Characterization of a Merkel Cell Polyomavirus-Positive Merkel Cell Carcinoma Cell Line CVG-1.

PubMed

Velásquez, Celestino; Amako, Yutaka; Harold, Alexis; Toptan, Tuna; Chang, Yuan; Shuda, Masahiro

2018-01-01

Merkel cell polyomavirus (MCV) plays a causal role in ∼80% of Merkel cell carcinomas (MCC). MCV is clonally integrated into the MCC tumor genome, which results in persistent expression of large T (LT) and small T (sT) antigen oncoproteins encoded by the early locus. In MCV-positive MCC tumors, LT is truncated by premature stop codons or deletions that lead to loss of the C-terminal origin binding (OBD) and helicase domains important for replication. The N-terminal Rb binding domain remains intact. MCV-positive cell lines derived from MCC explants have been valuable tools to study the molecular mechanism of MCV-induced Merkel cell carcinogenesis. Although all cell lines have integrated MCV and express truncated LT antigens, the molecular sizes of the LT proteins differ between cell lines. The copy number of integrated viral genome also varies across cell lines, leading to significantly different levels of viral protein expression. Nevertheless, these cell lines share phenotypic similarities in cell morphology, growth characteristics, and neuroendocrine marker expression. Several low-passage MCV-positive MCC cell lines have been established since the identification of MCV. We describe a new MCV-positive MCV cell line, CVG-1, with features distinct from previously reported cell lines. CVG-1 tumor cells grow in more discohesive clusters in loose round cell suspension, and individual cells show dramatic size heterogeneity. It is the first cell line to encode an MCV sT polymorphism resulting in a unique leucine (L) to proline (P) substitution mutation at amino acid 144. CVG-1 possesses a LT truncation pattern near identical to that of MKL-1 cells differing by the last two C-terminal amino acids and also shows an LT protein expression level similar to MKL-1. Viral T antigen knockdown reveals that, like other MCV-positive MCC cell lines, CVG-1 requires T antigen expression for cell proliferation.

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

PubMed

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

2015-12-21

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

Biological activity of Xanthium strumarium seed extracts on different cancer cell lines and Aedes caspius, Culex pipiens (Diptera: Culicidae).

PubMed

Al-Mekhlafi, Fahd A; Abutaha, Nael; Mashaly, Ashraf M A; Nasr, Fahd A; Ibrahim, Khalid E; Wadaan, Mohamed A

2017-05-01

Effects of methanol extracts of Xanthium strumarium on different cancer cell lines and on the mortality rates of Aedes caspius, Culex pipiens (Diptera: Culicidae) were investigated. Among the cell lines tested, the Jurkat cell line was the most sensitive to the methanol extract and ethyl acetate fraction, with reported LC 50 values of 50.18 and 48.73 μg/ml respectively. Conversely, methanol extracts were not that toxic to the A549 cell line though the toxicity increased on further purification. The percentage of growth inhibition was dose dependent for the methanol extract and ethyl acetate fraction. The ethyl acetate fraction showed higher toxicity to all cell lines tested when compared to the methanol extract. The results showed that methanol extracts of plant seeds caused 100% mortality of mosquito larvae at a concentration of 1000 μg/ml after 24 h of treatment. The LC 50 and LC 90 values of X. strumarium were found to be 531.07 and 905.95 μg/ml against Ae. caspius and 502.32 and 867.63 μg/ml against Cx. Pipiens, respectively. From the investigations, it was concluded that the crude extract of X. strumarium showed a weak potential for controlling the larval instars of Ae. caspius and Cx. pipiens . However, on further purification the extract lost the larvicidal activity. The ethyl acetate fraction showed higher toxicity to all cell lines tested when compared to the methanol extract. The ethyl acetate fraction investigated in this study appears to have a weak larvicidal activity but a promising cytotoxic activity. Future studies will include purification and investigation in further detail of the action of X. strumarium on Cancer Cell Lines and mosquitoes.

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.

Adhesion molecules affected by treatment of lung cancer cells with epidermal growth factor.

PubMed

Fonseca, Fernando L A; Azzalis, Ligia A; Feder, David; Nogoceke, Everson; Junqueira, Virginia B C; Valenti, Vitor E; de Abreu, Luiz Carlos

2011-10-01

Lung cancer is one of the leading causes of death in the world. Some tumor events are attributed to an important group of molecules (cadherins and integrins). We evaluated the interactions of cell adhesion molecules in cell lines from lung cancer. Two lung cancer cell lines were nonmetastatic (H358 and H441) and two were metastatic (H1299 and H292). All cell lines were treated with epidermal growth factor (EGF), and Western blot analysis was performed to assess the interactions between these proteins. The bronchoalveolar cells H358 showed the three analyzed proteins: E-cadherin, β-catenin, and p120 catenin. The adenocarcinoma cells H441 did not present p120 catenin, and carcinoma cells did not show E-cadherin (H1299) or p120 catenin (H292). FAK (pTyr925) was dephosphorylated in adenocarcinoma cells H441, absent in carcinoma cells H1299, and upregulated in the other carcinoma cells H292. p130Cas showed no difference when the cell lines were treated with EGF for 30 min; it was absent in the metastatic carcinoma cells H1299. Paxillin was dephosphorylated in adenocarcinoma cells H441 and also absent in other metastatic carcinoma cells H292. Vinculin showed the same results, and talin was downregulated in adenocarcinoma cells H441 when the cells were treated with EGF. Rap1 was downregulated and PYK2 was upregulated in the same cell line. Our data help to comprehend the mechanism involved in cell migration to the blood and metastasis generation. In conclusion, the expression patterns of cell-cell adhesion were not affected by EGF treatment but it affected cell-extracellular matrix adhesion.

The green tea catechin epigallocatechin gallate induces cell cycle arrest and shows potential synergism with cisplatin in biliary tract cancer cells.

PubMed

Mayr, Christian; Wagner, Andrej; Neureiter, Daniel; Pichler, Martin; Jakab, Martin; Illig, Romana; Berr, Frieder; Kiesslich, Tobias

2015-06-23

The green tea catechin epigallocatechin gallate (EGCG) was shown to effectively inhibit tumor growth in various types of cancer including biliary tract cancer (BTC). For most BTC patients only palliative therapy is possible, leading to a median survival of about one year. Chemoresistance is a major problem that contributes to the high mortality rates of BTC. The aim of this study was to investigate the cytotoxic effect of EGCG alone or in combination with cisplatin on eight BTC cell lines and to investigate the cellular anti-cancer mechanisms of EGCG. The effect of EGCG treatment alone or in combination with the standard chemotherapeutic cisplatin on cell viability was analyzed in eight BTC cell lines. Additionally, we analyzed the effects of EGCG on caspase activity, cell cycle distribution and gene expression in the BTC cell line TFK-1. EGCG significantly reduced cell viability in all eight BTC cell lines (p < 0.05 or p < 0.01, respectively, for most cell lines and EGCG concentrations > 5 μM). Combined EGCG and cisplatin treatment showed a synergistic cytotoxic effect in five cell lines and an antagonistic effect in two cell lines. Furthermore, EGCG reduced the mRNA levels of various cell cycle-related genes, while increasing the expression of the cell cycle inhibitor p21 and the apoptosis-related death receptor 5 (p < 0.05). This observation was accompanied by an increase in caspase activity and cells in the sub-G1 phase of the cell cycle, indicating induction of apoptosis. EGCG also induced a down-regulation of expression of stem cell-related genes and genes that are associated with an aggressive clinical character of the tumor, such as cd133 and abcg2. EGCG shows various anti-cancer effects in BTC cell lines and might therefore be a potential anticancer drug for future studies in BTC. Additionally, EGCG displays a synergistic cytotoxic effect with cisplatin in most tested BTC cell lines. Graphical abstract Summary illustration.

Acetonic Extract of Buxus sempervirens Induces Cell Cycle Arrest, Apoptosis and Autophagy in Breast Cancer Cells

PubMed Central

Ait-Mohamed, Ouardia; Battisti, Valentine; Joliot, Véronique; Fritsch, Lauriane; Pontis, Julien; Medjkane, Souhila; Redeuilh, Catherine; Lamouri, Aazdine; Fahy, Christine; Rholam, Mohamed; Atmani, Djebbar; Ait-Si-Ali, Slimane

2011-01-01

Plants are an invaluable source of potential new anti-cancer drugs. Here, we investigated the cytotoxic activity of the acetonic extract of Buxus sempervirens on five breast cancer cell lines, MCF7, MCF10CA1a and T47D, three aggressive triple positive breast cancer cell lines, and BT-20 and MDA-MB-435, which are triple negative breast cancer cell lines. As a control, MCF10A, a spontaneously immortalized but non-tumoral cell line has been used. The acetonic extract of Buxus sempervirens showed cytotoxic activity towards all the five studied breast cancer cell lines with an IC50 ranging from 7.74 µg/ml to 12.5 µg/ml. Most importantly, the plant extract was less toxic towards MCF10A with an IC50 of 19.24 µg/ml. Fluorescence-activated cell sorting (FACS) analysis showed that the plant extract induced cell death and cell cycle arrest in G0/G1 phase in MCF7, T47D, MCF10CA1a and BT-20 cell lines, concomitant to cyclin D1 downregulation. Application of MCF7 and MCF10CA1a respective IC50 did not show such effects on the control cell line MCF10A. Propidium iodide/Annexin V double staining revealed a pre-apoptotic cell population with extract-treated MCF10CA1a, T47D and BT-20 cells. Transmission electron microscopy analyses indicated the occurrence of autophagy in MCF7 and MCF10CA1a cell lines. Immunofluorescence and Western blot assays confirmed the processing of microtubule-associated protein LC3 in the treated cancer cells. Moreover, we have demonstrated the upregulation of Beclin-1 in these cell lines and downregulation of Survivin and p21. Also, Caspase-3 detection in treated BT-20 and T47D confirmed the occurrence of apoptosis in these cells. Our findings indicate that Buxus sempervirens extract exhibit promising anti-cancer activity by triggering both autophagic cell death and apoptosis, suggesting that this plant may contain potential anti-cancer agents for single or combinatory cancer therapy against breast cancer. PMID:21935420

Establishment and characterization of a novel dedifferentiated liposarcoma cell line, NDDLS-1.

PubMed

Ariizumi, Takashi; Ogose, Akira; Kawashima, Hiroyuki; Hotta, Tetsuo; Li, Guidong; Xu, Yongjun; Hirose, Takanori; Endo, Naoto

2011-08-01

We established a dedifferentiated liposarcoma cell line (NDDLS-1) that produces interleukin-6 (IL-6) and granulocyte-colony stimulating factor (G-CSF). The parental tumor showed high leukemoid reactions. The NDDLS-1 cell line was established from a pleural effusion associated with a lung metastasis. Pleomorphic tumor cells arranged in a haphazard growth pattern were seen in xenograft tumors. Numerous inflammatory cells including neutrophils or eosinophils were present throughout the tumor cells. This finding resembled the dedifferentiated area of the parental tumor. The mice bearing NDDLS-1 showed marked leukocytosis. In addition, the NDDLS-1 cells expressed IL-6 and G-CSF at both the mRNA and protein levels, while the NDDLS-1 cells produced near normal levels of tumor necrosis factor alpha (TNF-α). In the cytogenetic analysis, both the parental tumor and the NDDLS-1 cells showed a ring or giant marker chromosomes. The NDDLS-1 cell line demonstrated the amplification and expression of both MDM2 and CDK4 by fluorescence in situ hybridization and immunohistochemical analysis. The NDDLS-1 cell line is consistent with the parental dedifferentiated liposarcoma, and it should therefore be useful for further investigations of human dedifferentiated liposarcomas. © 2011 The Authors. Pathology International © 2011 Japanese Society of Pathology and Blackwell Publishing Asia Pty Ltd.

Morphological changes in human melanoma cells following irradiation with thermal neutrons.

PubMed

Barkla, D H; Allen, B J; Brown, J K; Mountford, M; Mishima, Y; Ichihashi, M

1989-01-01

Morphological changes in two human melanoma cell lines, MM96 and MM418, following irradiation with thermal neutrons, were studied using light and electron microscopy. The results show that the response of human malignant melanoma cells to neutron irradiation is both cell line dependent and dose dependent, and that in any given cell line, some cells are more resistant to irradiation than others, thus demonstrating heterogeneity in respect to radiosensitivity. Cells repopulating MM96 flasks after irradiation were morphologically similar to the cells of origin whereas in MM418 flasks cells differentiated into five morphologically distinct subgroups and showed increased melanization. The results also show that radiation causes distinctive morphological patterns of damage although ultrastructural changes unique to the high LET particles released from boron 10 neutron capture are yet to be identified.

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

PubMed Central

Cheng, J; Haas, M

1990-01-01

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

Synergistic effects of the sesquiterpene lactone, EPD, with cisplatin and paclitaxel in ovarian cancer cells.

PubMed

van Haaften, Caroline; Boot, Arnoud; Corver, Willem E; van Eendenburg, Jaap D H; Trimbos, Baptist J M Z; van Wezel, Tom

2015-04-25

Ovarian cancer remains still the leading cause of death of gynecological malignancy, in spite of first-line chemotherapy with cisplatin and paclitaxel. Although initial response is favorably, relapses are common and prognosis for women with advanced disease stays poor. Therefore efficacious approaches are needed. Previously, an anti-cancer agent, EPD exhibited potent cytotoxic effects towards ovarian cancer and not towards normal cells. Cell viability and cell cycle analysis studies were performed with EPD, in combination with cisplatin and/or paclitaxel, using the ovarian carcinoma cell lines: SK-OV-3, OVCAR-3, JC, JC-pl and normal fibroblasts. Cell viability was measured using Presto Blue and cell cycle analysis using a flow cytometer. Apoptosis was measured in JC and JC-pl , using the caspase 3 assay kit. In JC-pl, SK-OV-3 and JC, synergistic interactions between either EPD and cisplatin or EPD and paclitaxel were observed. For the first time the effects of EPD on the cell cycle of ovarian cancer cells and normal cells was studied. EPD and combinations of EPD with cisplatin and/ or paclitaxel showed cell cycle arrest in the G2/M phase. The combination of EPD and cisplatin showed a significant synergistic effect in cell line JC-pl, while EPD with paclitaxel showed synergistic interaction in JC. Additionally, synergistic drug combinations showed increased apoptosis. Our results showed a synergistic effect of EPD and cisplatin in an ovarian drug resistant cell line as well as a synergistic effect of EPD and paclitaxel in two other ovarian cell lines. These results might enhance clinical efficacy, compared to the existing regimen of paclitaxel and cisplatin.

Generation and characterization of two immortalized human osteoblastic cell lines useful for epigenetic studies.

PubMed

Pérez-Campo, Flor M; May, Tobias; Zauers, Jeannette; Sañudo, Carolina; Delgado-Calle, Jesús; Arozamena, Jana; Berciano, María T; Lafarga, Miguel; Riancho, José A

2017-03-01

Different model systems using osteoblastic cell lines have been developed to help understand the process of bone formation. Here, we report the establishment of two human osteoblastic cell lines obtained from primary cultures upon transduction of immortalizing genes. The resulting cell lines had no major differences to their parental lines in their gene expression profiles. Similar to primary osteoblastic cells, osteocalcin transcription increased following 1,25-dihydroxyvitamin D 3 treatment and the immortalized cells formed a mineralized matrix, as detected by Alizarin Red staining. Moreover, these human cell lines responded by upregulating ALPL gene expression after treatment with the demethylating agent 5-aza-2'-deoxycytidine (AzadC), as shown before for primary osteoblasts. We further demonstrate that these cell lines can differentiate in vivo, using a hydroxyapatite/tricalcium phosphate composite as a scaffold, to produce bone matrix. More importantly, we show that these cells respond to demethylating treatment, as shown by the increase in SOST mRNA levels, the gene encoding sclerostin, upon treatment of the recipient mice with AzadC. This also confirms, in vivo, the role of DNA methylation in the regulation of SOST expression previously shown in vitro. Altogether our results show that these immortalized cell lines constitute a particularly useful model system to obtain further insight into bone homeostasis, and particularly into the epigenetic mechanisms regulating sclerostin production.

Loss of N-Cadherin Expression in Tumor Transplants Produced From As+3- and Cd+2-Transformed Human Urothelial (UROtsa) Cell Lines.

PubMed

Sandquist, Elizabeth J; Somji, Seema; Dunlevy, Jane R; Garrett, Scott H; Zhou, Xu Dong; Slusser-Nore, Andrea; Sens, Donald A

2016-01-01

Epithelial to mesenchymal transition is a process in which a cell experiences a loss of epithelial cell characteristics and acquires a more mesenchymal cell phenotype. In cancer, epithelial to mesenchymal transition has been proposed to play an important role during specific stages of tumor progression. The role epithelial to mesenchymal transition and mesenchymal to epithelial transition might play in toxicant-induced urothelial cancer is unknown. Real-time PCR, Western blotting, immuno-histochemistry and immuno-fluorescence were used to determine the expression of E- and N-cadherin in the UROtsa parent, the As+3- and Cd+2-transformed cell lines, the spheroids isolated from these cell lines as well as the tumor heterotransplants that were produced by the injection of the transformed cells into immune compromised mice. This study showed that N-cadherin expression was increased in 6 As+3- and 7 Cd+2- transformed cell lines generated from human urothelial cells (UROtsa). The expression varied within each cell line, with 10% to 95% of the cells expressing N-cadherin. Tumors produced from these cell lines showed no expression of the N-cadherin protein. Spheroids which are made up of putative cancer initiating cells produced from these cell lines showed only background expression of N-cadherin mRNA, increased expression of aldehyde dehydrogenase 1 mRNA and produced tumors which did not express N-cadherin. There was no change in the expression of E-cadherin in the tumors, and the tumors formed by all the As+3 and Cd+2-transformed cell lines and cancer initiating cells stained intensely and uniformly for E-cadherin. The finding that the cells expressing N-cadherin gave rise to tumors with no expression of N-cadherin is in agreement with the classical view of epithelial to mesenchymal transition. Epithelial to mesenchymal transition and N-cadherin are associated with dissemination and not with the ability to establish new tumor growth. Mesenchymal to epithelial transition and E-cadherin are viewed as necessary for a cell to establish a new metastatic site. The lack of N-cadherin expression in tumor transplants is consistent with E-cadherin expressing cells "seeding" a site for tumor growth. The study shows that a minority population of cultured cells can be the initiators of tumor growth.

Loss of N-Cadherin Expression in Tumor Transplants Produced From As+3- and Cd+2-Transformed Human Urothelial (UROtsa) Cell Lines

PubMed Central

Sandquist, Elizabeth J.; Somji, Seema; Dunlevy, Jane R.; Garrett, Scott H.; Zhou, Xu Dong; Slusser-Nore, Andrea

2016-01-01

Background Epithelial to mesenchymal transition is a process in which a cell experiences a loss of epithelial cell characteristics and acquires a more mesenchymal cell phenotype. In cancer, epithelial to mesenchymal transition has been proposed to play an important role during specific stages of tumor progression. The role epithelial to mesenchymal transition and mesenchymal to epithelial transition might play in toxicant-induced urothelial cancer is unknown. Methods Real-time PCR, Western blotting, immuno-histochemistry and immuno-fluorescence were used to determine the expression of E- and N-cadherin in the UROtsa parent, the As+3- and Cd+2-transformed cell lines, the spheroids isolated from these cell lines as well as the tumor heterotransplants that were produced by the injection of the transformed cells into immune compromised mice. Results This study showed that N-cadherin expression was increased in 6 As+3- and 7 Cd+2- transformed cell lines generated from human urothelial cells (UROtsa). The expression varied within each cell line, with 10% to 95% of the cells expressing N-cadherin. Tumors produced from these cell lines showed no expression of the N-cadherin protein. Spheroids which are made up of putative cancer initiating cells produced from these cell lines showed only background expression of N-cadherin mRNA, increased expression of aldehyde dehydrogenase 1 mRNA and produced tumors which did not express N-cadherin. There was no change in the expression of E-cadherin in the tumors, and the tumors formed by all the As+3 and Cd+2-transformed cell lines and cancer initiating cells stained intensely and uniformly for E-cadherin. Conclusions The finding that the cells expressing N-cadherin gave rise to tumors with no expression of N-cadherin is in agreement with the classical view of epithelial to mesenchymal transition. Epithelial to mesenchymal transition and N-cadherin are associated with dissemination and not with the ability to establish new tumor growth. Mesenchymal to epithelial transition and E-cadherin are viewed as necessary for a cell to establish a new metastatic site. The lack of N-cadherin expression in tumor transplants is consistent with E-cadherin expressing cells “seeding” a site for tumor growth. The study shows that a minority population of cultured cells can be the initiators of tumor growth. PMID:27224422

Analysis and functional annotation of expressed sequence tags from in vitro cell lines of elasmobranchs: spiny dogfish shark (Squalus acanthias) and little skate (Leucoraja erinacea)

PubMed Central

Parton, Angela; Bayne, Christopher J.; Barnes, David W.

