in cancer. Conclusions Mutant EGFR enhances the oncogenic properties of MCF10A cell line, and increases sensitivity to gefitinib. Although the addition of EGFR E746-A750 renders the MCF10CA1a cells more tumourigenic in vivo it is not accompanied by increased gefitinib sensitivity, perhaps due to additional mutations, including the PIK3CA H1047R mutation, that the MCF10CA1a cell line has acquired. Screening TNBC/basal-like breast cancer for EGFR mutations may prove useful for directing therapy but, as in non-small cell lung cancer, accompanying mutations in PIK3CA may confer gefitinib resistance. PMID:25969993

Therapeutic efficacy of interleukin-2 activated killer cells against adriamycin resistant mouse B16-BL6 melanoma.

PubMed

Gautam, S C; Chikkala, N F; Lewis, I; Grabowski, D R; Finke, J H; Ganapathi, R

1992-01-01

Development of multidrug-resistance (MDR) remains a major cause of failure in the treatment of cancer with chemotherapeutic agents. In our efforts to explore alternative treatment regimens for multidrug-resistant tumors we have examined the sensitivity of MDR tumor cell lines to lymphokine activated killer (LAK) cells. Adriamycin (ADM) resistant B16-BL6 melanoma, L1210 and P388 leukemic cell lines were tested for sensitivity to lysis by LAK cells in vitro. While ADM-resistant B16-BL6 and L1210 sublines were found to exhibit at least 2-fold greater susceptibility to lysis by LAK cells, sensitivity of ADM-resistant P388 cell was similar to that of parental cells. Since ADM-resistant B16-BL6 cells were efficiently lysed by LAK cells in vitro, the efficacy of therapy with LAK cells against the ADM-resistant B16-BL6 subline in vivo was evaluated. Compared to mice bearing parental B16-BL6 tumor cells, the adoptive transfer of LAK cells and rIL2 significantly reduced formation of experimental metastases (P less than 0.009) and extended median survival time (P less than 0.001) of mice bearing ADM-resistant B16-BL6 tumor cells. Results suggest that immunotherapy with LAK cells and rIL2 may be a useful modality in the treatment of cancers with the MDR phenotype.

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.

Sensitization of interferon-γ induced apoptosis in human osteosarcoma cells by extracellular S100A4

PubMed Central

Pedersen, Kjetil Boye; Andersen, Kristin; Fodstad, Øystein; Mælandsmo, Gunhild Mari

2004-01-01

Background S100A4 is a small Ca2+-binding protein of the S100 family with metastasis-promoting properties. Recently, secreted S100A4 protein has been shown to possess a number of functions, including induction of angiogenesis, stimulation of cell motility and neurite extension. Methods Cell cultures from two human osteosarcoma cell lines, OHS and its anti-S100A4 ribozyme transfected counterpart II-11b, was treated with IFN-γ and recombinant S100A4 in order to study the sensitizing effects of extracellular S100A4 on IFN-γ mediated apoptosis. Induction of apoptosis was demonstrated by DNA fragmentation, cleavage of poly (ADP-ribose) polymerase and Lamin B. Results In the present work, we found that the S100A4-expressing human osteosarcoma cell line OHS was more sensitive to IFN-γ-mediated apoptosis than the II-11b cells. S100A4 protein was detected in conditioned medium from OHS cells, but not from II-11b cells, and addition of recombinant S100A4 to the cell medium sensitized II-11b cells to apoptosis induced by IFN-γ. The S100A4/IFN-γ-mediated induction of apoptosis was shown to be independent of caspase activation, but dependent on the formation of reactive oxygen species. Furthermore, addition of extracellular S100A4 was demonstrated to activate nuclear factor-κB (NF-κB). Conclusion In conclusion, we have shown that S100A4 sensitizes osteosarcoma cells to IFN-γ-mediated induction of apoptosis. Additionally, extracellular S100A4 activates NF-κB, but whether these events are causally related remains unknown. PMID:15318945

Sensitivity of imatinib-resistant T315I BCR-ABL CML to a synergistic combination of ponatinib and forskolin treatment.

PubMed

Oaxaca, Derrick M; Yang-Reid, Sun Ah; Ross, Jeremy A; Rodriguez, Georgialina; Staniswalis, Joan G; Kirken, Robert A

2016-09-01

Tyrosine kinase inhibitors (TKIs) have dramatically improved the life expectancy of patients suffering from chronic myeloid leukemia (CML); however, patients will eventually develop resistance to TKI therapy or adverse side effects due to secondary off-target mechanisms associated with TKIs. CML patients exhibiting TKI resistance are at greater risk of developing an aggressive and drug-insensitive disease. Drug-resistant CML typically arises in response to spontaneous mutations within the drug binding sites of the targeted oncoproteins. To better understand the mechanism of drug resistance in TKI-resistant CML patients, the BCR-ABL transformed cell line KCL22 was grown with increasing concentrations of imatinib for a period of 6 weeks. Subsequently, a drug-resistant derivative of the parental KCL22 cell line harboring the T315I gatekeeper mutation was isolated and investigated for TKI drug sensitivity via multi-agent drug screens. A synergistic combination of ponatinib- and forskolin-reduced cell viability was identified in this clinically relevant imatinib-resistant CML cell line, which also proved efficacious in other CML cell lines. In summary, this study provides new insight into the biological underpinnings of BCR-ABL-driven CML and potential rationale for investigating novel treatment strategies for patients with T315I CML.

Characterization of three human cell line models for high-throughput neuronal cytotoxicity screening.

PubMed

Tong, Zhi-Bin; Hogberg, Helena; Kuo, David; Sakamuru, Srilatha; Xia, Menghang; Smirnova, Lena; Hartung, Thomas; Gerhold, David

2017-02-01

More than 75 000 man-made chemicals contaminate the environment; many of these have not been tested for toxicities. These chemicals demand quantitative high-throughput screening assays to assess them for causative roles in neurotoxicities, including Parkinson's disease and other neurodegenerative disorders. To facilitate high throughput screening for cytotoxicity to neurons, three human neuronal cellular models were compared: SH-SY5Y neuroblastoma cells, LUHMES conditionally-immortalized dopaminergic neurons, and Neural Stem Cells (NSC) derived from human fetal brain. These three cell lines were evaluated for rapidity and degree of differentiation, and sensitivity to 32 known or candidate neurotoxicants. First, expression of neural differentiation genes was assayed during a 7-day differentiation period. Of the three cell lines, LUHMES showed the highest gene expression of neuronal markers after differentiation. Both in the undifferentiated state and after 7 days of neuronal differentiation, LUHMES cells exhibited greater cytotoxic sensitivity to most of 32 suspected or known neurotoxicants than SH-SY5Y or NSCs. LUHMES cells were also unique in being more susceptible to several compounds in the differentiating state than in the undifferentiated state; including known neurotoxicants colchicine, methyl-mercury (II), and vincristine. Gene expression results suggest that differentiating LUHMES cells may be susceptible to apoptosis because they express low levels of anti-apoptotic genes BCL2 and BIRC5/survivin, whereas SH-SY5Y cells may be resistant to apoptosis because they express high levels of BCL2, BIRC5/survivin, and BIRC3 genes. Thus, LUHMES cells exhibited favorable characteristics for neuro-cytotoxicity screening: rapid differentiation into neurons that exhibit high level expression neuronal marker genes, and marked sensitivity of LUHMES cells to known neurotoxicants. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Effect of carbamate pesticides on perforin, granzymes A-B-3/K, and granulysin in human natural killer cells.

PubMed

Li, Qing; Kobayashi, Maiko; Kawada, Tomoyuki

2015-09-01

We previously found that ziram, a carbamate pesticide, significantly reduced perforin, granzyme A (GrA), granzyme B (GrB), granzyme 3/K (Gr3/K), and granulysin (GRN) levels in NK-92MI cells, a human natural killer (NK) cell line. To investigate whether other carbamate pesticides also show similar toxicity on human NK cells, we conducted further experiments with NK-92CI cells, a human NK cell line, using a more sensitive assay. We previously confirmed that NK-92CI cells express CD56, perforin, GrA, GrB, Gr3/K, and GRN and are highly cytotoxic to K562 cells in a chromium release assay, which are more sensitive to organophosphorus pesticides and ziram than the NK-92MI cell line. NK-92CI cells were treated with ziram, thiram, maneb, or carbaryl at various concentrations for 4-24 h at 37°C in vitro. Thereafter, intracellular levels of perforin, GrA, GrB, Gr3/K, and GRN were determined by flow cytometry. It was found that all carbamate pesticides significantly reduced the intracellular levels of perforin, GrA, GrB, Gr3/K, and GRN in NK-92CI cells in a dose-dependent manner. However, the strength of the effect differed among the pesticides, and the order was thiram > ziram > maneb > carbaryl. In addition, it was also found that the degree of the reductions differed among the five proteins, with perforin more sensitive to pesticides than GRN, GrA, GrB, and Gr3/K, and the order was perforin > GRN > Gr3/K ≒ GrA ≒ GrB. © The Author(s) 2015.

Mapping genetic vulnerabilities reveals BTK as a novel therapeutic target in oesophageal cancer.

PubMed

Chong, Irene Yushing; Aronson, Lauren; Bryant, Hanna; Gulati, Aditi; Campbell, James; Elliott, Richard; Pettitt, Stephen; Wilkerson, Paul; Lambros, Maryou B; Reis-Filho, Jorge S; Ramessur, Anisha; Davidson, Michael; Chau, Ian; Cunningham, David; Ashworth, Alan; Lord, Christopher J

2017-08-22

Oesophageal cancer is the seventh most common cause of cancer-related death worldwide. Disease relapse is frequent and treatment options are limited. To identify new biomarker-defined therapeutic approaches for patients with oesophageal cancer, we integrated the genomic profiles of 17 oesophageal tumour-derived cell lines with drug sensitivity data from small molecule inhibitor profiling, identifying drug sensitivity effects associated with cancer driver gene alterations. We also interrogated recently described RNA interference screen data for these tumour cell lines to identify candidate genetic dependencies or vulnerabilities that could be exploited as therapeutic targets. By integrating the genomic features of oesophageal tumour cell lines with siRNA and drug screening data, we identified a series of candidate targets in oesophageal cancer, including a sensitivity to inhibition of the kinase BTK in MYC amplified oesophageal tumour cell lines. We found that this genetic dependency could be elicited with the clinical BTK/ERBB2 kinase inhibitor, ibrutinib. In both MYC and ERBB2 amplified tumour cells, ibrutinib downregulated ERK-mediated signal transduction, cMYC Ser-62 phosphorylation and levels of MYC protein, and elicited G 1 cell cycle arrest and apoptosis, suggesting that this drug could be used to treat biomarker-selected groups of patients with oesophageal cancer. BTK represents a novel candidate therapeutic target in oesophageal cancer that can be targeted with ibrutinib. On the basis of this work, a proof-of-concept phase II clinical trial evaluating the efficacy of ibrutinib in patients with MYC and/or ERBB2 amplified advanced oesophageal cancer is currently underway (NCT02884453). NCT02884453; Pre-results. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

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.

Overcoming Acquired Resistance to AZD9291, A Third-Generation EGFR Inhibitor, through Modulation of MEK/ERK-Dependent Bim and Mcl-1 Degradation.

PubMed

Shi, Puyu; Oh, You-Take; Deng, Liang; Zhang, Guojing; Qian, Guoqing; Zhang, Shuo; Ren, Hui; Wu, Grant; Legendre, Benjamin; Anderson, Emily; Ramalingam, Suresh S; Owonikoko, Taofeek K; Chen, Mingwei; Sun, Shi-Yong

2017-11-01

Purpose: The mechanisms accounting for anticancer activity of AZD9291 (osimertinib or TAGRISSO), an approved third-generation EGFR inhibitor, in EGFR-mutant non-small cell lung cancer (NSCLC) cells and particularly for the subsequent development of acquired resistance are unclear and thus are the focus of this study. Experimental Design: AZD9219-resistant cell lines were established by exposing sensitive cell lines to AZD9291. Protein alterations were detected with Western blotting. Apoptosis was measured with annexin V/flow cytometry. Growth-inhibitory effects of tested drugs were evaluated in vitro with cell number estimation and colony formation assay and in vivo with mouse xenograft models. Protein degradation was determined by comparing protein half-lives and inhibiting proteasome. Gene knockdown were achieved with siRNA or shRNA. Results: AZD9291 potently induced apoptosis in EGFR-mutant NSCLC cell lines, in which ERK phosphorylation was suppressed accompanied with Bim elevation and Mcl-1 reduction likely due to enhanced Mcl-1 degradation and increased Bim stability. Blocking Bim elevation by gene knockdown or enforcing Mcl-1 expression attenuated or abolished AZD9291-induced apoptosis. Moreover, AZD9291 lost its ability to modulate Bim and Mcl-1 levels in AZD9291-resistant cell lines. The combination of a MEK inhibitor with AZD9291 restores the sensitivity of AZD9291-resistant cells including those with C797S mutation to undergo apoptosis and growth regression in vitro and in vivo Conclusions: Modulation of MEK/ERK-dependent Bim and Mcl-1 degradation critically mediates sensitivity and resistance of EGFR-mutant NSCLC cells to AZD9291 and hence is an effective strategy to overcome acquired resistance to AZD9291. Clin Cancer Res; 23(21); 6567-79. ©2017 AACR . ©2017 American Association for Cancer Research.

Silencing expression of UO-44 (CUZD1) using small interfering RNA sensitizes human ovarian cancer cells to cisplatin in vitro.

PubMed

Leong, C T C; Ong, C K; Tay, S K; Huynh, H

2007-02-08

Ovarian cancer is currently the second leading cause of gynecological malignancy and cisplatin or cisplatin-based regimens have been the standard of care for the treatment of advance epithelial ovarian cancers. However, the efficacy of cisplatin treatment is often limited by the development of drug resistance either through the inhibition of apoptotic genes or activation of antiapoptotic genes. We have previously reported the overexpression of human UO-44 (HuUO-44) in ovarian cancers and the HuUO-44 antisera markedly inhibited NIH-OVCAR3 ovarian cancer cell attachment and proliferation (Oncogene 23: 5707-5718, 2004). In the present study, we observed through the cancer cell line profiling array that the expression of HuUO-44 was suppressed in the ovarian cancer cell line (SKOV-3) after treatment with several chemotherapeutic drugs. Similarly, this suppression in HuUO-44 expression was also correlated to the cisplatin sensitivity in two other ovarian cancer cell lines NIH-OVCAR3 and OV-90 in a dose-dependent manner. To elucidate the function of HuUO-44 in cisplatin chemoresistance in ovarian cancer cell, small interfering RNAs (siRNAs) were employed to mediate HuUO-44 silencing in ovarian cancer cell line, NIH-OVCAR3. HuUO-44 RNA interference (RNAi) resulted in the inhibition of cell growth and proliferation. Importantly, HuUO-44 RNAi significantly increased sensitivity of NIH-OVCAR3 to cytotoxic stress induced by cisplatin (P<0.01). Strikingly, we have also demonstrated that overexpression of HuUO-44 significantly conferred cisplatin resistance in NIH-OVCAR3 cells (P<0.05). Taken together, UO-44 is involved in conferring cisplatin resistance; the described HuUO-44-specific siRNA oligonucleotides that can potently silence HuUO-44 gene expression may prove to be valuable pretreatment targets for antitumor therapy or other pathological conditions that involves aberrant HuUO-44 expression.

An in vitro test to screen skin sensitizers using a stable THP-1-derived IL-8 reporter cell line, THP-G8.

PubMed

Takahashi, Toshiya; Kimura, Yutaka; Saito, Rumiko; Nakajima, Yoshihiro; Ohmiya, Yoshihiro; Yamasaki, Kenshi; Aiba, Setsuya

2011-12-01

Several studies have suggested that interleukin (IL)-8 can serve as a biomarker for discrimination of skin sensitizers from nonsensitizers. We established a stable THP-1-derived IL-8 reporter cell line, THP-G8, which harbors SLO and SLR luciferase genes under the control of IL-8 and glyceraldehyde 3-phosphate dehydrogenase promoters, respectively. After 6 h treatment with chemicals, normalized SLO luciferase activity (nSLO-LA) was calculated by dividing SLO-LA by SLR-LA, and the fold induction of nSLO-LA (FInSLO-LA) was calculated by dividing nSLO-LA of chemically treated cells by that of nontreated cells. The nSLO-LA of THP-G8 cells increased in response to lipopolysaccharide (LPS) and several sensitizers. The FInSLO-LA in THP-G8 cells induced by LPS or sensitizers positively correlated with their induction of IL-8 messenger RNA in THP-1 cells. The nSLO-LA value of THP-G8 cells was significantly increased (FInSLO-LA ≥ 1.4) by 13 of the 15 sensitizers as well as by 5 of the 7 nonsensitizers. Interestingly, pretreatment with N-acetylcysteine suppressed the increase in FInSLO-LA induced by all sensitizers (inhibition index (II) ≤ 0.8) but did not suppress that induced by most of the nonsensitizers. We then evaluated the performance of this assay using values of FInSLO-LA ≥ 1.4 and II ≤ 0.8 in at least two of three independent experiments as the criteria of a sensitizer, which resulted in test accuracies of 82% for the 22 chemicals used and of 88% for the chemicals proposed by European Center for the Validation of Alternative Methods. This newly developed assay is a candidate replacement for animal tests of skin sensitization because of its accuracy, convenience, and high throughput performance.

Dietary polyphenols influence antimetabolite agents: methotrexate, 6-mercaptopurine and 5-fluorouracil in leukemia cell lines

PubMed Central

Mahbub, Amani; Le Maitre, Christine; Haywood-Small, Sarah; Cross, Neil; Jordan-Mahy, Nicola

2017-01-01

Polyphenols have been previously shown to sensitize leukemia cell lines to topoisomerase inhibitors. Here, we assess the effects of five polyphenols when used alone and in combination with antimetabolites: methotrexate, 6-mercaptopurine and 5-fluorouracil; in lymphoid and myeloid leukemia cells lines, and non-tumor control cells. The effects of combined treatments were investigated on ATP and glutathione levels, cell-cycle progression, DNA damage and apoptosis. Polyphenols antagonized methotrexate and 6-mercaptopurine induced cell-cycle arrest and apoptosis in most leukemia cell lines. This was associated with reduced DNA damage and increased glutathione levels, greater than that seen following individual treatments alone. In contrast, 5-fluorouracil when combined with quercetin, apigenin and rhein caused synergistic decrease in ATP levels, induction of cell-cycle arrest and apoptosis in some leukemia cell lines. However, antagonistic effects were observed when 5-fluorouracil was combined with rhein and cis-stilbene in myeloid cell lines. The effects were dependant on polyphenol type and chemotherapy agent investigated, and cell type treated. Interestingly treatment of non-tumor control cells with polyphenols protected cells from antimetabolite treatments. This suggests that polyphenols modulate the action of antimetabolite agents; more importantly they antagonized methotrexate and 6-mercaptopurine actions, thus suggesting the requirement of polyphenol-exclusion during their use. PMID:29285220

Sensitive Detection of the Natural Killer Cell-Mediated Cytotoxicity of Anti-CD20 Antibodies and Its Impairment by B-Cell Receptor Pathway Inhibitors

PubMed Central

Knödgen, Eva; Göckeritz, Elisa; Vondey, Verena; Neumann, Lars; Herter, Sylvia; Klein, Christian; Hallek, Michael

2018-01-01

The antibody-dependent cell-mediated cytotoxicity (ADCC) of the anti-CD20 monoclonal antibodies (mAbs) rituximab and obinutuzumab against the cell line Raji and isolated CLL cells and its potential impairment by kinase inhibitors (KI) was determined via lactate dehydrogenase release or calcein retention, respectively, using genetically modified NK92 cells expressing CD16-176V as effector cells. Compared to peripheral blood mononuclear cells, recombinant effector cell lines showed substantial alloreactivity-related cytotoxicity without addition of mAbs but afforded determination of ADCC with reduced interassay variability. The cytotoxicity owing to alloreactivity was less susceptible to interference by KI than the ADCC of anti-CD20 mAbs, which was markedly diminished by ibrutinib, but not by idelalisib. Compared to rituximab, the ADCC of obinutuzumab against primary CLL cells showed approximately 30% higher efficacy and less interference with KI. Irreversible BTK inhibitors at a clinically relevant concentration of 1 μM only weakly impaired the ADCC of anti-CD20 mAbs, with less influence in combinations with obinutuzumab than with rituximab and by acalabrutinib than by ibrutinib or tirabrutinib. In summary, NK cell line-based assays permitted the sensitive detection of ADCC of therapeutic anti-CD20 mAbs against CLL cells and of the interference of KI with this important killing mechanism. PMID:29750146

The sonic hedgehog signaling pathway maintains the cancer stem cell self-renewal of anaplastic thyroid cancer by inducing snail expression.

PubMed

Heiden, Katherine B; Williamson, Ashley J; Doscas, Michelle E; Ye, Jin; Wang, Yimin; Liu, Dingxie; Xing, Mingzhao; Prinz, Richard A; Xu, Xiulong

2014-11-01

Cancer stem cells (CSCs) have been recently identified in thyroid neoplasm. Anaplastic thyroid cancer (ATC) contains a higher percentage of CSCs than well-differentiated thyroid cancer. The signaling pathways and the transcription factors that regulate thyroid CSC self-renewal remain poorly understood. The objective of this study is to use two ATC cell lines (KAT-18 and SW1736) as a model to study the role of the sonic hedgehog (Shh) pathway in maintaining thyroid CSC self-renewal and to understand its underlying molecular mechanisms. The expression and activity of aldehyde dehydrogenase (ALDH), a marker for thyroid CSCs, was analyzed by Western blot and ALDEFLUOR assay, respectively. The effect of three Shh pathway inhibitors (cyclopamine, HhAntag, GANT61), Shh, Gli1, Snail knockdown, and Gli1 overexpression on thyroid CSC self-renewal was analyzed by ALDEFLUOR assay and thyrosphere formation. The sensitivity of transfected KAT-18 cells to radiation was evaluated by a colony survival assay. Western blot analysis revealed that ALDH protein levels in five thyroid cancer cell lines (WRO82, a follicular thyroid cancer cell line; BCPAP and TPC1, two papillary thyroid cancer cell lines; KAT-18 and SW1736, two ATC cell lines) correlated with the percentage of the ALDH(High) cells as well as Gli1 and Snail expression. The Shh pathway inhibitors, Shh and Gli1 knockdown, in KAT-18 cells decreased thyroid CSC self-renewal and increased radiation sensitivity. In contrast, Gli1 overexpression led to increased thyrosphere formation, an increased percentage of ALDH(High) cells, and increased radiation resistance in KAT-18 cells. Inhibition of the Shh pathway by three specific inhibitors led to decreased Snail expression and a decreased number of ALDH(High) cells in KAT-18 and SW1736. Snail gene knockdown decreased the number of ALDH(High) cells in KAT-18 and SW1736 cells. The Shh pathway promotes the CSC self-renewal in ATC cell lines by Gli1-induced Snail expression.

Amine dependence of proliferative activity in two transplantable lines of mouse colonic carcinoma.

PubMed

Tutton, P J; Barkla, D H

1987-01-01

Serotonin, histamine and their antagonists have previously been shown to influence both the cell proliferation rate and the volumetric growth rate of colonic tumours. Of these earlier studies, those on cell proliferation could not distinguish between direct effects on tumour cells and indirect effects on the host, whereas those on the volumetric growth rate of tumours, whilst suggesting an outcome related to the individual properties of the tumour rather than the host, could not distinguish between influences on cell gain, cell loss or stromal changes. In an attempt to distinguish between these possibilities the current experiments on the mitotic rate in two lines of transplantable mouse colonic carcinoma were undertaken. One line of tumour proved to be sensitive to inhibition by a histamine H2 receptor antagonist and a dopamine D2 antagonist but resistant to serotonin antagonists; the inhibition by histamine antagonists was surmountable by co-administration of histamine. The other line proved to be highly sensitive to the inhibitory effects of serotonin antagonist and less so to antagonists of the other two amines and in this case the effect of serotonin antagonists was surmountable by serotonin. These results suggest that the variations between different colonic tumours in the response to amine antagonists is due to differences in the extent of inhibition of cell proliferation rather than differences in cell loss or stromal effects. Thus it appears likely that amine antagonists are able to directly interfere with the proliferation of some colonic tumour cells.

Limits to TYMS and TP53 genes as predictive determinants for fluoropyrimidine sensitivity and further evidence for an RNA-based toxicity as a major influence

PubMed Central

Brody, Jonathan R.; Hucl, Tomas; Costantino, Christina L.; Eshleman, James; Gallmeier, Eike; Zhu, Heng; Heijden, Michael S. van der; Winter, Jordan M; Wikiewicz, Agnieszka K.; Yeo, Charles J.; Kern, Scott E.