2010-01-01

Elasmobranchs are the most commonly used experimental models among the jawed, cartilaginous fish (Chondrichthyes). Previously we developed cell lines from embryos of two elasmobranchs, Squalus acanthias the spiny dogfish shark (SAE line), and Leucoraja erinacea the little skate (LEE-1 line). From these lines cDNA libraries were derived and expressed sequence tags (ESTs) generated. From the SAE cell line 4303 unique transcripts were identified, with 1848 of these representing unknown sequences (showing no BLASTX identification). From the LEE-1 cell line, 3660 unique transcripts were identified, and unknown, unique sequences totaled 1333. Gene Ontology (GO) annotation showed that GO assignments for the two cell lines were in general similar. These results suggest that the procedures used to derive the cell lines led to isolation of cell types of the same general embryonic origin from both species. The LEE-1 transcripts included GO categories “envelope” and “oxidoreductase activity” but the SAE transcripts did not. GO analysis of SAE transcripts identified the category “anatomical structure formation” that was not present in LEE-1 cells. Increased organelle compartments may exist within LEE-1 cells compared to SAE cells, and the higher oxidoreductase activity in LEE-1 cells may indicate a role for these cells in responses associated with innate immunity or in steroidogenesis. These EST libraries from elasmobranch cell lines provide information for assembly of genomic sequences and are useful in revealing gene diversity, new genes and molecular markers, as well as in providing means for elucidation of full-length cDNAs and probes for gene array analyses. This is the first study of this type with members of the Chondrichthyes. PMID:20471924

Analysis and functional annotation of expressed sequence tags from in vitro cell lines of elasmobranchs: Spiny dogfish shark (Squalus acanthias) and little skate (Leucoraja erinacea).

PubMed

Parton, Angela; Bayne, Christopher J; Barnes, David W

2010-09-01

Elasmobranchs are the most commonly used experimental models among the jawed, cartilaginous fish (Chondrichthyes). Previously we developed cell lines from embryos of two elasmobranchs, Squalus acanthias the spiny dogfish shark (SAE line), and Leucoraja erinacea the little skate (LEE-1 line). From these lines cDNA libraries were derived and expressed sequence tags (ESTs) generated. From the SAE cell line 4303 unique transcripts were identified, with 1848 of these representing unknown sequences (showing no BLASTX identification). From the LEE-1 cell line, 3660 unique transcripts were identified, and unknown, unique sequences totaled 1333. Gene Ontology (GO) annotation showed that GO assignments for the two cell lines were in general similar. These results suggest that the procedures used to derive the cell lines led to isolation of cell types of the same general embryonic origin from both species. The LEE-1 transcripts included GO categories "envelope" and "oxidoreductase activity" but the SAE transcripts did not. GO analysis of SAE transcripts identified the category "anatomical structure formation" that was not present in LEE-1 cells. Increased organelle compartments may exist within LEE-1 cells compared to SAE cells, and the higher oxidoreductase activity in LEE-1 cells may indicate a role for these cells in responses associated with innate immunity or in steroidogenesis. These EST libraries from elasmobranch cell lines provide information for assembly of genomic sequences and are useful in revealing gene diversity, new genes and molecular markers, as well as in providing means for elucidation of full-length cDNAs and probes for gene array analyses. This is the first study of this type with members of the Chondrichthyes. Copyright 2010 Elsevier Inc. All rights reserved.

Development and characterization of cell culture systems from Puntius (Tor) chelynoides (McClelland).

PubMed

Goswami, M; Sharma, B S; Tripathi, A K; Yadav, Kamalendra; Bahuguna, S N; Nagpure, N S; Lakra, W S; Jena, J K

2012-05-25

Puntius (Tor) chelynoides, commonly known as dark mahseer, is a commercially important coldwater fish species which inhabits fast-flowing hill-streams of India and Nepal. Cell culture systems were developed from eye, fin, heart and swim bladder tissues of P. chelynoides using explant method. The cell culture system developed from eye has been maintained towards a continuous cell line designated as PCE. The cells were grown in 25cm(2) tissue culture flasks with Leibovitz' L-15 media supplemented with 20 % fetal bovine serum (FBS) at 24°C. The PCE cell line consists of predominantly fibroblast-like cells and showed high plating efficiency. The monolayer formed from the fin and heart explants were comprised of epithelial as well as fibroblast-like cells, a prominent and rhythmic heartbeat was also observed in heart explants. Monolayer formed from swim bladder explants showed the morphology of fibroblast-like cells. All the cells from different tissues are able to grow at an optimum temperature of 24°C and growth rate increased as the FBS concentration increased. The PCE cell line was characterized using amplification of mitochondrial cytochrome oxidase subunit I (COI) & 16S rRNA genes which confirmed that the cell line originated from P. chelynoides. Cytogenetic analysis of PCE cell line and cells from fin revealed a diploid count of 100 chromosomes. Upon transfection with pEGFP-C1 plasmid, bright fluorescent signals were observed, suggesting that this cell line can be used for transgenic and genetic manipulation studies. Further, genotoxicity assessment of PCE cells illustrated the utility of this cell line as an in vitro model for aquatic toxicological studies. The PCE cell line was successfully cryopreserved and revived at different passage levels. The cell line and culture systems are being maintained to develop continuous cell lines for further studies. Copyright © 2012 Elsevier B.V. All rights reserved.

Assessment of biological effectiveness of boron neutron capture therapy in primary and metastatic melanoma cell lines.

PubMed

Rossini, Andrés E; Dagrosa, Maria A; Portu, Agustina; Saint Martin, Giselle; Thorp, Silvia; Casal, Mariana; Navarro, Aimé; Juvenal, Guillermo J; Pisarev, Mario A

2015-01-01

In order to optimize the effectiveness of Boron Neutron Capture Therapy (BNCT), Relative Biological Effectiveness (RBE) and Compound Biological Effectiveness (CBE) were determined in two human melanoma cell lines, M8 and Mel-J cells, using the amino acid p-boronophenylalanine (BPA) as boron carrier. The effects of BNCT on the primary amelanotic cell line M8 and on the metastatic pigmented melanoma cell line Mel-J were studied using colony formation assay. The RBE values were determined using both a gamma ray source, and the neutron beam from the Nuclear Reactor of the National Atomic Energy Commission (RA-3). For the determination of the RBE, cells were irradiated with increasing doses of both sources, between 1 and 8 Gy; and for the determination of CBE factors, the cells were pre-incubated with BPA before irradiation. Afterwards, the cell surviving fraction (SF) was determined for each treatment. Marked differences were observed between both cell lines. Mel-J cells were more radioresistant than the M8 cell line. The clonogenic assays showed that for a SF of 1%, the RBE values were 1.3 for M8 cells and 1.5 for Mel-J cells. Similarly, the CBE values for a 1% SF were 2.1 for M8 and 3 for Mel-J cell lines. For the endpoint of 0.1% of SF the RBE values obtained were 1.2 for M8 and 1.4 for Mel-J cells. Finally, CBE values calculated for a 0.1% were 2 and 2.6 for M8 and Mel-J cell lines respectively. In order to estimate the uptake of the non-radioactive isotope Boron 10 ((10)B), a neutron induced autoradiographic technique was performed showing discrepancies in (10)B uptake between both cell lines. These obtained in vitro results are the first effectiveness factors determined for human melanoma at the RA-3 nuclear reactor and show that BNCT dosimetry planning for patients could be successfully performed using these new factors.

Characterization and differentiation of human embryonic stem cells.

PubMed

Carpenter, M K; Rosler, E; Rao, M S

2003-01-01

Cell replacement therapies have been limited by the availability of sufficient quantities of cells for transplantation. Human ES (hES) cell lines have recently been generated by several laboratories. When maintained for over 1 year in vitro, they remain karyotypically and phenotypically stable and may therefore provide an excellent source material for cell therapies. Currently, data is available for 26 hES cell lines. Although limited characterization has been performed on most of these lines, there are remarkable similarities in expression of markers. hES cell lines derived in different laboratories show similar expression profiles of surface markers, including SSEA-4, Tra-1-60, and Tra-1-81. In addition, markers associated with pluripotent cells such as OCT-4 are expressed at in all cell lines tested. These cells express high levels of telomerase and appear to have indefinite growth potential. The generation of the large quantities of cells necessary for cell replacement therapies will require a cell population which is stable over long term culture. We have characterized the properties of multiple hES cell lines that have been maintained in culture for extended periods. Quantitative analyses demonstrate that all of the cell lines examined show consistent marker expression and retain a normal karyotype after long-term culture. hES cells have been differentiated into the derivatives of all three germ layers. Specifically this includes cardiomyocytes, neural cells, hepatocyte-like cells, endothelial cells and hematopoietic progenitor cells. These data demonstrating the karyotypic and phenotypic stability of hES cells and their extensive differentiative capacity indicate that they may be an appropriate source of cells for multiple regenerative medicine applications.

Human colon cancer HT-29 cell death responses to doxorubicin and Morus Alba leaves flavonoid extract.

PubMed

Fallah, S; Karimi, A; Panahi, G; Gerayesh Nejad, S; Fadaei, R; Seifi, M

2016-03-31

The mechanistic basis for the biological properties of Morus alba flavonoid extract (MFE) and chemotherapy drug of doxorubicin on human colon cancer HT-29 cell line death are unknown. The effect of doxorubicin and flavonoid extract on colon cancer HT-29 cell line death and identification of APC gene expression and PARP concentration of HT-29 cell line were investigated. The results showed that flavonoid extract and doxorubicin induce a dose dependent cell death in HT-29 cell line. MFE and doxorubicin exert a cytotoxic effect on human colon cancer HT-29 cell line by probably promoting or induction of apoptosis.

Synthesis and characterization of 2-substituted benzimidazoles and their evaluation as anticancer agent

NASA Astrophysics Data System (ADS)

Azam, Mohammad; Khan, Azmat Ali; Al-Resayes, Saud I.; Islam, Mohammad Shahidul; Saxena, Ajit Kumar; Dwivedi, Sourabh; Musarrat, Javed; Trzesowska-Kruszynska, Agata; Kruszynski, Rafal

2015-05-01

In this work, we report a series of benzimidazole derivatives synthesized from benzene-1,2-diamine and aryl-aldehydes at room temperature. The synthesized compounds have been characterized on the basis of elemental analysis and various spectroscopic studies viz., IR, 1H- and 13C-NMR, ESI-MS as well by X-ray single X-ray crystallographic study. Interaction of these compounds with CT-DNA has been examined with fluorescence experiments and showed significant binding ability. All the synthesized compounds have been screened for their antitumor activities against various human cancer cell lines viz., Human breast adenocarcinoma cell line (MCF-7), Human leukemia cell line (THP-1), Human prostate cancer cell lines (PC-3) and adenocarcinomic human alveolar basal epithelial cell lines (A-549). Interestingly, all the compounds showed significant anticancer activity.

Development, Characterization, and Pluripotency Analysis of Buffalo (Bubalus bubalis) Embryonic Stem Cell Lines Derived from In Vitro–Fertilized, Hand-Guided Cloned, and Parthenogenetic Embryos

PubMed Central

Shah, Syed Mohmad; Saini, Neha; Ashraf, Syma; Zandi, Mohammad; Manik, Radhey Sham; Singla, Suresh Kumar; Palta, Prabhat

2015-01-01

Abstract We present the derivation, characterization, and pluripotency analysis of three buffalo embryonic stem cell (buESC) lines, from in vitro–fertilized, somatic cell nuclear–transferred, and parthenogenetic blastocysts. These cell lines were developed for later differentiation into germ lineage cells and elucidation of the signaling pathways involved. The cell lines were established from inner cell masses (ICMs) that were isolated manually from the in vitro–produced blastocysts. Most of the ICMs (45–55%) resulted in formation of primary colonies that were subcultured after 8–10 days, leading subsequently to the formation of three buESC lines, one from each blastocyst type. All the cell lines expressed stem cell markers, such as Alkaline Phosphatase, OCT4, NANOG, SSEA1, SSEA4, TRA-1-60, TRA-1-81, SOX2, REX1, CD-90, STAT3, and TELOMERASE. They differentiated into all three germ layers as determined by ectodermal, mesodermal, and endodermal RNA and protein markers. All of the cell lines showed equal expression of pluripotency markers as well as equivalent differentiation potential into all the three germ layers. The static suspension culture–derived embryoid bodies (EBs) showed greater expression of all the three germ layer markers as compared to hanging drop culture–derived EBs. When analyzed for germ layer marker expression, EBs derived from 15% fetal bovine serum (FBS)-based spontaneous differentiation medium showed greater differentiation across all the three germ layers as compared to those derived from Knock-Out Serum Replacement (KoSR)-based differentiation medium. PMID:26168169

The transcriptional diversity of 25 Drosophila cell lines

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

Cherbas, Lucy; Willingham, Aarron; Zhang, Dayu

2010-12-22

Drosophila melanogaster cell lines are important resources for cell biologists. In this article, we catalog the expression of exons, genes, and unannotated transcriptional signals for 25 lines. Unannotated transcription is substantial (typically 19% of euchromatic signal). Conservatively, we identify 1405 novel transcribed regions; 684 of these appear to be new exons of neighboring, often distant, genes. Sixty-four percent of genes are expressed detectably in at least one line, but only 21% are detected in all lines. Each cell line expresses, on average, 5885 genes, including a common set of 3109. Expression levels vary over several orders of magnitude. Major signalingmore » pathways are well represented: most differentiation pathways are ‘‘off’’ and survival/growth pathways ‘‘on.’’ Roughly 50% of the genes expressed by each line are not part of the common set, and these show considerable individuality. Thirty-one percent are expressed at a higher level in at least one cell line than in any single developmental stage, suggesting that each line is enriched for genes characteristic of small sets of cells. Most remarkable is that imaginal disc-derived lines can generally be assigned, on the basis of expression, to small territories within developing discs. These mappings reveal unexpected stability of even fine-grained spatial determination. No two cell lines show identical transcription factor expression. We conclude that each line has retained features of an individual founder cell superimposed on a common ‘‘cell line‘‘ gene expression pattern. We report the transcriptional profiles of 25 Drosophila melanogaster cell lines, principally by whole-genome tiling microarray analysis of total RNA, carried out as part of the modENCODE project. The data produced in this study add to our knowledge of the cell lines and of the Drosophila transcriptome in several ways. We summarize the expression of previously annotated genes in each of the 25 lines with emphasis on what those patterns reveal about the origins of the lines and the stability of spatial expression patterns. In addition, we offer an initial analysis of previously unannotated transcripts in the cell lines.« less

A characterization of the ZFL cell line and primary hepatocytes as in vitro liver cell models for the zebrafish (Danio rerio).

PubMed

Eide, Marta; Rusten, Marte; Male, Rune; Jensen, Knut Helge Midtbø; Goksøyr, Anders

2014-02-01

The zebrafish (Danio rerio) is a widely used model species in biomedical research. The ZFL cell line, established from zebrafish liver, and freshly isolated primary hepatocytes from zebrafish have been used in several toxicological studies. However, no previous report has compared and characterized these two systems at the level of gene expression. The aim of this study was to evaluate the ZFL cell line in comparison to primary hepatocytes as in vitro models for studying effects of environmental contaminants in zebrafish liver. Using quantitative real-time PCR, the basal level and transcriptional induction potential of key genes involved in toxic responses in the ZFL cell line, primary hepatocytes and whole liver from zebrafish were compared. The study showed that the ZFL cells have lower levels of mRNA of most selected genes compared to zebrafish liver. The induced gene transcription following exposure to ligand was much lower in ZFL cells compared to zebrafish primary hepatocytes at the doses tested. Importantly, oestrogen receptor and vitellogenin genes showed low basal transcription and no induction response in the ZFL cell line. In conclusion, it appears that primary hepatocytes are well suited for studying environmental contaminants including xenoestrogens, but may show large sex-dependent differences in gene transcription. The ZFL cell line shows potential in toxicological studies involving the aryl hydrocarbon receptor pathway. However, low potential for transcriptional induction of genes in general should be expected, especially notable when studying estrogenic responses. Copyright © 2013 Elsevier B.V. All rights reserved.

Establishment and characterization of fetal and maternal mesenchymal stem/stromal cell lines from the human term placenta.

PubMed

Qin, Sharon Q; Kusuma, Gina D; Al-Sowayan, Batla; Pace, Rishika A; Isenmann, Sandra; Pertile, Mark D; Gronthos, Stan; Abumaree, Mohamed H; Brennecke, Shaun P; Kalionis, Bill

2016-03-01

Human placental mesenchymal stem/stromal cells (MSC) are an attractive source of MSC with great therapeutic potential. However, primary MSC are difficult to study in vitro due to their limited lifespan and patient-to-patient variation. Fetal and maternal MSC were prepared from cells of the chorionic and basal plates of the placenta, respectively. Fetal and maternal MSC were transduced with the human telomerase reverse transcriptase (hTERT). Conventional stem cell assays assessed the MSC characteristics of the cell lines. Functional assays for cell proliferation, cell migration and ability to form colonies in soft agar were used to assess the whether transduced cells retained properties of primary MSC. Fetal chorionic and maternal MSC were successfully transduced with hTERT to create the cell lines CMSC29 and DMSC23 respectively. The lifespans of CMSC29 and DMSC23 were extended in cell culture. Both cell lines retained important MSC characteristics including cell surface marker expression and multipotent differentiation potential. Neither of the cell lines was tumourigenic in vitro. Gene expression differences were observed between CMSC29 and DMSC23 cells and their corresponding parent, primary MSC. Both cell lines show similar migration potential to their corresponding primary, parent MSC. The data show that transduced MSC retained important functional properties of the primary MSC. There were gene expression and functional differences between cell lines CMSC29 and DMSC23 that reflect their different tissue microenvironments of the parent, primary MSC. CMSC29 and DMSC23 cell lines could be useful tools for optimisation and functional studies of MSC. Copyright © 2016 Elsevier Ltd. All rights reserved.

Visual selection and maintenance of the cell lines with high plant regeneration ability and low ploidy level in Dianthus acicularis by monitoring with flow cytometry analysis.

PubMed

Shiba, Tomonori; Mii, Masahiro

2005-12-01

Efficient plant regeneration system from cell suspension cultures was established in D. acicularis (2n=90) by monitoring ploidy level and visual selection of the cultures. The ploidy level of the cell cultures closely related to the shoot regeneration ability. The cell lines comprising original ploidy levels (2C+4C cells corresponding to DNA contents of G1 and G2 cells of diploid plant, respectively) showed high regeneration ability, whereas those containing the cells with 8C or higher DNA C-values showed low or no regeneration ability. The highly regenerable cell lines thus selected consisted of compact cell clumps with yellowish color and relatively moderate growth, suggesting that it is possible to select visually the highly regenerable cell lines with the original ploidy level. All the regenerated plantlets from the highly regenerable cell cultures exhibited normal phenotypes and no variations in ploidy level were observed by flow cytometry (FCM) analysis.

An Investigation of the Growth Inhibitory Capacity of Several Medicinal Plants From Iran on Tumor Cell Lines

PubMed Central

Esmaeilbeig, Maryam; Kouhpayeh, Seyed Amin; Amirghofran, Zahra

2015-01-01

Background: Traditional herbal medicine is a valuable resource that provides new drugs for cancer treatment. Objectives: In this study we aim to screen and investigate the in vitro anti-tumor activities of ten species of plants commonly grown in Southern Iran. Materials and Methods: We used the MTT colorimetric assay to evaluate the cytotoxic activities of the methanol extracts of these plants on various tumor cell lines. The IC50 was calculated as a scale for this evaluation. Results: Satureja bachtiarica, Satureja hortensis, Thymus vulgaris, Thymus daenensis and Mentha lonigfolia showed the inhibitoriest effects on Jurkat cells with > 80% inhibition at 200 µg/mL. Satureja hortensis (IC50: 66.7 µg/mL) was the most effective. These plants also strongly inhibited K562 cell growth; Satureja bachtiarica (IC50: 28.3 µg/mL), Satureja hortensis (IC50: 52 µg/mL) and Thymus vulgaris (IC50: 87 µg/mL) were the most effective extracts. Cichorium intybus, Rheum ribes, Alhagi pseudalhagi and Glycyrrihza glabra also showed notable effects on the leukemia cell lines. The Raji cell line was mostly inhibited by Satureja bachtiarica and Thymus vulgaris with approximately 40% inhibition at 200µg/ml. The influence of these extracts on solid tumor cell lines was not strong. Fen cells were mostly affected by Glycyrrihza glabra (IC50: 182 µg/mL) and HeLa cells by Satureja hortensis (31.6% growth inhibitory effect at 200 µg/mL). Conclusions: Leukemic cell lines were more sensitive to the extracts than the solid tumor cell lines; Satureja hortensis, Satureja bachtiarica, Thymus vulgaris, Thymus daenensis and Mentha lonigfolia showed remarkable inhibitory potential. PMID:26634114

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.

Cytogenetic damage, oncogenic transformation and p53 induction in human epithelial cells in response to irradiation

NASA Astrophysics Data System (ADS)

Armitage, Mark

Ionizing radiation can have several different effects on cells, some are almost instantaneous such as the generation of DNA damage, other cellular responses take a matter of minutes or hours - DNA repair protein induction/activation, and others may take months or even years to be manifested - carcinogenesis. Human epithelial cell lines derived from both normal, non-neoplastic tissues and from a malignant source were cultured in order to examine several effects of ionizing radiation on such cell types. Cells not from a malignant source were previously immortalized by viral infection or by transfection with viral sequences. Simian virus 40 immortalised uroepithelial cells (SV-HUC) were found to be approximately a factor of two fold more radioresistant than cells of malignant origin (T24) in terms of unrepaired clastogenic damage i.e. assessment of micronuclei levels following irradiation. SV-HUC lines unlike T24 cells are non-tumourigenic when inoculated into nude athymic mice. SV-HUC lines proved very resistant to full oncogenic transformation using radiation and chemical carcinogens. However, morphological alterations and decreased anchorage dependant growth was observed in post carcinogen treated cells after appropriate cell culture conditions were utilized. The progression from this phenotype to a fully tumourigenic one was not recorded in this study. The ability of ionizing radiation to induce increased levels of the nuclear phosphoprotein p53 was also assessed using several different cell lines. SV- HUC and T24 cell lines failed to exhibit any increased p53 stabilization following irradiation. One cell line, a human papilloma virus transformed line (HPV) did show an approximate two fold increase of the wild type p53 protein after treatment with radiation. Only the cell line HPV showed any cell cycle delay, resulting in accumulation of cells in the G2/M compartment in post irradiation cell cycle analysis. The status of p53 was also assessed i.e. wild type or mutant conformation in all the above cells lines and two other control lines HOS (a human osteosarcoma cell line) and H Tori-3 (SV40 immortalised thyroid epithelial cells).

Polymer encapsulated dopaminergic cell lines as "alternative neural grafts".

PubMed

Jaeger, C B; Greene, L A; Tresco, P A; Winn, S R; Aebischer, P

1990-01-01

Our preliminary findings (Jaeger et al., 1988; Aebischer et al., 1989; Tresco et al., 1989) and the studies in progress show that encapsulated dopaminergic cell lines survive enclosure within a semi-permeable membrane. The encapsulated cells remained viable for extended time periods when maintained in vitro. Moreover, encapsulated PC12 and T28 cells have the potential to survive following their implantation into the forebrain of rats. Cell lines are essentially "immortal" because they continue to divide indefinitely. This property allows perpetual "self-renewal" of a given cell population. However, the capacity of continuous uncontrolled cell division may also lead to tumor formation. This in fact is the case for unencapsulated PC12 cell implants placed into the brain of young Sprague Dawley rats (Jaeger, 1985). Cell line encapsulation has the potential to prevent tumor growth (Jaeger et al., 1988). Survival for 6 months in vitro suggests that encapsulation does not preclude long-term maintenance of an homogeneous cell line like PC12 cells. The presence of mitotic figures in the capsules further supports the likelihood of propagation and self renewal of the encapsulated population. Another significant property of cell lines is that they consist of a single, genetically homogeneous cell type. They do not require specific synaptic interactions for their survival. In the case of PC12 and T28 lines, the cells synthesize and release neurotransmitters. Our data show that PC12 and T28 cells continue to release dopamine spontaneously and to express specific transmitters and enzymes following encapsulation. Thus, cell lines such as these may constitute relatively simple "neural implants" exerting their function via humoral release.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis and characterization of 2-substituted benzimidazoles and their evaluation as anticancer agent.

PubMed

Azam, Mohammad; Khan, Azmat Ali; Al-Resayes, Saud I; Islam, Mohammad Shahidul; Saxena, Ajit Kumar; Dwivedi, Sourabh; Musarrat, Javed; Trzesowska-Kruszynska, Agata; Kruszynski, Rafal

2015-05-05

In this work, we report a series of benzimidazole derivatives synthesized from benzene-1,2-diamine and aryl-aldehydes at room temperature. The synthesized compounds have been characterized on the basis of elemental analysis and various spectroscopic studies viz., IR, (1)H- and (13)C-NMR, ESI-MS as well by X-ray single X-ray crystallographic study. Interaction of these compounds with CT-DNA has been examined with fluorescence experiments and showed significant binding ability. All the synthesized compounds have been screened for their antitumor activities against various human cancer cell lines viz., Human breast adenocarcinoma cell line (MCF-7), Human leukemia cell line (THP-1), Human prostate cancer cell lines (PC-3) and adenocarcinomic human alveolar basal epithelial cell lines (A-549). Interestingly, all the compounds showed significant anticancer activity. Copyright © 2015 Elsevier B.V. All rights reserved.

A Tetrameric Peptide Derived from Bovine Lactoferricin Exhibits Specific Cytotoxic Effects against Oral Squamous-Cell Carcinoma Cell Lines.

PubMed

Solarte, Víctor A; Rosas, Jaiver E; Rivera, Zuly J; Arango-Rodríguez, Martha L; García, Javier E; Vernot, Jean-Paul

2015-01-01

Several short linear peptides derived from cyclic bovine lactoferricin were synthesized and tested for their cytotoxic effect against the oral cavity squamous-cell carcinoma (OSCC) cell lines CAL27 and SCC15. As a control, an immortalized and nontumorigenic cell line, Het-1A, was used. Linear peptides based on the RRWQWR core sequence showed a moderate cytotoxic effect and specificity towards tumorigenic cells. A tetrameric peptide, LfcinB(20-25)4, containing the RRWQWR motif, exhibited greater cytotoxic activity (>90%) in both OSCC cell lines compared to the linear lactoferricin peptide or the lactoferrin protein. Additionally, this tetrameric peptide showed the highest specificity towards tumorigenic cells among the tested peptides. Interestingly, this effect was very fast, with cell shrinkage, severe damage to cell membrane permeability, and lysis within one hour of treatment. Our results are consistent with a necrotic effect rather than an apoptotic one and suggest that this tetrameric peptide could be considered as a new candidate for the therapeutic treatment of OSCC.

[Establishment and characterization of a new carcinoma cell line from uterine cervix of Uyghur women].

PubMed

Zhang, Lu; Aerziguli, Tursun; Guzalnur, Abliz

2012-04-01

To establish a uterine cervical carcinoma cell line of Uyghur ethnical background and to evaluate the related biological characteristics for future biomedical investigations of diseases in the Uyghur population. Poorly-differentiated squamous cell carcinoma specimens of Uyghur patients were obtained and cultured in vitro by enzymatic digestion method, followed by continuous passaging to reach a stable growth determined by cell viability and growth curve. Morphological study, cell cycling and chromosomal analysis were performed. Tumorigenesis study was conducted by inoculation of nude mice. Biomarker (CK17, CD44, Ki-67, CK14 and vimentin) expression was detected by immunofluorescence and immunocytochemical techniques. A cervical carcinoma cell line was successfully established and maintained for 12 months through 70 passages. The cell line had a stable growth with a population doubling time of 51.9 h. Flask method and double agar-agar assay showed that the cell line had colony-forming rates of 32.5% and 15.6%, respectively. Ultrastructural evaluation demonstrated numerous cell surface protrusions or microvilli, a large number of rod-shape structures in cytoplasm, typical desmosomes and nuclear atypia. Chromosomal analysis revealed human karyotype with the number of chromosomes per cell varying from 32 - 97 with a majority of 54 - 86 (60.3%). Xenogeneic tumors formed in nude mice showed histological structures identical to those of the primary tumor. The cells had high expression of CK17, CD44, Ki-67 and vimentin but no CK14 expression. A cervical carcinoma cell line from a female Uyghur patient is successfully established. The cell line has the characteristics of human cervical squamous cell carcinoma, and it is stable with maintaining the characteristic biological and morphological features in vitro for more than 12 months, therefore, qualified as a stable cell line for further biomedical research.

Characteristics of bovine inner cell mass-derived cell lines and their fate in chimeric conceptuses.

PubMed

Furusawa, Tadashi; Ohkoshi, Katsuhiro; Kimura, Koji; Matsuyama, Shuichi; Akagi, Satoshi; Kaneda, Masahiro; Ikeda, Mitsumi; Hosoe, Misa; Kizaki, Keiichiro; Tokunaga, Tomoyuki

2013-08-01

Bovine embryonic stem (ES) cells have the potential to provide significant benefits in a range of agricultural and biomedical applications. Here, we employed a combination of conventional methods using glycogen synthase kinase 3 and mitogen-activated protein kinase inhibitors to establish ES cell lines from in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT) bovine embryos. Five male cell lines were established from IVF embryos, and two female and three male cell lines from SCNT blastocysts; we named these lines bovine ES cell-like cells (bESLCs). The lines exhibited dome-shaped colonies, stained positively for alkaline phosphatase, and expressed pluripotent stem cell markers such as POU5F1, SOX2, and SSEA-1. The expression levels of these markers, especially for NANOG, varied among the cell lines. A DNA methylation assay showed the POU5F1 promoter region was hypomethylated compared to fibroblast cells. An in vitro differentiation assay showed that endoderm and ectoderm marker genes, but not mesoderm markers, were upregulated in differentiating bESLCs. To examine bESLCs in later embryonic stages, we created 22 chimeric blastocysts with a male bESLC line carrying a GFP marker gene and transferred these to a recipient cow. Four chimeric embryos were subsequently retrieved on Day 13 and retransferred to two recipient cows. One living fetus was obtained at Day 62. GFP signals were not identified in fetal cells by fluorescence microscopy; however, genomic PCR analysis detected the GFP gene in major organs. Clusters of GFP-positive cells were observed in amniotic membranes, suggesting that bESLCs can be categorized as a novel type of ICM-derived cells that can potentially differentiate into epiblast and hypoblast lineages.

Single-wall carbon nanohorns (SWNHs) inhibited proliferation of human glioma cells and promoted its apoptosis

NASA Astrophysics Data System (ADS)

Li, Yunjun; Zhang, Jinqian; Zhao, Ming; Shi, Zujin; Chen, Xin; He, Xihui; Han, Nanyin; Xu, Ruxiang

2013-08-01

Although single-wall carbon nanohorns (SWNHs) have been demonstrated to accumulate to cytotoxic levels within organs of various animal models and cell types, they have been exploited for cancer therapies. The role of SWNHs in human glioma cell lines was unclear. To address this question, the research about direct role of SWNHs on the growth, proliferation, and apoptosis of human glioma cell lines (U87, U251, and U373) had been performed. Our results indicate that particle size of SWNHs in water is between 342 and 712 nm, the films of SEM show that SWNHs on PS surface are individual particles. SWNHs significantly delayed mitotic entry of human glioma cell lines cells, and inhibited its proliferation in a time- and dose-dependent manner. SWNHs induced a significant increase in G1 phase and inhibition of S phase followed the gradually increasing concentrations. SWNHs in human glioma cell lines cells significantly induced apoptosis followed by their gradually increasing concentrations. The TEM images showed that individual spherical SWNHs particles smaller than 100 nm in diameters were localized inside lysosomes of human glioma cell lines. SWNHs inhibited mitotic entry, growth, and proliferation of human glioma cell lines, and promoted its apoptosis. SWNHs may be a novel opportunity or method for the research on treatment of human glioma.

DOK, a cell line established from human dysplastic oral mucosa, shows a partially transformed non-malignant phenotype.

PubMed

Chang, S E; Foster, S; Betts, D; Marnock, W E

1992-12-02

There are many reports of cell lines being established from human oral squamous-cell carcinomas but apparently none of cell lines from dysplastic or "pre-malignant" oral mucosa. We describe here the isolation and characterization of a cell line, DOK (dysplastic oral keratinocyte), from a piece of dorsal tongue showing epithelial dysplasia. The tissue was obtained from a 57-year-old man who was a heavy smoker prior to the appearance of a white patch on his tongue. Eleven years later a squamous-cell carcinoma developed at the site and was excised. Subsequently the remaining dysplasia was removed, and it was from a piece of this that the primary cell cultures which eventually gave rise to DOK were initiated. The DOK line has been single-cell cloned and is apparently immortal. It grows in the absence of 3T3 feeder cells, is anchorage-dependent for growth and is non-tumorigenic in nude mice. The keratin profile of the cells shows a striking similarity to that of the original tongue dysplasia. The karyotype of DOK is aneuploid and complex. By PCR and oligonucleotide hybridization on dot blots, codons 12, 13 and 61 of Ha-ras, Ki-ras and N-ras in DNA extracted from DOK cells were shown to be normal. Immunohistochemistry showed no abnormal, i.e., elevated expression of the onco-suppressor protein p53. Because of its origin and partially transformed phenotype, DOK presents an opportunity to study whether specific carcinogens associated with tobacco and areca nut can cause malignant transformation of oral keratinocytes in vitro.

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.

Downregulation of telomerase activity in human promyelocytic cell line using RNA interference.

PubMed

Miri-Moghaddam, E; Deezagi, A; Soheili, Z S

2009-12-01

Telomerase is a ribonucleoprotein complex. It consists of two main components, human telomerase reverse transcriptase (hTERT) and human telomerase RNA. High telomerase activity is present in most malignant cells, but it is barely detectable in majority of somatic cells. The direct correlation between telomerase reactivation and carcinogens has made hTERT a key target for anticancer therapeutic studies. In this study, for the first time, we evaluated the ability of the new generation of short interfering RNA (siRNA) to regulate telomerase activity in the human promyelocytic leukemia cell line (HL-60). Transient transfection cell line by hTERT siRNAs resulted in statistically significant suppression of hTERT messenger RNAs which were detected by quantitative real-time polymerase chain reaction, while the expressed hTERT protein levels were measured by flow cytometry. The results of telomeric repeat amplification protocol showed that telomerase activity was significantly reduced upon transfection of the HL-60 cell line with hTERT siRNAs. The results of this study showed that telomerase activity and cell proliferation were efficiently inhibited in the hTERT siRNA-treated leukemic cell line.

Antiproliferative activity of synthetic fatty acid amides from renewable resources.

PubMed

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

2015-01-15

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

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

Chemosensitivity of BRCA1-Mutated Ovarian Cancer Cells and Established Cytotoxic Agents.

PubMed

van Haaften, Caroline; van Eendenburg, Jaap; Boot, Arnoud; Corver, Willem E; Haans, Lucien; van Wezel, Tom; Trimbos, J Baptist

2017-10-01

Serous adenocarcinomas that arise in patients with inherited mutations in the tumor suppressor genes BRCA1 and BRCA2 are initially well treatable with platinum/paclitaxel. For recurrent disease in patients with BRCA1 or BRCA2 mutations, olaparib treatment is available. To study additional therapeutic regimens, a better understanding of the cellular and molecular mechanisms of the tumors in in vitro models is important. From a high-grade serous ovarian tumor of a BRCA1 mutation carrier, we established 3 distinct cell line subclones, OVCA-TR3.1, -2, and -3. Immunohistochemical characterization, flow cytometric analyses, chemosensitivity, and somatic mutation profiling were performed. The cell lines expressed AE1/AE3, Pax8, WT-1, OC125, estrogen receptor (ER), and p53, comparable to the primary tumor. Synergism could be shown in the combination treatment eremophila-1-(10)-11(13)-dien-12,8β-olide (EPD), with cisplatin, whereas combination with olaparib did not show synergism. Eremophila-1-(10)-11(13)-dien-12,8β-olide, a sesquiterpene lactone, is a novel chemotherapeutic agent. The inherited BRCA1 c.2989_2990dupAA mutation was confirmed in the cell lines. Loss of heterozygosity of BRCA1 was detected in each cell line, as well as a homozygous TP53 c.722C>A mutation. Flow cytometry showed that all cell lines had a distinct DNA index. Three new isogenic ovarian cancer cell lines were developed from a patient with a germ line BRCA1 mutation. Chemosensitivity profiling of the cell lines showed high tolerance for olaparib. Treatment with EPD proved synergistic with cisplatin. The effects of EPD will be further investigated for future clinical efficacy.

Establishment of cholangiocarcinoma cell lines from patients in the endemic area of liver fluke infection in Thailand.

PubMed

Saensa-Ard, Sunitta; Leuangwattanawanit, Saman; Senggunprai, Laddawan; Namwat, Nisana; Kongpetch, Sarinya; Chamgramol, Yaovalux; Loilome, Watcharin; Khansaard, Walaiporn; Jusakul, Apinya; Prawan, Auemduan; Pairojkul, Chawalit; Khantikeo, Narong; Yongvanit, Puangrat; Kukongviriyapan, Veerapol

2017-11-01

Cholangiocarcinoma is a rare type of cancer which is an increasingly discernible health threat. The disease is usually very difficult in diagnosis and various treatment modalities are typically not effective. Cholangiocarcinoma is a complex and very heterogeneous malignancy characterized by tumor location, different risk factors, molecular profiling, and prognosis. Cancer cell lines represent an important tool for investigation in various aspects of tumor biology and molecular therapeutics. We established two cell lines, KKU-452 and KKU-023, which were derived from patients residing in the endemic area of liver fluke infection in Thailand. Both of tumor tissues have gross pathology of perihilar and intrahepatic mass-forming cholangiocarcinoma. Two cell lines were characterized for their biological, molecular and genetic properties. KKU-452 and KKU-023 cells are both adherent cells with epithelium morphology, but have some differences in their growth pattern (a doubling time of 17.9 vs 34.8 h, respectively) and the expression of epithelial bile duct markers, CK7 and CK19. Cytogenetic analysis of KKU-452 and KKU-023 cells revealed their highly complex karyotypes; hypertriploid and hypotetraploid, respectively, with multiple chromosomal aberrations. Both cell lines showed mutations in p53 but not in KRAS. KKU-452 showed a very rapid migration and invasion properties in concert with low expression of E-cadherin and high expression of N-cadherin, whereas KKU-023 showed opposite characters. KKU-023, but not KKU-452, showed in vivo tumorigenicity in xenografted nude mice. Those two established cholangiocarcinoma cell lines with unique characters may be valuable for better understanding the process of carcinogenesis and developing new therapeutics for the patients.

Infection studies of nontarget mammalian cell lines with Bombyx mori macula-like virus.

PubMed

Innami, Katsuhisa; Aizawa, Takahiro; Tsukui, Toshihiro; Katsuma, Susumu; Imanishi, Shigeo; Kawasaki, Hideki; Iwanaga, Masashi

2016-03-01

Bombyx mori-derived cell lines are generally used for Bombyx mori nucleopolyhedrovirus (BmNPV)-based baculovirus expression vector system (BEVS). However, almost all of the B. mori-derived cell lines are persistently infected with Bombyx mori macula-like virus (BmMLV). In this study, nontarget mammalian cell lines were exposed to BmMLV, and their susceptibility was investigated. Real-time PCR showed that viral RNA in virus-inoculated nine mammalian cell lines decreased sharply at 7 days postinfection. Also, there was no significant effect on cell viability of mammalian cells after inoculation with BmMLV. These findings indicate that mammalian cell lines used in this study are not permissive to BmMLV, and BmMLV contamination might not affect the safety aspect of BmNPV-based BEVS. Copyright © 2015 Elsevier B.V. All rights reserved.

Variation of Keratin 7 Expression and Other Phenotypic Characteristics of Independent Isolates of Cadmium Transformed Human Urothelial Cells (UROtsa)

PubMed Central

Somji, Seema; Zhou, Xu Dong; Mehus, Aaron; Sens, Mary Ann; Garrett, Scott H.; Lutz, Krista L.; Dunlevy, Jane R.; Zheng, Yun; Sens, Donald. A.

2009-01-01

This laboratory has shown that a human urothelial cell line (UROtsa) transformed by cadmium (Cd+2) produced subcutaneous tumor heterotransplants that resemble human transitional cell carcinoma (TCC). In the present study, additional Cd+2 transformed cell lines were isolated to determine if independent exposures of the cell line to Cd+2 would result in malignantly transformed cell lines possessing similar phenotypic properties. Seven independent isolates were isolated and assessed for their doubling times, morphology, ability to heterotransplant subcutaneously and in the peritoneal cavity of nude mice and for the expression keratin 7. The 7 cell lines all displayed an epithelial morphology with no evidence of squamous differentiation. Doubling times were variable among the isolates, being significantly reduced or similar to the parental cells. All 7 isolates were able to form subcutaneous tumor heterotransplants with a TCC morphology and all heterotransplants displayed areas of squamous differentiation of the transitional cells. The degree of squamous differentiation varied among the isolates. In contrast to subcutaneous tumor formation, only 1 isolate of the Cd+2 transformed cells (UTCd#1) was able to effectively colonize multiple sites within the peritoneal cavity. An analysis of keratin 7 expression showed no correlation with squamous differentiation for the subcutaneous heterotransplants generated from the 7 cell lines. Keratin 7 was expressed in 6 of the 7 cell lines and their subcutaneous tumor heterotransplants. Keratin 7 was not expressed in the cell line that was able to form tumors within the peritoneal cavity. These results show that individual isolates of Cd+2 transformed cells have both similarities and differences in their phenotype. PMID:19921857

Characterization of cancer stem-like cells derived from a side population of a human gallbladder carcinoma cell line, SGC-996

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

Li, Xin-xing; Wang, Jian, E-mail: dr_wangjian@yahoo.com.cn; Wang, Hao-lu

2012-03-23

Highlights: Black-Right-Pointing-Pointer We sorted SP cells from a human gallbladder carcinoma cell lines, SGC-996. Black-Right-Pointing-Pointer SP cells displayed higher proliferation and stronger clonal-generating capability. Black-Right-Pointing-Pointer SP cells showed more migratory and invasive abilities. Black-Right-Pointing-Pointer SP cells were more resistant and tumorigenic than non-SP counterparts. Black-Right-Pointing-Pointer ABCG2 might be a candidate as a marker for SP cells. -- Abstract: The cancer stem cell (CSC) hypothesis proposes that CSCs, which can renew themselves proliferate infinitely, and escape chemotherapy, become the root of recurrence and metastasis. Previous studies have verified that side population (SP) cells, characterized by their ability to efflux lipophilic substratemore » Hoechst 33342, to share many characteristics of CSCs in multiplying solid tumors. The purpose of this study was to sort SP cells from a human gallbladder carcinoma cell line, SGC-996 and to preliminarily identify the biological characteristics of SP cells from the cell line. Using flow cytometry we effectively sorted SP cells from the cell line SGC-996. SP cells not only displayed higher proliferative, stronger clonal-generating, more migratory and more invasive capacities, but showed stronger resistance. Furthermore, our experiments demonstrated that SP cells were more tumorigenic than non-SP counterparts in vivo. Real-time PCR analysis and immunocytochemistry showed that the expression of ATP-binding cassette subfamily G member 2 (ABCG2) was significantly higher in SP cells. Hence, these results collectively suggest that SP cells are progenitor/stem-like cells and ABCG2 might be a candidate marker for SP cells in human gallbladder cancer.« less

Involvement of overexpressed wild-type BRAF in the growth of malignant melanoma cell lines.