2010-01-01

The major determinants of 5-flurouracil response would appear, based on accumulated literature, to be thymidylate synthase (TYMS, TS) expression levels, TS gene modifications, and TP53 status. We tested 5-fluorouracil sensitivity in yeast and human cancer cell models in which TS or TP53 alleles and expression were varied. Polymorphic TS tandem repeat status, TS expression levels reported, TS intragenic mutations, and TP53 status in outbred and experimental cancer cell lines did not predict 5-FU sensitivity or resistance. Novel observations included a dose-resistant persistence of unbound TS protein in many cancers and, upon 5-FU treatment of the colon cancer cell line, HCT116, evidence of allelic switching favoring transcripts of the mutant TS allele. The reported alleles having an intragenic mutation could not be causally associated with major degrees of 5-FU sensitivity. In yeast, TS protein was altered upon treatment with fluoro-deoxyuridine monophosphate, but 5-FU toxicity appeared largely to be RNA-based, being rescued by uridine rather than by thymidine. Cancer cell lines were also rescued from 5-FU toxicity with uridine rather than thymidine. Additionally, a TS (CDC21) knockout yeast strain, obviating any potential role for TS protein as a target, was hypersensitive to 5-FU. When denatured proteins from cancer cells treated with radio-labeled 5-FU were, labeled species with alternative molecular weights other than TS were visualized, providing further evidence for alternative 5-FU protein targets. These data emphasize that TS and TP53 status do not consistently explain the variance in responses of fluoropyrimidine-treated cancer cells, in part due to RNA-based toxicity. PMID:19155291

Pharmacological inhibition of poly(ADP-ribose) polymerase-1 modulates resistance of human glioblastoma stem cells to temozolomide

PubMed Central

2014-01-01

Background Chemoresistance of glioblastoma multiforme (GBM) has been attributed to the presence within the tumor of cancer stem cells (GSCs). The standard therapy for GBM consists of surgery followed by radiotherapy and the chemotherapeutic agent temozolomide (TMZ). However, TMZ efficacy is limited by O6-methylguanine-DNA-methyltransferase (MGMT) and Mismatch Repair (MMR) functions. Strategies to counteract TMZ resistance include its combination with poly(ADP-ribose) polymerase inhibitors (PARPi), which hamper the repair of N-methylpurines. PARPi are also investigated as monotherapy for tumors with deficiency of homologous recombination (HR). We have investigated whether PARPi may restore GSC sensitivity to TMZ or may be effective as monotherapy. Methods Ten human GSC lines were assayed for MMR proteins, MGMT and PARP-1 expression/activity, MGMT promoter methylation and sensitivity to TMZ or PARPi, alone and in combination. Since PTEN defects are frequently detected in GBM and may cause HR dysfunction, PTEN expression was also analyzed. The statistical analysis of the differences in drug sensitivity among the cell lines was performed using the ANOVA and Bonferroni’s post-test or the non-parametric Kruskal-Wallis analysis and Dunn’s post-test for multiple comparisons. Synergism between TMZ and PARPi was analyzed by the median-effect method of Chou and Talalay. Correlation analyses were done using the Spearman’s rank test. Results All GSCs were MMR-proficient and resistance to TMZ was mainly associated with high MGMT activity or low proliferation rate. MGMT promoter hypermethylation of GSCs correlated both with low MGMT activity/expression (Spearman’s test, P = 0.004 and P = 0.01) and with longer overall survival of GBM patients (P = 0.02). Sensitivity of each GSC line to PARPi as single agent did not correlate with PARP-1 or PTEN expression. Notably, PARPi and TMZ combination exerted synergistic antitumor effects in eight out of ten GSC lines and the TMZ dose reduction achieved significantly correlated with the sensitivity of each cell line to PARPi as single agent (P = 0.01). Conclusions The combination of TMZ with PARPi may represent a valuable strategy to reverse GSC chemoresistance. PMID:24593254

Culture of human cell lines by a pathogen-inactivated human platelet lysate.

PubMed

Fazzina, R; Iudicone, P; Mariotti, A; Fioravanti, D; Procoli, A; Cicchetti, E; Scambia, G; Bonanno, G; Pierelli, L

2016-08-01

Alternatives to the use of fetal bovine serum (FBS) have been investigated to ensure xeno-free growth condition. In this study we evaluated the efficacy of human platelet lysate (PL) as a substitute of FBS for the in vitro culture of some human cell lines. PL was obtained by pools of pathogen inactivated human donor platelet (PLT) concentrates. Human leukemia cell lines (KG-1, K562, JURKAT, HL-60) and epithelial tumor cell lines (HeLa and MCF-7) were cultured with either FBS or PL. Changes in cell proliferation, viability, morphology, surface markers and cell cycle were evaluated for each cell line. Functional characteristics were analysed by drug sensitivity test and cytotoxicity assay. Our results demonstrated that PL can support growth and expansion of all cell lines, although the cells cultured in presence of PL experienced a less massive proliferation compared to those grown with FBS. We found a comparable percentage of viable specific marker-expressing cells in both conditions, confirming lineage fidelity in all cultures. Functionality assays showed that cells in both FBS- and PL-supported cultures maintained their normal responsiveness to adriamycin and NK cell-mediated lysis. Our findings indicate that PL is a feasible serum substitute for supporting growth and propagation of haematopoietic and epithelial cell lines with many advantages from a perspective of process standardization, ethicality and product safety.

Influence of LOX/COX inhibitors on cell differentiation induced by all-trans retinoic acid in neuroblastoma cell lines.

PubMed

Redova, Martina; Chlapek, Petr; Loja, Tomas; Zitterbart, Karel; Hermanova, Marketa; Sterba, Jaroslav; Veselska, Renata

2010-02-01

We investigated the possible modulation by LOX/ COX inhibitors of all-trans retinoic acid (ATRA)-induced cell differentiation in two established neuroblastoma cell lines, SH-SY5Y and SK-N-BE(2). Caffeic acid, as an inhibitor of 5-lipoxygenase, and celecoxib, as an inhibitor of cyclooxygenase-2, were chosen for this study. The effects of the combined treatment with ATRA and LOX/COX inhibitors on neuroblastoma cells were studied using cell morphology assessment, detection of differentiation markers by immunoblotting, measurement of proliferation activity, and cell cycle analysis and apoptosis detection by flow cytometry. The results clearly demonstrated the potential of caffeic acid to enhance ATRA-induced cell differentiation, especially in the SK-N-BE(2) cell line, whereas application of celecoxib alone or with ATRA led predominantly to cytotoxic effects in both cell lines. Moreover, the higher sensitivity of the SK-N-BE(2) cell line to combined treatment with ATRA and LOX/COX inhibitors suggests that cancer stem cells are a main target for this therapeutic approach. Nevertheless, further detailed study of the phenomenon of enhanced cell differentiation by expression profiling is needed.

Response of transformed and normal mouse cell lines to anti-melanin compounds, hyperthermia, and radiation.

PubMed

Raaphorst, G P; Azzam, E I

1992-02-01

Five cell lines (one parental, two transformed melanin producing, and two transformed non-melanin producing) were evaluated for the responses to 2- and 4-hydroxyanisole (2HA, 4HA) alone or combined with hyperthermia or radiation. All cells exhibited a non-specific toxic response to the two compounds and the effect was exposure time and concentration dependent and was greater for 4HA compared to 2HA. In addition, the two melanin-producing cell lines were more sensitive, demonstrating specific toxicity to such cell lines. The treatment with either 2HA or 4HA combined with heat and radiation resulted mostly in additive or antagonistic effects, except for one combination of 2HA plus radiation in the melanin-producing R25 cells. Thus, while these compounds may be useful in therapy for pigmented melanomas, combined treatment with radiation is not recommended.

14-3-3 σ Expression Effects G2/M Response to Oxygen and Correlates with Ovarian Cancer Metastasis

PubMed Central

Ravi, Dashnamoorthy; Chen, Yidong; Karia, Bijal; Brown, Adam; Gu, Ting Ting; Li, Jie; Carey, Mark S.; Hennessy, Bryan T.; Bishop, Alexander J. R.

2011-01-01

Background In vitro cell culture experiments with primary cells have reported that cell proliferation is retarded in the presence of ambient compared to physiological O2 levels. Cancer is primarily a disease of aberrant cell proliferation, therefore, studying cancer cells grown under ambient O2 may be undesirable. To understand better the impact of O2 on the propagation of cancer cells in vitro, we compared the growth potential of a panel of ovarian cancer cell lines under ambient (21%) or physiological (3%) O2. Principal Findings Our observations demonstrate that similar to primary cells, many cancer cells maintain an inherent sensitivity to O2, but some display insensitivity to changes in O2 concentration. Further analysis revealed an association between defective G2/M cell cycle transition regulation and O2 insensitivity resultant from overexpression of 14-3-3 σ. Targeting 14-3-3 σ overexpression with RNAi restored O2 sensitivity in these cell lines. Additionally, we found that metastatic ovarian tumors frequently overexpress 14-3-3 σ, which in conjunction with phosphorylated RB, results in poor prognosis. Conclusions Cancer cells show differential proliferative sensitivity to changes in O2 concentration. Although a direct link between O2 insensitivity and metastasis was not determined, this investigation showed that an O2 insensitive phenotype in cancer cells to correlate with metastatic tumor progression. PMID:21249227

Heterogeneity in cancer cells: variation in drug response in different primary and secondary colorectal cancer cell lines in vitro.

PubMed

Arul, Melanie; Roslani, April Camilla; Cheah, Swee Hung

2017-05-01

Tumor heterogeneity may give rise to differential responses to chemotherapy drugs. Therefore, unraveling tumor heterogeneity has an implication for biomarker discovery and cancer therapeutics. To test this phenomenon, we investigated the differential responses of three secondary colorectal cancer cell lines of different origins (HCT116, HT29, and SW620 cells) and four novel primary cell lines obtained from different colorectal cancer patients to 5-fluorouracil (5-FU) and oxaliplatin (L-OHP) and explored the differences in gene expression among the primary cell lines in response to exposure to cytotoxic drugs. Cells were exposed to different doses of 5-FU and L-OHP separately or in combinations of equitoxic drug or equimolar drug ratios (median effect of Chou-Talalay principle). Cell viability was assessed using MTT assay and the respective IC 50 values were determined. Changes in gene expression in primary cell lines after exposure to the same drug doses were compared using real-time PCR array. The sensitivities (IC 50 ) of different cell lines, both secondary and primary, to 5-FU and L-OHP were significantly different, whether in monotherapy or combined treatment. Primary cell lines needed higher doses to reach IC 50 . There were variations in gene expression among the primary cell lines of different chemosensitivities to the challenge of the same combined dose of 5-FU and L-OHP. The results confirm the heterogeneous nature of colorectal cancer cells from different patient tumors. Studies using primary cancer cells established from patient's tumors rather than secondary cell lines will more closely reflect the actual character of the disease.

Endogenous pyrogen production by Hodgkin's disease and human histiocytic lymphoma cell lines in vitro.

PubMed

Bodel, P; Ralph, P; Wenc, K; Long, J C

1980-02-01

Fever not explained by infection may occur in patients with malignant lymphoma presumably caused by a release of endogenous pyrogen. Although pyrogen has been found in some tumors with a mixed cell population, production of endogenous pyrogen by the neoplastic cells has not been demonstrated. This report documents the apparently spontaneous synthesis and release of such pyrogen by two human tumor cell lines derived from patients with Hodgkin's disease and histiocytic lymphoma. The endogenous pyrogen from the two cell lines was similar and closely resembled that produced by normal human monocytes in antigenic properties as well as heat and pronase sensitivity. The Hodgkin's disease and histiocytic lymphoma cell lines do not require specific stimulation for the production of endogenous pyrogen suggesting that the mechanism of pyrogen release by neoplastic macrophage-related cells differs from that of normal phagocytic cells. The tumor-associated fever in some patients with malignant lymphoma may be caused by a release of endogenous pyrogen by proliferating neoplastic cells.

Endogenous pyrogen production by Hodgkin's disease and human histiocytic lymphoma cell lines in vitro.

PubMed Central

Bodel, P; Ralph, P; Wenc, K; Long, J C

1980-01-01

Fever not explained by infection may occur in patients with malignant lymphoma presumably caused by a release of endogenous pyrogen. Although pyrogen has been found in some tumors with a mixed cell population, production of endogenous pyrogen by the neoplastic cells has not been demonstrated. This report documents the apparently spontaneous synthesis and release of such pyrogen by two human tumor cell lines derived from patients with Hodgkin's disease and histiocytic lymphoma. The endogenous pyrogen from the two cell lines was similar and closely resembled that produced by normal human monocytes in antigenic properties as well as heat and pronase sensitivity. The Hodgkin's disease and histiocytic lymphoma cell lines do not require specific stimulation for the production of endogenous pyrogen suggesting that the mechanism of pyrogen release by neoplastic macrophage-related cells differs from that of normal phagocytic cells. The tumor-associated fever in some patients with malignant lymphoma may be caused by a release of endogenous pyrogen by proliferating neoplastic cells. PMID:6985918

Quantitative Chemical-Genetic Interaction Map Connects Gene Alterations to Drug Responses | Office of Cancer Genomics

Cancer.gov

In a recent Cancer Discovery report, CTD2 researchers at the University of California in San Francisco developed a new quantitative chemical-genetic interaction mapping approach to evaluate drug sensitivity or resistance in isogenic cell lines. Performing a high-throughput screen with isogenic cell lines allowed the researchers to explore the impact of a panel of emerging and established drugs on cells overexpressing a single cancer-associated gene in isolation.

Venetoclax Is Effective in Small-Cell Lung Cancers with High BCL-2 Expression.

PubMed

Lochmann, Timothy L; Floros, Konstantinos V; Naseri, Mitra; Powell, Krista M; Cook, Wade; March, Ryan J; Stein, Giovanna T; Greninger, Patricia; Maves, Yuki Kato; Saunders, Laura R; Dylla, Scott J; Costa, Carlotta; Boikos, Sosipatros A; Leverson, Joel D; Souers, Andrew J; Krystal, Geoffrey W; Harada, Hisashi; Benes, Cyril H; Faber, Anthony C

2018-01-15

Purpose: Small-cell lung cancer (SCLC) is an often-fatal neuroendocrine carcinoma usually presenting as extensive disease, carrying a 3% 5-year survival. Despite notable advances in SCLC genomics, new therapies remain elusive, largely due to a lack of druggable targets. Experimental Design: We used a high-throughput drug screen to identify a venetoclax-sensitive SCLC subpopulation and validated the findings with multiple patient-derived xenografts of SCLC. Results: Our drug screen consisting of a very large collection of cell lines demonstrated that venetoclax, an FDA-approved BCL-2 inhibitor, was found to be active in a substantial fraction of SCLC cell lines. Venetoclax induced BIM-dependent apoptosis in vitro and blocked tumor growth and induced tumor regressions in mice bearing high BCL-2-expressing SCLC tumors in vivo BCL-2 expression was a predictive biomarker for sensitivity in SCLC cell lines and was highly expressed in a subset of SCLC cell lines and tumors, suggesting that a substantial fraction of patients with SCLC could benefit from venetoclax. Mechanistically, we uncover a novel role for gene methylation that helped discriminate high BCL-2-expressing SCLCs. Conclusions: Altogether, our findings identify venetoclax as a promising new therapy for high BCL-2-expressing SCLCs. Clin Cancer Res; 24(2); 360-9. ©2017 AACR . ©2017 American Association for Cancer Research.

Low expression of pro-apoptotic Bcl-2 family proteins sets the apoptotic threshold in Waldenström Macroglobulinemia

PubMed Central

Gaudette, Brian T.; Dwivedi, Bhakti; Chitta, Kasyapa S.; Poulain, Stéphanie; Powell, Doris; Vertino, Paula; Leleu, Xavier; Lonial, Sagar; Chanan-Khan, Asher A.; Kowalski, Jeanne; Boise, Lawrence H.

2015-01-01

Waldenström Macroglobulinemia (WM) is a proliferative disorder of IgM secreting, lymphoplasmacytoid cells that inhabit the lymph nodes and bone marrow. The disease carries a high prevalence of activating mutations in MyD88 (91%) and CXCR4 (28%). Because signaling through these pathways leads to Bcl-xL induction, we examined Bcl-2 family expression in WM patients and cell lines. Unlike other B-lymphocyte-derived malignancies, which become dependent on expression of anti-apoptotic proteins to counter expression of pro-apoptotic proteins, WM samples expressed both pro- and anti-apoptotic Bcl-2 proteins at low levels similar to their normal B-cell and plasma cell counterparts. Three WM cell lines expressed pro-apoptotic Bcl-2 family members Bim or Bax and Bak at low levels which determined their sensitivity to inducers of intrinsic apoptosis. In two cell lines, miR-155 upregulation, which is common in WM, was responsible for inhibition of FOXO3a and Bim expression. Both antagonizing miR-155 to induce Bim and proteasome inhibition increased the sensitivity to ABT-737 in these lines indicating a lowering of the apoptotic threshold. In this manner, treatments that increase pro-apoptotic protein expression increase the efficacy of agents treated in combination in addition to direct killing. PMID:25893290

A gallotannin-rich fraction from Caesalpinia spinosa (Molina) Kuntze displays cytotoxic activity and raises sensitivity to doxorubicin in a leukemia cell line

PubMed Central

2012-01-01

Background Enhancement of tumor cell sensitivity may help facilitate a reduction in drug dosage using conventional chemotherapies. Consequently, it is worthwhile to search for adjuvants with the potential of increasing chemotherapeutic drug effectiveness and improving patient quality of life. Natural products are a very good source of such adjuvants. Methods The biological activity of a fraction enriched in hydrolysable polyphenols (P2Et) obtained from Caesalpinia spinosa was evaluated using the hematopoietic cell line K562. This fraction was tested alone or in combination with the conventional chemotherapeutic drugs doxorubicin, vincristine, etoposide, camptothecin and taxol. The parameters evaluated were mitochondrial depolarization, caspase 3 activation, chromatin condensation and clonogenic activity. Results We found that the P2Et fraction induced mitochondrial depolarization, activated caspase 3, induced chromatin condensation and decreased the clonogenic capacity of the K562 cell line. When the P2Et fraction was used in combination with chemotherapeutic drugs at sub-lethal concentrations, a fourfold reduction in doxorubicin inhibitory concentration 50 (IC50) was seen in the K562 cell line. This finding suggested that P2Et fraction activity is specific for the molecular target of doxorubicin. Conclusions Our results suggest that a natural fraction extracted from Caesalpinia spinosa in combination with conventional chemotherapy in combination with natural products on leukemia cells may increase therapeutic effectiveness in relation to leukemia. PMID:22490328

Human Monocytes in the Presence of Interferons Alpha2a and Gamma Are Potent Killers of Serous Ovarian Cancer Cell Lines in Combination with Paclitaxel and Carboplatin

PubMed Central

Johnson, Chase L.; Zoon, Kathryn C.

2015-01-01

Interferons (IFNs) play an important role in immune surveillance of tumors; however, their efficacy in the treatment of malignancies has been limited. Monocytes are mononuclear phagocytes that are critical to the generation of an innate immune response to tumors. The authors and others have shown that treatment of tumor cell lines in vitro and in vivo with human monocytes primed with type I and type II IFNs results in killing. We now expand on this work, in an extended panel of ovarian cancer cell lines. In this study, we hypothesized that there would be variable sensitivity amongst cell lines to the killing properties of monocytes and IFNs. To this end, we explored the interactions of IFN primed monocytes in conjunction with the standard of therapy for ovarian cancer, taxane, and platinum-based chemotherapeutics. Using 6 ovarian cancer cell lines, we demonstrated that there is variation from cell line to cell line in the ability of IFN-α2a and IFN-γ primed monocytes to synergistically kill target tumor cells, and further, there is an additive killing effect when target cells are treated with both IFN primed monocytes and chemotherapy. PMID:25068849

GENETICS OF HUMAN CELL LINES

PubMed Central

Djordjevic, B.; Szybalski, Waclaw

1960-01-01

The human cell line D98S can be cultivated indefinitely in the presence of up to 3 x 10–5 M 5-bromodeoxyuridine (BUDR), without loss of cell viability. During this time, BUDR is incorporated into both strands of the DNA molecules, replacing up to 45 per cent of the thymidine and thereby rendering the cells highly sensitive to UV light and to x-rays. Cells grown for a limited period of time in the presence of 5-iododeoxyuridine (IUDR) become UV-sensitized, while prolonged cultivation with IUDR results in the loss of cell viability. The properties of the BUDR label permitted the demonstration that: (a) human DNA replicates in a "semiconservative" manner; (b) the degree of radiosensitization of BUDR-treated cells depends on whether the DNA has been substituted in one strand only ("unifilarly") or in both strands ("bifilarly"); (c) functional human DNA is produced during partial inhibition of protein synthesis. The potential applicability of this new rational principle of radiosensitization to the radiotherapy of neoplastic diseases is discussed. PMID:13723177

Antitumor Effect of KX-01 through Inhibiting Src Family Kinases and Mitosis.

PubMed

Kim, Seongyeong; Min, Ahrum; Lee, Kyung-Hun; Yang, Yaewon; Kim, Tae-Yong; Lim, Jee Min; Park, So Jung; Nam, Hyun-Jin; Kim, Jung Eun; Song, Sang-Hyun; Han, Sae-Won; Oh, Do-Youn; Kim, Jee Hyun; Kim, Tae-You; Hangauer, David; Lau, Johnson Yiu-Nam; Im, Kyongok; Lee, Dong Soon; Bang, Yung-Jue; Im, Seock-Ah

2017-07-01

KX-01 is a novel dual inhibitor of Src and tubulin. Unlike previous Src inhibitors that failed to show clinical benefit during treatment of breast cancer, KX-01 can potentially overcome the therapeutic limitations of current Src inhibitors through inhibition of both Src and tubulin. The present study further evaluates the activity and mechanism of KX-01 in vitro and in vivo . The antitumor effect of KX-01 in triple negative breast cancer (TNBC) cell lines was determined by MTT assay. Wound healing and immunofluorescence assays were performed to evaluate the action mechanisms of KX-01. Changes in the cell cycle and molecular changes induced by KX-01 were also evaluated. A MDA-MB-231 mouse xenograft model was used to demonstrate the in vivo effects. KX-01 effectively inhibited the growth of breast cancer cell lines. The expression of phospho-Src and proliferative-signaling molecules were down-regulated in KX-01-sensitive TNBC cell lines. In addition, migration inhibition was observed by wound healing assay. KX-01-induced G2/M cell cycle arrest and increased the aneuploid cell population in KX-01-sensitive cell lines. Multi-nucleated cells were significantly increased after KX-01 treatment. Furthermore, KX-01 effectively delayed tumor growth in a MDA-MB-231 mouse xenograft model. KX-01 effectively inhibited cell growth and migration of TNBC cells. Moreover, this study demonstrated that KX-01 showed antitumor effects through the inhibition of Src signaling and the induction of mitotic catastrophe. The antitumor effects of KX-01 were also demonstrated in vivo using a mouse xenograft model.

Screening Fluorescent Voltage Indicators with Spontaneously Spiking HEK Cells

PubMed Central

Venkatachalam, Veena; Kralj, Joel M.; Dib-Hajj, Sulayman D.; Waxman, Stephen G.; Cohen, Adam E.

2013-01-01

Development of improved fluorescent voltage indicators is a key challenge in neuroscience, but progress has been hampered by the low throughput of patch-clamp characterization. We introduce a line of non-fluorescent HEK cells that stably express NaV 1.3 and KIR 2.1 and generate spontaneous electrical action potentials. These cells enable rapid, electrode-free screening of speed and sensitivity of voltage sensitive dyes or fluorescent proteins on a standard fluorescence microscope. We screened a small library of mutants of archaerhodopsin 3 (Arch) in spiking HEK cells and identified two mutants with greater voltage-sensitivity than found in previously published Arch voltage indicators. PMID:24391999

Impact of Procyanidins from Different Berries on Caspase 8 Activation in Colon Cancer.

PubMed

Minker, Carole; Duban, Livine; Karas, Daniel; Järvinen, Päivi; Lobstein, Annelise; Muller, Christian D

2015-01-01

The aim of this work is to identify which proapoptotic pathway is induced in human colon cancer cell lines, in contact with proanthocyanidins extracted from various berries. Proanthocyanidins (Pcys) extracted from 11 berry species are monitored for proapoptotic activities on two related human colon cancer cell lines: SW480-TRAIL-sensitive and SW620-TRAIL-resistant. Apoptosis induction is monitored by cell surface phosphatidylserine (PS) detection. Lowbush blueberry extract triggers the strongest activity. When tested on the human monocytic cell line THP-1, blueberry Pcys are less effective for PS externalisation and DNA fragmentation is absent, highlighting a specificity of apoptosis induction in gut cells. In Pcys-treated gut cell lines, caspase 8 (apoptosis extrinsic pathway) but not caspase 9 (apoptosis intrinsic pathway) is activated after 3 hours through P38 phosphorylation (90 min), emphasizing the potency of lowbush blueberry Pcys to eradicate gut TRAIL-resistant cancer cells. We highlight here that berries Pcys, especially lowbush blueberry Pcys, are of putative interest for nutritional chemoprevention of colorectal cancer in view of their apoptosis induction in a human colorectal cancer cell lines.

Comparison of test methods to screen for residual chemical contamination on medical device surfaces.

PubMed

Kulkarni, Prachi; Shoff, Megan; Lucas, Anne

2012-01-01

Reprocessing medical devices involves several steps including cleaning and disinfection or sterilization. Chemical residuals can occur at various stages of reprocessing. These residues could interfere with device function and potentially harm patients. These solutions are composed of a combination of various chemicals and their residues are highly diluted post rinsing, therefore, it is difficult to find a sensitive and rapid method to detect toxicity due to chemical residues. This study focused on (1) finding the levels of residues that are cytotoxic using two mammalian cell lines and Daphnia magna, and (2) evaluating two test methods, Total Organic Carbon (TOC) and the Luminescent Bacteria Test (LBT), to measure residual chemicals levels. The two mammalian cell lines were equal in their cytotoxicity responses. However, Daphnia were more sensitive to some chemical residue than the two mammalian cell lines. TOC and LBT were able to detect the presence of residue well below the levels that were determined to cause mammalian cytotoxicity. LBT was more sensitive for some chemicals and TOC for others, both in solution and in simulated cleaning and rinsing for the limited number of solutions tested in this study.