PubMed

Tanami, Hideaki; Imoto, Issei; Hirasawa, Akira; Yuki, Yasuhiro; Sonoda, Itaru; Inoue, Jun; Yasui, Kohichiro; Misawa-Furihata, Akiko; Kawakami, Yutaka; Inazawa, Johji

2004-11-18

Comparative genomic hybridization (CGH) using 40 cell lines derived from malignant melanomas (MMs) revealed frequent amplification at 7q33-q34 containing BRAF gene, which often is mutated in MM. We found this gene to be amplified to a remarkable degree in the MM cell lines that exhibited high-level gains at 7q33-q34 in CGH. Among 40 cell lines, the eight lines that revealed neither BRAF nor NRAS mutations showed even higher levels of BRAF mRNA expression than the 32 mutated lines, although DNA amplification at 7q33-q34 was not detected in every lines overexpressing BRAF. MM cells that carried wild-type BRAF and NRAS showed constitutive overexpression of B-Raf protein and phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), even after serum starvation. Not only downregulation of the endogenously overexpressed wild-type B-Raf by antisense oligonucleotide but also a treatment with an inhibitor of mitogen-activated protein kinase kinase (MAPKK, MEK) reduced phosphorylated ERK1/2 and cell growth, whereas the exogenously expressed wild-type B-Raf promoted cell growth in MM cells. Our results provide the evidence that overexpression of wild-type B-Raf, in part but not always as a result of gene amplification, is one of the mechanisms underlying constitutive activation of the MAPK pathway that stimulates growth of MM cells.

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

PubMed

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

2012-10-12

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

Uveal Melanoma Cell Lines: Where do they come from? (An American Ophthalmological Society Thesis).

PubMed

Jager, Martine J; Magner, J Antonio Bermudez; Ksander, Bruce R; Dubovy, Sander R

2016-08-01

To determine whether some of the most often used uveal melanoma cell lines resemble their original tumor. Analysis of the literature, patient charts, histopathology, mutations, chromosome status, HLA type, and expression of melanocyte markers on cell lines and their primary tumors. We examined five cell lines and the primary tumors from which they were derived. Four of the five examined primary tumors were unusual: one occupied the orbit, two were recurrences after prior irradiation, and one developed in an eye with a nevus of Ota. One cell line did not contain the GNA11 mutation, but it was present in the primary tumor. Three of the primary tumors had monosomy 3 (two of these lacked BAP1 expression); however, all five cell lines showed disomy 3 and BAP1 expression. All of the cell lines had gain of 8q. Two cell lines lacked expression of melanocyte markers, although these were present in the corresponding primary tumor. All cell lines could be traced back to their original uveal melanoma. Four of the five primary tumors were unusual. Cell lines often differed from their primary tumor in chromosome status and melanocyte markers. However, their specific chromosome aberrations and capacity to continue proliferation characterize them as uveal melanoma cell lines.

Uveal Melanoma Cell Lines: Where do they come from? (An American Ophthalmological Society Thesis)

PubMed Central

Jager, Martine J.; Magner, J. Antonio Bermudez; Ksander, Bruce R.; Dubovy, Sander R.

2016-01-01

Purpose To determine whether some of the most often used uveal melanoma cell lines resemble their original tumor. Methods Analysis of the literature, patient charts, histopathology, mutations, chromosome status, HLA type, and expression of melanocyte markers on cell lines and their primary tumors. We examined five cell lines and the primary tumors from which they were derived. Results Four of the five examined primary tumors were unusual: one occupied the orbit, two were recurrences after prior irradiation, and one developed in an eye with a nevus of Ota. One cell line did not contain the GNA11 mutation, but it was present in the primary tumor. Three of the primary tumors had monosomy 3 (two of these lacked BAP1 expression); however, all five cell lines showed disomy 3 and BAP1 expression. All of the cell lines had gain of 8q. Two cell lines lacked expression of melanocyte markers, although these were present in the corresponding primary tumor. Conclusions All cell lines could be traced back to their original uveal melanoma. Four of the five primary tumors were unusual. Cell lines often differed from their primary tumor in chromosome status and melanocyte markers. However, their specific chromosome aberrations and capacity to continue proliferation characterize them as uveal melanoma cell lines. PMID:28018010

[Study on garlic oil combined with 5-FU induced apoptosis of adenoid cystic carcinoma cell line ACC-M].

PubMed

Wu, Fayin; Zhou, Hefeng; Fan, Zhiying; Zhu, Yawen; Li, Yongye; Yao, Yukun; Ran, Dan

2014-02-01

To observe the effect of garlic oil combined with 5-FU induced apoptosis of adenoid cystic carcinoma cell line ACC-M. Human salivary in adenoid cystic carcinoma cell line AC-M was cultured, divided into the experimental group (5-FU group, garlic oil group, garlic oil + 5-FU group) and the control group, to observe the growth activity of tumor cells by MTT methods; to analyse the changes of cell cycle and apoptosis rate by flow cytometry. MTT experiments showed that 5-FU, garlic oil, garlic oil and 5-FU on ACC-M cells have inhibition in different concentration, with the increase of concentration and action time of the rise; Cell cycle analysis showed significant changes in flow cytometry. With the increase of concentration and the acting time, the G0/G1, phase of the cell ratio increased, S had no significant change, but G2/M phase cells decreased. Apoptosis rate display showed garlic oil combined with 5-FU induced apoptosis of ACC-M cells was significantly stronger than single group. Garlic oil can effectively induce the apoptosis of adenoid cystic carcinoma cell line ACC-M. The effect of garlic oil combined with 5-FU on ACC-M cells was stronger than the garlic oil, 5-FU used alone.

Establishment of dermal sheath cell line from Cashmere goat and characterizing cytokeratin 13 as its novel biomarker.

PubMed

Zhu, Bing; Guo, Zhili; Jin, Muzi; Bai, Yujuan; Yang, Wenliang; Hui, Lihua

2018-05-01

To establish a dermal sheath cell line, a dermal papilla cell line and a outer root sheath cell line from Cashmere goat and clarify the similarities and differences among them. We established a dermal sheath cell line, a dermal papilla cell line and a outer root sheath cell line from the pelage skin hair follicles of Cashmere goat. The growth rate of dermal sheath cells was intermediate between that of dermal papilla cells and outer root sheath cells. Immunofluorescence experiments and reverse transcription-polymerase chain reaction analysis showed that at both the transcriptional and translational levels, the dermal sheath cells were alpha-smooth muscle actin (α-SMA) + /cytokeratin 13 + , while the dermal papilla cells were α-SMA + /cytokeratin 13 - and the outer root sheath cells were α-SMA - /cytokeratin 13 + . Patterns of cytokeratin 13 expression could distinguish the dermal sheath cells from the dermal papilla cells. These results suggest that cytokeratin 13 could serve as a novel biomarker for dermal sheath cells of Cashmere goat, and should prove useful for researchers investigating dermal stem cells or interaction of different types of cells during hair cycle.

Comparative label-free LC-MS/MS analysis of colorectal adenocarcinoma and metastatic cells treated with 5-fluorouracil.

PubMed

Bauer, Kerry M; Lambert, Paul A; Hummon, Amanda B

2012-06-01

A label-free mass spectrometric strategy was used to examine the effect of 5-fluorouracil (5-FU) on the primary and metastatic colon carcinoma cell lines, SW480 and SW620, with and without treatment. 5-FU is the most common chemotherapeutic treatment for colon cancer. Pooled biological replicates were analyzed by nanoLC-MS/MS and protein quantification was determined via spectral counting. Phenotypic and proteomic changes were evident and often similar in both cell lines. The SW620 cells were more resistant to 5-FU treatment, with an IC(50) 2.7-fold higher than that for SW480. In addition, both cell lines showed pronounced abundance changes in pathways relating to antioxidative stress response and cell adhesion remodeling due to 5-FU treatment. For example, the detoxification enzyme NQO1 was increased with treatment in both cell lines, while disparate members of the peroxiredoxin family, PRDX2 or PRDX5 and PRDX6, were elevated with 5-FU exposure in either SW480 or SW620, respectively. Cell adhesion-associated proteins CTNNB1 and RhoA showed decreased expression with 5-FU treatment in both cell lines. The differential quantitative response in the proteomes of these patient-matched cell lines to drug treatment underscores the subtle molecular differences separating primary and metastatic cancer cells. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

Gopalan, Vinod; Smith, Robert A.; Lam, Alfred K.-Y., E-mail: a.lam@griffith.edu.au

miR-498 is a non-coding RNA located intergenically in 19q13.41. Due to its predicted targeting of several genes involved in control of cellular growth, we examined the expression of miR-498 in colon cancer cell lines and a large cohort of patients with colorectal adenocarcinoma. Two colon cancer cancer cell lines (SW480 and SW48) and one normal colonic epithelial cell line (FHC) were recruited. The expression of miR-498 was tested in these cell lines by using quantitative real-time polymerase chain reaction (qRT-PCR). Tissues from 80 patients with surgical resection of colorectum (60 adenocarcinomas and 20 non-neoplastic tissues) were tested for miR-498 expressionmore » by qRT-PCR. In addition, an exogenous miR-498 (mimic) was used to detect the miRNA's effects on cell proliferation and cell cycle events in SW480 using MTT calorimetric assay and flow cytometry respectively. The colon cancer cell lines showed reduced expression of miR-498 compared to a normal colonic epithelial cell line. Mimic driven over expression of miR-498 in the SW480 cell line resulted in reduced cell proliferation and increased proportions of G2-M phase cells. In tissues, miR-498 expression was too low to be detected in all colorectal adenocarcinoma compared to non-neoplastic tissues. This suggests that the down regulation of miR-498 in colorectal cancer tissues and the direct suppressive cellular effect noted in cancer cell lines implies that miR-498 has some direct or indirect role in the pathogenesis of colorectal adenocarcinomas. - Highlights: • miR-498 is a non-coding RNA located in 19q13.41. • Colon cancer cell lines showed reduced expression of miR-498. • Mimic driven over expression of miR-498 in colon cancer cells resulted in lower cell proliferation. • miR-498 expression was down regulated in all colorectal adenocarcinoma tissues.« less

[Establishment of fibroblast cell line and its biological characteristics in Matou goat].

PubMed

Li, Tianda; Liu, Chousheng; Wang, Zhigang; Zhang, Liping; Sun, Xiuzhu; Zhao, Junjin; Meng, Fei; Luo, Guihe; Zhu, Jinqing

2008-12-01

Taking Matou goat ear margin as the study material, we succeeded in established a fibroblast cell line by the method of explant culture directly. Observations on morphology, dynamic growth, determination of viability, analysis of karyotype, test of microorganism and other characteristics were detected. Results showed: Population Doubling Time (PDT) of cells was approximately 36 h; Cell viability was 96.7% after thawing; The status of cell After passage was constant; Analysis of chromosomal karyotyps indicated that diploid (2n=60) account for 98% in the cell line. Every index in the cell line met all the standard quality controls of ATCC in USA. The established of Matou goat ear fibroblast cell line has not only important genetic resources preserved at the cell level, but also valuable material for genome, postgenome and somatic cell nuclear transfer research.

Use of deferasirox, an iron chelator, to overcome imatinib resistance of chronic myeloid leukemia cells.

PubMed

Kim, Dae Sik; Na, Yoo Jin; Kang, Myoung Hee; Yoon, Soo-Young; Choi, Chul Won

2016-03-01

The treatment of chronic myeloid leukemia (CML) has achieved impressive success since the development of the Bcr-Abl tyrosine kinase inhibitor, imatinib mesylate. Nevertheless, resistance to imatinib has been observed, and a substantial number of patients need alternative treatment strategies. We have evaluated the effects of deferasirox, an orally active iron chelator, and imatinib on K562 and KU812 human CML cell lines. Imatinib-resistant CML cell lines were created by exposing cells to gradually increasing concentrations of imatinib. Co-treatment of cells with deferasirox and imatinib induced a synergistic dose-dependent inhibition of proliferation of both CML cell lines. Cell cycle analysis showed an accumulation of cells in the subG1 phase. Western blot analysis of apoptotic proteins showed that co-treatment with deferasirox and imatinib induced an increased expression of apoptotic proteins. These tendencies were clearly identified in imatinib-resistant CML cell lines. The results also showed that co-treatment with deferasirox and imatinib reduced the expression of BcrAbl, phosphorylated Bcr-Abl, nuclear factor-κB (NF-κB) and β-catenin. We observed synergistic effects of deferasirox and imatinib on both imatinib-resistant and imatinib-sensitive cell lines. These effects were due to induction of apoptosis and cell cycle arrest by down-regulated expression of NF-κB and β-catenin levels. Based on these results, we suggest that a combination treatment of deferasirox and imatinib could be considered as an alternative treatment option for imatinib-resistant CML.

Experimental infection of Leishmania (L.) chagasi in a cell line derived from Lutzomyia longipalpis (Diptera:Psychodidae).

PubMed

Bello, Felio J; Mejía, Astrid J; Corena, María del Pilar; Ayala, Martha; Sarmiento, Ladys; Zuñiga, Claudio; Palau, María T

2005-10-01

The present work describes the in vitro infection of a cell line Lulo, derived from Lutzomyia longipalpis embryonic tissue, by Leishmania chagasi promastigotes. This infection process is compared with a parallel one developed using the J774 cell line. The L. chagasi MH/CO/84/CI-044B strain was used for experimental infection in two cell lines. The cells were seeded on glass coverslips in 24-well plates to reach a final number of 2 x 10(5) cells/well. Parasites were added to the adhered Lulo and J774 cells in a 10:1 ratio and were incubated at 28 and 37 masculineC respectively. After 2, 4, 6, 8, and 10 days post-infection, the cells were extensively washed with PBS, fixed with methanol, and stained with Giemsa. The number of internalized parasites was determined by counting at least 400 cultured cells on each coverslip. The results showed continuous interaction between L. chagasi promastigotes with the cell lines. Some ultrastructural characteristics of the amastigote forms were observed using transmission electron microscopy. The highest percentage of infection in Lulo cells was registered on day 6 post-infection (29.6%) and on day 4 in the J774 cells (51%). This work shows similarities and differences in the L. chagasi experimental infection process in the two cell lines. However, Lulo cells emerge as a new model to study the life-cycle of this parasite.

Tailoring Mathematical Models to Stem-Cell Derived Cardiomyocyte Lines Can Improve Predictions of Drug-Induced Changes to Their Electrophysiology.

PubMed

Lei, Chon Lok; Wang, Ken; Clerx, Michael; Johnstone, Ross H; Hortigon-Vinagre, Maria P; Zamora, Victor; Allan, Andrew; Smith, Godfrey L; Gavaghan, David J; Mirams, Gary R; Polonchuk, Liudmila

2017-01-01

Human induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) have applications in disease modeling, cell therapy, drug screening and personalized medicine. Computational models can be used to interpret experimental findings in iPSC-CMs, provide mechanistic insights, and translate these findings to adult cardiomyocyte (CM) electrophysiology. However, different cell lines display different expression of ion channels, pumps and receptors, and show differences in electrophysiology. In this exploratory study, we use a mathematical model based on iPSC-CMs from Cellular Dynamic International (CDI, iCell), and compare its predictions to novel experimental recordings made with the Axiogenesis Cor.4U line. We show that tailoring this model to the specific cell line, even using limited data and a relatively simple approach, leads to improved predictions of baseline behavior and response to drugs. This demonstrates the need and the feasibility to tailor models to individual cell lines, although a more refined approach will be needed to characterize individual currents, address differences in ion current kinetics, and further improve these results.

Dehydroleucodine, a Sesquiterpene Lactone from Gynoxys verrucosa, Demonstrates Cytotoxic Activity against Human Leukemia Cells.

PubMed

Ordóñez, Paola E; Sharma, Krishan K; Bystrom, Laura M; Alas, Maria A; Enriquez, Raul G; Malagón, Omar; Jones, Darin E; Guzman, Monica L; Compadre, Cesar M

2016-04-22

The sesquiterpene lactones dehydroleucodine (1) and leucodine (2) were isolated from Gynoxys verrucosa, a species used in traditional medicine in southern Ecuador. The activity of these compounds was determined against eight acute myeloid leukemia (AML) cell lines and compared with their activity against normal peripheral blood mononuclear cells. Compound 1 showed cytotoxic activity against the tested cell lines, with LD50 values between 5.0 and 18.9 μM. Compound 2 was inactive against all of the tested cell lines, demonstrating that the exocyclic methylene in the lactone ring is required for cytotoxic activity. Importantly, compound 1 induced less toxicity to normal blood cells than to AML cell lines and was active against human AML cell samples from five patients, with an average LD50 of 9.4 μM. Mechanistic assays suggest that compound 1 has a similar mechanism of action to parthenolide (3). Although these compounds have significant structural differences, their lipophilic surface signatures show striking similarities.

Molluscan cells in culture: primary cell cultures and cell lines

PubMed Central

Yoshino, T. P.; Bickham, U.; Bayne, C. J.

2013-01-01

In vitro cell culture systems from molluscs have significantly contributed to our basic understanding of complex physiological processes occurring within or between tissue-specific cells, yielding information unattainable using intact animal models. In vitro cultures of neuronal cells from gastropods show how simplified cell models can inform our understanding of complex networks in intact organisms. Primary cell cultures from marine and freshwater bivalve and gastropod species are used as biomonitors for environmental contaminants, as models for gene transfer technologies, and for studies of innate immunity and neoplastic disease. Despite efforts to isolate proliferative cell lines from molluscs, the snail Biomphalaria glabrata Say, 1818 embryonic (Bge) cell line is the only existing cell line originating from any molluscan species. Taking an organ systems approach, this review summarizes efforts to establish molluscan cell cultures and describes the varied applications of primary cell cultures in research. Because of the unique status of the Bge cell line, an account is presented of the establishment of this cell line, and of how these cells have contributed to our understanding of snail host-parasite interactions. Finally, we detail the difficulties commonly encountered in efforts to establish cell lines from molluscs and discuss how these difficulties might be overcome. PMID:24198436

The neuroendocrine phenotype, genomic profile and therapeutic sensitivity of GEPNET cell lines

PubMed Central

Hofving, Tobias; Arvidsson, Yvonne; Almobarak, Bilal; Inge, Linda; Pfragner, Roswitha; Persson, Marta; Stenman, Göran; Kristiansson, Erik; Johanson, Viktor; Nilsson, Ola

2018-01-01

Experimental models of neuroendocrine tumour disease are scarce, and no comprehensive characterisation of existing gastroenteropancreatic neuroendocrine tumour (GEPNET) cell lines has been reported. In this study, we aimed to define the molecular characteristics and therapeutic sensitivity of these cell lines. We therefore performed immunophenotyping, copy number profiling, whole-exome sequencing and a large-scale inhibitor screening of seven GEPNET cell lines. Four cell lines, GOT1, P-STS, BON-1 and QGP-1, displayed a neuroendocrine phenotype while three others, KRJ-I, L-STS and H-STS, did not. Instead, these three cell lines were identified as lymphoblastoid. Characterisation of remaining authentic GEPNET cell lines by copy number profiling showed that GOT1, among other chromosomal alterations, harboured losses on chromosome 18 encompassing the SMAD4 gene, while P-STS had a loss on 11q. BON-1 had a homozygous loss of CDKN2A and CDKN2B, and QGP-1 harboured amplifications of MDM2 and HMGA2. Whole-exome sequencing revealed both disease-characteristic mutations (e.g. ATRX mutation in QGP-1) and, for patient tumours, rare genetic events (e.g. TP53 mutation in P-STS, BON-1 and QGP-1). A large-scale inhibitor screening showed that cell lines from pancreatic NETs to a greater extent, when compared to small intestinal NETs, were sensitive to inhibitors of MEK. Similarly, neuroendocrine NET cells originating from the small intestine were considerably more sensitive to a group of HDAC inhibitors. Taken together, our results provide a comprehensive characterisation of GEPNET cell lines, demonstrate their relevance as neuroendocrine tumour models and explore their therapeutic sensitivity to a broad range of inhibitors. PMID:29444910

Establishment and characterization of a human uterine endometrial undifferentiated carcinoma cell line, TMG-L.

PubMed

Hasegawa, Kiyoshi; Suzuki, Machiko; Ishikawa, Kunimi; Yasue, Akira; Kato, Rina; Nakamura, Azumi; Kuroki, Jun; Udagawa, Yasuhiro

2003-03-01

A new cell line of human uterine endometrial undifferentiated carcinoma, designated as TMG-L, was established from the metastatic lymph node of 56-year-old patient TMG-L cells have been cultured with Ham's F-12 medium supplemented with 10% FCS and grew as a loosely adherent monolayer with polygonal or spindle-shaped cells exhibiting poor cell-cell contact and piled up against each other, showing a tendency to grow as floating cells. The doubling time of this cell line was about 48 hours, and chromosomal analysis revealed aneuploidy at passage 25. The cells formed tumors in SCID mouse, the histology of which was similar to that of undifferentiated carcinoma component of primary tumor. TMG-L cells showed the loss of expression and membranous localization of either E-cadherin or alpha-catenin, implied corresponding loss of their adhesive function. And this dysfunction implicated the biological aggressive behavior of uterine endometrial undifferentiated carcinoma. This cell line appears to provide a useful system for studying uterine undifferentiated carcinoma in vivo and in vitro.

The establishment and characterization of the first canine hepatocellular carcinoma cell line, which resembles human oncogenic expression patterns

PubMed Central

Boomkens, Sacha Y; Spee, Bart; IJzer, Jooske; Kisjes, Ronald; Egberink, Herman F; van den Ingh, Ted SGAM; Rothuizen, Jan; Penning, Louis C

2004-01-01

Background Hepatocellular carcinoma (HCC) is one of the most worldwide frequent primary carcinomas resulting in the death of many cirrhotic patients. Unfortunately, the molecular mechanisms of this cancer are not well understood; therefore, we need a good model system to study HCC. The dog is recognized as a promising model for human medical research, namely compared with rodents. The objective of this study was to establish and characterize a spontaneous canine tumor cell line as a potential model for studies on HCC. Results Histomorphological, biochemical, molecular biological and quantitative assays were performed to characterize the canine HCC cell line that originated from a dog with a spontaneous liver tumor. Morphological investigations provided strong evidence for the hepatocytic and neoplastic nature of the cell line, while biochemical assays showed that they produced liver-specific enzymes. PCR analysis confirmed expression of ceruloplasmin, alpha-fetoprotein and serum albumin. Quantitative RT-PCR showed that the canine HCC cell line resembles human HCC based on the measurements of expression profiles of genes involved in cell proliferation and apoptosis. Conclusions We have developed a novel, spontaneous tumor liver cell line of canine origin that has many characteristics of human HCC. Therefore, the canine HCC cell line might be an excellent model for comparative studies on the molecular pathogenesis of HCC. PMID:15566568

Cytotoxic and Antimicrobial Activity of Dehydrozingerone based Cyclopropyl Derivatives.