Antitumor activity of pan-HER inhibitors in HER2-positive gastric cancer.

PubMed

Yoshioka, Takahiro; Shien, Kazuhiko; Namba, Kei; Torigoe, Hidejiro; Sato, Hiroki; Tomida, Shuta; Yamamoto, Hiromasa; Asano, Hiroaki; Soh, Junichi; Tsukuda, Kazunori; Nagasaka, Takeshi; Fujiwara, Toshiyoshi; Toyooka, Shinichi

2018-04-01

Molecularly targeted therapy has enabled outstanding advances in cancer treatment. Whereas various anti-human epidermal growth factor receptor 2 (HER2) drugs have been developed, trastuzumab is still the only anti-HER2 drug presently available for gastric cancer. In this study, we propose novel treatment options for patients with HER2-positive gastric cancer. First, we determined the molecular profiles of 12 gastric cancer cell lines, and examined the antitumor effect of the pan-HER inhibitors afatinib and neratinib in those cell lines. Additionally, we analyzed HER2 alteration in 123 primary gastric cancers resected from Japanese patients to clarify possible candidates with the potential to respond to these drugs. In the drug sensitivity analysis, both afatinib and neratinib produced an antitumor effect in most of the HER2-amplified cell lines. However, some cells were not sensitive to the drugs. When the molecular profiles of the cells were compared based on the drug sensitivities, we found that cancer cells with lower mRNA expression levels of IGFBP7, a tumor suppressor gene that inhibits the activation of insulin-like growth factor-1 receptor (IGF-1R), were less sensitive to pan-HER inhibitors. A combination therapy consisting of pan-HER inhibitors and an IGF-1R inhibitor, picropodophyllin, showed a notable synergistic effect. Among 123 clinical samples, we found 19 cases of HER2 amplification and three cases of oncogenic mutations. In conclusion, afatinib and neratinib are promising therapeutic options for the treatment of HER2-amplified gastric cancer. In addition to HER2 amplification, IGFBP7 might be a biomarker of sensitivity to these drugs, and IGF-1R-targeting therapy can overcome drug insensitiveness in HER2-amplified gastric cancer. © 2018 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Computational discovery of pathway-level genetic vulnerabilities in non-small-cell lung cancer

PubMed Central

Young, Jonathan H.; Peyton, Michael; Seok Kim, Hyun; McMillan, Elizabeth; Minna, John D.; White, Michael A.; Marcotte, Edward M.

2016-01-01

Motivation: Novel approaches are needed for discovery of targeted therapies for non-small-cell lung cancer (NSCLC) that are specific to certain patients. Whole genome RNAi screening of lung cancer cell lines provides an ideal source for determining candidate drug targets. Results: Unsupervised learning algorithms uncovered patterns of differential vulnerability across lung cancer cell lines to loss of functionally related genes. Such genetic vulnerabilities represent candidate targets for therapy and are found to be involved in splicing, translation and protein folding. In particular, many NSCLC cell lines were especially sensitive to the loss of components of the LSm2-8 protein complex or the CCT/TRiC chaperonin. Different vulnerabilities were also found for different cell line subgroups. Furthermore, the predicted vulnerability of a single adenocarcinoma cell line to loss of the Wnt pathway was experimentally validated with screening of small-molecule Wnt inhibitors against an extensive cell line panel. Availability and implementation: The clustering algorithm is implemented in Python and is freely available at https://bitbucket.org/youngjh/nsclc_paper. Contact: marcotte@icmb.utexas.edu or jon.young@utexas.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:26755624

Computational discovery of pathway-level genetic vulnerabilities in non-small-cell lung cancer.

PubMed

Young, Jonathan H; Peyton, Michael; Seok Kim, Hyun; McMillan, Elizabeth; Minna, John D; White, Michael A; Marcotte, Edward M

2016-05-01

Novel approaches are needed for discovery of targeted therapies for non-small-cell lung cancer (NSCLC) that are specific to certain patients. Whole genome RNAi screening of lung cancer cell lines provides an ideal source for determining candidate drug targets. Unsupervised learning algorithms uncovered patterns of differential vulnerability across lung cancer cell lines to loss of functionally related genes. Such genetic vulnerabilities represent candidate targets for therapy and are found to be involved in splicing, translation and protein folding. In particular, many NSCLC cell lines were especially sensitive to the loss of components of the LSm2-8 protein complex or the CCT/TRiC chaperonin. Different vulnerabilities were also found for different cell line subgroups. Furthermore, the predicted vulnerability of a single adenocarcinoma cell line to loss of the Wnt pathway was experimentally validated with screening of small-molecule Wnt inhibitors against an extensive cell line panel. The clustering algorithm is implemented in Python and is freely available at https://bitbucket.org/youngjh/nsclc_paper marcotte@icmb.utexas.edu or jon.young@utexas.edu Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press.

IRS-1: essential for insulin- and IL-4-stimulated mitogenesis in hematopoietic cells.

PubMed

Wang, L M; Myers, M G; Sun, X J; Aaronson, S A; White, M; Pierce, J H

1993-09-17

Although several interleukin-3 (IL-3)-dependent cell lines proliferate in response to IL-4 or insulin, the 32D line does not. Insulin and IL-4 sensitivity was restored to 32D cells by expression of IRS-1, the principal substrate of the insulin receptor. Although 32D cells possessed receptors for both factors, they lacked the IRS-1--related protein, 4PS, which becomes phosphorylated by tyrosine in insulin- or IL-4--responsive lines after stimulation. These results indicate that factors that bind unrelated receptors can use similar mitogenic signaling pathways in hematopoietic cells and that 4PS and IRS-1 are functionally similar proteins that are essential for insulin- and IL-4--induced proliferation.

INTEGRATED MODULATION OF PHORBOL ESTER-INDUCED RAF ACTIVATION IN EL4 LYMPHOMA CELLS

PubMed Central

Han, Shujie; Meier, Kathryn E.

2009-01-01

The EL4 murine lymphoma cell line exists in variant phenotypes that differ with respect to responses to the tumor promoter phorbol 12-myristate 13-acetate (PMA1). Previous work showed that “PMA-sensitive” cells, characterized by a high magnitude of PMA-induced Erk activation, express RasGRP, a phorbol ester receptor that directly activates Ras. In “PMA-resistant” and “intermediate” EL4 cell lines, PMA induces Erk activation to lesser extents, but with a greater response in intermediate cells. In the current study, these cell lines were used to examine mechanisms of Raf-1 modulation. Phospho-specific antibodies were utilized to define patterns and kinetics of Raf-1 phosphorylation on several sites. Further studies showed that Akt is constitutively activated to a greater extent in PMA-resistant than in PMA-sensitive cells, and also to a greater extent in resistant than intermediate cells. Akt negatively regulates Raf-1 activation (Ser259), partially explaining the difference between resistant and intermediate cells. Erk activation exerts negative feedback on Raf-1 (Ser289/296/301), thus resulting in earlier termination of the signal in cells with a higher level of Erk activation. RKIP, a Raf inhibitory protein, is expressed at higher levels in resistant cells than in sensitive or intermediate cells. Knockdown of RKIP increases Erk activation and also negative feedback. In conclusion, this study delineates Raf-1 phosphorylation events occurring in response to PMA in cell lines with different extents of Erk activation. Variations in the levels of expression and activation of multiple signaling proteins work in an integrated fashion to modulate the extent and duration of Erk activation. PMID:19263515

Fusogenic pH sensitive liposomal formulation for rapamycin: improvement of antiproliferative effect.

PubMed

Ghanbarzadeh, Saeed; Khorrami, Arash; Mohamed Khosroshahi, Leila; Arami, Sanam

2014-07-01

Liposomes are increasingly employed to deliver chemotherapeutic agents, antisense oligonucleotides, and genes to various therapeutic targets. The present investigation evaluates the ability of fusogenic pH-sensitive liposomes of rapamycin in increasing its antiproliferative effect on human breast adenocarcinoma (MCF-7) cell line. Cholesterol (Chol) and dipalmitoylphosphatidylcholine (DPPC) (DPPC:Chol, 7:3) were used to prepare conventional rapamycin liposomes by a modified ethanol injection method. Dioleoylphosphatidylethanolamine (DOPE) was used to produce fusogenic and pH-sensitive properties in liposomes simultaneously (DPPC:Chol:DOPE, 7:3:4.2). The prepared liposomes were characterized by their size, zeta potential, encapsulation efficiency percent (EE%), and chemical stability during 6 months. The antiproliferative effects of both types of rapamycin liposomes (10, 25, and 50 nmol/L) with optimized formulations were assessed on MCF-7 cells, as cancerous cells, and human umbilical vein endothelial cells (HUVEC), as healthy cells, employing the diphenyltetrazolium bromide (MTT) assay for 72 h. The particle size, zeta potential, and EE% of the liposomes were 165 ± 12.3 and 178 ± 15.4 nm, -39.6 ± 1.3, and -41.2 ± 2.1 mV as well as 76.9 ± 2.6 and 76.9 ± 2.6% in conventional and fusogenic pH-sensitive liposomes, respectively. Physicochemical stability results indicated that both liposome types were relatively stable at 4 °C than 25 °C. In vitro antiproliferative evaluation showed that fusogenic pH-sensitive liposomes had better antiproliferative effects on MCF-7 cells compared to the conventional liposomes. Conversely, fusogenic pH-sensitive liposomes had less cytotoxicity on HUVEC cell line.

MiR-224 expression increases radiation sensitivity of glioblastoma cells

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

Upraity, Shailendra; Kazi, Sadaf; Padul, Vijay

Highlights: • MiR-224 expression in established glioblastoma cell lines and sporadic tumor tissues is low. • Exogenous miR-224 expression was found to increase radiation sensitivity of glioblastoma cells. • MiR-224 expression brought about 55–60% reduction in API5 expression levels. • Transfection with API5 siRNA increased radiation sensitivity of glioblastoma cells. • Low miR-224 and high API5 expression correlated with worse survival of GBM patients. - Abstract: Glioblastoma (GBM) is the most common and highly aggressive primary malignant brain tumor. The intrinsic resistance of this brain tumor limits the efficacy of administered treatment like radiation therapy. In the present study, effectmore » of miR-224 expression on growth characteristics of established GBM cell lines was analyzed. MiR-224 expression in the cell lines as well as in primary GBM tumor tissues was found to be low. Exogenous transient expression of miR-224 using either synthetic mimics or stable inducible expression using doxycycline inducible lentiviral vector carrying miR-224 gene, was found to bring about 30–55% reduction in clonogenic potential of U87 MG cells. MiR-224 expression reduced clonogenic potential of U87 MG cells by 85–90% on irradiation at a dose of 6 Gy, a dose that brought about 50% reduction in clonogenic potential in the absence of miR-224 expression. MiR-224 expression in glioblastoma cells resulted in 55–65% reduction in the expression levels of API5 gene, a known target of miR-224. Further, siRNA mediated down-regulation of API5 was also found to have radiation sensitizing effect on glioblastoma cell lines. Analysis of the Cancer Genome Atlas data showed lower miR-224 expression levels in male GBM patients to correlate with poorer survival. Higher expression levels of miR-224 target API5 also showed significant correlation with poorer survival of GBM patients. Up-regulation of miR-224 or down-regulation of its target API5 in combination with radiation therapy, therefore appear as promising options for the treatment of glioblastoma, which is refractory to the existing treatment strategies.« less

Disturbance of DKK1 level is partly involved in survival of lung cancer cells via regulation of ROMO1 and γ-radiation sensitivity

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

Kim, In Gyu, E-mail: igkim@kaeri.re.kr; Department of Radiation Biotechnology and Applied Radioisotope, University of Science and Technology; Kim, Seo Yoen

2014-01-03

Highlights: •DKK1 was expressed differently among non-small-cell lung cancer cell lines. •DKK1 negatively regulated ROMO1 gene expression. •Disturbance of DKK1 level induced the imbalance of cellular ROS. •DKK1/ROMO1-induced ROS imbalance is involved in cell survival in NSCLC. -- Abstract: Dickkopf1 (DKK1), a secreted protein involved in embryonic development, is a potent inhibitor of the Wnt signaling pathway and has been postulated to be a tumor suppressor or tumor promoter depending on the tumor type. In this study, we showed that DKK1 was expressed differently among non-small-cell lung cancer cell lines. The DKK1 expression level was much higher in A549 cellsmore » than in H460 cells. We revealed that blockage of DKK1 expression by silencing RNA in A549 cells caused up-regulation of intracellular reactive oxygen species (ROS) modulator (ROMO1) protein, followed by partial cell death, cell growth inhibition, and loss of epithelial–mesenchymal transition property caused by ROS, and it also increased γ-radiation sensitivity. DKK1 overexpression in H460 significantly inhibited cell survival with the decrease of ROMO1 level, which induced the decrease of cellular ROS. Thereafter, exogenous N-acetylcysteine, an antioxidant, or hydrogen peroxide, a pro-oxidant, partially rescued cells from death and growth inhibition. In each cell line, both overexpression and blockage of DKK1 not only elevated p-RB activation, which led to cell growth arrest, but also inactivated AKT/NF-kB, which increased radiation sensitivity and inhibited cell growth. This study is the first to demonstrate that strict modulation of DKK1 expression in different cell types partially maintains cell survival via tight regulation of the ROS-producing ROMO1 and radiation resistance.« less

Glioblastoma cells deficient in DNA-dependent protein kinase are resistant to cell death.

PubMed

Chen, George G; Sin, Fanny L F; Leung, Billy C S; Ng, Ho K; Poon, Wai S

2005-04-01

DNA-dependent protein kinase (DNA-PK), a nuclear serine/threonine kinase, is responsible for the DNA double-strand break repair. Cells lacking or with dysfunctional DNA-PK are often associated with mis-repair, chromosome aberrations, and complex exchanges, all of which are known to contribute to the development of human cancers including glioblastoma. Two human glioblastoma cell lines were used in the experiment, M059J cells lacking the catalytic subunit of DNA-PK, and their isogenic but DNA-PK proficient counterpart, M059K. We found that M059K cells were much more sensitive to staurosporine (STS) treatment than M059J cells, as demonstrated by MTT assay, TUNEL detection, and annexin-V and propidium iodide (PI) staining. A possible mechanism responsible for the different sensitivity in these two cell lines was explored by the examination of Bcl-2, Bax, Bak, and Fas. The cell death stimulus increased anti-apoptotic Bcl-2 and decreased pro-apoptotic Bcl-2 members (Bak and Bax) and Fas in glioblastoma cells deficient in DNA-PK. Activation of DNA-PK is known to promote cell death of human tumor cells via modulation of p53, which can down-regulate the anti-apoptotic Bcl-2 member proteins, induce pro-apoptotic Bcl-2 family members and promote a Bax-Bak interaction. Our experiment also demonstrated that the mode of glioblastoma cell death induced by STS consisted of both apoptosis and necrosis and the percentage of cell death in both modes was similar in glioblastoma cell lines either lacking DNA-PK or containing intact DNA-PK. Taken together, our findings suggest that DNA-PK has a positive role in the regulation of apoptosis in human glioblastomas. The aberrant expression of Bcl-2 family members and Fas was, at least in part, responsible for decreased sensitivity of DNA-PK deficient glioblastoma cells to cell death stimuli. 2004 Wiley-Liss, Inc.

MEK and TAK1 Regulate Apoptosis in Colon Cancer Cells with KRAS-Dependent Activation of Proinflammatory Signaling.

PubMed

McNew, Kelsey L; Whipple, William J; Mehta, Anita K; Grant, Trevor J; Ray, Leah; Kenny, Connor; Singh, Anurag

2016-12-01

MEK inhibitors have limited efficacy in treating RAS-RAF-MEK pathway-dependent cancers due to feedback pathway compensation and dose-limiting toxicities. Combining MEK inhibitors with other targeted agents may enhance efficacy. Here, codependencies of MEK, TAK1, and KRAS in colon cancer were investigated. Combined inhibition of MEK and TAK1 potentiates apoptosis in KRAS-dependent cells. Pharmacologic studies and cell-cycle analyses on a large panel of colon cancer cell lines demonstrate that MEK/TAK1 inhibition induces cell death, as assessed by sub-G 1 accumulation, in a distinct subset of cell lines. Furthermore, TAK1 inhibition causes G 2 -M cell-cycle blockade and polyploidy in many of the cell lines. MEK plus TAK1 inhibition causes reduced G 2 -M/polyploid cell numbers and additive cytotoxic effects in KRAS/TAK1-dependent cell lines as well as a subset of BRAF-mutant cells. Mechanistically, sensitivity to MEK/TAK1 inhibition can be conferred by KRAS and BMP receptor activation, which promote expression of NF-κB-dependent proinflammatory cytokines, driving tumor cell survival and proliferation. MEK/TAK1 inhibition causes reduced mTOR, Wnt, and NF-κB signaling in TAK1/MEK-dependent cell lines concomitant with apoptosis. A Wnt/NF-κB transcriptional signature was derived that stratifies primary tumors into three major subtypes: Wnt-high/NF-κB-low, Wnt-low/NF-κB-high and Wnt-high/NF-κB-high, designated W, N, and WN, respectively. These subtypes have distinct characteristics, including enrichment for BRAF mutations with serrated carcinoma histology in the N subtype. Both N and WN subtypes bear molecular hallmarks of MEK and TAK1 dependency seen in cell lines. Therefore, N and WN subtype signatures could be utilized to identify tumors that are most sensitive to anti-MEK/TAK1 therapeutics. This study describes a potential therapeutic strategy for a subset of colon cancers that are dependent on oncogenic KRAS signaling pathways, which are currently difficult to block with selective agents. Mol Cancer Res; 14(12); 1204-16. ©2016 AACR. ©2016 American Association for Cancer Research.

The sensitivity of glioma cells to pyropheophorbide-αmethyl ester-mediated photodynamic therapy is enhanced by inhibiting ABCG2.

PubMed

Pan, Li; Lin, Haidan; Tian, Si; Bai, Dingqun; Kong, Yuhan; Yu, Lehua

2017-09-01

To study the mechanisms of human glioblastoma cell resistance to methyl ester pyropheophorbide-a-mediated photodynamic therapy (MPPa-PDT) and the relationship between the cells and adenosine triphosphate-binding cassette superfamily G member 2 (ABCG2). The sensitivity of four human glioma cell lines (U87, A172, SHG-44, and U251) to MPPa-PDT was detected with a CCK-8 assay. Cell apoptosis, intracellular MPPa, and singlet oxygen were tested with flow cytometry. The mRNA and protein expression of ATP-binding cassette transporters (ABCG2, MRP1, and MDR1) were detected by PCR and Western blot, respectively. Both the sensitivity to MPPa-PDT and intracellular MPPa in A172 were the lowest among the four cell lines, while expression of ABCG2 mRNA and protein in A172 were the highest. The intracellular MPPa and ROS in A172 receiving MPPa-PDT significantly increased after using the ABCG2 inhibitor fumitremorgin C (FTC). Both cell viability and apoptosis in A172 cells undergoing MPPa-PDT were significantly improved with FTC. ABCG2 plays a significant role in the resistance of A172 to MPPa-PDT. Lasers Surg. Med. 49:719-726, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Skin sensitizer identification by IL-8 secretion and CD86 expression on THP-1 cells.

PubMed

Parise, Carolina Bellini; Sá-Rocha, Vanessa Moura; Moraes, Jane Zveiter

2015-12-25

Substantial progress has been made in the development of alternative methods for skin sensitization in the last decade in several countries around the world. Brazil is experiencing an increasing concern about using animals for product development, since the publication of the Law 9605/1998, which prohibits the use of animals when an alternative method is available. In this way, an in vitro test to evaluate allergenic potential is a pressing need.This preliminary study started setting the use of myelomonocytic THP-1 cell line, according to the human cell line activation test (h-CLAT), already under validation process. We found that 48-h chemical exposure was necessary to identify 22 out of 23 sensitizers by the analyses of CD86 expression. In addition, the CD54 expression analyses presented a poor efficiency to discriminate sensitizers from non-sensitizers in our conditions. In view of these results, we looked for changes of pro-inflammatory interleukin profile. The IL-8 secretion analyses after 24-h chemical incubation seemed to be an alternative for CD54 expression assessing.Altogether, our findings showed that the combination of the analyses of CD86 expression and IL-8 secretion allowed predicting allergenicity.

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.

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.

Quantitative Evaluation of Cisplatin Uptake in Sensitive and Resistant Individual Cells by Single-Cell ICP-MS (SC-ICP-MS).

PubMed

Corte Rodríguez, M; Álvarez-Fernández García, R; Blanco, E; Bettmer, J; Montes-Bayón, M

2017-11-07

One of the main limitations to the Pt-therapy in cancer is the development of associated drug resistance that can be associated with a significant reduction of the intracellular platinum concentration. Thus, intracellular Pt concentration could be considered as a biomarker of cisplatin resistance. In this work, an alternative method to address intracellular Pt concentration in individual cells is explored to permit the evaluation of different cell models and alternative therapies in a relatively fast way. For this aim, total Pt analysis in single cells has been implemented using a total consumption nebulizer coupled to inductively coupled plasma mass spectrometric detection (ICP-MS). The efficiency of the proposed device has been evaluated in combination with flow cytometry and turned out to be around 25% (cells entering the ICP-MS from the cells in suspension). Quantitative uptake studies of a nontoxic Tb-containing compound by individual cells were conducted and the results compared to those obtained by bulk analysis of the same cells. Both sets of data were statistically comparable. Thus, final application of the developed methodology to the comparative uptake of Pt-species in cisplatin resistant and sensitive cell lines (A2780cis and A2780) was conducted. The results obtained revealed the potential of this analytical strategy to differentiate between different cell lines of different sensitivity to the drug which might be of high medical interest.

Functional assessment of sodium chloride cotransporter NCC mutants in polarized mammalian epithelial cells.

PubMed

Rosenbaek, Lena L; Rizzo, Federica; MacAulay, Nanna; Staub, Olivier; Fenton, Robert A

2017-08-01

The thiazide-sensitive sodium chloride cotransporter NCC is important for maintaining serum sodium (Na + ) and, indirectly, serum potassium (K + ) levels. Functional studies on NCC have used cell lines with native NCC expression, transiently transfected nonpolarized cell lines, or Xenopus laevis oocytes. Here, we developed the use of polarized Madin-Darby canine kidney type I (MDCKI) mammalian epithelial cell lines with tetracycline-inducible human NCC expression to study NCC activity and membrane abundance in the same system. In radiotracer assays, induced cells grown on filters had robust thiazide-sensitive and chloride dependent sodium-22 ( 22 Na) uptake from the apical side. To minimize cost and maximize throughput, assays were modified to use cells grown on plastic. On plastic, cells had similar thiazide-sensitive 22 Na uptakes that increased following preincubation of cells in chloride-free solutions. NCC was detected in the plasma membrane, and both membrane abundance and phosphorylation of NCC were increased by incubation in chloride-free solutions. Furthermore, in cells exposed for 15 min to low or high extracellular K + , the levels of phosphorylated NCC increased and decreased, respectively. To demonstrate that the system allows rapid and systematic assessment of mutated NCC, three phosphorylation sites in NCC were mutated, and NCC activity was examined. 22 Na fluxes in phosphorylation-deficient mutants were reduced to baseline levels, whereas phosphorylation-mimicking mutants were constitutively active, even without chloride-free stimulation. In conclusion, this system allows the activity, cellular localization, and abundance of wild-type or mutant NCC to be examined in the same polarized mammalian expression system in a rapid, easy, and low-cost fashion. Copyright © 2017 the American Physiological Society.

Inward rectifier potassium channels in the HL-1 cardiomyocyte-derived cell line.

PubMed

Goldoni, Dana; Zhao, YouYou; Green, Brian D; McDermott, Barbara J; Collins, Anthony

2010-11-01

HL-1 is a line of immortalized cells of cardiomyocyte origin that are a useful complement to native cardiomyocytes in studies of cardiac gene regulation. Several types of ion channel have been identified in these cells, but not the physiologically important inward rectifier K(+) channels. Our aim was to identify and characterize inward rectifier K(+) channels in HL-1 cells. External Ba(2+) (100 µM) inhibited 44 ± 0.05% (mean ± s.e.m., n = 11) of inward current in whole-cell patch-clamp recordings. The reversal potential of the Ba(2+)-sensitive current shifted with external [K(+)] as expected for K(+)-selective channels. The slope conductance of the inward Ba(2+)-sensitive current increased with external [K(+)]. The apparent Kd for Ba(2+) was voltage dependent, ranging from 15 µM at -150  mV to 148 µM at -75  mV in 120  mM external K(+). This current was insensitive to 10 µM glybenclamide. A component of whole-cell current was sensitive to 150 µM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), although it did not correspond to the Ba(2+)-sensitive component. The effect of external 1 mM Cs(+) was similar to that of Ba(2+). Polymerase chain reaction using HL-1 cDNA as template and primers specific for the cardiac inward rectifier K(ir)2.1 produced a fragment of the expected size that was confirmed to be K(ir)2.1 by DNA sequencing. In conclusion, HL-1 cells express a current that is characteristic of cardiac inward rectifier K(+) channels, and express K(ir)2.1 mRNA. This cell line may have use as a system for studying inward rectifier gene regulation in a cardiomyocyte phenotype. © 2010 Wiley-Liss, Inc.