PubMed

Burmudžija, Adrijana Z; Muškinja, Jovana M; Kosanić, Marijana M; Ranković, Branislav R; Novaković, Slađana B; Đorđević, Snežana B; Stanojković, Tatjana P; Baskić, Dejan D; Ratković, Zoran R

2017-08-01

A small series of 1-acetyl-2-(4-alkoxy-3-methoxyphenyl)cyclopropanes was prepared, starting from dehydrozingerone (4-(4-hydroxy-3-methoxyphenyl)-3-buten-2-one) and its O-alkyl derivatives. Their microbiological activities toward some strains of bacteria and fungi were tested, as well as their in vitro cytotoxic activity against some cancer cell lines (HeLa, LS174 and A549). All synthesized compounds showed significant antimicrobial activity and expressed cytotoxic activity against tested carcinoma cell lines, but they showed no significant influence on normal cell line (MRC5). Butyl derivative is the most active on HeLa cells (IC 50 = 8.63 μm), while benzyl one is active against LS174 and A549 cell lines (IC 50 = 10.17 and 12.15 μm, respectively). © 2017 Wiley-VHCA AG, Zurich, Switzerland.

Evaluation of the drug sensitivity and expression of 16 drug resistance-related genes in canine histiocytic sarcoma cell lines

PubMed Central

ASADA, Hajime; TOMIYASU, Hirotaka; GOTO-KOSHINO, Yuko; FUJINO, Yasuhito; OHNO, Koichi; TSUJIMOTO, Hajime

2015-01-01

Canine histiocytic sarcoma (HS) is an aggressive tumor type originating from histiocytic cell lineages. This disease is characterized by poor response to chemotherapy and short survival time. Therefore, it is of critical importance to identify and develop effective antitumor drugs against HS. The objectives of this study were to examine the drug sensitivities of 10 antitumor drugs. Using a real-time RT-PCR system, the mRNA expression levels of 16 genes related to drug resistance in 4 canine HS cell lines established from dogs with disseminated HS were determined and compared to 2 canine lymphoma cell lines (B-cell and T-cell). These 4 canine HS cell lines showed sensitivities toward microtubule inhibitors (vincristine, vinblastine and paclitaxel), comparable to those in the canine B-cell lymphoma cell line. Moreover, it was shown that P-gp in the HS cell lines used in this study did not have enough function to efflux its substrate. Sensitivities to melphalan, nimustine, methotrexate, cytarabine, doxorubicin and etoposide were lower in the 4 HS cell lines than in the 2 canine lymphoma cell lines. The data obtained in this study using cultured cell lines could prove helpful in the developing of advanced and effective chemotherapies for treating dogs that are suffering from HS. PMID:25715778

Evaluation of the drug sensitivity and expression of 16 drug resistance-related genes in canine histiocytic sarcoma cell lines.

PubMed

Asada, Hajime; Tomiyasu, Hirotaka; Goto-Koshino, Yuko; Fujino, Yasuhito; Ohno, Koichi; Tsujimoto, Hajime

2015-06-01

Canine histiocytic sarcoma (HS) is an aggressive tumor type originating from histiocytic cell lineages. This disease is characterized by poor response to chemotherapy and short survival time. Therefore, it is of critical importance to identify and develop effective antitumor drugs against HS. The objectives of this study were to examine the drug sensitivities of 10 antitumor drugs. Using a real-time RT-PCR system, the mRNA expression levels of 16 genes related to drug resistance in 4 canine HS cell lines established from dogs with disseminated HS were determined and compared to 2 canine lymphoma cell lines (B-cell and T-cell). These 4 canine HS cell lines showed sensitivities toward microtubule inhibitors (vincristine, vinblastine and paclitaxel), comparable to those in the canine B-cell lymphoma cell line. Moreover, it was shown that P-gp in the HS cell lines used in this study did not have enough function to efflux its substrate. Sensitivities to melphalan, nimustine, methotrexate, cytarabine, doxorubicin and etoposide were lower in the 4 HS cell lines than in the 2 canine lymphoma cell lines. The data obtained in this study using cultured cell lines could prove helpful in the developing of advanced and effective chemotherapies for treating dogs that are suffering from HS.

Authentication of Primordial Characteristics of the CLBL-1 Cell Line Prove the Integrity of a Canine B-Cell Lymphoma in a Murine In Vivo Model

PubMed Central

Reimann-Berg, Nicola; Walter, Ingrid; Fuchs-Baumgartinger, Andrea; Wagner, Siegfried; Kovacic, Boris; Essler, Sabine E.; Schwendenwein, Ilse; Nolte, Ingo; Saalmüller, Armin; Escobar, Hugo Murua

2012-01-01

Cell lines are key tools in cancer research allowing the generation of neoplasias in animal models resembling the initial tumours able to mimic the original neoplasias closely in vivo. Canine lymphoma is the major hematopoietic malignancy in dogs and considered as a valuable spontaneous large animal model for human Non-Hodgkin's Lymphoma (NHL). Herein we describe the establishment and characterisation of an in vivo model using the canine B-cell lymphoma cell line CLBL-1 analysing the stability of the induced tumours and the ability to resemble the original material. CLBL-1 was injected into Rag2−/−γc −/− mice. The generated tumor material was analysed by immunophenotyping and histopathology and used to establish the cell line CLBL-1M. Both cell lines were karyotyped for detection of chromosomal aberrations. Additionally, CLBL-1 was stimulated with IL-2 and DSP30 as described for primary canine B-cell lymphomas and NHL to examine the stimulatory effect on cell proliferation. CLBL-1 in vivo application resulted in lymphoma-like disease and tumor formation. Immunophenotypic analysis of tumorous material showed expression of CD45+, MHCII+, CD11a+ and CD79αcy+. PARR analysis showed positivity for IgH indicating a monoclonal character. These cytogenetic, molecular, immunophenotypical and histological characterisations of the in vivo model reveal that the induced tumours and thereof generated cell line resemble closely the original material. After DSP30 and IL-2 stimulation, CLBL-1 showed to respond in the same way as primary material. The herein described CLBL-1 in vivo model provides a highly stable tool for B-cell lymphoma research in veterinary and human medicine allowing various further in vivo studies. PMID:22761949

Authentication of primordial characteristics of the CLBL-1 cell line prove the integrity of a canine B-cell lymphoma in a murine in vivo model.

PubMed

Rütgen, Barbara C; Willenbrock, Saskia; Reimann-Berg, Nicola; Walter, Ingrid; Fuchs-Baumgartinger, Andrea; Wagner, Siegfried; Kovacic, Boris; Essler, Sabine E; Schwendenwein, Ilse; Nolte, Ingo; Saalmüller, Armin; Murua Escobar, Hugo

2012-01-01

Cell lines are key tools in cancer research allowing the generation of neoplasias in animal models resembling the initial tumours able to mimic the original neoplasias closely in vivo. Canine lymphoma is the major hematopoietic malignancy in dogs and considered as a valuable spontaneous large animal model for human Non-Hodgkin's Lymphoma (NHL). Herein we describe the establishment and characterisation of an in vivo model using the canine B-cell lymphoma cell line CLBL-1 analysing the stability of the induced tumours and the ability to resemble the original material. CLBL-1 was injected into Rag2(-/-)γ(c) (-/-) mice. The generated tumor material was analysed by immunophenotyping and histopathology and used to establish the cell line CLBL-1M. Both cell lines were karyotyped for detection of chromosomal aberrations. Additionally, CLBL-1 was stimulated with IL-2 and DSP30 as described for primary canine B-cell lymphomas and NHL to examine the stimulatory effect on cell proliferation. CLBL-1 in vivo application resulted in lymphoma-like disease and tumor formation. Immunophenotypic analysis of tumorous material showed expression of CD45(+), MHCII(+), CD11a(+) and CD79αcy(+). PARR analysis showed positivity for IgH indicating a monoclonal character. These cytogenetic, molecular, immunophenotypical and histological characterisations of the in vivo model reveal that the induced tumours and thereof generated cell line resemble closely the original material. After DSP30 and IL-2 stimulation, CLBL-1 showed to respond in the same way as primary material. The herein described CLBL-1 in vivo model provides a highly stable tool for B-cell lymphoma research in veterinary and human medicine allowing various further in vivo studies.

Expression of MUC1, MUC2, MUC3 and MUC4 mucin mRNAs in human pancreatic and intestinal tumor cell lines.

PubMed

Hollingsworth, M A; Strawhecker, J M; Caffrey, T C; Mack, D R

1994-04-15

We examined the steady-state expression levels of mRNA for the MUC1, MUC2, MUC3 and MUC4 gene products in 12 pancreatic tumor cell lines, 6 colon tumor cell lines, and one ileocecal tumor cell line. The results showed that 10 of 12 pancreatic tumor cell lines expressed MUC1 mRNA and that 7 of these 12 lines also expressed relatively high levels of MUC4 mRNA. In contrast, MUC2 mRNA was expressed at only low levels and MUC3 was not detected in the pancreatic tumor cell lines. All 7 intestinal tumor cell lines examined expressed MUC2, and 5 of 7 expressed MUC3; however only one expressed significant levels of MUC1 and 2 expressed low levels of MUC4 mRNA. This report of high levels of MUC4 mRNA expression by pancreatic tumor cells raises the possibility that mucin carbohydrate epitopes defined by antibodies such as DuPan 2 may be expressed on a second mucin core protein produced by pancreatic tumor cells.

In vitro culture of human osteosarcoma cell lines: a comparison of functional characteristics for cell lines cultured in medium without and with fetal calf serum.

PubMed

Bruserud, Oystein; Tronstad, Karl Johan; Berge, Rolf

2005-06-01

Experimental in vitro models including well-characterised cell lines can be used to identify possible new therapeutic targets for the treatment of osteosarcoma. Culture media including inactivated serum is often recommended for in vitro culture of osteosarcoma cells, but the serum component then represents a nonstandardised parameter including a wide range of unidentified mediators. To improve the standardisation we have investigated whether serum-free culture media can be used in experimental in vitro studies of osteosarcoma cell lines. The seven osteosarcoma cell lines Cal72, SJSA-1, Saos-2, SK-ES-1, U2OS, 143.98.2, and KHOS-32IH were cultured in vitro in various serum-free media and media supplemented with 10% heat-inactivated fetal calf serum (FCS). Although proliferation often was relatively low in serum-free media (X-vivo 10, X-vivo 15, X-vivo 20, Stem Span SFEM), some cell lines (Cal72, KHOS-32IH, Saos-2) showed proliferation comparable with the recommended FCS-containing media even when using serum-free conditions. The optimal serum-free medium then varied between cell lines. We also compared 6 different FCS-containing media (including Stem Span with 10% FCS) and the optimal FCS-containing medium varied between cell lines. However, all cell lines proliferated well in Stem Span with FCS, and this medium was regarded as optimal for four of the lines. FCS could not be replaced by fatty acids or low density lipoprotein when testing the Stem Span medium. The release of a wide range of soluble mediators showed only minor differences when using serum-free and FCS-containing media (including Stem Span with and without FCS), and serum-free Stem Span could also be used for in vitro studies of mitogen-stimulated T cell activation in the presence of accessory osteosarcoma cells. The use of Stem Span with 10% FCS allowed the release of a wide range of chemokines by osteosarcoma cell lines (Cal72, SJSA-1), and the chemokine release profile was very similar to the fibroblast lines Hs27 and HFL1. Serum-free culture media can be used for in vitro studies of several osteosarcoma cell lines, but the optimal medium varies between cell lines and thus depends on: (i) the cell lines to be investigated/compared; (ii) the functional characteristic that is evaluated (proliferation, cytokine release); and (iii) whether coculture experiments are included.

Synergistic Effect of Curcumin in Combination with Anticancer Agents in Human Retinoblastoma Cancer Cell Lines.

PubMed

Sreenivasan, Seethalakshmi; Krishnakumar, Subramanian

2015-01-01

Curcumin (diferuloylmethane), a phenolic compound obtained from the rhizome of the herb Curcuma longa, is known to have anti-proliferative and anti-tumor properties. In this study, we evaluated the cytotoxic effect of curcumin alone and in combination with individual drugs like carboplatin, etoposide, or vincristine in a human retinoblastoma (RB) cancer cell line. A drug-drug interaction was analyzed using the median effect/isobologram method and combination index values were used to characterize the interaction as synergistic or additive. We also performed the apoptosis and cell-cycle kinetics study with single drugs in combination with curcumin in a human RB cell lines (Y79 and Weri-Rb1). Curcumin caused concentration-dependent decrease in cell proliferation, cell kinetics, and also induced apoptosis in both the RB cell lines. When combination of curcumin with individual drugs like carboplatin or etoposide or vincristine was treated on to RB cells, both cell viability and cell cycling were reduced and increased apoptosis was noted, in comparison with single drug treatment. These effects were significant in both the cell lines, indicating the ability of curcumin to increase the sensitivity of RB cells to chemotherapy drugs. Our in vitro findings showed that the combination of curcumin with single drug treatment showed marked synergistic inhibitory effect against RB cell lines. These results suggest that curcumin can be used as a modulator which may have a potential therapeutic value for the treatment of RB cancer patients.

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.

Anticancer activity of Cynodon dactylon and Oxalis corniculata on Hep2 cell line.

PubMed

Salahuddin, H; Mansoor, Q; Batool, R; Farooqi, A A; Mahmood, T; Ismail, M

2016-04-30

Bioactive chemicals isolated from plants have attracted considerable attention over the years and overwhelmingly increasing laboratory findings are emphasizing on tumor suppressing properties of these natural agents in genetically and chemically induced animal carcinogenesis models. We studied in vitro anticancer activity of organic extracts of Cynodon dactylon and Oxalis corniculata on Hep2 cell line and it was compared with normal human corneal epithelial cells (HCEC) by using MTT assay. Real Time PCR was conducted for p53 and PTEN genes in treated cancer cell line. DNA fragmentation assay was also carried out to note DNA damaging effects of the extracts. The minimally effective concentration of ethanolic extract of Cynodon dactylon and methanolic extract of Oxalis corniculata that was nontoxic to HCEC but toxic to Hep2 was recorded (IC50) at a concentration of 0.042mg/ml (49.48 % cell death) and 0.048mg/ml (47.93% cell death) respectively, which was comparable to the positive control. Our results indicated dose dependent increase in cell death. P53 and PTEN did not show significant increase in treated cell line. Moreover, DNA damaging effects were also not detected in treated cancer cell line. Anticancer activity of these plants on the cancer cell line showed the presence of anticancer components which should be characterized to be used as anticancer therapy.

A distinct group of CpG islands shows differential DNA methylation between replicas of the same cell line in vitro

PubMed Central

2013-01-01

Background CpG dinucleotide-rich genomic DNA regions, known as CpG islands (CGIs), can be methylated at their cytosine residues as an epigenetic mark that is stably inherited during cell mitosis. Differentially methylated regions (DMRs) are genomic regions showing different degrees of DNA methylation in multiple samples. In this study, we focused our attention on CGIs showing different DNA methylation between two culture replicas of the same cell line. Results We used methylation data of 35 cell lines from the Encyclopedia of DNA Elements (ENCODE) consortium to identify CpG islands that were differentially methylated between replicas of the same cell line and denoted them Inter Replicas Differentially Methylated CpG islands (IRDM-CGIs). We identified a group of IRDM-CGIs that was consistently shared by different cell lines, and denoted it common IRDM-CGIs. X chromosome CGIs were overrepresented among common IRDM-CGIs. Autosomal IRDM-CGIs were preferentially located in gene bodies and intergenic regions had a lower G + C content, a smaller mean length, and a reduced CpG percentage. Functional analysis of the genes associated with autosomal IRDM-CGIs showed that many of them are involved in DNA binding and development. Conclusions Our results show that several specific functional and structural features characterize common IRDM-CGIs. They may represent a specific subset of CGIs that are more prone to being differentially methylated for their intrinsic characteristics. PMID:24106769

Ex vivo expanded natural killer cells from breast cancer patients and healthy donors are highly cytotoxic against breast cancer cell lines and patient-derived tumours.

PubMed

Shenouda, Mira M; Gillgrass, Amy; Nham, Tina; Hogg, Richard; Lee, Amanda J; Chew, Marianne V; Shafaei, Mahsa; Aarts, Craig; Lee, Dean A; Hassell, John; Bane, Anita; Dhesy-Thind, Sukhbinder; Ashkar, Ali A

2017-07-01

Natural killer (NK) cells play a critical role in cancer immunosurveillance. Recent developments in NK cell ex-vivo expansion makes it possible to generate millions of activated NK cells from a small volume of peripheral blood. We tested the functionality of ex vivo expanded NK cells in vitro against breast cancer cell lines and in vivo using a xenograft mouse model. The study aim was to assess functionality and phenotype of expanded NK cells from breast cancer patients against breast cancer cell lines and autologous primary tumours. We used a well-established NK cell co-culture system to expand NK cells ex vivo from healthy donors and breast cancer patients and examined their surface marker expression. Moreover, we tested the ability of expanded NK cells to lyse the triple negative breast cancer and HER2-positive breast cancer cell lines MDA-MB-231 and MDA-MB-453, respectively. We also tested their ability to prevent tumour growth in vivo using a xenograft mouse model. Finally, we tested the cytotoxicity of expanded NK cells against autologous and allogeneic primary breast cancer tumours in vitro. After 3 weeks of culture we observed over 1000-fold expansion of NK cells isolated from either breast cancer patients or healthy donors. We also showed that the phenotype of expanded NK cells is comparable between those from healthy donors and cancer patients. Moreover, our results confirm the ability of ex vivo expanded NK cells to lyse tumour cell lines in vitro. While the cell lines examined had differential sensitivity to NK cell killing we found this was correlated with level of major histocompatibility complex (MHC) class I expression. In our in vivo model, NK cells prevented tumour establishment and growth in immunocompromised mice. Finally, we showed that NK cells expanded from the peripheral blood of breast cancer patients show high cytotoxicity against allogeneic and autologous patient-derived tumour cells in vitro. NK cells from breast cancer patients can be expanded similarly to those from healthy donors, have a high cytotoxic ability against breast cancer cell lines and patient-derived tumour cells, and can be compatible with current cancer treatments to restore NK cell function in cancer patients.

Malignant hematopoietic cell lines: in vitro models for the study of natural killer cell leukemia-lymphoma.

PubMed

Drexler, H G; Matsuo, Y

2000-05-01

Malignancies involving natural killer (NK) cells are rare disorders. The complexity of NK cell-involving disorders has only recently been appreciated. Modern classifications discern immature (precursor) from mature NK cell leukemias-lymphomas. Continuous NK leukemia-lymphoma cell lines represent important model systems to study these neoplasms. While there are a number of putative NK cell lines which are, however, either not characterized, not immortalized, non-malignant, non-NK, or plain false cell lines, six bona fide malignant NK cell lines have been established and are sufficiently well characterized: HANK1, KHYG-1, NK-92, NKL, NK-YS and YT. Except for YT which was derived from a not further defined acute lymphoblastic lymphoma, these cell lines were established from patients with various NK cell malignancies. Five of the six cell lines are constitutively interleukin-2-dependent. Their immunoprofile is remarkably similar: CD1-, CD2+, surface CD3 (but cytoplasmic CD3epsilon+), CD4-, CD5-, CD7+, CD8-, CD16-, CD56+, CD57-, TCRalphabeta-, TCRgammadelta-, negative for B cell and myelomonocytic markers. The immunoglobulin heavy chain and T cell receptor genes are all in germline configuration. All six lines show complex chromosomal alterations, with both numerical and structural aberrations, attesting to their malignant and monoclonal nature. Functionally, these cells which contain azurophilic granules in their cytoplasm are nearly universally positive in NK activity assays. Three of five cell lines are Epstein-Barr virus-positive (type II latency). The composite data on these six cell lines allow for the operational definition of a typical malignant NK cell line profile. NK leukemia-lymphoma cell lines will prove invaluable for studies of normal and malignant NK cell biology.

Establishment of stable cell line for inducing KAP1 protein expression.

PubMed

Liu, Xiaoyan; Khan, Md Asaduzzaman; Cheng, Jingliang; Wei, Chunli; Zhang, Lianmei; Fu, Junjiang

2015-06-01

Generation of the stable cell lines is a highly efficient tool in functional studies of certain genes or proteins, where the particular genes or proteins are inducibly expressed. The KRAB-associated protein-1 (KAP1) is an important transcription regulatory protein, which is investigated in several molecular biological studies. In this study, we have aimed to generate a stable cell line for inducing KAP1 expression. The recombinant plasmid pcDNA5/FRT/TO-KAP1 was constructed at first, which was then transfected into Flp-In™T-REx™-HEK293 cells to establish an inducible pcDNA5/FRT/TO-KAP1-HEK293 cell line. The Western blot analysis showed that the protein level of KAP1 is over-expressed in the established stable cell line by doxycycline induction, both dose and time dependently. Thus we have successfully established stable pcDNA5/FRT/TO-KAP1-HEK293 cell line, which can express KAP1 inducibly. This inducible cell line might be very useful for KAP1 functional studies.

A comparative genomic hybridization approach to study gene copy number variations among Chinese hamster cell lines.

PubMed

Vishwanathan, Nandita; Bandyopadhyay, Arpan; Fu, Hsu-Yuan; Johnson, Kathryn C; Springer, Nathan M; Hu, Wei-Shou

2017-08-01

Chinese Hamster Ovary (CHO) cells are aneuploid in nature. The genome of recombinant protein producing CHO cell lines continuously undergoes changes in its structure and organization. We analyzed nine cell lines, including parental cell lines, using a comparative genomic hybridization (CGH) array focused on gene-containing regions. The comparison of CGH with copy-number estimates from sequencing data showed good correlation. Hierarchical clustering of the gene copy number variation data from CGH data revealed the lineage relationships between the cell lines. On analyzing the clones of a clonal population, some regions with altered genomic copy number status were identified indicating genomic changes during passaging. A CGH array is thus an effective tool in quantifying genomic alterations in industrial cell lines and can provide insights into the changes in the genomic structure during cell line derivation and long term culture. Biotechnol. Bioeng. 2017;114: 1903-1908. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

A Tetrameric Peptide Derived from Bovine Lactoferricin Exhibits Specific Cytotoxic Effects against Oral Squamous-Cell Carcinoma Cell Lines

PubMed Central

Solarte, Víctor A.; Rosas, Jaiver E.; Rivera, Zuly J.; Arango-Rodríguez, Martha L.; García, Javier E.; Vernot, Jean-Paul

2015-01-01

Several short linear peptides derived from cyclic bovine lactoferricin were synthesized and tested for their cytotoxic effect against the oral cavity squamous-cell carcinoma (OSCC) cell lines CAL27 and SCC15. As a control, an immortalized and nontumorigenic cell line, Het-1A, was used. Linear peptides based on the RRWQWR core sequence showed a moderate cytotoxic effect and specificity towards tumorigenic cells. A tetrameric peptide, LfcinB(20–25)4, containing the RRWQWR motif, exhibited greater cytotoxic activity (>90%) in both OSCC cell lines compared to the linear lactoferricin peptide or the lactoferrin protein. Additionally, this tetrameric peptide showed the highest specificity towards tumorigenic cells among the tested peptides. Interestingly, this effect was very fast, with cell shrinkage, severe damage to cell membrane permeability, and lysis within one hour of treatment. Our results are consistent with a necrotic effect rather than an apoptotic one and suggest that this tetrameric peptide could be considered as a new candidate for the therapeutic treatment of OSCC. PMID:26609531

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.

Intrinsic anticarcinogenic effects of Piper sarmentosum ethanolic extract on a human hepatoma cell line

PubMed Central

Zainal Ariffin, Shahrul Hisham; Wan Omar, Wan Haifa Haryani; Zainal Ariffin, Zaidah; Safian, Muhd Fauzi; Senafi, Sahidan; Megat Abdul Wahab, Rohaya

2009-01-01

Background Piper sarmentosum, locally known as kaduk is belonging to the family of Piperaceae. It is our interest to evaluate their effect on human hepatoma cell line (HepG2) for the potential of anticarcinogenic activity. Results The anticarcinogenic activity of an ethanolic extract from Piper sarmentosum in HepG2 and non-malignant Chang's liver cell lines has been previously determined using (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) (MTT) assays, where the IC50 value was used as a parameter for cytotoxicity. The ethanolic extract that showed anticarcinogenic properties in HepG2 cells had an IC50 of 12.5 μg mL-1, while IC50 values in the non-malignant Chang's liver cell line were greater than 30 μg mL-1. Apoptotic morphological changes in HepG2 cells were observed using an inverted microscope and showed chromatin condensation, cell shrinkage and apoptotic bodies following May-Grunwald-Giemsa's staining. The percentage of apoptotic cells in the overall population (apoptotic index) showed a continuously significant increase (p < 0.05) in 12.5 μg mL-1 ethanolic extract-treated cells at 24, 48 and 72 hours compared to controls (untreated cells). Following acridine orange and ethidium bromide staining, treatment with 10, 12 and 14 μg mL-1 of ethanolic extracts caused typical apoptotic morphological changes in HepG2 cells. Molecular analysis of DNA fragmentation was used to examine intrinsic apoptosis induced by the ethanolic extracts. These results showed a typical intrinsic apoptotic characterisation, which included fragmentation of nuclear DNA in ethanolic extract-treated HepG2 cells. However, the non-malignant Chang's liver cell line produced no DNA fragmentation. In addition, the DNA genome was similarly intact for both the untreated non-malignant Chang's liver and HepG2 cell lines. Conclusion Therefore, our results suggest that the ethanolic extract from P. sarmentosum induced anticarcinogenic activity through an intrinsic apoptosis pathway in HepG2 cells in vitro. PMID:19257877

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

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

Nanostructured polysaccharidic microcapsules for intracellular release of cisplatin.

PubMed

Vergaro, Viviana; Papadia, Paride; Petrini, Paola; Fanizzi, Francesco Paolo; De Pascali, Sandra A; Baldassarre, Francesca; Pastorino, Laura; Ciccarella, Giuseppe

2017-06-01

Carbohydrate polimeric microcapsules were assembled using a LbL approach onto a CaCO 3 core. The microcapsules were used to delivery the anticancer drug cisplatin into HeLa and MCF-7 cancer cell lines. Drug encapsulation, measured by ICP spectroscopy, was around 50% of the charging solution. Fluorimetric measurements showed an efficient cellular uptake of polysacchardic microcapsules in both cell lines. The drug-loaded capsules demonstrated a better efficiency against cell viability than the free drug. Specifically, the amount of platinum reaching genomic DNA was measured, showing that encapsulation improves the nuclear delivery of the drug for both cell lines. Copyright © 2017 Elsevier B.V. All rights reserved.

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

PubMed

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

2017-05-06

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

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

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.

Cold-induced retrotransposition of fish LINEs.

PubMed

Chen, Shue; Yu, Mengchao; Chu, Xu; Li, Wenhao; Yin, Xiujuan; Chen, Liangbiao

2017-08-20

Classes of retrotransposons constitute a large portion of metazoan genome. There have been cases reported that genomic abundance of retrotransposons is correlated with the severity of low environmental temperatures. However, the molecular mechanisms underlying such correlation are unknown. We show here by cell transfection assays that retrotransposition (RTP) of a long interspersed nuclear element (LINE) from an Antarctic notothenioid fish Dissostichus mawsoni (dmL1) could be activated by low temperature exposure, causing increased dmL1 copies in the host cell genome. The cold-induced dmL1 propagation was demonstrated to be mediated by the mitogen-activated protein kinases (MAPK)/p38 signaling pathway, which is activated by accumulation of reactive oxygen species (ROS) in cold-stressed conditions. Surprisingly, dmL1 transfected cells showed an increase in the number of viable cells after prolonged cold exposures than non-transfected cells. Features of cold inducibility of dmL1 were recapitulated in LINEs of zebrafish origin both in cultured cell lines and tissues, suggesting existence of a common cold-induced LINE amplification in fishes. The findings reveal an important function of LINEs in temperature adaptation and provid insights into the MAPK/p38 stress responsive pathway that shapes LINE composition in fishes facing cold stresses. Copyright © 2017. Published by Elsevier Ltd.

Establishment of a novel immortalized human prostatic epithelial cell line stably expressing androgen receptor and its application for the functional screening of androgen receptor modulators

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

Yu, Shan; Wang, Ming-Wei; Yao, Xiaoqiang

2009-05-15

In this study, we developed a human prostatic epithelial cell line BPH-1-AR stably expressing AR by lentiviral transduction. Characterization by immunoblot and RT-PCR showed that AR was stably expressed in all representative BPH-1-AR clones. Androgen treatment induced a secretory differentiation phenotype in BPH-1-AR cells but suppressed their cell proliferation. Treatments with AR agonists induced transactivation of a transfected PSA-gene promoter reporter in BPH-1-AR cells, whereas this transactivation was suppressed by an AR antagonist flutamide, indicating that the transduced AR in BPH-1-AR cells was functional. Finally, we utilized BPH-1-AR cells to evaluate the androgenic activities and growth effects of five newlymore » developed non-steroidal compounds. Results showed that these compounds showed androgenic activities and growth-inhibitory effects on BPH-1-AR cells. Our results showed that BPH-1-AR cell line would be a valuable in vitro model for the study of androgen-regulated processes in prostatic epithelial cells and identification of compounds with AR-modulating activities.« less

Synthesis, characterization and biological evaluation of bile acid-aromatic/heteroaromatic amides linked via amino acids as anti-cancer agents.

PubMed

Agarwal, Devesh S; Anantaraju, Hasitha Shilpa; Sriram, Dharmarajan; Yogeeswari, Perumal; Nanjegowda, Shankara H; Mallu, P; Sakhuja, Rajeev

2016-03-01

A series of bile acid (Cholic acid and Deoxycholic acid) aryl/heteroaryl amides linked via α-amino acid were synthesized and tested against 3 human cancer cell-lines (HT29, MDAMB231, U87MG) and 1 human normal cell line (HEK293T). Some of the conjugates showed promising results to be new anticancer agents with good in vitro results. More specifically, Cholic acid derivatives 6a (1.35 μM), 6c (1.41 μM) and 6m (4.52 μM) possessing phenyl, benzothiazole and 4-methylphenyl groups showed fairly good activity against the breast cancer cell line with respect to Cisplatin (7.21 μM) and comparable with respect to Doxorubicin (1 μM), while 6e (2.49μM), 6i (2.46 μM) and 6m (1.62 μM) showed better activity against glioblastoma cancer cell line with respect to both Cisplatin (2.60 μM) and Doxorubicin (3.78 μM) drugs used as standards. Greater than 65% of the compounds were found to be safer on human normal cell line. Copyright © 2016 Elsevier Inc. All rights reserved.

Coexistence of mucous retention cyst and basal cell adenoma arising from the lining epithelium of the cyst. Report of two cases.

PubMed

Antoniades, D; Epivatianos, A; Markopoulos, A; Kolokotronis, A; Zaraboukas, T

2009-01-01

To report 2 cases of coexisting mucous retention cyst and basal cell adenoma arising from the lining epithelium of the cyst. Two cases of painless swellings, well-demarcated, soft to palpation, and located in the submucosa of the upper lip were clinically examined with the provisional diagnosis of mucocele or salivary gland tumor. Histological examination showed the presence of a large unilocular cystic cavity in many parts surrounded by single or bilayered lining epithelium composed of flattened to cuboidal cells, and in other parts surrounded by projections of cells arranged in a trabecular pattern far into the cystic cavity. The trabeculae were composed of basal and low columnar cells that sometimes formed small duct-like structures. Immunohistochemistry showed that the lining epithelium of the cystic cavity and the cells of the projections expressed cytokeratin 7 and high-molecular-weight cytokeratins. The cells of the projections were weakly positive for S-100 protein and negative for vimentin and alpha-smooth muscle actin. Based on the results, a diagnosis of coexisting mucous retention cysts and basal cell adenomas arising from the lining epithelium of cysts was made. The coexistence of mucous retention cysts and basal cell adenomas arising from the lining epithelium of the cyst is reported. Copyright 2009 S. Karger AG, Basel.

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

PubMed

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

2017-06-01

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

Identification of a unique hepatocellular carcinoma line, Li-7, with CD13(+) cancer stem cells hierarchy and population change upon its differentiation during culture and effects of sorafenib.

PubMed

Yamada, Takeshi; Abei, Masato; Danjoh, Inaho; Shirota, Ryoko; Yamashita, Taro; Hyodo, Ichinosuke; Nakamura, Yukio

2015-04-11

Cancer stem cell (CSC) research has highlighted the necessity of developing drugs targeting CSCs. We investigated a hepatocellular carcinoma (HCC) cell line that not only has CSC hierarchy but also shows phenotypic changes (population changes) upon differentiation of CSC during culture and can be used for screening drugs targeting CSC. Based on a hypothesis that the CSC proportion should decrease upon its differentiation into progenitors (population change), we tested HCC cell lines (HuH-7, Li-7, PLC/PRF/5, HLF, HLE) before and after 2 months culture for several markers (CD13, EpCAM, CD133, CD44, CD90, CD24, CD166). Tumorigenicity was tested using nude mice. To evaluate the CSC hierarchy, we investigated reconstructivity, proliferation, ALDH activity, spheroid formation, chemosensitivity and microarray analysis of the cell populations sorted by FACS. Only Li-7 cells showed a population change during culture: the proportion of CD13 positive cells decreased, while that of CD166 positive cells increased. The high tumorigenicity of the Li-7 was lost after the population change. CD13(+)/CD166(-) cells showed slow growth and reconstructed the bulk Li-7 populations composed of CD13(+)/CD166(-), CD13(-)/CD166(-) and CD13(-)/CD166(+) fractions, whereas CD13(-)/CD166(+) cells showed rapid growth but could not reproduce any other population. CD13(+)/CD166(-) cells showed high ALDH activity, spheroid forming ability and resistance to 5-fluorouracil. Microarray analysis demonstrated higher expression of stemness-related genes in CD166(-) than CD166(+) fraction. These results indicated a hierarchy in Li-7 cells, in which CD13(+)/CD166(-) and CD13(-)/CD166(+) cells serve as slow growing CSCs and rapid growing progenitors, respectively. Sorafenib selectively targeted the CD166(-) fraction, including CD13(+) CSCs, which exhibited higher mRNA expression for FGF3 and FGF4, candidate biomarkers for sorafenib. 5-fluorouracil followed by sorafenib inhibited the growth of bulk Li-7 cells more effectively than the reverse sequence or either alone. We identified a unique HCC line, Li-7, which not only shows heterogeneity for a CD13(+) CSC hierarchy, but also undergoes a "population change" upon CSC differentiation. Sorafenib targeted the CSC in vitro, supporting the use of this model for screening drugs targeting the CSC. This type of "heterogeneous, unstable" cell line may prove more useful in the CSC era than conventional "homogeneous, stable" cell lines.

The Reverse Transcription Inhibitor Abacavir Shows Anticancer Activity in Prostate Cancer Cell Lines

PubMed Central

Molinari, Agnese; Parisi, Chiara; Bozzuto, Giuseppina; Toccacieli, Laura; Formisano, Giuseppe; De Orsi, Daniela; Paradisi, Silvia; Grober, OlÌ Maria Victoria; Ravo, Maria; Weisz, Alessandro; Arcieri, Romano; Vella, Stefano; Gaudi, Simona

2010-01-01

Background Transposable Elements (TEs) comprise nearly 45% of the entire genome and are part of sophisticated regulatory network systems that control developmental processes in normal and pathological conditions. The retroviral/retrotransposon gene machinery consists mainly of Long Interspersed Nuclear Elements (LINEs-1) and Human Endogenous Retroviruses (HERVs) that code for their own endogenous reverse transcriptase (RT). Interestingly, RT is typically expressed at high levels in cancer cells. Recent studies report that RT inhibition by non-nucleoside reverse transcriptase inhibitors (NNRTIs) induces growth arrest and cell differentiation in vitro and antagonizes growth of human tumors in animal model. In the present study we analyze the anticancer activity of Abacavir (ABC), a nucleoside reverse transcription inhibitor (NRTI), on PC3 and LNCaP prostate cancer cell lines. Principal Findings ABC significantly reduces cell growth, migration and invasion processes, considerably slows S phase progression, induces senescence and cell death in prostate cancer cells. Consistent with these observations, microarray analysis on PC3 cells shows that ABC induces specific and dose-dependent changes in gene expression, involving multiple cellular pathways. Notably, by quantitative Real-Time PCR we found that LINE-1 ORF1 and ORF2 mRNA levels were significantly up-regulated by ABC treatment. Conclusions Our results demonstrate the potential of ABC as anticancer agent able to induce antiproliferative activity and trigger senescence in prostate cancer cells. Noteworthy, we show that ABC elicits up-regulation of LINE-1 expression, suggesting the involvement of these elements in the observed cellular modifications. PMID:21151977

Development of a chemically defined platform fed-batch culture media for monoclonal antibody-producing CHO cell lines with optimized choline content.

PubMed

Kuwae, Shinobu; Miyakawa, Ichiko; Doi, Tomohiro

2018-01-11

A chemically defined platform basal medium and feed media were developed using a single Chinese hamster ovary (CHO) cell line that produces a monoclonal antibody (mAb). Cell line A, which showed a peak viable cell density of 5.9 × 10 6  cells/mL and a final mAb titer of 0.5 g/L in batch culture, was selected for the platform media development. Stoichiometrically balanced feed media were developed using glucose as an indicator of cell metabolism to determine the feed rates of all other nutrients. A fed-batch culture of cell line A using the platform fed-batch medium yielded a 6.4 g/L mAb titer, which was 12-fold higher than that of the batch culture. To examine the applicability of the platform basal medium and feed media, three other cell lines (A16, B, and C) that produce mAbs were cultured using the platform fed-batch medium, and they yielded mAb titers of 8.4, 3.3, and 6.2 g/L, respectively. The peak viable cell densities of the three cell lines ranged from 1.3 × 10 7 to 1.8 × 10 7  cells/mL. These results show that the nutritionally balanced fed-batch medium and feeds worked well for other cell lines. During the medium development, we found that choline limitation caused a lower cell viability, a lower mAb titer, a higher mAb aggregate content, and a higher mannose-5 content. The optimal choline chloride to glucose ratio for the CHO cell fed-batch culture was determined. Our platform basal medium and feed media will shorten the medium-development time for mAb-producing cell lines.

Hematopoietic Cancer Cell Lines Can Support Replication of Sabin Poliovirus Type 1

PubMed Central

van Eikenhorst, Gerco; de Gruijl, Tanja D.; van der Pol, Leo A.; Bakker, Wilfried A. M.

2015-01-01

Viral vaccines can be produced in adherent or in suspension cells. The objective of this work was to screen human suspension cell lines for the capacity to support viral replication. As the first step, it was investigated whether poliovirus can replicate in such cell lines. Sabin poliovirus type 1 was serially passaged on five human cell lines, HL60, K562, KG1, THP-1, and U937. Sabin type 1 was capable of efficiently replicating in three cell lines (K562, KG1, and U937), yielding high viral titers after replication. Expression of CD155, the poliovirus receptor, did not explain susceptibility to replication, since all cell lines expressed CD155. Furthermore, we showed that passaged virus replicated more efficiently than parental virus in KG1 cells, yielding higher virus titers in the supernatant early after infection. Infection of cell lines at an MOI of 0.01 resulted in high viral titers in the supernatant at day 4. Infection of K562 with passaged Sabin type 1 in a bioreactor system yielded high viral titers in the supernatant. Altogether, these data suggest that K562, KG1, and U937 cell lines are useful for propagation of poliovirus. PMID:25815312

Manipulating mammalian cell morphologies using chemical-mechanical polished integrated circuit chips

NASA Astrophysics Data System (ADS)

Moussa, Hassan I.; Logan, Megan; Siow, Geoffrey C.; Phann, Darron L.; Rao, Zheng; Aucoin, Marc G.; Tsui, Ting Y.

2017-12-01

Tungsten chemical-mechanical polished integrated circuits were used to study the alignment and immobilization of mammalian (Vero) cells. These devices consist of blanket silicon oxide thin films embedded with micro- and nano-meter scale tungsten metal line structures on the surface. The final surfaces are extremely flat and smooth across the entire substrate, with a roughness in the order of nanometers. Vero cells were deposited on the surface and allowed to adhere. Microscopy examinations revealed that cells have a strong preference to adhere to tungsten over silicon oxide surfaces with up to 99% of cells adhering to the tungsten portion of the surface. Cells self-aligned and elongated into long threads to maximize contact with isolated tungsten lines as thin as 180 nm. The orientation of the Vero cells showed sensitivity to the tungsten line geometric parameters, such as line width and spacing. Up to 93% of cells on 10 μm wide comb structures were aligned within ± 20° of the metal line axis. In contrast, only 22% of cells incubated on 0.18 μm comb patterned tungsten lines were oriented within the same angular interval. This phenomenon is explained using a simple model describing cellular geometry as a function of pattern width and spacing, which showed that cells will rearrange their morphology to maximize their contact to the embedded tungsten. Finally, it was discovered that the materials could be reused after cleaning the surfaces, while maintaining cell alignment capability.

Manipulating mammalian cell morphologies using chemical-mechanical polished integrated circuit chips.

PubMed

Moussa, Hassan I; Logan, Megan; Siow, Geoffrey C; Phann, Darron L; Rao, Zheng; Aucoin, Marc G; Tsui, Ting Y

2017-01-01

Tungsten chemical-mechanical polished integrated circuits were used to study the alignment and immobilization of mammalian (Vero) cells. These devices consist of blanket silicon oxide thin films embedded with micro- and nano-meter scale tungsten metal line structures on the surface. The final surfaces are extremely flat and smooth across the entire substrate, with a roughness in the order of nanometers. Vero cells were deposited on the surface and allowed to adhere. Microscopy examinations revealed that cells have a strong preference to adhere to tungsten over silicon oxide surfaces with up to 99% of cells adhering to the tungsten portion of the surface. Cells self-aligned and elongated into long threads to maximize contact with isolated tungsten lines as thin as 180 nm. The orientation of the Vero cells showed sensitivity to the tungsten line geometric parameters, such as line width and spacing. Up to 93% of cells on 10 μm wide comb structures were aligned within ± 20° of the metal line axis. In contrast, only ~22% of cells incubated on 0.18 μm comb patterned tungsten lines were oriented within the same angular interval. This phenomenon is explained using a simple model describing cellular geometry as a function of pattern width and spacing, which showed that cells will rearrange their morphology to maximize their contact to the embedded tungsten. Finally, it was discovered that the materials could be reused after cleaning the surfaces, while maintaining cell alignment capability.

Manipulating mammalian cell morphologies using chemical-mechanical polished integrated circuit chips

PubMed Central

Moussa, Hassan I.; Logan, Megan; Siow, Geoffrey C.; Phann, Darron L.; Rao, Zheng; Aucoin, Marc G.; Tsui, Ting Y.