Apigenin sensitizes BEL-7402/ADM cells to doxorubicin through inhibiting miR-101/Nrf2 pathway

PubMed Central

Gao, Ai-Mei; Zhang, Xiao-Yu; Ke, Zun-Ping

2017-01-01

Chemo-resistance is one of the main obstacle in hepatocellular carcinoma therapy. Apigenin as a natural bioflavonoid has been exhibited anti-cancer properties in various malignant cancers. The aim of this study is to evaluate the potential chemo-sensitization effect of apigenin in doxorubicin-resistant hepatocellular carcinoma cell line BEL-7402/ADM and to investigate its possible mechanism. We found that apigenin significantly reversed doxorubicin sensitivity and induced caspase-dependent apoptosis in BEL-7402/ADM cells. Furthermore, apigenin induced miR-101 expression, and overexpression of miR-101 mimicked the doxorubicin-sensitizing effect of apigenin. Importantly, we showed that miR-101 was able to target the 3′-UTR of Nrf2. The results suggested that apigenin sensitizes BEL-7402/ADM cells to doxorubicin through miR-101/Nrf2 pathway, which furtherly supports apigenin as a potential chemo-sensitizer for hepatocellular carcinoma. PMID:29137246

SiMa Cells for a Serotype Specific and Sensitive Cell-Based Neutralization Test for Botulinum Toxin A and E.

PubMed

Bak, Nicola; Rajagopal, Shalini; Stickings, Paul; Sesardic, Dorothea

2017-07-20

Botulinum toxins (BoNTs), of which there are seven serotypes, are among the most potent neurotoxins, with serotypes A, B and E causing human botulism. Antitoxins form the first line of treatment for botulism, and functional, highly sensitive in vitro methods for toxin neutralization are needed to replace the current in vivo methods used for determination of antitoxin potency. In this preliminary proof of concept study, we report the development of a neutralization test using the neuroblastoma SiMa cell line. The assay is serotype specific for either BoNT/A or BoNT/E, which both cleave unique sequences on SNAP-25 within SiMa cells. The end point is simple immunodetection of cleaved SNAP-25 from cell lysates with antibodies detecting only the newly exposed sequence on SNAP-25. Neutralizing antibodies prevent the toxin-induced cleavage of SNAP-25. The toxin neutralization assay, with an EC50 of ~2 mIU/mL determined with a standardized reference antiserum, is more sensitive than the mouse bioassays. Relevance was demonstrated with commercial and experimental antitoxins targeting different functional domains, and of known in vivo neutralizing activities. This is the first report describing a simple, specific, in vitro cell-based assay for the detection of neutralizing antibodies against BoNT/A and BoNT/E with a sensitivity exceeding that of the mouse bioassay.

SiMa Cells for a Serotype Specific and Sensitive Cell-Based Neutralization Test for Botulinum Toxin A and E

PubMed Central

Bak, Nicola; Rajagopal, Shalini; Stickings, Paul; Sesardic, Dorothea

2017-01-01

Botulinum toxins (BoNTs), of which there are seven serotypes, are among the most potent neurotoxins, with serotypes A, B and E causing human botulism. Antitoxins form the first line of treatment for botulism, and functional, highly sensitive in vitro methods for toxin neutralization are needed to replace the current in vivo methods used for determination of antitoxin potency. In this preliminary proof of concept study, we report the development of a neutralization test using the neuroblastoma SiMa cell line. The assay is serotype specific for either BoNT/A or BoNT/E, which both cleave unique sequences on SNAP-25 within SiMa cells. The end point is simple immunodetection of cleaved SNAP-25 from cell lysates with antibodies detecting only the newly exposed sequence on SNAP-25. Neutralizing antibodies prevent the toxin-induced cleavage of SNAP-25. The toxin neutralization assay, with an EC50 of ~2 mIU/mL determined with a standardized reference antiserum, is more sensitive than the mouse bioassays. Relevance was demonstrated with commercial and experimental antitoxins targeting different functional domains, and of known in vivo neutralizing activities. This is the first report describing a simple, specific, in vitro cell-based assay for the detection of neutralizing antibodies against BoNT/A and BoNT/E with a sensitivity exceeding that of the mouse bioassay. PMID:28726719

Increased acid ceramidase expression depends on upregulation of androgen-dependent deubiquitinases, USP2, in a human prostate cancer cell line, LNCaP.

PubMed

Mizutani, Naoki; Inoue, Minami; Omori, Yukari; Ito, Hiromi; Tamiya-Koizumi, Keiko; Takagi, Akira; Kojima, Tetsuhito; Nakamura, Mitsuhiro; Iwaki, Soichiro; Nakatochi, Masahiro; Suzuki, Motoshi; Nozawa, Yoshinori; Murate, Takashi

2015-10-01

Acid ceramidase (ACDase) metabolizes ceramide to sphingosine, leading to sphingosine 1-phosphate production. Reportedly, ACDase has been upregulated in prostate cancer. However, its regulatory mechanism remains unclear. LNCaP (androgen-sensitive prostate cancer cell line) but not PC3 and DU-145, (androgen-unresponsive cell lines) exhibited the highest ACDase protein. Among three cell lines, ASAH1 mRNA level was not correlated with ACDase protein expression, and the 5'-promoter activity did not show androgen dependency, suggesting the post-transcriptional regulation of ACDase in LNCaP cells. Based on these results, LNCaP was analysed further. Casodex, androgen receptor antagonist, and charcoal-stripped FCS (CS-FCS) decreased ACDase protein and activity, whereas dihydrotestosterone in CS-FCS culture increased ACDase protein and enzyme activity. MG132, a proteasome inhibitor, prevented the decrease of ACDase protein when cultured in CS-FCS, suggesting the involvement of ubiquitin/proteasome system. Reportedly, USP2, a deubiquitinase, plays an important role in LNCaP cells. USP2 siRNA decreased ACDase protein, whereas USP2 overexpression increased ACDase protein of LNCaP cells. However, SKP2, an ubiquitin E3 ligase known to be active in prostate cancer, did not affect androgen-dependent ACDase expression in LNCaP cells. Thus, ACDase regulation by androgen in androgen-sensitive LNCaP cells is mainly due to its prolonged protein half-life by androgen-stimulated USP2 expression. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

A dual reporter cell assay for identifying serotype and drug susceptibility of herpes simplex virus.

PubMed

Lu, Wen-Wen; Sun, Jun-Ren; Wu, Szu-Sian; Lin, Wan-Hsuan; Kung, Szu-Hao

2011-08-15

A dual reporter cell assay (DRCA) that allows real-time detection of herpes simplex virus (HSV) infection was developed. This was achieved by stable transfection of cells with an expression cassette that contains the dual reporter genes, secreted alkaline phosphatase (SEAP) and enhanced green fluorescent protein (EGFP), under the control of an HSV early gene promoter. Baby hamster kidney (BHK) and Chinese hamster ovary (CHO) cell lines were used as parental cell lines because the former is permissive for both HSV serotypes, HSV-1 and HSV-2, whereas the latter is susceptible to infection only by HSV-2. The DRCA permitted differential detection of HSV-1 and HSV-2 by observation of EGFP-positive cells, as substantiated by screening a total of 35 samples. The BHK-based cell line is sensitive to a viral titer as low as a single plaque-forming unit with a robust assay window as measured by a chemiluminescent assay. Evaluations of the DRCA with representative acyclovir-sensitive and acyclovir-resistant HSV strains demonstrated that their drug susceptibilities were accurately determined by a 48-h format. In summary, this novel DRCA is a useful means for serotyping of HSV in real time as well as a rapid screening method for determining anti-HSV susceptibilities. Copyright © 2011 Elsevier Inc. All rights reserved.

Anticancer drugs are synergistic with freezing in induction of apoptosis in HCC cells.

PubMed

Yuan, FangJun; Zhou, Wenbo; Zhang, Jifa; Zhang, Zhiyun; Zou, Can; Huang, Ling; Zhang, YouShun; Dai, Zongqing

2008-08-01

Cryotherapy has been shown to be an important therapeutic alternative to surgery in the treatment of hepatocellular carcinoma (HCC). Here, the influence of cryo-chemotherapy on HCC was examined in vitro using the human HCC cell line Bel-7402, a drug-resistant HCC cell line originating from Bel-7402 cells (Bel-7402/R), as well as two control cell lines, the HCC cell line SMMC-7721 and a colorectal tumor cell line HIC-251. Cells were treated with either exposure to different freezing temperatures (ranging from -15 to -80 degrees C for 20 min), exposure to sub-lethal concentrations of anticancer chemotherapy drugs or a combination of cryotherapy and chemotherapy. Cell viability and apoptosis under each condition were investigated. We found that the combined treatment resulted in increases in both cell death and apoptosis compared to either treatment alone. The increased level of apoptosis observed in Bel-7402 cells after cryo-chemotherapy was inhibited in the presence of caspase inhibitors. Furthermore, Bax expression was increased 2- to 3-fold in cells exposed to the combination treatment compared with cells treated by freezing or drugs alone. In contrast, Bcl-2 levels remained constant. Although Bel-7402/R cells originated from the Bel-7402 cell line, they were more sensitive to the freezing procedure than the parental cell line. The level of Bax expression in Bel-7402/R cells was also higher than that observed in the parental cell line. In addition, we found that Bel-7402/R cells had lower levels of survivin mRNA than the parental Bel-7402 cells, in both untreated and treated cells. In conclusion, our data show that in HCC cells, apoptosis induced by cryotherapy can be synergistically enhanced using anticancer drugs.

Identifying anti-cancer drug response related genes using an integrative analysis of transcriptomic and genomic variations with cell line-based drug perturbations.

PubMed

Sun, Yi; Zhang, Wei; Chen, Yunqin; Ma, Qin; Wei, Jia; Liu, Qi

2016-02-23

Clinical responses to anti-cancer therapies often only benefit a defined subset of patients. Predicting the best treatment strategy hinges on our ability to effectively translate genomic data into actionable information on drug responses. To achieve this goal, we compiled a comprehensive collection of baseline cancer genome data and drug response information derived from a large panel of cancer cell lines. This data set was applied to identify the signature genes relevant to drug sensitivity and their resistance by integrating CNVs and the gene expression of cell lines with in vitro drug responses. We presented an efficient in-silico pipeline for integrating heterogeneous cell line data sources with the simultaneous modeling of drug response values across all the drugs and cell lines. Potential signature genes correlated with drug response (sensitive or resistant) in different cancer types were identified. Using signature genes, our collaborative filtering-based drug response prediction model outperformed the 44 algorithms submitted to the DREAM competition on breast cancer cells. The functions of the identified drug response related signature genes were carefully analyzed at the pathway level and the synthetic lethality level. Furthermore, we validated these signature genes by applying them to the classification of the different subtypes of the TCGA tumor samples, and further uncovered their in vivo implications using clinical patient data. Our work may have promise in translating genomic data into customized marker genes relevant to the response of specific drugs for a specific cancer type of individual patients.

Harnessing Connectivity in a Large-Scale Small-Molecule Sensitivity Dataset | Office of Cancer Genomics

Cancer.gov

Identifying genetic alterations that prime a cancer cell to respond to a particular therapeutic agent can facilitate the development of precision cancer medicines. Cancer cell-line (CCL) profiling of small-molecule sensitivity has emerged as an unbiased method to assess the relationships between genetic or cellular features of CCLs and small-molecule response. Here, we developed annotated cluster multidimensional enrichment analysis to explore the associations between groups of small molecules and groups of CCLs in a new, quantitative sensitivity dataset.

Experimental Model to Study the Role of Retinoblastoma Gene Product (pRb) for Determination of Adipocyte Differentiation.

PubMed

Popov, B V; Shilo, P S; Zhidkova, O V; Zaichik, A M; Petrov, N S

2015-06-01

Using stable constitutive expression of retinoblastoma gene product (pRb) in polypotent mesenchymal 10T1/2 cells we obtained stable cell lines hyperexpressing functionally active or inactive mutant pRb. The cells producing active exogenous pRb demonstrated high sensitivity to adipocyte differentiation inductors, whereas production of inactive form of the exogenous protein suppressed adipocyte differentiation. The obtained lines can serve as the experimental model for studying the role of pRb in determination of adipocyte differentiation.

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

The in-line measurement of plant cell biomass using radio frequency impedance spectroscopy as a component of process analytical technology.

PubMed

Holland, Tanja; Blessing, Daniel; Hellwig, Stephan; Sack, Markus

2013-10-01

Radio frequency impedance spectroscopy (RFIS) is a robust method for the determination of cell biomass during fermentation. RFIS allows non-invasive in-line monitoring of the passive electrical properties of cells in suspension and can distinguish between living and dead cells based on their distinct behavior in an applied radio frequency field. We used continuous in situ RFIS to monitor batch-cultivated plant suspension cell cultures in stirred-tank bioreactors and compared the in-line data to conventional off-line measurements. RFIS-based analysis was more rapid and more accurate than conventional biomass determination, and was sensitive to changes in cell viability. The higher resolution of the in-line measurement revealed subtle changes in cell growth which were not accessible using conventional methods. Thus, RFIS is well suited for correlating such changes with intracellular states and product accumulation, providing unique opportunities for employing systems biotechnology and process analytical technology approaches to increase product yield and quality. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Identification of heat-sensitive QTL derived from common wild rice (Oryza rufipogon Griff.).

PubMed

Lei, Dongyang; Tan, Lubin; Liu, Fengxia; Chen, Liyun; Sun, Chuanqing

2013-03-01

Understanding the responses of rice plants to heat-stress is a challenging, yet crucial, endeavor. A set of introgression lines was previously developed using an advanced backcrossing strategy that involved the elite indica cultivar Teqing as the recipient and an accession of common wild rice (Oryza rufipongon Griff.) as the donor. In this study, we evaluated the responses of 90 of these previously developed introgression lines to heat stress. Five quantitative trait loci (QTLs) related to heat response were detected. The phenotypic variances explained by these QTLs ranged from 6.83% to 14.63%, and O. rufipogon-derived alleles at one locus reduced sensitivity to heat. A heat-sensitive introgression line, YIL106, was identified and characterized. Genotypic analysis demonstrated that YIL106 contained four introgressed segments derived from O. rufipongon and two QTLs (qHTS1-1 and qHTS3) related to heat response. Physiological tests, including measurements of chlorophyll content, electrolyte leakage, malondialdehyde content, and soluble sugar content, were consistent with the heat sensitivity observed in YIL106. Ultrastructural analysis of YIL106 mesophyll cells showed that they were severely damaged following heat stress. This suggests that modification of the cell membrane system is a primary response to heat stress in plants. Identification and characterization of the heat-sensitive line YIL106 may facilitate the isolation of genes associated with the response of rice plants to heat stress. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

In vitro antileukaemic activity of extracts from chokeberry (Aronia melanocarpa [Michx] Elliott) and mulberry (Morus alba L.) leaves against sensitive and multidrug resistant HL60 cells.

PubMed

Skupień, Katarzyna; Kostrzewa-Nowak, Dorota; Oszmiański, Jan; Tarasiuk, Jolanta

2008-05-01

The aim of the present study was to determine in vitro antileukaemic activities of extracts obtained from chokeberry (Aronia melanocarpa [Michx] Elliot) and mulberry (Morus alba L.) leaves against promyelocytic HL60 cell line and its multidrug resistant sublines exhibiting two different MDR phenotypes: HL60/VINC (overexpressing P-glycoprotein) and HL60/DOX (overexpressing MRP1 protein). It was found that the extracts from chokeberry and mulberry leaves were active against the sensitive leukaemic cell line HL60 and retained the in vitro activity against multidrug resistant sublines (HL60/VINC and HL60/DOX). The values of resistance factor (RF) found for these extracts were very low lying in the range 1.2-1.6.

A highly reproducible quantitative viral outgrowth assay for the measurement of the replication-competent latent HIV-1 reservoir.

PubMed

Fun, Axel; Mok, Hoi Ping; Wills, Mark R; Lever, Andrew M

2017-02-24

Cure of Human Immunodeficiency Virus (HIV) infection remains elusive due to the persistence of HIV in a latent reservoir. Strategies to eradicate latent infection can only be evaluated with robust, sensitive and specific assays to quantitate reactivatable latent virus. We have taken the standard peripheral blood mononuclear cell (PBMC) based viral outgrowth methodology and from it created a logistically simpler and more highly reproducible assay to quantify replication-competent latent HIV in resting CD4 + T cells, both increasing accuracy and decreasing cost and labour. Purification of resting CD4 + T cells from whole PBMC is expedited and achieved in 3 hours, less than half the time of conventional protocols. Our indicator cell line, SupT1-CCR5 cells (a clonal cell line expressing CD4, CXCR4 and CCR5) provides a readily available standardised readout. Reproducibility compares favourably to other published assays but with reduced cost, labour and assay heterogeneity without compromising sensitivity.

INF-γ sensitizes head and neck squamous cell carcinoma cells to chemotherapy-induced apoptosis and necroptosis through up-regulation of Egr-1.

PubMed

Xu, Bei; Shu, Yongqian; Liu, Peng

2014-11-01

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. Acquired resistance to standard chemotherapy accounts for most of treatment failure. Here we demonstrate that Interferon-γ (INF-γ) may up-regulate Egr-1 gene expression in HNSCC cell line SCC-25. Forced expression of Egr-1 sensitizes SCC-25 cells to chemotherapy-induced apoptosis and necroptosis, a novel form of programmed cell death. Egr-1 up-regulation also significantly increases the production of Thrombospondin-1 (TSP-1), a matricellular glycoprotein which has been described to induce cell death in HNSCC. Moreover, INF-γ-induced sensitization of cells to chemotherapy-mediated cell death and TSP-1 production could be markedly abolished by Egr-1 silencing. The present investigation provides the first evidence that INF-γ may sensitize HNSCC cells to chemotherapy-induced apoptosis and necroptosis through up-regulation of Egr-1. These data support the combination use of INF-γ and cytotoxic drugs for HNSCC Therapy.

Intracellular flow cytometry may be combined with good quality and high sensitivity RT-qPCR analysis.

PubMed

Sandstedt, Mikael; Jonsson, Marianne; Asp, Julia; Dellgren, Göran; Lindahl, Anders; Jeppsson, Anders; Sandstedt, Joakim

2015-12-01

Flow cytometry (FCM) has become a well-established method for analysis of both intracellular and cell-surface proteins, while quantitative RT-PCR (RT-qPCR) is used to determine gene expression with high sensitivity and specificity. Combining these two methods would be of great value. The effects of intracellular staining on RNA integrity and RT-qPCR sensitivity and quality have not, however, been fully examined. We, therefore, intended to assess these effects further. Cells from the human lung cancer cell line A549 were fixed, permeabilized and sorted by FCM. Sorted cells were analyzed using RT-qPCR. RNA integrity was determined by RNA quality indicator analysis. A549 cells were then mixed with cells of the mouse cardiomyocyte cell line HL-1. A549 cells were identified by the cell surface marker ABCG2, while HL-1 cells were identified by intracellular cTnT. Cells were sorted and analyzed by RT-qPCR. Finally, cell cultures from human atrial biopsies were used to evaluate the effects of fixation and permeabilization on RT-qPCR analysis of nonimmortalized cells stored prior to analysis by FCM. A large amount of RNA could be extracted even when cells had been fixed and permeabilized. Permeabilization resulted in increased RNA degradation and a moderate decrease in RT-qPCR sensitivity. Gene expression levels were also affected to a moderate extent. Sorted populations from the mixed A549 and HL-1 cell samples showed gene expression patterns that corresponded to FCM data. When samples were stored before FCM sorting, the RT-qPCR analysis could still be performed with high sensitivity and quality. In summary, our results show that intracellular FCM may be performed with only minor impairment of the RT-qPCR sensitivity and quality when analyzing sorted cells; however, these effects should be considered when comparing RT-qPCR data of not fixed samples with those of fixed and permeabilized samples. © 2015 International Society for Advancement of Cytometry.

Systematic Identification of Combinatorial Drivers and Targets in Cancer Cell Lines

PubMed Central

Tabchy, Adel; Eltonsy, Nevine; Housman, David E.; Mills, Gordon B.

2013-01-01

There is an urgent need to elicit and validate highly efficacious targets for combinatorial intervention from large scale ongoing molecular characterization efforts of tumors. We established an in silico bioinformatic platform in concert with a high throughput screening platform evaluating 37 novel targeted agents in 669 extensively characterized cancer cell lines reflecting the genomic and tissue-type diversity of human cancers, to systematically identify combinatorial biomarkers of response and co-actionable targets in cancer. Genomic biomarkers discovered in a 141 cell line training set were validated in an independent 359 cell line test set. We identified co-occurring and mutually exclusive genomic events that represent potential drivers and combinatorial targets in cancer. We demonstrate multiple cooperating genomic events that predict sensitivity to drug intervention independent of tumor lineage. The coupling of scalable in silico and biologic high throughput cancer cell line platforms for the identification of co-events in cancer delivers rational combinatorial targets for synthetic lethal approaches with a high potential to pre-empt the emergence of resistance. PMID:23577104

Systematic identification of combinatorial drivers and targets in cancer cell lines.

PubMed

Tabchy, Adel; Eltonsy, Nevine; Housman, David E; Mills, Gordon B

2013-01-01

There is an urgent need to elicit and validate highly efficacious targets for combinatorial intervention from large scale ongoing molecular characterization efforts of tumors. We established an in silico bioinformatic platform in concert with a high throughput screening platform evaluating 37 novel targeted agents in 669 extensively characterized cancer cell lines reflecting the genomic and tissue-type diversity of human cancers, to systematically identify combinatorial biomarkers of response and co-actionable targets in cancer. Genomic biomarkers discovered in a 141 cell line training set were validated in an independent 359 cell line test set. We identified co-occurring and mutually exclusive genomic events that represent potential drivers and combinatorial targets in cancer. We demonstrate multiple cooperating genomic events that predict sensitivity to drug intervention independent of tumor lineage. The coupling of scalable in silico and biologic high throughput cancer cell line platforms for the identification of co-events in cancer delivers rational combinatorial targets for synthetic lethal approaches with a high potential to pre-empt the emergence of resistance.

Convenient method of establishing permanent lines of xeroderma pigmentosum cells. [Ultraviolet radiation

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

Tohda, H.; Oikawa, A.; Katsuki, T.

Nine lymphoblastoid cell lines were established after transformation by Epstein-Barr virus of peripheral lymphocytes from four xeroderma pigmentosum (XP) patients, the parents of one XP patient, and three normal donors. All these cell lines proliferate as suspension in Roswell Park Memorial Institute Medium 1640 supplemented with 20% fetal bovine serum, without detectable release of infectious Epstein-Barr virus. Some characteristics of these cell lines, such as growth rates, chromosome numbers, uv sensitivities, and activities of unscheduled DNA syntheses induced by uv, 4-nitroquinoline 1-oxide, and N-methyl-N'-nitro-N-nitrosoguanidine, were determined. Results confirm that the properties related to XP are not altered by transformation withmore » Epstein-Barr virus and are the same in degrees of defect as are those of dermal fibroblasts from the respective individuals. These XP and normal lymphoblastoid cell lines should be especially useful for biochemical studies on the mechanism of DNA repair, because they are easy to grow in mass culture.« less

Downregulation of indoleamine-2,3-dioxygenase in cervical cancer cells suppresses tumor growth by promoting natural killer cell accumulation

PubMed Central

SATO, NAOTO; SAGA, YASUSHI; MIZUKAMI, HIROAKI; WANG, DONGDONG; TAKAHASHI, SUZUYO; NONAKA, HIROAKI; FUJIWARA, HIROYUKI; TAKEI, YUJI; MACHIDA, SHIZUO; TAKIKAWA, OSAMU; OZAWA, KEIYA; SUZUKI, MITSUAKI

2012-01-01

This study examined the role of the immunosuppressive enzyme indoleamine-2,3-dioxygenase (IDO) in cervical cancer progression and the possible use of this enzyme for cervical cancer therapy. We analyzed IDO protein expression in 9 cervical cancer cell lines (SKG-I, -II, -IIIa, -IIIb, SiHa, CaSki, BOKU, HCS-2 and ME-180) stimulated with interferon-γ. IDO expression was observed in all cell lines except for SKG-IIIb. We transfected the human cervical cancer cell line CaSki that constitutively expresses IDO with a short hairpin RNA vector targeting IDO, and established an IDO-downregulated cell line to determine whether inhibition of IDO mediates cervical cancer progression. IDO downregulation suppressed tumor growth in vivo, without influencing cancer cell growth in vitro. Moreover, IDO downregulation enhanced the sensitivity of cervical cancer cells to natural killer (NK) cells in vitro and promoted NK cell accumulation in the tumor stroma in vivo. These findings indicate that downregulation of IDO controls cervical cancer progression by activating NK cells, suggesting IDO as a potential therapy for cervical cancer. PMID:22923135

Different sensitivity of germinal center B cell-like diffuse large B cell lymphoma cells towards ibrutinib treatment

PubMed Central

2014-01-01

Background Although rituximab in the combination of CHOP chemotherapy has been widely used as the standard treatment for several kinds of B-cell non-Hodgkin lymphoma (B-NHL), a great number of B-NHL patients treated with this immunotherapy still develop primary and secondary resistance. Recently Bruton’s tyrosine kinase (Btk) inhibitor ibrutinib showed promising therapeutic effect in relapsed/refractory CLL and B-cell NHL, which provided essential alternatives for these patients. Methods The proliferation and apoptosis induction of tumor cells were measured by cell viability assay and Annexin-V staining. Western Blotting analysis and real-time PCR were used to detect the expression level of target proteins and chemokines production. Results We demonstrated that ibrutinib inhibited the proliferation and induced apoptosis of GCB-DLBCL cell lines through suppression of BCR signaling pathway and activation of caspase-3. Furthermore, the chemokines CCL3 and CCL4 production from tumor cells were also found to be attenuated by ibrutinib treatment. But different cell lines exhibited distinct sensitivity after ibrutinib treatment. Interestingly, the decreasing level of p-ERK after ibrutinib treatment, but not the basal expression level of Btk, correlated with different drug sensitivity. Conclusions Ibrutinib could be a potentially useful therapy for GCB-DLBCL and the decreasing level of p-ERK could become a useful biomarker to predict related therapeutic response. PMID:24693884

Complementation of a Fanconi anemia group A cell line by UbA{sup 52}

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

Moses, R.E.; Heina, J.A.; Jakobs, P.M.

1994-09-01

Cells from patients with Fanconi anemia (FA) display chromosomal instability and increased sensitivity to mitomycin C (MMC) and diepoxybutane (DEB) relative to normal cells. Several genes act in this pathway of DNA damage processing based upon four known complementation groups in FA. We have made a cDNA expression library in a vector with a G418 selectable marker to identify FA genes other than the FA-C group. Approximately 1 x 10{sup 6} independent cDNA clones were isolated with an average cDNA size of 1.5 kb. Five cell lines resistant to MMC and DEB were isolated from 6 x 10{sup 6} G418-resistantmore » transfectants from 65 individual transfections of the FA-A fibroblast line GM6914. The isolated cell lines also showed normal chromosome stability. The same cDNA (600 bp) was recovered from three independent cell lines by PCR using flanking sequence primers. The gene has sequence identity with a known gene, the ubiquitin fusion gene, UbA{sub 52}. Interestingly, each of the cDNAs were inserted in antisense orientation relative to the cytomegalovirus (CMV) promoter as determined by sequencing and PCR using UbA{sub 52}-specific internal primers. Southern blot analysis indicated the cell lines had distinct chromosomal insertion sites. Mutation analysis by chemical cleavage showed no reading frame mutations, indicating that UbA{sub 52} is not the FA-A gene. Re-transfection with the UbA{sub 52} gene in antisense gave complementation for MMC, DEB and chromosome stability to varying degrees. Re-transfection of the antisense construct with the CMV promotor removed or with a sense construct did not alter the MMC sensitivity. We conclude that the antisense UbA{sub 52} gene has a non-specific effect, perhaps acting by altering the cell cycle or susceptibility to apoptosis.« less

Networking of differentially expressed genes in human cancer cells resistant to methotrexate

PubMed Central

2009-01-01

Background The need for an integrated view of data obtained from high-throughput technologies gave rise to network analyses. These are especially useful to rationalize how external perturbations propagate through the expression of genes. To address this issue in the case of drug resistance, we constructed biological association networks of genes differentially expressed in cell lines resistant to methotrexate (MTX). Methods Seven cell lines representative of different types of cancer, including colon cancer (HT29 and Caco2), breast cancer (MCF-7 and MDA-MB-468), pancreatic cancer (MIA PaCa-2), erythroblastic leukemia (K562) and osteosarcoma (Saos-2), were used. The differential expression pattern between sensitive and MTX-resistant cells was determined by whole human genome microarrays and analyzed with the GeneSpring GX software package. Genes deregulated in common between the different cancer cell lines served to generate biological association networks using the Pathway Architect software. Results Dikkopf homolog-1 (DKK1) is a highly interconnected node in the network generated with genes in common between the two colon cancer cell lines, and functional validations of this target using small interfering RNAs (siRNAs) showed a chemosensitization toward MTX. Members of the UDP-glucuronosyltransferase 1A (UGT1A) family formed a network of genes differentially expressed in the two breast cancer cell lines. siRNA treatment against UGT1A also showed an increase in MTX sensitivity. Eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) was overexpressed among the pancreatic cancer, leukemia and osteosarcoma cell lines, and siRNA treatment against EEF1A1 produced a chemosensitization toward MTX. Conclusions Biological association networks identified DKK1, UGT1As and EEF1A1 as important gene nodes in MTX-resistance. Treatments using siRNA technology against these three genes showed chemosensitization toward MTX. PMID:19732436

Roscovitine strongly enhances the effect of olaparib on radiosensitivity for HPV neg. but not for HPV pos. HNSCC cell lines.

PubMed

Ziemann, Frank; Seltzsam, Steve; Dreffke, Kristin; Preising, Stefanie; Arenz, Andrea; Subtil, Florentine S B; Rieckmann, Thorsten; Engenhart-Cabillic, Rita; Dikomey, Ekkehard; Wittig, Andrea

2017-12-01

At present, advanced stage human Papillomavirus (HPV) negative and positive head and neck squamous cell carcinoma (HNSCC) are treated by intense multimodal therapy that includes radiochemotherapy, which are associated with relevant side effects. Patients with HPV positive tumors possess a far better prognosis than those with HPV negative cancers. Therefore, new therapeutic strategies are needed to improve the outcome especially of the latter one as well as quality of life for all HNSCC patients. Here we tested whether roscovitine, an inhibitor of cyclin-dependent kinases (CDKs), which hereby also blocks homologous recombination (HR), can be used to enhance the radiation sensitivity of HNSCC cell lines. In all five HPV negative and HPV positive cell lines tested, roscovitine caused inhibition of CDK1 and 2. Surprisingly, all HPV positive cell lines were found to be defective in HR. In contrast, HPV negative strains demonstrated efficient HR, which was completely suppressed by roscovitine. In line with this, for HPV negative but not for HPV positive cell lines, treatment with roscovitine resulted in a pronounced enhancement of the radiation-induced G2 arrest as well as a significant increase in radiosensitivity. Due to a defect in HR, all HPV positive cell lines were efficiently radiosensitized by the PARP-1 inhibitor olaparib. In contrast, in HPV negative cell lines a significant radiosensitization by olaparib was only achieved when combined with roscovitine.

Intestinal T cell responses to cereal proteins in celiac disease.

PubMed

Kilmartin, C; Wieser, H; Abuzakouk, M; Kelly, J; Jackson, J; Feighery, C

2006-01-01

Celiac disease is caused by sensitivity to wheat gluten in genetically susceptible individuals. The etiological role of the other wheat-related cereals, barley, rye, and oats, is still debated. In order to investigate this issue further, in this study we examined the immune response of celiac mucosal T cell lines to fractions from all four cereals. Cell stimulation was assessed by measuring proliferation (employing (3)H-thymidine incorporation) or cytokine (IL-2, IFN-gamma) production. All five T cell lines demonstrated immunoreactivity to protein fractions from the four related cereals. In some cell lines, reactivity to wheat, barley, and rye was only evident when these cereal fractions had been pretreated with tissue transglutaminase. This study confirms the similar T cell antigenic reactivity of these four related cereals and has implications for their exclusion in the gluten-free diet. However, despite oats stimulation of T cell lines, this cereal does not activate a mucosal lesion in most celiac patients.

Establishment of paclitaxel-resistant breast cancer cell line and nude mice models, and underlying multidrug resistance mechanisms in vitro and in vivo.

PubMed

Chen, Si-Ying; Hu, Sa-Sa; Dong, Qian; Cai, Jiang-Xia; Zhang, Wei-Peng; Sun, Jin-Yao; Wang, Tao-Tao; Xie, Jiao; He, Hai-Rong; Xing, Jian-Feng; Lu, Jun; Dong, Ya-Lin

2013-01-01

Breast cancer is a common malignant tumor which affects health of women and multidrug resistance (MDR) is one of the main factors leading to failure of chemotherapy. This study was conducted to establish paclitaxel-resistant breast cancer cell line and nude mice models to explore underlying mechanisms of MDR. The breast cancer drug-sensitive cell line MCF-7 (MCF-7/S) was exposed in stepwise escalating paclitaxel (TAX) to induce a resistant cell line MCF-7/TAX. Cell sensitivity to drugs and growth curves were measured by MTT assay. Changes of cell morphology and ultrastructure were examined by optical and electron microscopy. The cell cycle distribution was determined by flow cytometry. Furthermore, expression of proteins related to breast cancer occurrence and MDR was tested by immunocytochemistry. In Vivo, nude mice were injected with MCF-7/S and MCF-7/TAX cells and weights and tumor sizes were observed after paclitaxel treatment. In addition, proteins involved breast cancer and MDR were detected by immunohistochemistry. Compared to MCF-7/S, MCF-7/TAX cells had a higher resistance to paclitaxel, cross-resistance and prolonged doubling time. Moreover, MCF-7/TAX showed obvious alterations of ultrastructure. Estrogen receptor (ER) expression was low in drug resistant cells and tumors while expression of human epidermal growth factor receptor 2 (HER2) and Ki-67 was up-regulated. P-glycoprotein (P-gp), lung resistance-related protein (LRP) and glutathione-S-transferase-π (GST-π) involved in the MDR phenotype of resistant cells and tumors were all overexpressed. The underlying MDR mechanism of breast cancer may involve increased expression of P-gp, LRP and GST-π.

Homoharringtonine targets Smad3 and TGF-β pathway to inhibit the proliferation of acute myeloid leukemia cells.

PubMed

Chen, Jian; Mu, Qitian; Li, Xia; Yin, Xiufeng; Yu, Mengxia; Jin, Jing; Li, Chenying; Zhou, Yile; Zhou, Jiani; Suo, Shanshan; Lu, Demin; Jin, Jie

2017-06-20

Homoharringtonine (HHT) has long and widely been used in China for the treatment of acute myeloid leukemia (AML), the clinical therapeutic effect is significant but the working mechanism is poorly understood. The purpose of this study is to screen the possible target for HHT with virtual screening and verify the findings by cell experiments. Software including Autodock, Python, and MGL tools were used, with HHT being the ligand and proteins from PI3K-Akt pathway, Jak-stat pathway, TGF-β pathway and NK-κB pathway as the receptors. Human AML cell lines including U937, KG-1, THP-1 were cultured and used as the experiment cell lines. MTT assay was used for proliferation detection, flowcytometry was used to detect apoptosis and cell cycle arrest upon HHT functioning, western blotting was used to detect the protein level changes, viral shRNA transfection was used to suppress the expression level of the target protein candidate, and viral mRNA transfection was used for over-expression. Virtual screening revealed that smad3 from TGF-β pathway might be the candidate for HHT binding. In AML cell line U937 and KG-1, HHT can induce the Ser423/425 phosphorylation of smad3, and this phosphorylation can subsequently activate the TGF-β pathway, causing cell cycle arrest at G1 phase in U937 cells and apoptosis in KG-1 cells, knockdown of smad3 can impair the sensitivity of U937 cell to HHT, and over-expression of smad3 can re-establish the sensitivity in both cell lines. We conclude that smad3 is the probable target protein of HHT and plays an important role in the functioning mechanism of HHT.

Homoharringtonine targets Smad3 and TGF-β pathway to inhibit the proliferation of acute myeloid leukemia cells

PubMed Central

Yin, Xiufeng; Yu, Mengxia; Jin, Jing; Li, Chenying; Zhou, Yile; Zhou, Jiani; Suo, Shanshan; Lu, Demin; Jin, Jie

2017-01-01

Homoharringtonine (HHT) has long and widely been used in China for the treatment of acute myeloid leukemia (AML), the clinical therapeutic effect is significant but the working mechanism is poorly understood. The purpose of this study is to screen the possible target for HHT with virtual screening and verify the findings by cell experiments. Software including Autodock, Python, and MGL tools were used, with HHT being the ligand and proteins from PI3K-Akt pathway, Jak-stat pathway, TGF-β pathway and NK-κB pathway as the receptors. Human AML cell lines including U937, KG-1, THP-1 were cultured and used as the experiment cell lines. MTT assay was used for proliferation detection, flowcytometry was used to detect apoptosis and cell cycle arrest upon HHT functioning, western blotting was used to detect the protein level changes, viral shRNA transfection was used to suppress the expression level of the target protein candidate, and viral mRNA transfection was used for over-expression. Virtual screening revealed that smad3 from TGF-β pathway might be the candidate for HHT binding. In AML cell line U937 and KG-1, HHT can induce the Ser423/425 phosphorylation of smad3, and this phosphorylation can subsequently activate the TGF-β pathway, causing cell cycle arrest at G1 phase in U937 cells and apoptosis in KG-1 cells, knockdown of smad3 can impair the sensitivity of U937 cell to HHT, and over-expression of smad3 can re-establish the sensitivity in both cell lines. We conclude that smad3 is the probable target protein of HHT and plays an important role in the functioning mechanism of HHT. PMID:28454099

Evaluation of the sensitizing potential of antibiotics in vitro using the human cell lines THP-1 and MUTZ-LC and primary monocyte‐derived dendritic cells

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

Sebastian, Katrin, E-mail: ksebastian@ukaachen.de; Ott, Hagen; Zwadlo-Klarwasser, Gabriele

Since the 7th amendment to the EU cosmetics directive foresees a complete ban on animal testing, alternative in vitro methods have been established to evaluate the sensitizing potential of small molecular weight compounds. To find out whether these novel in vitro assays are also capable to predict the sensitizing potential of small molecular weight drugs, model compounds such as beta-lactams and sulfonamides – which are the most frequent cause of adverse drug reactions – were co-incubated with THP-1, MUTZ-LC, or primary monocyte‐derived dendritic cells for 48 h and subsequent expression of selected marker genes (IL-8, IL-1β, CES1, NQO1, GCLM, PIRmore » and TRIM16) was studied by real time PCR. Benzylpenicillin and phenoxymethylpenicillin were recognized as sensitizing compounds because they are capable to induce the mRNA expression of these genes in moDCs and, except for IL-8, in THP-1 cells but not in MUTZ-LC. Ampicillin stimulated the expression of some marker genes in moDCs and THP-1 cells. SMX did not affect the expression of these genes in THP-1, however, in moDCs, at least PIR was enhanced and there was an increase of the release of IL-8. These data reveal that novel in vitro DC based assays might play a role in the evaluation of the allergenic potential of novel drug compounds, but these systems seem to lack the ability to detect the sensitizing potential of prohaptens that require metabolic activation prior to sensitization and moDCs seem to be superior with regard to the sensitivity compared with THP-1 and MUTZ-3 cell lines. -- Highlights: ► We tested the sensitizing potential of small molecular weight drugs in vitro. ► In vitro assays were performed with moDCs and THP-1 cells. ► Beta-lactam antibiotics can be recognized as sensitizing compounds. ► They affect the expression of metabolic enzymes, cytokines and transcription factors. ► Sulfamethoxazole has no measurable effect on THP-1 cells and moDCs.« less

Hepatitis B X-interacting protein promotes cisplatin resistance and regulates CD147 via Sp1 in ovarian cancer.

PubMed

Zou, Wei; Ma, Xiangdong; Yang, Hong; Hua, Wei; Chen, Biliang; Cai, Guoqing

2017-03-01

Ovarian cancer is the highest mortality rate of all female reproductive malignancies. Drug resistance is a major cause of treatment failure in malignant tumors. Hepatitis B X-interacting protein acts as an oncoprotein, regulates cell proliferation, and migration in breast cancer. We aimed to investigate the effects and mechanisms of hepatitis B X-interacting protein on resistance to cisplatin in human ovarian cancer cell lines. The mRNA and protein levels of hepatitis B X-interacting protein were detected using RT-PCR and Western blotting in cisplatin-resistant and cisplatin-sensitive tissues, cisplatin-resistant cell lines A2780/CP and SKOV3/CP, and cisplatin-sensitive cell lines A2780 and SKOV3. Cell viability and apoptosis were measured to evaluate cellular sensitivity to cisplatin in A2780/CP cells. Luciferase reporter gene assay was used to determine the relationship between hepatitis B X-interacting protein and CD147. The in vivo function of hepatitis B X-interacting protein on tumor burden was assessed in cisplatin-resistant xenograft models. The results showed that hepatitis B X-interacting protein was highly expressed in ovarian cancer of cisplatin-resistant tissues and cells. Notably, knockdown of hepatitis B X-interacting protein significantly reduced cell viability in A2780/CP compared with cisplatin treatment alone. Hepatitis B X-interacting protein and cisplatin cooperated to induce apoptosis and increase the expression of c-caspase 3 as well as the Bax/Bcl-2 ratio. We confirmed that hepatitis B X-interacting protein up-regulated CD147 at the protein expression and transcriptional levels. Moreover, we found that hepatitis B X-interacting protein was able to activate the CD147 promoter through Sp1. In vivo, depletion of hepatitis B X-interacting protein decreased the tumor volume and weight induced by cisplatin. Taken together, these results indicate that hepatitis B X-interacting protein promotes cisplatin resistance and regulated CD147 via Sp1 in ovarian cancer cell lines. Impact statement We found that hepatitis B X-interacting protein (HBXIP) was able to activate the CD147 promoter through Sp1. In vivo, depletion of HBXIP decreased the tumor volume and weight induced by CP. Taken together, these results indicate that HBXIP promotes cisplatin resistance and regulated CD147 via Sp1 in ovarian cancer cell lines.

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

Wolfe, Adam R.; Morgan Welch Inflammatory Breast Cancer Research Program and Clinic, University of Texas MD Anderson Cancer Center, Houston, Texas; Atkinson, Rachel L.

Purpose: We previously demonstrated that cholesterol-lowering agents regulate radiation sensitivity of inflammatory breast cancer (IBC) cell lines in vitro and are associated with less radiation resistance among IBC patients who undergo postmastectomy radiation. We hypothesized that decreasing IBC cellular cholesterol induced by treatment with lipoproteins would increase radiation sensitivity. Here, we examined the impact of specific transporters of cholesterol (ie lipoproteins) on the responses of IBC cells to self-renewal and to radiation in vitro and on clinical outcomes in IBC patients. Methods and Materials: Two patient-derived IBC cell lines, SUM 149 and KPL4, were incubated with low-density lipoproteins (LDL), very-low-density lipoproteins (VLDL),more » or high-density lipoproteins (HDL) for 24 hours prior to irradiation (0-6 Gy) and mammosphere formation assay. Cholesterol panels were examined in a cohort of patients with primary IBC diagnosed between 1995 and 2011 at MD Anderson Cancer Center. Lipoprotein levels were then correlated to patient outcome, using the log rank statistical model, and examined in multivariate analysis using Cox regression. Results: VLDL increased and HDL decreased mammosphere formation compared to untreated SUM 149 and KPL4 cells. Survival curves showed enhancement of survival in both of the IBC cell lines when pretreated with VLDL and, conversely, radiation sensitization in all cell lines when pretreated with HDL. In IBC patients, higher VLDL values (>30 mg/dL) predicted a lower 5-year overall survival rate than normal values (hazard ratio [HR] = 1.9 [95% confidence interval [CI]: 1.05-3.45], P=.035). Lower-than-normal patient HDL values (<60 mg/dL) predicted a lower 5-year overall survival rate than values higher than 60 mg/dL (HR = 3.21 [95% CI: 1.25-8.27], P=.015). Conclusions: This study discovered a relationship among the plasma levels of lipoproteins, overall patient response, and radiation resistance in IBC patients and IBC patient-derived cell lines. A more expansive study is needed to verify these observations.« less

Oncogenic PIK3CA gene mutations and HER2/neu gene amplifications determine the sensitivity of uterine serous carcinoma cell lines to GDC-0980, a selective inhibitor of Class I PI3 kinase and mTOR kinase (TORC1/2).

PubMed

English, Diana P; Bellone, Stefania; Cocco, Emiliano; Bortolomai, Ileana; Pecorelli, Sergio; Lopez, Salvatore; Silasi, Dan-Arin; Schwartz, Peter E; Rutherford, Thomas; Santin, Alessandro D

2013-11-01

To evaluate PIK3CA mutational status and c-erbB2 gene amplification in a series of primary uterine serous carcinomas (USC) cell lines. To assess the efficacy of GDC-0980, a potent inhibitor of Class I PI3 kinase and mTOR kinase (TORC1/2), against primary USC harboring HER2/neu gene amplification and/or PIK3CA mutations. Twenty-two primary USC cell lines were evaluated for c-erbB2 oncogene amplification by fluorescence in situ hybridization (FISH) assays and for PIK3CA gene mutations by direct DNA sequencing of exons 9 and 20. In vitro sensitivity to GDC-0980 was evaluated by flow-cytometry-based viability and proliferation assays. Downstream cellular responses to GDC-0980 were assessed by measuring phosphorylation of the 4-EBP1 protein by flow-cytometry. Five of 22 (22.7%) USC cell lines contained oncogenic PIK3CA mutations although 9 (40.9%) harbored c-erbB2 gene amplification by FISH. GDC-0980 caused a strong differential growth inhibition in FISH+ USC when compared with FISH- (GDC-0980 IC50 mean ± SEM = 0.29 ± 0.05 μM in FISH+ vs 1.09 ± 0.20 μM in FISH- tumors, P = .02). FISH+ USC harboring PIK3CA mutations were significantly more sensitive to GDC-0980 exposure when compared with USC cell lines harboring wild-type PIK3CA (P = .03). GDC-0980 growth-inhibition was associated with a significant and dose-dependent decline in phosphorylated 4-EBP1 levels. Oncogenic PIK3CA mutations and c-erbB2 gene amplification may represent biomarkers to identify patients harboring USC who may benefit most from the use of GDC-0980. Copyright © 2013 Mosby, Inc. All rights reserved.

Energy metabolism of leukemia cells: glycolysis versus oxidative phosphorylation.

PubMed

Suganuma, Kazuto; Miwa, Hiroshi; Imai, Norikazu; Shikami, Masato; Gotou, Mayuko; Goto, Mineaki; Mizuno, Shohei; Takahashi, Miyuki; Yamamoto, Hidesuke; Hiramatsu, Akihito; Wakabayashi, Motohiro; Watarai, Masaya; Hanamura, Ichiro; Imamura, Akira; Mihara, Hidetsugu; Nitta, Masakazu

2010-11-01

For generation of energy, cancer cells utilize glycolysis more vigorously than oxidative phosphorylation in mitochondria (Warburg effect). We examined the energy metabolism of four leukemia cell lines by using glycolysis inhibitor, 2-deoxy-d-glucose (2-DG) and inhibitor of oxidative phosphorylation, oligomycin. NB4 was relatively sensitive to 2-DG (IC(50): 5.75 mM), consumed more glucose and produced more lactate (waste product of glycolysis) than the three other cell lines. Consequently, NB4 was considered as a "glycolytic" leukemia cell line. Dependency on glycolysis in NB4 was confirmed by the fact that glucose (+) FCS (-) medium showed more growth and survival than glucose (-) FCS (+) medium. Alternatively, THP-1, most resistant to 2-DG (IC(50): 16.14 mM), was most sensitive to oligomycin. Thus, THP-1 was recognized to be dependent on oxidative phosphorylation. In THP-1, glucose (-) FCS (+) medium showed more growth and survival than glucose (+) FCS (-) medium. The dependency of THP-1 on FCS was explained, at least partly, by fatty acid oxidation because inhibitor of fatty acid β-oxidation, etomoxir, augmented the growth suppression of THP-1 by 2-DG. We also examined the mechanisms by which THP-1 was resistant to, and NB4 was sensitive to 2-DG treatment. In THP-1, AMP kinase (AMPK), which is activated when ATP becomes limiting, was rapidly phosphorylated by 2-DG, and expression of Bcl-2 was augmented, which might result in resistance to 2-DG. On the other hand, AMPK phosphorylation and augmentation of Bcl-2 expression by 2-DG were not observed in NB4, which is 2-DG sensitive. These results will facilitate the future leukemia therapy targeting metabolic pathways.

Insulin-Like Growth Factor 2 Silencing Restores Taxol Sensitivity in Drug Resistant Ovarian Cancer

PubMed Central

Brouwer-Visser, Jurriaan; Lee, Jiyeon; McCullagh, KellyAnne; Cossio, Maria J.; Wang, Yanhua; Huang, Gloria S.

2014-01-01

Drug resistance is an obstacle to the effective treatment of ovarian cancer. We and others have shown that the insulin-like growth factor (IGF) signaling pathway is a novel potential target to overcome drug resistance. The purpose of this study was to validate IGF2 as a potential therapeutic target in drug resistant ovarian cancer and to determine the efficacy of targeting IGF2 in vivo. An analysis of The Cancer Genome Atlas (TCGA) data in the serous ovarian cancer cohort showed that high IGF2 mRNA expression is significantly associated with shortened interval to disease progression and death, clinical indicators of drug resistance. In a genetically diverse panel of ovarian cancer cell lines, the IGF2 mRNA levels measured in cell lines resistant to various microtubule-stabilizing agents including Taxol were found to be significantly elevated compared to the drug sensitive cell lines. The effect of IGF2 knockdown on Taxol resistance was investigated in vitro and in vivo. Transient IGF2 knockdown significantly sensitized drug resistant cells to Taxol treatment. A Taxol-resistant ovarian cancer xenograft model, developed from HEY-T30 cells, exhibited extreme drug resistance, wherein the maximal tolerated dose of Taxol did not delay tumor growth in mice. Blocking the IGF1R (a transmembrane receptor that transmits signals from IGF1 and IGF2) using a monoclonal antibody did not alter the response to Taxol. However, stable IGF2 knockdown using short-hairpin RNA in HEY-T30 effectively restored Taxol sensitivity. These findings validate IGF2 as a potential therapeutic target in drug resistant ovarian cancer and show that directly targeting IGF2 may be a preferable strategy compared with targeting IGF1R alone. PMID:24932685

Potential role of mTORC2 as a therapeutic target in clear cell carcinoma of the ovary.