2017-01-01

Abstract Tungsten chemical-mechanical polished integrated circuits were used to study the alignment and immobilization of mammalian (Vero) cells. These devices consist of blanket silicon oxide thin films embedded with micro- and nano-meter scale tungsten metal line structures on the surface. The final surfaces are extremely flat and smooth across the entire substrate, with a roughness in the order of nanometers. Vero cells were deposited on the surface and allowed to adhere. Microscopy examinations revealed that cells have a strong preference to adhere to tungsten over silicon oxide surfaces with up to 99% of cells adhering to the tungsten portion of the surface. Cells self-aligned and elongated into long threads to maximize contact with isolated tungsten lines as thin as 180 nm. The orientation of the Vero cells showed sensitivity to the tungsten line geometric parameters, such as line width and spacing. Up to 93% of cells on 10 μm wide comb structures were aligned within ± 20° of the metal line axis. In contrast, only ~22% of cells incubated on 0.18 μm comb patterned tungsten lines were oriented within the same angular interval. This phenomenon is explained using a simple model describing cellular geometry as a function of pattern width and spacing, which showed that cells will rearrange their morphology to maximize their contact to the embedded tungsten. Finally, it was discovered that the materials could be reused after cleaning the surfaces, while maintaining cell alignment capability. PMID:29152017

Use of deferasirox, an iron chelator, to overcome imatinib resistance of chronic myeloid leukemia cells

PubMed Central

Kim, Dae Sik; Na, Yoo Jin; Kang, Myoung Hee; Yoon, Soo-Young; Choi, Chul Won

2016-01-01

Background/Aims: The treatment of chronic myeloid leukemia (CML) has achieved impressive success since the development of the Bcr-Abl tyrosine kinase inhibitor, imatinib mesylate. Nevertheless, resistance to imatinib has been observed, and a substantial number of patients need alternative treatment strategies. Methods: We have evaluated the effects of deferasirox, an orally active iron chelator, and imatinib on K562 and KU812 human CML cell lines. Imatinib-resistant CML cell lines were created by exposing cells to gradually increasing concentrations of imatinib. Results: Co-treatment of cells with deferasirox and imatinib induced a synergistic dose-dependent inhibition of proliferation of both CML cell lines. Cell cycle analysis showed an accumulation of cells in the subG1 phase. Western blot analysis of apoptotic proteins showed that co-treatment with deferasirox and imatinib induced an increased expression of apoptotic proteins. These tendencies were clearly identified in imatinib-resistant CML cell lines. The results also showed that co-treatment with deferasirox and imatinib reduced the expression of BcrAbl, phosphorylated Bcr-Abl, nuclear factor-κB (NF-κB) and β-catenin. Conclusions: We observed synergistic effects of deferasirox and imatinib on both imatinib-resistant and imatinib-sensitive cell lines. These effects were due to induction of apoptosis and cell cycle arrest by down-regulated expression of NF-κB and β-catenin levels. Based on these results, we suggest that a combination treatment of deferasirox and imatinib could be considered as an alternative treatment option for imatinib-resistant CML. PMID:26874514

Derivation and characterization of Chinese human embryonic stem cell line with high potential to differentiate into pancreatic and hepatic cells.

PubMed

Shi, Cheng; Shen, Huan; Jiang, Wei; Song, Zhi-Hua; Wang, Cheng-Yan; Wei, Li-Hui

2011-04-01

Human embryonic stem cells have prospective uses in regenerative medicine and drug screening. Every human embryonic stem cell line has its own genetic background, which determines its specific ability for differentiation as well as susceptibility to drugs. It is necessary to compile many human embryonic stem cell lines with various backgrounds for future clinical use, especially in China due to its large population. This study contributes to isolating new Chinese human embryonic stem cell lines with clarified directly differentiation ability. Donated embryos that exceeded clinical use in our in vitro fertilization-embryo transfer (IVF-ET) center were collected to establish human embryonic stem cells lines with informed consent. The classic growth factors of basic fibroblast growth factor (bFGF) and recombinant human leukaemia inhibitory factor (hLIF) for culturing embryonic stem cells were used to capture the stem cells from the plated embryos. Mechanical and enzymetic methods were used to propagate the newly established human embryonic stem cells line. The new cell line was checked for pluripotent characteristics with detecting the expression of stemness genes and observing spontaneous differentiation both in vitro and in vivo. Finally similar step-wise protocols from definitive endoderm to target specific cells were used to check the cell line's ability to directly differentiate into pancreatic and hepatic cells. We generated a new Chinese human embryonic stem cells line, CH1. This cell line showed the same characteristics as other reported Chinese human embryonic stem cells lines: normal morphology, karyotype and pluripotency in vitro and in vivo. The CH1 cells could be directly differentiated towards pancreatic and hepatic cells with equal efficiency compared to the H1 cell line. This newly established Chinese cell line, CH1, which is pluripotent and has high potential to differentiate into pancreatic and hepatic cells, will provide a useful tool for embryo development research, along with clinical treatments for diabetes and some hepatic diseases.

Identification of a Novel Rhabdovirus in Spodoptera frugiperda Cell Lines

PubMed Central

Ma, Hailun; Galvin, Teresa A.; Glasner, Dustin R.; Shaheduzzaman, Syed

2014-01-01

ABSTRACT The Sf9 cell line, derived from Spodoptera frugiperda, is used as a cell substrate for biological products, and no viruses have been reported in this cell line after extensive testing. We used degenerate PCR assays and massively parallel sequencing (MPS) to identify a novel RNA virus belonging to the order Mononegavirales in Sf9 cells. Sequence analysis of the assembled virus genome showed the presence of five open reading frames (ORFs) corresponding to the genes for the N, P, M, G, and L proteins in other rhabdoviruses and an unknown ORF of 111 amino acids located between the G- and L-protein genes. BLAST searches indicated that the S. frugiperda rhabdovirus (Sf-rhabdovirus) was related in a limited region of the L-protein gene to Taastrup virus, a newly discovered member of the Mononegavirales from a leafhopper (Hemiptera), and also to plant rhabdoviruses, particularly in the genus Cytorhabdovirus. Phylogenetic analysis of sequences in the L-protein gene indicated that Sf-rhabdovirus is a novel virus that branched with Taastrup virus. Rhabdovirus morphology was confirmed by transmission electron microscopy of filtered supernatant samples from Sf9 cells. Infectivity studies indicated potential transient infection by Sf-rhabdovirus in other insect cell lines, but there was no evidence of entry or virus replication in human cell lines. Sf-rhabdovirus sequences were also found in the Sf21 parental cell line of Sf9 cells but not in other insect cell lines, such as BT1-TN-5B1-4 (Tn5; High Five) cells and Schneider's Drosophila line 2 [D.Mel.(2); SL2] cells, indicating a species-specific infection. The results indicate that conventional methods may be complemented by state-of-the-art technologies with extensive bioinformatics analysis for identification of novel viruses. IMPORTANCE The Spodoptera frugiperda Sf9 cell line is used as a cell substrate for the development and manufacture of biological products. Extensive testing has not previously identified any viruses in this cell line. This paper reports on the identification and characterization of a novel rhabdovirus in Sf9 cells. This was accomplished through the use of next-generation sequencing platforms, de novo assembly tools, and extensive bioinformatics analysis. Rhabdovirus identification was further confirmed by transmission electron microscopy. Infectivity studies showed the lack of replication of Sf-rhabdovirus in human cell lines. The overall study highlights the use of a combinatorial testing approach including conventional methods and new technologies for evaluation of cell lines for unexpected viruses and use of comprehensive bioinformatics strategies for obtaining confident next-generation sequencing results. PMID:24672045

MicroRNA-190 regulates FOXP2 genes in human gastric cancer.

PubMed

Jia, Wen-Zhuo; Yu, Tao; An, Qi; Yang, Hua; Zhang, Zhu; Liu, Xiao; Xiao, Gang

2016-01-01

To investigate how microRNA-190 (miR-190) regulates FOXP2 genes in gastric cancer (GC) cell line SGC7901. We identified that miR-190 could target FOXP2 genes by using dual luciferase enzyme assay. Precursor fragment transfection of miR-190 was performed with GC cell line SGC7901 and human gastric mucosal cell line GES-1. miR-190 expression was detected by reverse transcription-polymerase chain reaction (RT-PCR) and FOXP2 protein expression was measured by Western blotting. FOXP2-3'-untranslated region (UTR) in miR-190 transfection group was significantly decreased as compared with other groups. There were no significant differences in fluorescence signals of FOXP2mut-3'-UTR in each group. Therefore, it was assumed that miR-190 can target FOXP2 genes. Through RT-PCR verification, it was observed that the expression level of miR-190 was significantly higher in GC cell line SGC7901 than in human gastric mucosa cell line GES-1 after transfection with miR-190 mimics. The expression level of miR-190 was significantly higher in GES-1 cells than in SGC7901 cells after transfection with miR-190 inhibitors. Western blotting results showed the expression level of FOXP2 was significantly lower in GC cell line SGC7901 than in GES-1 cells. Compared with blank, mimics control, and inhibitors control groups, the miR-190 mimics group showed significantly enhanced proliferation, migration, and invasion abilities, while miR-190 inhibitors group showed decreased abilities toward proliferation, migration, and invasion (P<0.05). The transcription level of miR-190 and the expression level of FOXP2 in tumor tissues and adjacent normal tissues in GC patients were verified to be consistent with those of cell line experiments. Upregulation of miR-190 can lead to downregulation of FOXP2 protein expression. miR-190 may serve as a potential target for GC diagnosis.

Newly-derived neuroblastoma cell lines propagated in serum-free media recapitulate the genotype and phenotype of primary neuroblastoma tumours.

PubMed

Bate-Eya, Laurel T; Ebus, Marli E; Koster, Jan; den Hartog, Ilona J M; Zwijnenburg, Danny A; Schild, Linda; van der Ploeg, Ida; Dolman, M Emmy M; Caron, Huib N; Versteeg, Rogier; Molenaar, Jan J

2014-02-01

Recently protocols have been devised for the culturing of cell lines from fresh tumours under serum-free conditions in defined neural stem cell medium. These cells, frequently called tumour initiating cells (TICs) closely retained characteristics of the tumours of origin. We report the isolation of eight newly-derived neuroblastoma TICs from six primary neuroblastoma tumours and two bone marrow metastases. The primary tumours from which these TICs were generated have previously been fully typed by whole genome sequencing (WGS). Array comparative genomic hybridisation (aCGH) analysis showed that TIC lines retained essential characteristics of the primary tumours and exhibited typical neuroblastoma chromosomal aberrations such as MYCN amplification, gain of chromosome 17q and deletion of 1p36. Protein analysis showed expression for neuroblastoma markers MYCN, NCAM, CHGA, DBH and TH while haematopoietic markers CD19 and CD11b were absent. We analysed the growth characteristics and confirmed tumour-forming potential using sphere-forming assays, subcutaneous and orthotopic injection of these cells into immune-compromised mice. Affymetrix mRNA expression profiling of TIC line xenografts showed an expression pattern more closely mimicking primary tumours compared to xenografts from classical cell lines. This establishes that these neuroblastoma TICs cultured under serum-free conditions are relevant and useful neuroblastoma tumour models. Copyright © 2013 Elsevier Ltd. All rights reserved.

Delayed Cell Cycle Progression and Apoptosis Induced by Hemicellulase-Treated Agaricus blazei

PubMed Central

Kasai, Hirotake

2007-01-01

We examined the effects of hemicellulase-treated Agaricus blazei (AB fraction H, ABH) on growth of several tumor cell lines. ABH inhibited the proliferation of some cell lines without cytotoxic effects. It markedly prolonged the S phase of the cell cycle. ABH also induced mitochondria-mediated apoptosis in different cell lines. However, it had no impact on the growth of other cell lines. ABH induced strong activation of p38 mitogen-activated protein kinase (MAPK) in the cells in which it evoked apoptosis. On the other hand, ABH showed only a weak p38 activation effect in those cell lines in which it delayed cell cycle progression with little induction of apoptosis. However, p38 MAPK-specific inhibitor inhibited both ABH-induced effects, and ABH also caused apoptosis in the latter cells under conditions of high p38 MAPK activity induced by combined treatment with TNF-α. These results indicate that the responsiveness of p38 MAPK to ABH, which differs between cell lines, determines subsequent cellular responses on cell growth. PMID:17342245

Bis-demethoxy curcumin analog nanoparticles: synthesis, characterization, and anticancer activity in vitro.

PubMed

Francis, Arul Prakash; Murthy, Prakhya Balakishna; Devas, Thiyagarajan

2014-07-01

We have optimized a protocol for the preparation of bisdemethoxy curcumin analog nanoparticles (BDMCA-NP) by the solvent assisted process. The structural similarities between bulk and nano BDMCA were determined by Co-TLC, NMR and F-TIR. This shows that our synthesis protocol enhanced the dispersibility and reduce the size of BDMCA without altering the integrity of functional moieties and structure, which is crucial for anticancer and antioxidant activities. The morphology and size of BDMCA-NP as determined by SEM, HRTEM and DLS was found to be around 80 nm. BDMCA-NP treated breast cancer cell lines (MCF 7) showed cell death as characterized by MTT assay. Flow cytometric analysis of BDMCA-NP treated MCF 7 cell lines showed an increase of cell count in G2/M phase indicates the cell cycle arrest. Western blot analysis revealed the presence of caspase 3, caspase 9, cleaved fragments of PARP and Bax proteins in the BDMCA-NP treated MCF 7 cell lines, but not in untreated cell lines. To recap, we have prepared BDMCA-NP by solvent assisted process, which exerted anticancer activity against breast cancer cells, which may be due to (i) enhanced dispersibility and surface: volume ratio, (ii) apoptosis (iii) mitochondrial pathway induced cell death, (iv) G2/M phase cell cycle arrest and (v) disassembly of mitotic spindle of the cancer cells. Thus, nano BDMCA can be used as a potent anticancer agent.

ESCDL-1, a new cell line derived from chicken embryonic stem cells, supports efficient replication of Mardiviruses

PubMed Central

Jean, Christian; Fragnet-Trapp, Laetitia; Rémy, Sylvie; Chabanne-Vautherot, Danièle; Montillet, Guillaume; Fuet, Aurélie; Denesvre, Caroline; Pain, Bertrand

2017-01-01

Marek’s disease virus is the etiological agent of a major lymphoproliferative disorder in poultry and the prototype of the Mardivirus genus. Primary avian somatic cells are currently used for virus replication and vaccine production, but they are largely refractory to any genetic modification compatible with the preservation of intact viral susceptibility. We explored the concept of induction of viral replication permissiveness in an established pluripotent chicken embryonic stem cell-line (cES) in order to derive a new fully susceptible cell-line. Chicken ES cells were not permissive for Mardivirus infection, but as soon as differentiation was triggered, replication of Marek’s disease virus was detected. From a panel of cyto-differentiating agents, hexamethylene bis (acetamide) (HMBA) was found to be the most efficient regarding the induction of permissiveness. These initial findings prompted us to analyse the effect of HMBA on gene expression, to derive a new mesenchymal cell line, the so-called ESCDL-1, and monitor its susceptibility for Mardivirus replication. All Mardiviruses tested so far replicated equally well on primary embryonic skin cells and on ESCDL-1, and the latter showed no variation related to its passage number in its permissiveness for virus infection. Viral morphogenesis studies confirmed efficient multiplication with, as in other in vitro models, no extra-cellular virus production. We could show that ESCDL-1 can be transfected to express a transgene and subsequently cloned without any loss in permissiveness. Consequently, ESCDL-1 was genetically modified to complement viral gene deletions thus yielding stable trans-complementing cell lines. We herein claim that derivation of stable differentiated cell-lines from cES cell lines might be an alternative solution to the cultivation of primary cells for virology studies. PMID:28406989

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

PubMed

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

2016-08-01

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

Synthesis of 2',4-dihydroxy-3-methoxychalcone and 2',4',4-trihydroxy-3-methoxychalcone as a candidate anticancer against cervical (WiDr), colon (HeLa), and breast (T47d) cancer cell lines in vitro

NASA Astrophysics Data System (ADS)

Matsjeh, Sabirin; Swasono, Respati Tri; Anwar, Chairil; Solikhah, Eti Nurwening; Lestari, Endang

2017-03-01

The compound 2',4-dihydroxy-3-methoxychalcone and 2',4',4-trihydroxy-3-methoxychalcone have been synthesized through Claisen-Schmidt reaction from 2-hydroxyacetophenone and 2,4-dihydroxyacetophenone with 4-hydroxy-3-methoxy benzaldehida (vanillin) in aqueous KOH 40% and KSF montmorillonite as catalyst in methanol. All these products were characterized by FT-IR, TLC Scanner, GC-MS, MS-Direct, and 1H-NMR and 13C-NMR spectrometer. Both of these compounds were tested citotoxycity activity as an anticancer against cervical, colon, and breast cancer cells (Hela, WiDr, and T47D cell lines) using MTT assay in vitro. Dose series given test solution concentration on Hela, WiDr, and T47D cells started from 6,25; 25; 50 and 100 µg/mL with incubation treatment for 24 hours. The result of study showed that the 2',4-dihydroxy-3-methoxychalcone as bright yellow crystal with the melting point of 114-115 °C and the yield of 13.77% and the 2',4',4-trihydroxy-3-methoxychalcone as bright yellow crystals with the melting point of 195-197 °C and the yield of 6%. Other 2',4-dihydroxy-3-methoxychalcone and 2',4',4-trihydroxy-3-methoxychalcone also exhibited cytotoxic activity against the cancer cell lines, with the 2',4',4-trihydroxy-3-methoxychalcone showed greater activities than the 2',4-dihydroxy-3-methoxychalcone in WiDr cell lines. The 2',4-dihydroxy-3-methoxychalcone and 2',4',4-trihydroxy-3-methoxychalcone exhibited strong anticancer activities with IC50 value below 20 µg/mL. The activity of 2',4',4-trihydroxy-3-methoxychalcone showed the most active against Hela and WiDr cell lines with IC50 value 8.53 and 2.66 µg/mL respectively, than T47D cell lines with IC50 value 24.61 µg/mL. The test results cytotoxic of 2',4-dihydroxy-3-methoxychalcone showed the most active against Hela and WiDr cell lines with IC50 value 12.80, 19.57 µg/mL than T47D cell lines with IC50 value of 20.73 µg/mL. IC50 value indicated that 2',4-dihydroxy-3-methoxychalcone and 2',4',4-trihydroxy-3-methoxychalcone potential as inhibitors in Hela, WiDr and T47D cell lines.

Relative quantification of beta-casein expression in primary goat mammary epithelial cell lines.

PubMed

Ogorevc, J; Dovč, P

2015-04-15

Primary mammary epithelial cell cultures were established from mammary tissue of lactating and non-lactating goats to assess the expression of beta-casein (CSN2) in vitro. Primary cell cultures were established by enzymatic digestion of mammary tissue and characterized using antibodies against cytokeratin 14, cytokeratin 18, and vimentin. The established primary cell lines in the second passage were grown in basal medium on plastic and in hormone-supplemented (lactogenic) medium on plastic and on an extracellular matrix-covered surface, respectively. CSN2 gene expression was evaluated using quantitative reverse transcription PCR. The presence of CSN2 transcripts was detected in all samples, including cells originating from non-lactating goat, grown in basal medium. The presence of CSN2 protein was confirmed using immunofluorescence. Response to the hormonal treatment and cell morphology differed between the cell lines and treatments. In 2 cell lines supplemented with lactogenic hormones in the medium, CSN2 expression was increased, while CSN2 levels in one of the cell lines remained constant, regardless of the treatment. Addition of extracellular matrix showed positive effects on CSN2 transcription activity in 1 of the cell lines, while in the other 2 showed no statistically significant effects. CSN2 expression appeared to depend on subtle differences in physiological state of the starting tissue material, growth conditions, cell types present in the culture, and methods used for cell culture establishment. Further studies are necessary to identify factors that determine hormone-responsiveness and transcriptional activity of milk protein genes in goat primary mammary cell cultures.

β-Cell Replacement in Mice Using Human Type 1 Diabetes Nuclear Transfer Embryonic Stem Cells.

PubMed

Sui, Lina; Danzl, Nichole; Campbell, Sean R; Viola, Ryan; Williams, Damian; Xing, Yuan; Wang, Yong; Phillips, Neil; Poffenberger, Greg; Johannesson, Bjarki; Oberholzer, Jose; Powers, Alvin C; Leibel, Rudolph L; Chen, Xiaojuan; Sykes, Megan; Egli, Dieter

2018-01-01

β-Cells derived from stem cells hold great promise for cell replacement therapy for diabetes. Here we examine the ability of nuclear transfer embryonic stem cells (NT-ESs) derived from a patient with type 1 diabetes to differentiate into β-cells and provide a source of autologous islets for cell replacement. NT-ESs differentiate in vitro with an average efficiency of 55% into C-peptide-positive cells, expressing markers of mature β-cells, including MAFA and NKX6.1. Upon transplantation in immunodeficient mice, grafted cells form vascularized islet-like structures containing MAFA/C-peptide-positive cells. These β-cells adapt insulin secretion to ambient metabolite status and show normal insulin processing. Importantly, NT-ES-β-cells maintain normal blood glucose levels after ablation of the mouse endogenous β-cells. Cystic structures, but no teratomas, were observed in NT-ES-β-cell grafts. Isogenic induced pluripotent stem cell lines showed greater variability in β-cell differentiation. Even though different methods of somatic cell reprogramming result in stem cell lines that are molecularly indistinguishable, full differentiation competence is more common in ES cell lines than in induced pluripotent stem cell lines. These results demonstrate the suitability of NT-ES-β-cells for cell replacement for type 1 diabetes and provide proof of principle for therapeutic cloning combined with cell therapy. © 2017 by the American Diabetes Association.