PubMed

Hisamatsu, Takeshi; Mabuchi, Seiji; Matsumoto, Yuri; Kawano, Mahiru; Sasano, Tomoyuki; Takahashi, Ryoko; Sawada, Kenjiro; Ito, Kimihiko; Kurachi, Hirohisa; Schilder, Russell J; Testa, Joseph R; Kimura, Tadashi

2013-07-01

The goal of this study was to examine the role of mTOR complex 2 (mTORC2) as a therapeutic target in ovarian clear cell carcinoma (CCC), which is regarded as an aggressive, chemoresistant histologic subtype. Using tissue microarrays of 98 primary ovarian cancers [52 CCCs and 46 serous adenocarcinomas (SAC)], activation of mTORC2 was assessed by immunohistochemistry. Then, the growth-inhibitory effect of mTORC2-targeting therapy, as well as the role of mTORC2 signaling as a mechanism for acquired resistance to the mTOR complex 1 (mTORC1) inhibitor RAD001 in ovarian CCC, were examined using two pairs of RAD001-sensitive parental (RMG2 and HAC2) and RAD001-resistant CCC cell lines (RMG2-RR and HAC2-RR). mTORC2 was more frequently activated in CCCs than in SACs (71.2% vs. 45.7%). Simultaneous inhibition of mTORC1 and mTORC2 by AZD8055 markedly inhibited the proliferation of both RAD001-sensitive and -resistant cells in vitro. Treatment with RAD001 induced mTORC2-mediated AKT activation in RAD001-sensitive CCC cells. Moreover, increased activation of mTORC2-AKT signaling was observed in RAD001-resistant CCC cells compared with the respective parental cells. Inhibition of mTORC2 during RAD001 treatment enhanced the antitumor effect of RAD001 and prevented CCC cells from acquiring resistance to RAD001. In conclusion, mTORC2 is frequently activated, and can be a promising therapeutic target, in ovarian CCCs. Moreover, mTORC2-targeted therapy may be efficacious in a first-line setting as well as for second-line treatment of recurrent disease developing after RAD001-treatment.

Phenformin enhances the therapeutic effect of selumetinib in KRAS-mutant non-small cell lung cancer irrespective of LKB1 status.

PubMed

Zhang, Jun; Nannapaneni, Sreenivas; Wang, Dongsheng; Liu, Fakeng; Wang, Xu; Jin, Rui; Liu, Xiuju; Rahman, Mohammad Aminur; Peng, Xianghong; Qian, Guoqing; Chen, Zhuo G; Wong, Kwok-Kin; Khuri, Fadlo R; Zhou, Wei; Shin, Dong M

2017-08-29

MEK inhibition is potentially valuable in targeting KRAS-mutant non-small cell lung cancer (NSCLC). Here, we analyzed whether concomitant LKB1 mutation alters sensitivity to the MEK inhibitor selumetinib, and whether the metabolism drug phenformin can enhance the therapeutic effect of selumetinib in isogenic cell lines with different LKB1 status. Isogenic pairs of KRAS-mutant NSCLC cell lines A549, H460 and H157, each with wild-type and null LKB1, as well as genetically engineered mouse-derived cell lines 634 ( kras G12D/wt /p53 -/- /lkb1 wt/wt ) and t2 ( kras G12D/wt /p53 -/- / lkb1 -/- ) were used in vitro to analyze the activities of selumetinib, phenformin and their combination. Synergy was measured and potential mechanisms investigated. The in vitro findings were then confirmed in vivo using xenograft models. The re-expression of wild type LKB1 increased phospho-ERK level, suggesting that restored dependency on MEK->ERK->MAPK signaling might have contributed to the enhanced sensitivity to selumetinib. In contrast, the loss of LKB1 sensitized cells to phenformin. At certain combination ratios, phenformin and selumetinib showed synergistic activity regardless of LKB1 status. Their combination reduced phospho-ERK and S6 levels and induced potent apoptosis, but was likely through different mechanisms in cells with different LKB1 status. Finally, in xenograft models bearing isogenic A549 cells, we confirmed that loss of LKB1 confers resistance to selumetinib, and phenformin significantly enhances the therapeutic effect of selumetinib. Irrespective of LKB1 status, phenformin may enhance the anti-tumor effect of selumetinib in KRAS-mutant NSCLC. The dual targeting of MEK and cancer metabolism may provide a useful strategy to treat this subset of lung cancer.

The selective PI3Kα inhibitor BYL719 as a novel therapeutic option for neuroendocrine tumors: Results from multiple cell line models

PubMed Central

Rentsch, Jakob; Freitag, Helma; Detjen, Katharina; Briest, Franziska; Möbs, Markus; Weissmann, Victoria; Siegmund, Britta; Auernhammer, Christoph J.; Aristizabal Prada, Elke Tatjana; Lauseker, Michael; Grossman, Ashley; Exner, Samantha; Fischer, Christian; Grötzinger, Carsten

2017-01-01

Background/Aims The therapeutic options for metastatic neuroendocrine tumors (NETs) are limited. As PI3K signaling is often activated in NETs, we have assessed the effects of selective PI3Kp110α inhibition by the novel agent BYL719 on cell viability, colony formation, apoptosis, cell cycle, signaling pathways, differentiation and secretion in pancreatic (BON-1, QGP-1) and pulmonary (H727) NET cell lines. Methods Cell viability was investigated by WST-1 assay, colony formation by clonogenic assay, apoptosis by caspase3/7 assay, the cell cycle by FACS, cell signaling by Western blot analysis, expression of chromogranin A and somatostatin receptors 1/2/5 by RT-qPCR, and chromogranin A secretion by ELISA. Results BYL719 dose-dependently decreased cell viability and colony formation with the highest sensitivity in BON-1, followed by H727, and lowest sensitivity in QGP-1 cells. BYL719 induced apoptosis and G0/G1 cell cycle arrest associated with increased p27 expression. Western blots showed inhibition of PI3K downstream targets to a varying degree in the different cell lines, but IGF1R activation. The most sensitive BON-1 cells displayed a significant, and H727 cells a non-significant, GSK3 inhibition after BYL719 treatment, but these effects do not appear to be mediated through the IGF1R. In contrast, the most resistant QGP-1 cells showed no GSK3 inhibition, but a modest activation, which would partially counteract the other anti-proliferative effects. Accordingly, BYL719 enhanced neuroendocrine differentiation with the strongest effect in BON-1, followed by H727 cells indicated by induction of chromogranin A and somatostatin receptor 1/2 mRNA-synthesis, but not in QGP-1 cells. In BON-1 and QGP-1 cells, the BYL719/everolimus combination was synergistic through simultaneous AKT/mTORC1 inhibition, and significantly increased somatostatin receptor 2 transcription compared to each drug separately. Conclusion Our results suggest that the agent BYL719 could be a novel therapeutic approach to the treatment of NETs that may sensitize NET cells to somatostatin analogs, and that if there is resistance to its action this may be overcome by combination with everolimus. PMID:28800359

The selective PI3Kα inhibitor BYL719 as a novel therapeutic option for neuroendocrine tumors: Results from multiple cell line models.

PubMed

Nölting, Svenja; Rentsch, Jakob; Freitag, Helma; Detjen, Katharina; Briest, Franziska; Möbs, Markus; Weissmann, Victoria; Siegmund, Britta; Auernhammer, Christoph J; Aristizabal Prada, Elke Tatjana; Lauseker, Michael; Grossman, Ashley; Exner, Samantha; Fischer, Christian; Grötzinger, Carsten; Schrader, Jörg; Grabowski, Patricia

2017-01-01

The therapeutic options for metastatic neuroendocrine tumors (NETs) are limited. As PI3K signaling is often activated in NETs, we have assessed the effects of selective PI3Kp110α inhibition by the novel agent BYL719 on cell viability, colony formation, apoptosis, cell cycle, signaling pathways, differentiation and secretion in pancreatic (BON-1, QGP-1) and pulmonary (H727) NET cell lines. Cell viability was investigated by WST-1 assay, colony formation by clonogenic assay, apoptosis by caspase3/7 assay, the cell cycle by FACS, cell signaling by Western blot analysis, expression of chromogranin A and somatostatin receptors 1/2/5 by RT-qPCR, and chromogranin A secretion by ELISA. BYL719 dose-dependently decreased cell viability and colony formation with the highest sensitivity in BON-1, followed by H727, and lowest sensitivity in QGP-1 cells. BYL719 induced apoptosis and G0/G1 cell cycle arrest associated with increased p27 expression. Western blots showed inhibition of PI3K downstream targets to a varying degree in the different cell lines, but IGF1R activation. The most sensitive BON-1 cells displayed a significant, and H727 cells a non-significant, GSK3 inhibition after BYL719 treatment, but these effects do not appear to be mediated through the IGF1R. In contrast, the most resistant QGP-1 cells showed no GSK3 inhibition, but a modest activation, which would partially counteract the other anti-proliferative effects. Accordingly, BYL719 enhanced neuroendocrine differentiation with the strongest effect in BON-1, followed by H727 cells indicated by induction of chromogranin A and somatostatin receptor 1/2 mRNA-synthesis, but not in QGP-1 cells. In BON-1 and QGP-1 cells, the BYL719/everolimus combination was synergistic through simultaneous AKT/mTORC1 inhibition, and significantly increased somatostatin receptor 2 transcription compared to each drug separately. Our results suggest that the agent BYL719 could be a novel therapeutic approach to the treatment of NETs that may sensitize NET cells to somatostatin analogs, and that if there is resistance to its action this may be overcome by combination with everolimus.

Bcl-XL small interfering RNA suppresses the proliferation of 5-fluorouracil-resistant human colon cancer cells.

PubMed

Zhu, Hongbo; Guo, Wei; Zhang, Lidong; Davis, John J; Teraishi, Fuminori; Wu, Shuhong; Cao, Xiaobo; Daniel, Jonathan; Smythe, W Roy; Fang, Bingliang

2005-03-01

5-Fluorouracil (5-FU) is commonly used to treat human colon cancers but resistance to this compound is frequently observed in clinics. To characterize mechanisms of resistance to 5-FU and to develop new strategies for overcoming it, we established two cell lines that were resistant to 5-FU but not other chemotherapeutic agents from parental 5-FU-sensitive cell lines. Western blot analysis revealed that these resistant cells overexpressed the proteins Bcl-XL, Bcl-Xs, and Bik, and further data showed that the cells were resistant to 5-FU-induced DNA damage and cell cycle disorder. However, in parental cells, enforced expression of Bcl-XL protein provided only limited protection from 5-FU-induced apoptosis and overexpression of Bcl-XL protein did not affect 5-FU-induced DNA damage or cell cycle changes; these findings suggested that overexpression of Bcl-XL protein was not the major contributor to 5-FU resistance in any of our cells lines. Even so, knockdown of Bcl-XL protein expression by Bcl-XL-specific small interfering RNA could inhibit proliferation more effectively in 5-FU-resistant cells than in 5-FU-sensitive cells, and the combination of Bcl-XL-specific small interfering RNA and 5-FU had additive effect on the inhibition of 5-FU-resistant cells. These results suggest that down-regulation of Bcl-XL protein expression might provide a new treatment strategy for human 5-FU-resistant colon cancer therapy.

Etoposide radiosensitizes p53-defective cholangiocarcinoma cell lines independent of their G2 checkpoint efficacies

PubMed Central

Hematulin, Arunee; Meethang, Sutiwan; Utapom, Kitsana; Wongkham, Sopit; Sagan, Daniel

2018-01-01

Radiotherapy has been accounted as the most comprehensive cancer treatment modality over the past few decades. However, failure of this treatment modality occurs in several malignancies due to the resistance of cancer cells to radiation. It was previously reported by the present authors that defective cell cycle checkpoints could be used as biomarkers for predicting the responsiveness to radiation in individual patients with cholangiocarcinoma (CCA). However, identification of functional defective cell cycle checkpoints from cells from a patient's tissues is cumbersome and not applicable in the clinic. The present study evaluated the radiosensitization potential of etoposide in p53-defective CCA KKU-M055 and KKU-M214 cell lines. Treatment with etoposide enhanced the responsiveness of two p53-defective CCA cell lines to radiation independent of G2 checkpoint function. In addition, etoposide treatment increased radiation-induced cell death without altering the dominant mode of cell death of the two cell lines. These findings indicate that etoposide could be used as a radiation sensitizer for p53-defective tumors, independent of the function of G2 checkpoint. PMID:29541168

Identification of chrysoplenetin from Vitex negundo as a potential cytotoxic agent against PANC-1 and a panel of 39 human cancer cell lines (JFCR-39).

PubMed

Awale, Suresh; Linn, Thein Zaw; Li, Feng; Tezuka, Yasuhiro; Myint, Aung; Tomida, Akihiro; Yamori, Takao; Esumi, Hiroyasu; Kadota, Shigetoshi

2011-12-01

Human pancreatic cancer is known to be the most deadly disease with the lowest 5-year survival rate and is resistant to well known conventional chemotherapeutic drugs in clinical use. Screening of medicinal plants from Myanmar utilizing antiausterity strategy led to the identification of Vitex negundo as one of the medicinal plants having potent preferential cytotoxic activity against PANC-1 human pancreatic cancer cells. Bioactivity-guided phytochemical investigation led to the isolation of chrysoplenetin (1) and chrysosplenol D (2) as the active constituents with a PC(50) value of 3.4 μg/mL and 4.6 μg/mL, respectively, against PANC-1 cells. Both these compounds induced apoptosis-like morphological changes in PANC-1 cells. Chrysoplenetin was further tested against a panel of 39 human cancer cell lines (JFCR-39) at the Japanese Foundation for Cancer Research, and 25 cell lines belonging to lung, breast, CNS, colon, melanoma, ovarian, prostate cancer and stomach cancer cell lines were found to be highly sensitive to chrysoplenetin at a submicromolar range. In the JFCR-39 panel, lung NCI-H522, ovarian OVCAR-3 and prostate PC-3 cells were found to be most sensitive with GI(50) of 0.12, 0.18 and 0.17 μm, respectively. The COMPARE analysis suggested that the molecular mode of action of chrysoplenetin was unique compared with the existing anticancer drugs. Copyright © 2011 John Wiley & Sons, Ltd.

In- and ex-vivo molecular imaging of apoptosis to assess sensitivity of non-small cell lung cancer to EGFR inhibitors using probe-based confocal laser endomicroscopy.

PubMed

Guisier, Florian; Bohn, Pierre; Patout, Maxime; Piton, Nicolas; Farah, Insaf; Vera, Pierre; Thiberville, Luc; Salaün, Mathieu

2017-01-01

Prediction of treatment outcome of non-small cell lung cancer (NSCLC) with EGFR inhibitors on the basis of the genetic analysis of the tumor can be incorrect in case of rare or complex mutations, bypass molecular activation pathways, or pharmacodynamic variations. The aim of this study was to develop an ex vivo and in vivo real-time quantitative imaging test for EGFR inhibitors sensitivity assessment. Erlotinib resistant (A549, H460, H1975), insensitive (H1650) and hypersensitive (HCC827) cell lines were injected subcutaneously in Nude mice. Tumor xenografts from mice treated with Erlotinib were imaged ex vivo and in vivo using probe-based confocal laser endomicroscopy (pCLE) and NucView 488 Caspase 3 substrate, a fluorescent probe specific for the activated caspase 3. Assessment of apoptosis at 24h post treatment, both ex vivo in explanted tumor xenografts and in vivo, showed a significant difference between resistant cell lines (A549, H460 and H1975) and insensitive (H1650) or hypersensitive (HCC827) ones (p<0.05 for ex vivo imaging, p≤0.02 for in vivo imaging). There was also a significant difference between insensitive and hypersensitive cell lines, both ex vivo (p<0.05) and in vivo (p = 0.01). Real-time in vivo and ex vivo assessment of apoptosis using pCLE differentiates resistant from sensitive NSCLC xenografts to Erlotinib.

53BP1 depletion causes PARP inhibitor resistance in ATM-deficient breast cancer cells.

PubMed

Hong, Ruoxi; Ma, Fei; Zhang, Weimin; Yu, Xiying; Li, Qing; Luo, Yang; Zhu, Changjun; Jiang, Wei; Xu, Binghe

2016-09-09

Mutations in DNA damage response factors BRCA1 and BRCA2 confer sensitivity to poly(ADP-ribose) polymerase (PARP) inhibitors in breast and ovarian cancers. BRCA1/BRCA2-defective tumors can exhibit resistance to PARP inhibitors via multiple mechanisms, one of which involves loss of 53BP1. Deficiency in the DNA damage response factor ataxia-telangiectasia mutated (ATM) can also sensitize tumors to PARP inhibitors, raising the question of whether the presence or absence of 53BP1 can predict sensitivity of ATM-deficient breast cancer to these inhibitors. Cytotoxicity of PARP inhibitor and ATM inhibitor in breast cancer cell lines was assessed by MTS, colony formation and apoptosis assays. ShRNA lentiviral vectors were used to knockdown 53BP1 expression in breast cancer cell lines. Phospho-ATM and 53BP1 protein expressions were determined in human breast cancer tissues by immunohistochemistry (IHC). We show that inhibiting ATM increased cytotoxicity of PARP inhibitor in triple-negative and non-triple-negative breast cancer cell lines, and depleting the cells of 53BP1 reduced this cytotoxicity. Inhibiting ATM abrogated homologous recombination induced by PARP inhibitor, and down-regulating 53BP1 partially reversed this effect. Further, overall survival was significantly better in triple-negative breast cancer patients with lower levels of phospho-ATM and tended to be better in patients with negative 53BP1. These results suggest that 53BP1 may be a predictor of PARP inhibitor resistance in patients with ATM-deficient tumors.

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.

Acquisition of relative interstrand crosslinker resistance and PARP inhibitor sensitivity in Fanconi anemia head and neck cancers

PubMed Central

Lombardi, Anne J.; Hoskins, Elizabeth E.; Foglesong, Grant D.; Wikenheiser-Brokamp, Kathryn A.; Wiesmüller, Lisa; Hanenberg, Helmut; Andreassen, Paul R.; Jacobs, Allison J.; Olson, Susan B.; Keeble, Winifred W.; Hays, Laura E.; Wells, Susanne I.

2015-01-01

Purpose Fanconi anemia (FA) is an inherited disorder associated with a constitutional defect in the FA DNA repair machinery that is essential for resolution of DNA interstrand crosslinks. Individuals with FA are predisposed to formation of head and neck squamous cell carcinomas (HNSCCs) at a young age. Prognosis is poor, partly due to patient intolerance of chemotherapy and radiation requiring dose reduction, which may lead to early recurrence of disease. Experimental Design Using HNSCC cell lines derived from the tumors of FA patients, and murine HNSCC cell lines derived from the tumors of wild type and Fancc−/− mice, we sought to define FA-dependent chemosensitivity and DNA repair characteristics. We utilized DNA repair reporter assays to explore the preference of FA HNSCC cells for non-homologous end joining (NHEJ). Results Surprisingly, interstrand crosslinker (ICL) sensitivity was not necessarily FA-dependent in human or murine cell systems. Our results suggest that the increased Ku-dependent NHEJ that is expected in FA cells did not mediate relative ICL resistance. ICL exposure resulted in increased DNA damage sensing and repair by poly(ADP-ribose) polymerase (PARP) in FA-deficient cells. Moreover, human and murine FA HNSCC cells were sensitive to PARP inhibition, and sensitivity of human cells was attenuated by FA gene complementation. Conclusions The observed reliance upon PARP-mediated mechanisms reveals a means by which FA HNSCCs can acquire relative resistance to the ICL-based chemotherapy that is a foundation of HNSCC treatment, as well as a potential target for overcoming chemoresistance in the chemosensitive individual. PMID:25609062

Precision Oncology beyond Targeted Therapy: Combining Omics Data with Machine Learning Matches the Majority of Cancer Cells to Effective Therapeutics.

PubMed

Ding, Michael Q; Chen, Lujia; Cooper, Gregory F; Young, Jonathan D; Lu, Xinghua

2018-02-01

Precision oncology involves identifying drugs that will effectively treat a tumor and then prescribing an optimal clinical treatment regimen. However, most first-line chemotherapy drugs do not have biomarkers to guide their application. For molecularly targeted drugs, using the genomic status of a drug target as a therapeutic indicator has limitations. In this study, machine learning methods (e.g., deep learning) were used to identify informative features from genome-scale omics data and to train classifiers for predicting the effectiveness of drugs in cancer cell lines. The methodology introduced here can accurately predict the efficacy of drugs, regardless of whether they are molecularly targeted or nonspecific chemotherapy drugs. This approach, on a per-drug basis, can identify sensitive cancer cells with an average sensitivity of 0.82 and specificity of 0.82; on a per-cell line basis, it can identify effective drugs with an average sensitivity of 0.80 and specificity of 0.82. This report describes a data-driven precision medicine approach that is not only generalizable but also optimizes therapeutic efficacy. The framework detailed herein, when successfully translated to clinical environments, could significantly broaden the scope of precision oncology beyond targeted therapies, benefiting an expanded proportion of cancer patients. Mol Cancer Res; 16(2); 269-78. ©2017 AACR . ©2017 American Association for Cancer Research.

Effect of simulated microgravity on oxidation-sensitive gene expression in PC12 cells

NASA Astrophysics Data System (ADS)

Kwon, Ohwon; Sartor, Maureen; Tomlinson, Craig R.; Millard, Ronald W.; Olah, Mark E.; Sankovic, John M.; Banerjee, Rupak K.

2006-01-01

Oxygen utilization by and oxygen dependence of cellular processes may be different in biological systems that are exposed to microgravity (micro-g). A baseline in which cellular changes in oxygen sensitive molecular processes occur during micro-g conditions would be important to pursue this question. The objective of this research is to analyze oxidation-sensitive gene expression in a model cell line [rat pheochromocytoma (PC12)] under simulated micro-g conditions. The PC12 cell line is well characterized in its response to oxygen, and is widely recognized as a sensitive model for studying the responses of oxygen-sensitive molecular and cellular processes. This study uses the rotating wall vessel bioreactor (RWV) designed at NASA to simulate micro-g. Gene expression in PC12 cells in response to micro-g was analyzed by DNA microarray technology. The microarray analysis of PC12 cells cultured for 4 days under simulated micro-g under standardized oxygen environment conditions revealed more than 100 genes whose expression levels were changed at least twofold (up-regulation of 65 genes and down-regulation of 39 genes) compared with those from cells in the unit gravity (unit-g) control. This study observed that genes involved in the oxidoreductase activity category were most significantly differentially expressed under micro-g conditions. Also, known oxidation-sensitive transcription factors such as hypoxia-inducible factor-2α, c-myc, and the peroxisome proliferator-activated receptor-γ were changed significantly. Our initial results from the gene expression microarray studies may provide a context in which to evaluate the effect of varying oxygen environments on the background of differential gene regulation of biological processes under variable gravity conditions.

Effect of simulated microgravity on oxidation-sensitive gene expression in PC12 cells

PubMed Central

Kwon, Ohwon; Sartor, Maureen; Tomlinson, Craig R.; Millard, Ronald W.; Olah, Mark E.; Sankovic, John M.; Banerjee, Rupak K.

2008-01-01

Oxygen utilization by and oxygen dependence of cellular processes may be different in biological systems that are exposed to microgravity (micro-g). A baseline in which cellular changes in oxygen sensitive molecular processes occur during micro-g conditions would be important to pursue this question. The objective of this research is to analyze oxidation-sensitive gene expression in a model cell line [rat pheochromocytoma (PC12)] under simulated micro-g conditions. The PC12 cell line is well characterized in its response to oxygen, and is widely recognized as a sensitive model for studying the responses of oxygen-sensitive molecular and cellular processes. This study uses the rotating wall vessel bioreactor (RWV) designed at NASA to simulate micro-g. Gene expression in PC12 cells in response to micro-g was analyzed by DNA microarray technology. The microarray analysis of PC12 cells cultured for 4 days under simulated micro-g under standardized oxygen environment conditions revealed more than 100 genes whose expression levels were changed at least twofold (up-regulation of 65 genes and down-regulation of 39 genes) compared with those from cells in the unit gravity (unit-g) control. This study observed that genes involved in the oxidoreductase activity category were most significantly differentially expressed under micro-g conditions. Also, known oxidation-sensitive transcription factors such as hypoxia-inducible factor-2α, c-myc, and the peroxisome proliferator-activated receptor-γ were changed significantly. Our initial results from the gene expression microarray studies may provide a context in which to evaluate the effect of varying oxygen environments on the background of differential gene regulation of biological processes under variable gravity conditions. PMID:19081771

Regulation of D-cyclin translation inhibition in myeloma cells treated with mTOR inhibitors: Rationale for combined treatment with ERK inhibitors and rapamycin

PubMed Central

Frost, Patrick; Shi, Yijiang; Hoang, Bao; Gera, Joseph; Lichtenstein, Alan

2009-01-01

We have shown that heightened AKT activity sensitized multiple myeloma (MM) cells to the anti-tumor effects of the mTOR-inhibitor, CCI-779. To test the mechanism of AKT’s regulatory role, we stably transfected U266 MM cell lines with an activated AKT allele or empty vector. The AKT-transfected cells were more sensitive to cytostasis induced in vitro by rapamycin or in vivo by its analog, CCI-779, whereas cells with quiescent AKT were resistant. The ability of mTOR inhibitors to downregulate D-cyclin expression was significantly greater in AKT-transfected MM cells, due in part, to AKT’s ability to curtail cap-independent translation and internal ribosome entry site (IRES) activity of D-cyclin transcripts. Similar AKT-dependent regulation of rapamycin responsiveness was demonstrated in a second myeloma model: the PTEN-null OPM-2 cell line transfected with wild type PTEN. As ERK/p38 activity facilitates IRES-mediated translation of some transcripts, we investigated ERK/p38 as regulators of AKT-dependent effects on rapamycin sensitivity. AKT-transfected U266 cells demonstrated significantly decreased ERK and p38 activity. However, only an ERK inhibitor prevented D-cyclin IRES activity in resistant “low AKT” myeloma cells. Furthermore, the ERK inhibitor successfully sensitized myeloma cells to rapamycin in terms of down regulated D-cyclin protein expression and G1 arrest. However, ectopic over-expression of an activated MEK gene did not increase cap-independent translation of D-cyclin in “high AKT” myeloma cells indicating that MEK/ERK activity was required but not sufficient for activation of the IRES. These data support a scenario where heightened AKT activity down-regulates D-cyclin IRES function in MM cells and ERK facilitates activity. PMID:19139116

Repeated cisplatin treatment can lead to a multiresistant tumor cell population with stem cell features and sensitivity to 3-bromopyruvate.

PubMed

Wintzell, My; Löfstedt, Lina; Johansson, Joel; Pedersen, Anne B; Fuxe, Jonas; Shoshan, Maria

2012-12-01

Cisplatin is used in treatment of several types of cancer, including epithelial ovarian carcinoma (EOC). In order to mimic clinical treatment and to investigate longterm effects of cisplatin in surviving cancer cells, two EOC cell lines were repeatedly treated with low doses. In the SKOV-3 cell line originating from malignant ascites, but not in A2780 cells from a primary tumor, this led to emergence of a stable population (SKOV-3-R) which in the absence of cisplatin showed increased motility, epithelial-mesenchymal transition (EMT) and expression of cancer stem cell markers CD117, CD44 and ALDH1. Accordingly, the cells formed self-renewing spheres in serum-free stem cell medium. Despite upregulation of mitochondrial mass and cytochrome c, and no upregulation of Bcl-2/Bcl-xL, SKOV-3-R were multiresistant to antineoplastic drugs. Cancer stem cells, or tumor-initiating cells (TICs) are highly chemoresistant and are believed to cause relapse into disseminated and resistant EOC. Our second aim was therefore to target resistance in these TIC-like cells. Resistance could be correlated with upregulation of hexokinase-II and VDAC, which are known to form a survival-promoting mitochondrial complex. The cells were thus sensitive to 3-bromopyruvate, which dissociates hexokinase-II from this complex, and were particularly sensitive to combination treatment with cisplatin at doses down to 0.1 x IC 50. 3-bromopyruvate might thus be of use in targeting the especially aggressive TIC populations.

Repeated cisplatin treatment can lead to a multiresistant tumor cell population with stem cell features and sensitivity to 3-bromopyruvate

PubMed Central

Wintzell, My; Löfstedt, Lina; Johansson, Joel; Pedersen, Anne B.; Fuxe, Jonas; Shoshan, Maria

2012-01-01

Cisplatin is used in treatment of several types of cancer, including epithelial ovarian carcinoma (EOC). In order to mimic clinical treatment and to investigate longterm effects of cisplatin in surviving cancer cells, two EOC cell lines were repeatedly treated with low doses. In the SKOV-3 cell line originating from malignant ascites, but not in A2780 cells from a primary tumor, this led to emergence of a stable population (SKOV-3-R) which in the absence of cisplatin showed increased motility, epithelial-mesenchymal transition (EMT) and expression of cancer stem cell markers CD117, CD44 and ALDH1. Accordingly, the cells formed self-renewing spheres in serum-free stem cell medium. Despite upregulation of mitochondrial mass and cytochrome c, and no upregulation of Bcl-2/Bcl-xL, SKOV-3-R were multiresistant to antineoplastic drugs. Cancer stem cells, or tumor-initiating cells (TICs) are highly chemoresistant and are believed to cause relapse into disseminated and resistant EOC. Our second aim was therefore to target resistance in these TIC-like cells. Resistance could be correlated with upregulation of hexokinase-II and VDAC, which are known to form a survival-promoting mitochondrial complex. The cells were thus sensitive to 3-bromopyruvate, which dissociates hexokinase-II from this complex, and were particularly sensitive to combination treatment with cisplatin at doses down to 0.1 x IC50. 3-bromopyruvate might thus be of use in targeting the especially aggressive TIC populations. PMID:22954696

BLIND TRIALS EVALUATING IN VITRO INFECTIVITY OF CRYPTOSPORIDIUM PARVUM OOCYSTS USING CELL CULTURE IMMUNOFLUORESCENCE

EPA Science Inventory

An optimized cell culture-immunofluorescence (IFA) procedure, using the HCT-8 cell line, was evaluated in 'blind' trials to determine the sensitivity and reproducibility for measuring infectivity of flow cytometry prepared inocula of C. parvum oocysts. In separate trials, suspens...

In Vitro Study of Influence of Au Nanoparticles on HT29 and SPEV Cell Lines

NASA Astrophysics Data System (ADS)

Pavlovich, Elena; Volkova, Nataliia; Yakymchuk, Elena; Perepelitsyna, Olena; Sydorenko, Michail; Goltsev, Anatoliy

2017-08-01

Cell culture models are excellent tools for potential toxicity of nanoparticles and fundamental investigations in cancer research. Thus, information about AuNP potential toxicity and effects on human health is necessary for the use of nanomaterials in clinical settings. The aim of our research is to examine the effects of AuNPs on the epithelial origin cell lines: continuous and oncogenic. Embryonic porcine kidney epithelial inoculated (SPEV) cell line and colorectal carcinoma cell line (HT29) were used. In the test cultures, the cell proliferation, necrosis/apoptosis, and multicellular spheroids generation were evaluated. We demonstrated that AuNP concentrations of 6-12 μg/ml reduced the proliferation of SPEV and HT29 cells and increased the cell number at early and late stages of apoptosis and necrosis. It was shown that small concentrations of AuNPs (1-3 μg/ml) stimulate multicellular spheroid formation by HT29 and SPEV cells. However, higher AuNP concentrations (6-12 μg/ml) had both cytotoxic and anti-cohesive effects on cell in suspension. The large sensitiveness to the action of AuNPs was shown by the line of HT29 (6 μg/ml) as compared to the SPEV cells (12 μg/ml). This experimental study of the effect of AuNPs on SPEV and HT29 cell lines will justify their further application in AuNP-mediated anticancer treatment.

Novel derivatives of 6-mercaptopurine: synthesis, characterization and antiproliferative activities of S-allylthio-mercaptopurines.

PubMed

Miron, T; Arditti, F; Konstantinovski, L; Rabinkov, A; Mirelman, D; Berrebi, A; Wilchek, M

2009-02-01

Biologically active S-allylthio derivatives of 6-mercaptopurine (6-MP) and 6-mercaptopurine riboside (6-MPR) were synthesized. The products, S-allylthio-6-mercaptopurine (SA-6MP) and S-allylthio-6-mercaptopurine riboside (SA-6MPR) were characterized. The antiproliferative activity of the new prodrugs was tested on human leukemia and monolayer cell lines, and compared to that of their parent reactants. The new prodrugs acted by a concentration-dependent mechanism. They inhibited cell proliferation and induced-apoptosis more efficiently than the parent molecules. Leukemia cell lines were more sensitive to the new prodrugs than monolayer cell lines. Higher hydrophobicity of the derivatives improves their penetration into cells, where upon reaction with glutathione, S-allylthioglutathione (GSSA) is formed, and 6-MP or 6-MPR is released for further processing.

The sigma-2 receptor as a therapeutic target for drug delivery in triple negative breast cancer

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

Makvandi, Mehran; Tilahun, Estifanos D.; Lieberman, Brian P.

Background: Triple-negative breast cancer (TNBC) is associated with high relapse rates and increased mortality when compared with other breast cancer subtypes. In contrast to receptor positive breast cancers, there are no approved targeted therapies for TNBC. Identifying biomarkers for TNBC is of high importance for the advancement of patient care. The sigma-2 receptor has been shown to be overexpressed in triple negative breast cancer in vivo and has been characterized as a marker of proliferation. The aim of the present study was to define the sigma-2 receptor as a target for therapeutic drug delivery and biomarker in TNBC. Methods: Three TNBCmore » cell lines were evaluated: MDA-MB-231, HCC1937 and HCC1806. Sigma-2 compounds were tested for pharmacological properties specific to the sigma-2 receptor through competitive inhibition assays. Sigma-2 receptor expression was measured through radioligand receptor saturation studies. Drug sensitivity for taxol was compared to a sigma-2 targeting compound conjugated to a cytotoxic payload, SW IV-134. Cell viability was assessed after treatments for 2 or 48 h. Sigma-2 blockade was assessed to define sigma-2 mediated cytotoxicity of SW IV-134. Caspase 3/7 activation induced by SW IV-134 was measured at corresponding treatment time points. Results: SW IV-134 was the most potent compound tested in two of the three cell lines and was similarly effective in all three. MDA-MB-231 displayed a statistically significant higher sigma-2 receptor expression and also was the most sensitive cell line evaluated to SW IV-134. Conclusion: Targeting the sigma-2 receptor with a cytotoxic payload was effective in all the three cell lines evaluated and provides the proof of concept for future development of a therapeutic platform for the treatment of TNBC. - Highlights: • TNBC cells are sensitive to sigma-2 receptor targeted drug conjugate SW IV-134. • MDA-MB-231 displayed the highest amount of sigma-2 receptors and corresponded well with sensitivity. • The sigma-2 receptor is a potential biomarker in TNBC for prognosis and therapy.« less

Evidence of a cellular immune response against sialyl-Tn in breast and ovarian cancer patients after high-dose chemotherapy, stem cell rescue, and immunization with Theratope STn-KLH cancer vaccine.

PubMed

Sandmaier, B M; Oparin, D V; Holmberg, L A; Reddish, M A; MacLean, G D; Longenecker, B M

1999-01-01

Seven ovarian and 33 breast high-risk stage II/III and stage IV cancer patients received high-dose chemotherapy followed by stem cell rescue. Thirty to 151 days after stem cell transplantation, the patients received their first immunotherapy treatment with Theratope STn-KLH cancer vaccine. Most patients developed increasing IgG anti-STn titers to a sustained peak after the fourth or fifth immunizations. Only one patient had elevated CA27.29 (MUC1 mucin) serum levels at trial entry. Five of the seven patients with preimmunotherapy elevated serum CA125 levels demonstrated decreasing CA125 levels during immunotherapy, consistent with an antitumor response. Evidence of STn antigen-specific T-cell proliferation was obtained from 17 of the 27 evaluable patients who received at least three immunotherapy treatments. Eleven of the 26 patients tested had evidence of an anti-STn TH1 antigen-specific T-cell response as determined by interferon-gamma, but not interleukin (IL)-4, production. After immunization, lytic activity of peripheral blood lymphocytes (PBLs) tested against a lymphokine activated killer (LAK)-sensitive cell line, a natural killer (NK)-sensitive cell line, and an STn-expressing cancer cell line (OVCAR) increased significantly. In vitro IL-2 treatment of the PBLs after vaccination greatly enhanced killing of the STn+ cancer cell line. Evidence of the development of OVCAR specific killing activity, over and above that seen due to LAK or NK killing, is presented. These studies provide the strongest evidence in humans of the development of an antitumor T-cell response after immunization with a cancer-associated carbohydrate antigen.

Acquisition of resistance toward HYD1 correlates with a reduction in cleaved α4 integrin expression and a compromised CAM-DR phenotype.

PubMed

Emmons, Michael F; Gebhard, Anthony W; Nair, Rajesh R; Baz, Rachid; McLaughlin, Mark L; Cress, Anne E; Hazlehurst, Lori A

2011-12-01

We recently reported that the β1 integrin antagonist, referred to as HYD1, induces necrotic cell death in myeloma cell lines as a single agent using in vitro and in vivo models. In this article, we sought to delineate the determinants of sensitivity and resistance toward HYD1-induced cell death. To this end, we developed an HYD1 isogenic resistant myeloma cell line by chronically exposing H929 myeloma cells to increasing concentrations of HYD1. Our data indicate that the acquisition of resistance toward HYD1 correlates with reduced levels of the cleaved α4 integrin subunit. Consistent with reduced VLA-4 (α4β1) expression, the resistant variant showed ablated functional binding to fibronectin, VCAM-1, and the bone marrow stroma cell line HS-5. The reduction in binding of the resistant cell line to HS-5 cells translated to a compromised cell adhesion-mediated drug resistant phenotype as shown by increased sensitivity to melphalan- and bortezomib-induced cell death in the bone marrow stroma coculture model of drug resistance. Importantly, we show that HYD1 is more potent in relapsed myeloma specimens than newly diagnosed patients, a finding that correlated with α4 integrin expression. Collectively, these data indicate that this novel d-amino acid peptide may represent a good candidate for pursuing clinical trials in relapsed myeloma and in particular patients with high levels of α4 integrin. Moreover, our data provide further rationale for continued preclinical development of HYD1 and analogues of HYD1 for the treatment of multiple myeloma and potentially other tumors that home and/or metastasize to the bone.

Use of Humanized RS-ATL8 Reporter System for Detection of Allergen-Specific IgE Sensitization in Human Food Allergy.

PubMed

Ali, Eman Ali; Nakamura, Ryosuke; Falcone, Franco H

2017-01-01

Allergen-specific Immunoglobulin E (IgE) determination lies at the heart of diagnosis of sensitization to food and other allergens. In the past few years, reporter systems capable of detecting the presence of allergen-specific IgE have been developed by several labs. These rely on humanized rat basophil leukemia cell lines stably transfected with reporter genes such as firefly luciferase. In this chapter, we describe protocols for the use of the RS-ATL8 cell line (IgE cross-linking-induced luciferase expression; EXiLE) in 96-well and 384-well formats. We also describe optional treatment steps for enveloped virus and complement inactivation.

Radiation enhanced efficiency of combined electromagnetic hyperthermia and chemotherapy of lung carcinoma using cisplatin functionalized magnetic nanoparticles.

PubMed

Babincová, M; Kontrisova, K; Durdík, S; Bergemann, C; Sourivong, P

2014-02-01

The effect of trimodality treatment consisting of hyperthermia, cisplatin and radiation was investigated in two non-small lung carcinoma cell lines with different sensitivities to cisplatin. Hyperthermia treatment was performed using heat released via Neél and Brown relaxation of magnetic nanoparticles in an alternating magnetic field. Radiation with dose 1.5 Gy was performed after 15 min electromagnetic hyperthermia and cisplatin treatment. Electromagnetic hyperthermia enhanced cisplatin-induced radiosensitization in both the cisplatin-sensitive H460 (viability 11.2 +/- 1.8 %) and cisplatin-resistant A549 (viability 14.5 +/- 2.3 %) lung carcinoma cell line. Proposed nanotechnology based trimodality cancer treatment may have therefore important clinical applications.

Targeting mitochondrial respiration as a therapeutic strategy for cervical cancer.

PubMed

Tian, Shenglan; Chen, Heng; Tan, Wei

2018-05-23

Targeting mitochondrial respiration has been documented as an effective therapeutic strategy in cancer. However, the impact of mitochondrial respiration inhibition on cervical cancer cells are not well elucidated. Using a panel of cervical cancer cell lines, we show that an existing drug atovaquone is active against the cervical cancer cells with high profiling of mitochondrial biogenesis. Atovaquone inhibited proliferation and induced apoptosis with varying efficacy among cervical cancer cell lines regardless of HPV infection, cellular origin and their sensitivity to paclitaxel. We further demonstrated that atovaquone acts on cervical cancer cells via inhibiting mitochondrial respiration. In particular, atovaquone specifically inhibited mitochondrial complex III but not I, II or IV activity, leading to respiration inhibition and energy crisis. Importantly, we found that the different sensitivity of cervical cancer cell lines to atovaquone were due to their differential level of mitochondrial biogenesis and dependency to mitochondrial respiration. In addition, we demonstrated that the in vitro observations were translatable to in vivo cervical cancer xenograft mouse model. Our findings suggest that the mitochondrial biogenesis varies among patients with cervical cancer. Our work also suggests that atovaquone is a useful addition to cervical cancer treatment, particularly to those with high dependency on mitochondrial respiration. Copyright © 2018 Elsevier Inc. All rights reserved.

In vitro and in vivo sensitization of SW620 metastatic colon cancer cells to CDDP-induced apoptosis by the nitric oxide donor DETANONOate: Involvement of AIF.

PubMed

Huerta, Sergio; Baay-Guzman, Guillermina; Gonzalez-Bonilla, Cesar R; Livingston, Edward H; Huerta-Yepez, Sara; Bonavida, Benjamin

2009-05-01

Tumor cells develop mechanisms that dysregulate apoptotic pathways resulting in resistance to cytotoxic stimuli. Primary SW480 and metastatic SW620 colon cancer cells are resistant to CDDP-induced apoptosis. Apoptosis-inducing factor (AIF) was significantly downregulated in SW620 compared to SW480 cells; while apoptotic mediators such as Bax, Bcl-2, and Bcl(XL) were not altered in these cell lines. Examination of tumor tissues from patients with colon cancer demonstrated a significant downregulation of AIF in patients with advanced disease. The role of AIF expression in resistance was examined. Several lines of evidence suggest the involvement of AIF expression level in the sensitivity of SW620 to CDDP-induced apoptosis: (1) sensitization of SW620 by the NO donor DETANONOate to CDDP-induced apoptosis correlated with the induction of AIF as assessed by RT-PCR and Western blot analysis, (2) treatment of SW620 cells with siRNA AIF, but not with control siRNAs, inhibited DETANONOate-induced sensitization to CDDP apoptosis, (3) sensitization by DETANONOate observed in vitro was corroborated in vivo in nude mice bearing SW620 tumor xenografts and treated with the combination of DETANONOate and CDDP, and (4) tumor tissues derived from the SW620 xenografts revealed significant upregulation of AIF and increased apoptosis by DETANONOate and CDDP combination treatment. Altogether, these findings underscore the potential therapeutic application of NO donors and subtoxic chemotherapeutic drugs in the treatment of advanced colon cancer resistant to conventional chemotherapeutic agents.

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

Establishment and characterization of a novel lymphangiosarcoma cell line (MO-LAS) compared with the hemangiosarcoma cell line (ISO-HAS).

PubMed

Masuzawa, Mikio; Masuzawa, Mamiko; Hamada, Yuhko; Arakawa, Nobuko; Mori, Mari; Ishii, Masako; Nishiyama, Shigeo

2012-08-01

The concept of "lymphangiosarcoma" remains obscure. Therefore, we reported a patient with lymphangiosarcoma, resistant to immunotherapy. The patient presented with impressive and discriminative features: clinically an ill-defined edematous lesion with lymphorrhea and pathologically atypical vascular channel formation without extravasation of blood, clearly distinguished from common angiosarcoma with hemorrhage. From this case, a lymphangiosarcoma cell line, MO-LAS, was established and its characteristics were compared with the hemangiosarcoma cell line, ISO-HAS. Flow cytometric analysis revealed that MO-LAS was negative for factor VIII-related antigen, but positive for CD31, D2-40, NZ-1, and vascular endothelial growth factor receptor-3 (VEGFR-3), similar to ISO-HAS. However, MO-LAS expressed a much higher level of homeobox gene PROX1, indicating a lymphatic phenotype, compared with ISO-HAS. Furthermore, MO-LAS showed a much lesser expression of oncogenes and much lower sensitivity against lymphokine-activated killer (LAK) cells. Lymphangiosarcoma may be difficult to recognize by the immune system. Conclusively, the establishment of MO-LAS, a novel angiosarcoma cell line bearing lymphatic characters, strongly suggests the entity of lymphangiosarcoma.

Bortezomib sensitises TRAIL-resistant HPV-positive head and neck cancer cells to TRAIL through a caspase-dependent, E6-independent mechanism.

PubMed

Bullenkamp, J; Raulf, N; Ayaz, B; Walczak, H; Kulms, D; Odell, E; Thavaraj, S; Tavassoli, M

2014-10-23

Human papillomavirus (HPV) is causative for a new and increasing form of head and neck squamous cell carcinomas (HNSCCs). Although localised HPV-positive cancers have a favourable response to radio-chemotherapy (RT/CT), the impact of HPV in advanced or metastatic HNSCC remains to be defined and targeted therapeutics need to be tested for cancers resistant to RT/CT. To this end, we investigated the sensitivity of HPV-positive and -negative HNSCC cell lines to TRAIL (tumour necrosis factor-related apoptosis-inducing ligand), which induces tumour cell-specific apoptosis in various cancer types. A clear correlation was observed between HPV positivity and resistance to TRAIL compared with HPV-negative head and neck cancer cell lines. All TRAIL-resistant HPV-positive cell lines tested were sensitised to TRAIL-induced cell death by treatment with bortezomib, a clinically approved proteasome inhibitor. Bortezomib-mediated sensitisation to TRAIL was associated with enhanced activation of caspase-8, -9 and -3, elevated membrane expression levels of TRAIL-R2, cytochrome c release and G2/M arrest. Knockdown of caspase-8 significantly blocked cell death induced by the combination therapy, whereas the BH3-only protein Bid was not required for induction of apoptosis. XIAP depletion increased the sensitivity of both HPV-positive and -negative cells to TRAIL alone or in combination with bortezomib. In contrast, restoration of p53 following E6 knockdown in HPV-positive cells had no effect on their sensitivity to either single or combination therapy, suggesting a p53-independent pathway for the observed response. In summary, bortezomib-mediated proteasome inhibition sensitises previously resistant HPV-positive HNSCC cells to TRAIL-induced cell death through a mechanism involving both the extrinsic and intrinsic pathways of apoptosis. The cooperative effect of these two targeted anticancer agents therefore represents a promising treatment strategy for RT/CT-resistant HPV-associated head and neck cancers.

Curcumin improves the efficacy of cisplatin by targeting cancer stem-like cells through p21 and cyclin D1-mediated tumour cell inhibition in non-small cell lung cancer cell lines

PubMed Central

BAHARUDDIN, PUTERI; SATAR, NAZILAH; FAKIRUDDIN, KAMAL SHAIK; ZAKARIA, NORASHIKIN; LIM, MOON NIAN; YUSOFF, NARAZAH MOHD; ZAKARIA, ZUBAIDAH; YAHAYA, BADRUL HISHAM

2016-01-01

Natural compounds such as curcumin have the ability to enhance the therapeutic effectiveness of common chemotherapy agents through cancer stem-like cell (CSC) sensitisation. In the present study, we showed that curcumin enhanced the sensitivity of the double-positive (CD166+/EpCAM+) CSC subpopulation in non-small cell lung cancer (NSCLC) cell lines (A549 and H2170) to cisplatin-induced apoptosis and inhibition of metastasis. Our results revealed that initial exposure of NSCLC cell lines to curcumin (10–40 µM) markedly reduced the percentage of viability to an average of ~51 and ~54% compared to treatment with low dose cisplatin (3 µM) with only 94 and 86% in both the A549 and H2170 cells. Moreover, sensitisation of NSCLC cell lines to curcumin through combined treatment enhanced the single effect induced by low dose cisplatin on the apoptosis of the double-positive CSC subpopulation by 18 and 20% in the A549 and H2170 cells, respectively. Furthermore, we found that curcumin enhanced the inhibitory effects of cisplatin on the highly migratory CD166+/EpCAM+ subpopulation, marked by a reduction in cell migration to 9 and 21% in the A549 and H2170 cells, respectively, indicating that curcumin may increase the sensitivity of CSCs to cisplatin-induced migratory inhibition. We also observed that the mRNA expression of cyclin D1 was downregulated, while a substantial increased in p21 expression was noted, followed by Apaf1 and caspase-9 activation in the double-positive (CD166+/EpCAM+) CSC subpopulation of A549 cells, suggested that the combined treatments induced cell cycle arrest, therefore triggering CSC growth inhibition via the intrinsic apoptotic pathway. In conclusion, we provided novel evidence of the previously unknown therapeutic effects of curcumin, either alone or in combination with cisplatin on the inhibition of the CD166+/EpCAM+ subpopulation of NSCLC cell lines. This finding demonstrated the potential therapeutic approach of using curcumin that may enhance the effects of cisplatin by targeting the CSC subpopulation in NSCLC. PMID:26531053

BTG2 Is Down-Regulated and Inhibits Cancer Stem Cell-Like Features of Side Population Cells in Hepatocellular Carcinoma.

PubMed

Huang, Chen-Song; Zhai, Jing-Ming; Zhu, Xiao-Xu; Cai, Jian-Peng; Chen, Wei; Li, Jian-Hui; Yin, Xiao-Yu

2017-12-01

Our previous study found that B cell translocation gene 2 (BTG2) was hyper-methylated and down-regulated in side population (SP) cells of hepatocellular carcinoma (HCC) cell line. However, its clinical significances and biological impacts on HCC SP cells remained unclear. To investigate the prognostic value of BTG2 gene in HCC and its influences on cancer stem cells (CSCs)-like traits of HCC cell line SP cells. BTG2 expression in human HCC and adjacent non-cancerous tissues was detected by immunohistochemical staining and quantitative real-time PCR, and also obtained from GEO and TCGA data. Its prognostic values were assessed. Its biological influences on HCC cell line SP cells were evaluated using cell viability, cell cycle, plate clone-forming assay, and chemoresistance in vitro and tumorigenicity in vivo. BTG2 expression was significantly suppressed in human HCC compared to adjacent non-cancerous tissues. BTG2 expression was correlated with TNM stage, tumor size and vascular invasion. Lower expression of BTG2 was associated with poorer overall survival and disease-free survival. In vitro, overexpression of BTG2 substantially suppressed cell proliferation and accumulation of HCC cell line SP cells in G0/G1 phase. Colony formation ability was markedly suppressed by BTG2 overexpression. Moreover, sensitivity of HCC cell line SP cells to 5-fluorouracil was substantially increased by overexpression of BTG2. Furthermore, tumorigenicity of HCC cell line SP cells transfected with BTG2 plasmids was significantly reduced in vivo. BTG2 gene could regulate the CSC-like traits of HCC cell line SP cells, and it represented as a molecular prognostic marker for HCC.

Impact of Procyanidins from Different Berries on Caspase 8 Activation in Colon Cancer

PubMed Central

Minker, Carole; Duban, Livine; Karas, Daniel; Järvinen, Päivi; Lobstein, Annelise; Muller, Christian D.

2015-01-01

Scope. The aim of this work is to identify which proapoptotic pathway is induced in human colon cancer cell lines, in contact with proanthocyanidins extracted from various berries. Methods and Results. Proanthocyanidins (Pcys) extracted from 11 berry species are monitored for proapoptotic activities on two related human colon cancer cell lines: SW480-TRAIL-sensitive and SW620-TRAIL-resistant. Apoptosis induction is monitored by cell surface phosphatidylserine (PS) detection. Lowbush blueberry extract triggers the strongest activity. When tested on the human monocytic cell line THP-1, blueberry Pcys are less effective for PS externalisation and DNA fragmentation is absent, highlighting a specificity of apoptosis induction in gut cells. In Pcys-treated gut cell lines, caspase 8 (apoptosis extrinsic pathway) but not caspase 9 (apoptosis intrinsic pathway) is activated after 3 hours through P38 phosphorylation (90 min), emphasizing the potency of lowbush blueberry Pcys to eradicate gut TRAIL-resistant cancer cells. Conclusion. We highlight here that berries Pcys, especially lowbush blueberry Pcys, are of putative interest for nutritional chemoprevention of colorectal cancer in view of their apoptosis induction in a human colorectal cancer cell lines. PMID:26180579

Lack of association between deletion polymorphism of BIM gene and in vitro drug sensitivity in B-cell precursor acute lymphoblastic leukemia.

PubMed

Huang, Meixian; Miyake, Kunio; Kagami, Keiko; Abe, Masako; Shinohara, Tamao; Watanabe, Atsushi; Somazu, Shinpei; Oshiro, Hiroko; Goi, Kumiko; Goto, Hiroaki; Minegishi, Masayoshi; Iwamoto, Shotaro; Kiyokawa, Nobutaka; Sugita, Kanji; Inukai, Takeshi

2017-09-01

A deletion polymorphism in the BIM gene was identified as an intrinsic mechanism for resistance to tyrosine kinase inhibitor in chronic myeloid leukemia patients in East Asia. BIM is also involved in the responses to glucocorticoid and chemotherapy in acute lymphoblastic leukemia (ALL), suggesting a possible association between deletion polymorphism of BIM and the chemosensitivity of ALL. Thus, we analyzed 72 B-cell precursor (BCP)-ALL cell lines established from Japanese patients. Indeed, higher BIM gene expression was associated with good in vitro sensitivities to glucocorticoid and chemotherapeutic agents used in induction therapy. We also analyzed the methylation status of the BIM gene promoter by next generation sequencing of genome bisulfite PCR products, since genetic polymorphism could be insignificant when epigenetically inactivated. Hypermethylation of the BIM gene promoter was associated with lower BIM gene expression and poorer sensitivity to vincristine. Of note, however, the prevalence of a deletion polymorphism was not associated with the BIM gene expression level or drug sensitivities in BCP-ALL cell lines, in which the BIM gene was unmethylated. These observations suggest that an association of a deletion polymorphism of BIM and the response to induction therapy in BCP-ALL may be clinically minimal. Copyright © 2017. Published by Elsevier Ltd.

Generation and functional analysis of T cell lines and clones specific for schistosomula released products (SRP-A).

PubMed

Damonneville, M; Velge, F; Verwaerde, C; Pestel, J; Auriault, C; Capron, A

1987-08-01

Antigens present in the products released by the larval stage of schistosome (SRP-A) were shown to induce a strong cytotoxic and protective IgE response both in the rat and the monkey. T cell lines and clones specific for SRP-A or 26 kD antigens which are the main target of the cytotoxic IgE have been derived. The passive transfer of SRP-A specific T lymphocytes into infected rats led to an increase of the IgE response, conferring a significant level of protection to the rats. In coculture assays in vitro, these cell lines significantly enhanced the production of IgE by SRP-A sensitized rat spleen cells. This helper effect on the IgE response was confirmed with 26 kD T cell clone supernatants. Moreover, supernatants obtained after stimulation with phorbol myristate acetate were able to enhance the IgE production of a hybridoma B cell line (B48-14) producing a monoclonal IgE antibody, cytotoxic for the schistosomula.

Cell diameter measurements obtained with a handheld cell counter could be used as a surrogate marker of G2/M arrest and apoptosis in colon cancer cell lines exposed to SN-38

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

Tahara, Makiko; Department of Gastrointestinal Surgery, Jichi Medical University, Shimotsuke, Tochigi; Inoue, Takeshi

2013-05-17

Highlights: •Chemo-sensitivity to SN-38 was assayed by the automated cell counter. •Colon cancer cell line, HCT116 cells were more sensitive to SN-38 than HT29 cells. •Increase of cell size reflects G2/M arrest. •Appearance of small particles indicates cell apoptosis. -- Abstract: In vitro assessment of chemosensitivity are important for experiments evaluating cancer therapies. The Scepter 2.0 cell counter, an automated handheld device based on the Coulter principle of impedance-based particle detection, enables the accurate discrimination of cell populations according to cell size and volume. In this study, the effects of SN-38, the active metabolite of irinotecan, on the colon cancermore » cell lines HCT116 and HT29 were evaluated using this device. The cell count data obtained with the Scepter counter were compared with those obtained with the {sup 3}H-thymidine uptake assay, which has been used to measure cell proliferation in many previous studies. In addition, we examined whether the changes in the size distributions of these cells reflected alterations in the frequency of cell cycle arrest and/or apoptosis induced by SN-38 treatment. In our experiments using the Scepter 2.0 cell counter, the cell counts were demonstrated to be accurate and reproducible measure and alterations of cell diameter reflected G2/M cell cycle arrest and apoptosis. Our data show that easy-to-use cell counting tools can be utilized to evaluate the cell-killing effects of novel treatments on cancer cells in vitro.« less

Lung Squamous Cell Carcinoma Stem Cells as Immunotherapy Targets

DTIC Science & Technology

2017-08-01

will examine the importance of candidate genes in CSC activity using blocking antibodies, knockdown or CRISPR strategies coupled with transplantation...Aim 1. Engineer isogenic human and murine BRG1 mutant cell lines using CRISPR -Cas9 to dissect the mechanisms behind the sensitivity to combined EZH2

Effects of thymidine phosphorylase on tumor aggressiveness and 5-fluorouracil sensitivity in cholangiocarcinoma

PubMed Central

Thanasai, Jongkonnee; Limpaiboon, Temduang; Jearanaikoon, Patcharee; Sripa, Banchob; Pairojkul, Chawalit; Tantimavanich, Srisurang; Miwa, Masanao

2010-01-01

AIM: To evaluate the role of thymidine phosphorylase (TP) in cholangiocarcinoma using small interfering RNA (siRNA). METHODS: A human cholangiocarcinoma-derived cell line KKU-M139, which has a naturally high level of endogenous TP, had TP expression transiently knocked down using siRNA. Cell growth, migration, in vitro angiogenesis, apoptosis, and cytotoxicity were assayed in TP knockdown and wild-type cell lines. RESULTS: TP mRNA and protein expression were decreased by 87.1% ± 0.49% and 72.5% ± 3.2%, respectively, compared with control cells. Inhibition of TP significantly decreased migration of KKU-M139, and suppressed migration and tube formation of human umbilical vein endothelial cells. siRNA also reduced the ability of TP to resist hypoxia-induced apoptosis, while suppression of TP reduced the sensitivity of KKU-M139 to 5-fluorouracil. CONCLUSION: Inhibition of TP may be beneficial in decreasing angiogenesis-dependent growth and migration of cholangiocarcinoma but may diminish the response to 5-fluorouracil chemotherapy. PMID:20355241

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

Mavrakis, K. J.; McDonald, E. R.; Schlabach, M. R.

5-Methylthioadenosine phosphorylase (MTAP) is a key enzyme in the methionine salvage pathway. The MTAP gene is frequently deleted in human cancers because of its chromosomal proximity to the tumor suppressor gene CDKN2A. By interrogating data from a large-scale short hairpin RNA–mediated screen across 390 cancer cell line models, we found that the viability of MTAP-deficient cancer cells is impaired by depletion of the protein arginine methyltransferase PRMT5. MTAP-deleted cells accumulate the metabolite methylthioadenosine (MTA), which we found to inhibit PRMT5 methyltransferase activity. Deletion of MTAP in MTAP-proficient cells rendered them sensitive to PRMT5 depletion. Conversely, reconstitution of MTAP in anmore » MTAP-deficient cell line rescued PRMT5 dependence. Thus, MTA accumulation in MTAP–deleted cancers creates a hypomorphic PRMT5 state that is selectively sensitized toward further PRMT5 inhibition. Inhibitors of PRMT5 that leverage this dysregulated metabolic state merit further investigation as a potential therapy for MTAP/CDKN2A-deleted tumors.« less

A hybrid of B and T lymphoblastic cell line could potentially substitute dendritic cells to efficiently expand out Her-2/neu-specific cytotoxic T lymphocytes from advanced breast cancer patients in vitro.

PubMed

Chen, Sheng; Gu, Feifei; Li, Kang; Zhang, Kai; Liu, Yangyang; Liang, Jinyan; Gao, Wei; Wu, Gang; Liu, Li

2017-02-28

Adoptive transfer of cytotoxic T lymphocytes (CTLs) holds promises to cure cancer. However, this treatment is hindered by lacking a robust way to specifically expand out CTLs. Here, we developed a hybrid of B lymphoblastic cell line and T lymphoblastic cell line (T2 cells) as a substitute of dendritic cells, together with irradiated autologous peripheral blood mononuclear cell (PBMC) as feeder cells and rhIL-2, to activate and expand Her-2/neu-specific CD8 + T cells from human epidermal growth factor receptor 2 (Her-2/neu) and human leukocyte antigen (HLA)-A2 double positive advanced breast cancer patients in vitro. These Her-2/neu-loaded T2 cells reproducibly activated and expanded out Her-2/neu-specific CD8 + T cells to 10 7 in 8 weeks. Furthermore, these Her-2/neu-specific CD8 + T cells had good sensitivity of recognition and killing Her-2/neu-overexpressed breast cancer cell line SK.BR.3. This technique gives us another insight on how to rapidly obtain sufficient CTLs for adoptive cancer immunotherapy.

GRIDSS: sensitive and specific genomic rearrangement detection using positional de Bruijn graph assembly

PubMed Central

Do, Hongdo; Molania, Ramyar

2017-01-01

The identification of genomic rearrangements with high sensitivity and specificity using massively parallel sequencing remains a major challenge, particularly in precision medicine and cancer research. Here, we describe a new method for detecting rearrangements, GRIDSS (Genome Rearrangement IDentification Software Suite). GRIDSS is a multithreaded structural variant (SV) caller that performs efficient genome-wide break-end assembly prior to variant calling using a novel positional de Bruijn graph-based assembler. By combining assembly, split read, and read pair evidence using a probabilistic scoring, GRIDSS achieves high sensitivity and specificity on simulated, cell line, and patient tumor data, recently winning SV subchallenge #5 of the ICGC-TCGA DREAM8.5 Somatic Mutation Calling Challenge. On human cell line data, GRIDSS halves the false discovery rate compared to other recent methods while matching or exceeding their sensitivity. GRIDSS identifies nontemplate sequence insertions, microhomologies, and large imperfect homologies, estimates a quality score for each breakpoint, stratifies calls into high or low confidence, and supports multisample analysis. PMID:29097403

Krüppel-like factor 4 promotes c-Met amplification-mediated gefitinib resistance in non-small-cell lung cancer.

PubMed

Feng, Wei; Xie, Qianyi; Liu, Suo; Ji, Ying; Li, Chunyun; Wang, Chunle; Jin, Longyu

2018-06-01

Gefitinib has been widely used in the first-line treatment of advanced EGFR-mutated non-small-cell lung cancer (NSCLC). However, many NSCLC patients will acquire resistance to gefitinib after 9-14 months of treatment. This study revealed that Krüppel-like factor 4 (KLF4) contributes to the formation of gefitinib resistance in c-Met-overexpressing NSCLC cells. We observed that KLF4 was overexpressed in c-Met-overexpressing NSCLC cells and tissues. Knockdown of KLF4 increased tumorigenic properties in gefitinib-resistant NSCLC cell lines without c-Met overexpression, but it reduced tumorigenic properties and increased gefitinib sensitivity in gefitinib-resistant NSCLC cells with c-Met overexpression, whereas overexpression of KLF4 reduced gefitinib sensitivity in gefitinib-sensitive NSCLC cells. Furthermore, Western blot analysis revealed that KLF4 contributed to the formation of gefitinib resistance in c-Met-overexpressing NSCLC cells by inhibiting the expression of apoptosis-related proteins under gefitinib treatment and activating the c-Met/Akt signaling pathway by decreasing the inhibition of β-catenin on phosphorylation of c-Met to prevent blockade by gefitinib. In summary, this study's results suggest that KLF4 is a promising candidate molecular target for both prevention and therapy of NSCLC with c-Met overexpression. © 2018 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Different sensitivities of cultured mammalian cells towards aphidicolin-enhanced DNA effects in the comet assay.

PubMed

Speit, Günter; Schütz, Petra; Bausinger, Julia

2016-06-01

The comet assay in combination with the polymerase inhibitor aphidicolin (APC) has been used to measure DNA excision repair activity, DNA repair kinetics and individual DNA repair capacity. Since APC can enhance genotoxic effects of mutagens measured by the comet assay, this approach has been proposed for increasing the sensitivity of the comet assay in human biomonitoring. The APC-modified comet assay has mainly been performed with human blood and it was shown that it not only enhances the detection of DNA damage repaired by nucleotide excision repair (NER) but also damage typically repaired by base excision repair (BER). Recently, we reported that in contrast to blood leukocytes, A549 cells (a human lung adenocarcinoma cell line) seem to be insensitive towards the repair-inhibiting action of APC. To further elucidate the general usefulness of the APC-modified comet assay for studying repair in cultured mammalian cells, we comparatively investigated further cell lines (HeLa, TK6, V79). DNA damage was induced by BPDE (benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide) and MMS (methyl methanesulfonate) in the absence and presence of APC (3 or 15μM). APC was either added for 2h together with the mutagen or cells were pre-incubated for 30min with APC before the mutagen was added. The results indicate that the cell lines tested differ fundamentally with regard to their sensitivity and specificity towards the repair-inhibiting effect of APC. The actual cause for these differences is still unclear but potential molecular explanations are discussed. Irrespective of the underlying mechanism(s), our study revealed practical limitations of the use of the APC-modified comet assay. Copyright © 2016 Elsevier B.V. All rights reserved.

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

Ultrasound-mediated structural changes in cells revealed by FTIR spectroscopy: A contribution to the optimization of gene and drug delivery

NASA Astrophysics Data System (ADS)

Grimaldi, Paola; Di Giambattista, Lucia; Giordani, Serena; Udroiu, Ion; Pozzi, Deleana; Gaudenzi, Silvia; Bedini, Angelico; Giliberti, Claudia; Palomba, Raffaele; Congiu Castellano, Agostina

2011-12-01

Ultrasound effects on biological samples are gaining a growing interest concerning in particular, the intracellular delivery of drugs and genes in a safe and in a efficient way. Future progress in this field will require a better understanding of how ultrasound and acoustic cavitation affect the biological system properties. The morphological changes of cells due to ultrasound (US) exposure have been extensively studied, while little attention has been given to the cells structural changes. We have exposed two different cell lines to 1 MHz frequency ultrasound currently used in therapy, Jurkat T-lymphocytes and NIH-3T3 fibroblasts, both employed as models respectively in the apoptosis and in the gene therapy studies. The Fourier Transform Infrared (FTIR) Spectroscopy was used as probe to reveal the structural changes in particular molecular groups belonging to the main biological systems. The genotoxic damage of cells exposed to ultrasound was ascertained by the Cytokinesis-Block Micronucleus (CBMN) assay. The FTIR spectroscopy results, combined with multivariate statistical analysis, regarding all cellular components (lipids, proteins, nucleic acids) of the two cell lines, show that Jurkat cells are more sensitive to therapeutic ultrasound in the lipid and protein regions, whereas the NIH-3T3 cells are more sensitive in the nucleic acids region; a meaningful genotoxic effect is present in both cell lines only for long sonication times while in the Jurkat cells also a significant cytotoxic effect is revealed for long times of exposure to ultrasound.

Detection and quantification of Epstein-Barr virus EBER1 in EBV-infected cells by fluorescent in situ hybridization and flow cytometry

NASA Technical Reports Server (NTRS)

Stowe, R. P.; Cubbage, M. L.; Sams, C. F.; Pierson, D. L.; Barrett, A. D.

1998-01-01

A rapid and highly sensitive fluorescent in situ hybridization (FISH) assay was developed to detect Epstein Barr virus (EBV)-infected cells in peripheral blood. Multiple fluorescein-labeled antisense oligonucleotide probes were designed to hybridize to the EBER1 transcript, which is highly expressed in latently infected cells. After a rapid (30 min) hybridization, the cells were analyzed by flow cytometry. EBER1 was detected in several positive control cell lines that have variable numbers of EBV genome copies. No EBER1 was detected in two known EBV-negative cell lines. Northern blot analyses confirmed the presence and quantity of EBER1 transcripts in each cell line. This method was used to quantify the number of EBV-infected cells in peripheral blood from a patient with chronic mononucleosis. These results indicate that EBV-infected cells can be detected at the single cell level, and that this assay can be used to quantify the number of EBV-infected cells in clinical samples.

The effects of baicalein on canine osteosarcoma cell proliferation and death.

PubMed

Helmerick, E C; Loftus, J P; Wakshlag, J J

2014-12-01

Flavonoids are a group of modified triphenolic compounds from plants with medicinal properties. Baicalein, a specific flavone primarily isolated from plant roots (Scutellaria baicalensis), is commonly used in Eastern medicine for its anti-inflammatory and antineoplastic properties. Previous research shows greater efficacy for baicalein than most flavonoids; however, there has been little work examining their effects on sarcoma cells, let alone canine cells. Three canine osteosarcoma cell lines (HMPOS, D17 and OS 2.4) were treated with baicalein to examine cell viability, cell cycle kinetics, anchorage-independent growth and apoptosis. Results showed that osteosarcoma cells were sensitive to baicalein at concentrations from approximately 1 to 25 μM. Modest cell cycle changes were observed in one cell line. Baicalein was effective in inducing apoptosis and did not prevent doxorubicin cell proliferation inhibition in all the cell lines. The mechanism for induction of apoptosis has not been fully elucidated; however, changes in mitochondrial permeability supersede the apoptotic response. © 2012 Blackwell Publishing Ltd.

Identification of different ALK mutations in a pair of neuroblastoma cell lines established at diagnosis and relapse.

PubMed

Chen, Lindi; Humphreys, Angharad; Turnbull, Lisa; Bellini, Angela; Schleiermacher, Gudrun; Salwen, Helen; Cohn, Susan L; Bown, Nick; Tweddle, Deborah A

2016-12-27

Anaplastic Lymphoma Kinase (ALK) is a transmembrane receptor kinase that belongs to the insulin receptor superfamily and has previously been shown to play a role in cell proliferation, migration and invasion in neuroblastoma. Activating ALK mutations are reported in both hereditary and sporadic neuroblastoma tumours, and several ALK inhibitors are currently under clinical evaluation as novel treatments for neuroblastoma. Overall, mutations at codons F1174, R1275 and F1245 together account for ~85% of reported ALK mutations in neuroblastoma. NBLW and NBLW-R are paired cell lines originally derived from an infant with metastatic MYCN amplified Stage IVS (Evans Criteria) neuroblastoma, at diagnosis and relapse, respectively. Using both Sanger and targeted deep sequencing, this study describes the identification of distinct ALK mutations in these paired cell lines, including the rare R1275L mutation, which has not previously been reported in a neuroblastoma cell line. Analysis of the sensitivity of NBLW and NBLW-R cells to a panel of ALK inhibitors (TAE-684, Crizotinib, Alectinib and Lorlatinib) revealed differences between the paired cell lines, and overall NBLW-R cells with the F1174L mutation were more resistant to ALK inhibitor induced apoptosis compared with NBLW cells. This pair of cell lines represents a valuable pre-clinical model of clonal evolution of ALK mutations associated with neuroblastoma progression.

Expression profiles and functional associations of endogenous androgen receptor and caveolin-1 in prostate cancer cell lines.

PubMed

Bennett, Nigel C; Hooper, John D; Johnson, David W; Gobe, Glenda C

2014-05-01

In prostate cancer (PCa) patients, the protein target for androgen deprivation and blockade therapies is androgen receptor (AR). AR interacts with many proteins that function to either co-activate or co-repress its activity. Caveolin-1 (Cav-1) is not found in normal prostatic epithelium, but is found in PCa, and may be an AR co-regulator protein. We investigated cell line-specific signatures and associations of endogenous AR and Cav-1 in six PCa cell lines of known androgen sensitivity: LNCaP (androgen sensitive); 22Rv1 (androgen responsive); PC3, DU145, and ALVA41 (androgen non-reliant); and RWPE1 (non-malignant). Protein and mRNA expression profiles were compared and electron microscopy used to identify cells with caveolar structures. For cell lines expressing both AR and Cav-1, knockdown techniques using small interfering RNA against AR or Cav-1 were used to test whether diminished expression of one affected the other. Co-sedimentation of AR and Cav-1 was used to test their association. A reporter assay for AR genomic activity was utilized following Cav-1 knockdown. AR-expressing LNCaP and 22Rv1 cells had low endogenous Cav-1 mRNA and protein. Cell lines that expressed little or no AR (DU145, PC3, ALVA41, and RWPE1) expressed high endogenous levels of Cav-1. AR knockdown in LNCaP cells had little effect on Cav-1, but Cav-1 knockdown inhibited AR expression and genomic activity. These data show endogenous AR and Cav-1 mRNA and protein expression is inversely related in PCa cells, with Cav-1 acting on the androgen/AR signaling axis possibly as an AR co-activator, demonstrated by diminished AR genomic activity following Cav-1 knockdown. © 2013 Wiley Periodicals, Inc.

Early spatiotemporal-specific changes in intermediate signals are predictive of cytotoxic sensitivity to TNFα and co-treatments

NASA Astrophysics Data System (ADS)

Loo, Lit-Hsin; Bougen-Zhukov, Nicola Michelle; Tan, Wei-Ling Cecilia

2017-03-01

Signaling pathways can generate different cellular responses to the same cytotoxic agents. Current quantitative models for predicting these differential responses are usually based on large numbers of intracellular gene products or signals at different levels of signaling cascades. Here, we report a study to predict cellular sensitivity to tumor necrosis factor alpha (TNFα) using high-throughput cellular imaging and machine-learning methods. We measured and compared 1170 protein phosphorylation events in a panel of human lung cancer cell lines based on different signals, subcellular regions, and time points within one hour of TNFα treatment. We found that two spatiotemporal-specific changes in an intermediate signaling protein, p90 ribosomal S6 kinase (RSK), are sufficient to predict the TNFα sensitivity of these cell lines. Our models could also predict the combined effects of TNFα and other kinase inhibitors, many of which are not known to target RSK directly. Therefore, early spatiotemporal-specific changes in intermediate signals are sufficient to represent the complex cellular responses to these perturbations. Our study provides a general framework for the development of rapid, signaling-based cytotoxicity screens that may be used to predict cellular sensitivity to a cytotoxic agent, or identify co-treatments that may sensitize or desensitize cells to the agent.