Spontaneous lung metastasis formation of human Merkel cell carcinoma cell lines transplanted into scid mice.

PubMed

Knips, Jill; Czech-Sioli, Manja; Spohn, Michael; Heiland, Max; Moll, Ingrid; Grundhoff, Adam; Schumacher, Udo; Fischer, Nicole

2017-07-01

Merkel cell carcinoma (MCC) is an aggressive skin cancer entity that frequently leads to rapid death due to its high propensity to metastasize. The etiology of most MCC cases is linked to Merkel cell polyomavirus (MCPyV), a virus which is monoclonally integrated in up to 95% of tumors. While there are presently no animal models to study the role of authentic MCPyV infection on transformation, tumorigenesis or metastasis formation, xenograft mouse models employing engrafted MCC-derived cell lines (MCCL) represent a promising approach to study certain aspects of MCC pathogenesis. Here, the two MCPyV-positive MCC cell lines WaGa and MKL-1 were subcutaneously engrafted in scid mice. Engraftment of both MCC cell lines resulted in the appearance of circulating tumor cells and metastasis formation, with WaGa-engrafted mice showing a significantly shorter survival time as well as increased numbers of spontaneous lung metastases compared to MKL-1 mice. Interestingly, explanted tumors compared to parental cell lines exhibit an upregulation of MCPyV sT-Antigen expression in all tumors, with WaGa tumors showing significantly higher sT-Antigen expression than MKL-1 tumors. RNA-Seq analysis of explanted tumors and parental cell lines furthermore revealed that in the more aggressive WaGa tumors, genes involved in inflammatory response, growth factor activity and Wnt signalling pathway are significantly upregulated, suggesting that sT-Antigen is the driver of the observed differences in metastasis formation. © 2017 UICC.

Perturbation of nucleo-cytoplasmic transport affects size of nucleus and nucleolus in human cells.

PubMed

Ganguly, Abira; Bhattacharjee, Chumki; Bhave, Madhura; Kailaje, Vaishali; Jain, Bhawik K; Sengupta, Isha; Rangarajan, Annapoorni; Bhattacharyya, Dibyendu

2016-03-01

Size regulation of human cell nucleus and nucleolus are poorly understood subjects. 3D reconstruction of live image shows that the karyoplasmic ratio (KR) increases by 30-80% in transformed cell lines compared to their immortalized counterpart. The attenuation of nucleo-cytoplasmic transport causes the KR value to increase by 30-50% in immortalized cell lines. Nucleolus volumes are significantly increased in transformed cell lines and the attenuation of nucleo-cytoplasmic transport causes a significant increase in the nucleolus volume of immortalized cell lines. A cytosol and nuclear fraction swapping experiment emphasizes the potential role of unknown cytosolic factors in nuclear and nucleolar size regulation. © 2016 Federation of European Biochemical Societies.

Comparative evaluation of PLGA nanoparticle delivery system for 5-fluorouracil and curcumin on squamous cell carcinoma.

PubMed

Masloub, Shaimaa M; Elmalahy, Mohamed H; Sabry, Dina; Mohamed, Wael S; Ahmed, Sahar H

2016-04-01

The purpose of this study is to assess the effect of 5-fluorouracil nanoparticles and curcumin naoparticles on cell proliferation and the expression of the apoptotic marker (caspase 3) in squamous cell carcinoma cell line. PLGA 5-fluorouracil nanopartciles and PLGA curcumin nanoparticles were prepared and applied for 24 and 48h on human laryngeal squamous carcinoma cell line (Hep-2) as regard IC 50 concentration. MTT assay was used for evaluation of cytotoxicity of prepared nanoparticles. Quantitaive reverse transcriptase polymerase chain reaction (QRT-PCR) was used for the assessment of caspase-3 expression in the treated cell line. The drug release rate profiles was dependent upon polymer to drug ratio, noting that the higher PLGA polymer ratio to 5-fluprouracil or curcumin drug showed faster release rates. On the other hand, the least PLGA polymer ratio to 5-fluprouracil or curcumin drug showed the slowest release rates. MTT assay revelaed that 5-fluorouracil nanoparticels or curcumin nanoparticels showed a clear cytotoxic effect on Hep-2 cell line compared to non treated cancer cells. The RT-PCR assessment of caspase-3 expression revealed that there was a significant increase in caspase-3 expression in Hep-2 cell line treated with 5-fluorouracil nanoparticles or curcumin compared to non treated cancer cells. Curcumin nanoparticles could be more active in inducing apoptosis in short term assays (24h) than long term assays (48h) due to differential cellular uptake. While 5-fluorouracil nanoparticles induced higher significant apoptosis in long term (48h) compared to curcumin group. Copyright © 2015 Elsevier Ltd. All rights reserved.

Overexpression of phosphoserine aminotransferase PSAT1 stimulates cell growth and increases chemoresistance of colon cancer cells

PubMed Central

Vié, Nadia; Copois, Virginie; Bascoul-Mollevi, Caroline; Denis, Vincent; Bec, Nicole; Robert, Bruno; Fraslon, Caroline; Conseiller, Emmanuel; Molina, Franck; Larroque, Christian; Martineau, Pierre; Del Rio, Maguy; Gongora, Céline

2008-01-01

Background Colorectal cancer (CRC) is one of the most common causes of cancer death throughout the world. In this work our aim was to study the role of the phosphoserine aminotransferase PSAT1 in colorectal cancer development. Results We first observed that PSAT1 is overexpressed in colon tumors. In addition, we showed that after drug treatment, PSAT1 expression level in hepatic metastases increased in non responder and decreased in responder patients. In experiments using human cell lines, we showed that ectopic PSAT1 overexpression in colon carcinoma SW480 cell line resulted in an increase in its growth rate and survival. In addition, SW480-PSAT1 cells presented a higher tumorigenic potential than SW480 control cells in xenografted mice. Moreover, the SW480-PSAT1 cell line was more resistant to oxaliplatin treatment than the non-transfected SW480 cell line. This resistance resulted from a decrease in the apoptotic response and in the mitotic catastrophes induced by the drug treatment. Conclusion These results show that an enzyme playing a role in the L-serine biosynthesis could be implicated in colon cancer progression and chemoresistance and indicate that PSAT1 represents a new interesting target for CRC therapy. PMID:18221502

Induced Pluripotent Stem Cells from Patients with Huntington’s Disease Show CAG Repeat Expansion Associated Phenotypes

PubMed Central

Mattis, Virginia B; Svendsen, Soshana P; Ebert, Allison; Svendsen, Clive N; King, Alvin R; Casale, Malcolm; Winokur, Sara T; Batugedara, Gayani; Vawter, Marquis; Donovan, Peter J; Lock, Leslie F; Thompson, Leslie M; Zhu, Yu; Fossale, Elisa; Singh Atwal, Ranjit; Gillis, Tammy; Mysore, Jayalakshmi; Li, Jian-hong; Seong, IhnSik; Shen, Yiping; Chen, Xiaoli; Wheeler, Vanessa C; MacDonald, Marcy E; Gusella, James F; Akimov, Sergey; Arbez, Nicolas; Juopperi, Tarja; Ratovitski, Tamara; Chiang, Jason H; Kim, Woon Roung; Chighladze, Eka; Watkin, Erin; Zhong, Chun; Makri, Georgia; Cole, Robert N; Margolis, Russell L; Song, Hongjun; Ming, Guoli; Ross, Christopher A; Kaye, Julia A; Daub, Aaron; Sharma, Punita; Mason, Amanda R; Finkbeiner, Steven; Yu, Junying; Thomson, James A; Rushton, David; Brazier, Stephen P; Battersby, Alysia A; Redfern, Amanda; Tseng, Hsui-Er; Harrison, Alexander W; Kemp, Paul J; Allen, Nicholas D; Onorati, Marco; Castiglioni, Valentina; Cattaneo, Elena; Arjomand, Jamshid

2013-01-01

Huntington's disease (HD) is an inherited neurodegenerative disorder caused by an expanded stretch of CAG trinucleotide repeats that results in neuronal dysfunction and death. Here, the HD consortium reports the generation and characterization of 14 induced pluripotent stem cell (iPSC) lines from HD patients and controls. Microarray profiling revealed CAG expansion-associated gene expression patterns that distinguish patient lines from controls, and early onset versus late onset HD. Differentiated HD neural cells showed disease associated changes in electrophysiology, metabolism, cell adhesion, and ultimately cell death for lines with both medium and longer CAG repeat expansions. The longer repeat lines were however the most vulnerable to cellular stressors and BDNF withdrawal using a range of assays across consortium laboratories. The HD iPSC collection represents a unique and well-characterized resource to elucidate disease mechanisms in HD and provides a novel human stem cell platform for screening new candidate therapeutics. PMID:22748968

Herpesvirus papio: state and properties of intracellular viral DNA in baboon lymphoblastoid cell lines.

PubMed

Falk, L; Lindahl, T; Bjursell, G; Klein, G

1979-07-15

Herpesvirus papio (HVP) is an indigenous B-lymphotropic virus of baboons (Papio sp.) present in latent form in baboon lymphoblastoid cell lines. It shares cross-reacting viral capsid and early antigens with the Epstein-Barr virus (EBV), and HVP DNA and EBV DNA show partial sequence homology. EBV-specific complementary RNA was employed here as a probe to investigate the physical state of the HVP DNA component in baboon lymphoblastoid cells after fractionation of cellular DNA by density gradient centrifugation. Five virus-producing cultures contained both free and integrated HVP DNA sequences while one non-producing cell line had two or three viral genome equivalents per cell in an apparently integrated form. Further analysis of one virus-producing line showed that the free HVP DNA fraction was composed of both linear and circular viral DNA. Contour length measurements of HVP circular DNA molecules by electron microscopy revealed that they were similar in length to the EBV circular DNA present in human lymphoblastoid cells.

Investigation of imatinib loaded surface decorated biodegradable nanocarriers against glioblastoma cell lines: Intracellular uptake and cytotoxicity studies.

PubMed

Khan, Abrar M; Ahmad, Farhan Jalees; Panda, Amulya K; Talegaonkar, Sushama

2016-06-30

Overexpression of P-glycoprotein (P-gp) efflux transporter in glioma cells thwarts the build-up of therapeutic concentration of drugs usually resulting into poor therapeutic outcome. To surmount aforesaid challenge, Imatinib (IMM) loaded Poly-lactide-co-glycolic acid nanoparticles (IMM-PLGA-NPs) were developed and optimized by Box Behnken Design as a new treatment stratagem in malignant glioma. Optimized NPs were functionalized with Pluronic(®) P84, P-gp inhibitor (IMM-PLGA-P84-NPs) which showed size, PDI, zeta potential, drug loading, 182.63±13.56nm, 0.196±0.021, -15.2±1.49mV, 40.63±2.04μg/mg, respectively. Intracellular uptake study conducted on A172, U251MG and C6 glioma cells demonstrated significantly high uptake of IMM through NPs when compared with IMM solution (IMM-S), p<0.001. IMM-PLGA-P84-NPs showed better uptake in P-gp expressing cell line (U251MG and C6) while uncoated NPs showed higher uptake in non-P-gp expressing cell line (A-172). Cytotoxicity studies demonstrated significantly low IC50 for both IMM-PLGA-NPs and IMM-PLGA-P84-NPs when compared with IC50 of IMM-S. IMM-PLGA-P84-NPs showed a significantly low IC50 against P-gp overexpressing cell lines when compared with IC50 of IMM-PLGA-NPs. In contrary, IMM-PLGA-NPs showed lower IC50 against non P-gp expressing cell line. This study demonstrated the feasibility of targeting surface decorated NPs to multidrug resistant gliomas. However, to address its clinical utility extensive in vivo studies are required. Copyright © 2016 Elsevier B.V. All rights reserved.

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

PubMed

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

2007-11-01

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

Establishment of a new human pre-B acute lymphoblastic leukemia cell line (KMO-90) with 1;19 translocation carrying p53 gene alterations.

PubMed

Sotomatsu, M; Hayashi, Y; Kawamura, M; Yugami, S; Shitara, T

1993-10-01

A new human pre-B acute lymphoblastic leukemia cell line (KMO-90) was established from the bone marrow sample of a 12-year-old girl with acute lymphoblastic leukemia (ALL) carrying 1;19 chromosome translocation. KMO-90 cells expressed HLA-DR, CD10, CD19, and CD22 antigens. These cells had also cytoplasmic immunoglobulin lacking surface immunoglobulin, indicating that these had a pre-B phenotype. Chromosome analysis of this cell line showed 48, XX, +8, +19, t(1;19)(q23;p13). Southern blot analysis showed the same sized rearrangements of the E2A gene in KMO-90 cells as those in the original leukemic cells. By means of reverse transcriptase-polymerase chain reaction analysis, we detected E2A/PBX1 fusion transcripts in KMO-90 cells. KMO-90 is useful when studying the role of the 1;19 translocation in the etiology of pre-B ALL. Furthermore, we studied alterations of the p53 gene in this cell line by polymerase chain reaction, single-strand conformation polymorphism analysis. KMO-90 cells were identified to have a point mutation at codon 177 (CCC-->TCC) of the p53 gene, suggesting that alterations of the p53 gene may have an important role in the establishment of this cell line.

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

PubMed Central

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

2015-01-01

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

6-Gingerol Mediates its Anti Tumor Activities in Human Oral and Cervical Cancer Cell Lines through Apoptosis and Cell Cycle Arrest.

PubMed

Kapoor, Vaishali; Aggarwal, Sadhna; Das, Satya N

2016-04-01

6-Gingerol, a potent nutraceutical, has been shown to have antitumor activity in different tumors, although its mechanism of action is not well understood. In this study, we evaluated antitumor activities of 6-gingerol on human oral (SCC4, KB) and cervical cancer (HeLa) cell lines with or without wortmannin, rapamycin, and cisplatin. Tumor cell proliferation was observed using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium, inner salt assay, cell cycle analysis by propidium iodide labeling and flow cytometry, apoptosis by Annexin-V binding assay, and caspase activity by chemiluminescence assay. 6-Gingerol showed dose-dependent cytotoxicity in all three cell lines. Combinations of 6-gingerol with wortmannin and cisplatin showed additive effects, while with rapamycin, it showed 50% cytotoxicity that was equivalent to IC50 of 6-gingerol alone. Treatment with 6-gingerol resulted in G2-phase arrest in KB and HeLa cells and S-phase arrest in SCC4 cells. 6-Gingerol, wortmannin, and rapamycin treatment showed almost two-fold higher expression of caspase 3 in all cell lines. The results imply that 6-gingerol either alone or in combination with PI-3 K inhibitor and cisplatin may provide better therapeutic effects in oral and cervical carcinoma. Thus, 6-gingerol appears to be a safe and potent chemotherapeutic/chemopreventive compound acting through cell cycle arrest and induction of apoptosis in human oral and cervical tumor cells. Copyright © 2016 John Wiley & Sons, Ltd.

[Expression of Chemokine receptor CXCR6 and its significance in breast cancer cell lines].

PubMed

Cheng, Hao; Chen, Nian-yong

2014-05-01

To detect the expression of Chemokine receptor CXCR6 in invasive breast cancer cell lines and normal mammary epithelial cell line, and assess the relationship between CXCR6 expression and malignant behavior of breast cancer cells. Expression level of CXCR6 in different invasive breast cancer cell lines (SK-BR-3, MCF-7, MDA-MB-231) and normal mammary epithelial cell line (MCF-10A)was detected by real time reverse transcription-polymerase chain reaction (real time-PCR) and Western blot. Lentivirus was employed to interfere CXCR6 expression in MDA-MB-231. MTT assay and transwell chamber were used to study proliferative and invasive ability of those cells respectively. Vascular enothelial growth factor (VEGF) expression was detected to study the role of CXCR6 in angiogenesis. At both mRNA level and protein level, normal mammary epithelial cell line MCF-10A showed the weakest CXCR6 expression. The breast cancer cell lines expressed CXCR6 in different levels, the expression level of CXCR6 in highly invasive cell line MDA-MB-231 was significantly higher than that in two low-invasive cell lines SK-BR-3 and MCF-7 (P < 0.05). Silencing CXCR6 gene by Lentivirus-mediated RNA interference in MDA-MB-231 inhibited its proliferation ability, invasion ability and angiogenesis ability in vitro (P < 0.05). Different invasive breast cancer cell lines express CXCR6 at different levels, positively correlated with its invasive ability.

The antiproliferative effect of acridone alkaloids on several cancer cell lines.

PubMed

Kawaii, S; Tomono, Y; Katase, E; Ogawa, K; Yano, M; Takemura, Y; Ju-ichi, M; Ito, C; Furukawa, H

1999-04-01

Fifteen acridone alkaloids were examined for their antiproliferative activity toward monolayers and suspension of several types of cancer and normal human cell lines. As a result, atalaphyllidine (9), 5-hydroxy-N-methylseverifoline (11), atalaphyllinine (12), and des-N-methylnoracronycine (13) showed potent antiproliferative activity against tumor cell lines, whereas they have weak cytotoxicity on normal human cell lines. The structure-activity relationship established from the results revealed that a secondary amine, hydroxyl groups at C-1 and C-5, and a prenyl group at C-2 played an important role for antiproliferative activities of the tetracyclic acridones.

Human metastatic melanoma cell lines express high levels of growth hormone receptor and respond to GH treatment

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

Sustarsic, Elahu G.; Department of Biological Sciences, Ohio University, Athens, OH; Junnila, Riia K.

2013-11-08

Highlights: •Most cancer types of the NCI60 have sub-sets of cell lines with high GHR expression. •GHR is highly expressed in melanoma cell lines. •GHR is elevated in advanced stage IV metastatic tumors vs. stage III. •GH treatment of metastatic melanoma cell lines alters growth and cell signaling. -- Abstract: Accumulating evidence implicates the growth hormone receptor (GHR) in carcinogenesis. While multiple studies show evidence for expression of growth hormone (GH) and GHR mRNA in human cancer tissue, there is a lack of quantification and only a few cancer types have been investigated. The National Cancer Institute’s NCI60 panel includesmore » 60 cancer cell lines from nine types of human cancer: breast, CNS, colon, leukemia, melanoma, non-small cell lung, ovarian, prostate and renal. We utilized this panel to quantify expression of GHR, GH, prolactin receptor (PRLR) and prolactin (PRL) mRNA with real-time RT qPCR. Both GHR and PRLR show a broad range of expression within and among most cancer types. Strikingly, GHR expression is nearly 50-fold higher in melanoma than in the panel as a whole. Analysis of human metastatic melanoma biopsies confirmed GHR gene expression in melanoma tissue. In these human biopsies, the level of GHR mRNA is elevated in advanced stage IV tumor samples compared to stage III. Due to the novel finding of high GHR in melanoma, we examined the effect of GH treatment on three NCI60 melanoma lines (MDA-MB-435, UACC-62 and SK-MEL-5). GH increased proliferation in two out of three cell lines tested. Further analysis revealed GH-induced activation of STAT5 and mTOR in a cell line dependent manner. In conclusion, we have identified cell lines and cancer types that are ideal to study the role of GH and PRL in cancer, yet have been largely overlooked. Furthermore, we found that human metastatic melanoma tumors express GHR and cell lines possess active GHRs that can modulate multiple signaling pathways and alter cell proliferation. Based on this data, GH could be a new therapeutic target in melanoma.« less

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.

Delayed luminescence to monitor programmed cell death induced by berberine on thyroid cancer cells

NASA Astrophysics Data System (ADS)

Scordino, Agata; Campisi, Agata; Grasso, Rosaria; Bonfanti, Roberta; Gulino, Marisa; Iauk, Liliana; Parenti, Rosalba; Musumeci, Francesco

2014-11-01

Correlation between apoptosis and UVA-induced ultraweak photon emission delayed luminescence (DL) from tumor thyroid cell lines was investigated. In particular, the effects of berberine, an alkaloid that has been reported to have anticancer activities, on two cancer cell lines were studied. The FTC-133 and 8305C cell lines, as representative of follicular and anaplastic thyroid human cancer, respectively, were chosen. The results show that berberine is able to arrest cell cycle and activate apoptotic pathway as shown in both cell lines by deoxyribonucleic acid fragmentation, caspase-3 cleavage, p53 and p27 protein overexpression. In parallel, changes in DL spectral components after berberine treatment support the hypothesis that DL from human cells originates mainly from mitochondria, since berberine acts especially at the mitochondrial level. The decrease of DL blue component for both cell lines could be related to the decrease of intra-mitochondrial nicotinamide adenine dinucleotide and may be a hallmark of induced apoptosis. In contrast, the response in the red spectral range is different for the two cell lines and may be ascribed to a different iron homeostasis.

Establishment, characterization, virus susceptibility and transfection of cell lines from cobia, Rachycentron canadum (L.), brain and fin.

PubMed

Cheng, T-C; Lai, Y-S; Lin, I-Y; Wu, C-P; Chang, S-L; Chen, T-I; Su, M-S

2010-02-01

Establishment and characterization of two cobia, Rachycentron canadum, cell lines derived from cobia brain (CB) and cobia fin (CF) are described. Caudal fin and brain from juvenile cobia were dissociated for 30 and 10 min, respectively, in phosphate-buffered saline containing 0.25% trypsin at 25 degrees C. The optimal culture condition for both dissociated cells (primary cell culture) was at 28 degrees C in Leibovitz-15 medium containing 10% foetal bovine serum. The cells have been sub-cultured at a ratio of 1:2 for more than 160 passages over a period of 3 years. Origin of the cultured cells was verified by comparison of their sequences of mitochondrial cytochrome oxidase subunit I genes (cox I) with the cox 1 sequence from cobia muscle tissue. The cell lines showed polyploidy. No mycoplasma contamination was detected. Susceptibility to grouper iridovirus was observed for the CB cell line but not the CF cell line. Both cell lines expressed green fluorescent protein after being transfected with green fluorescent reporter gene driven by the cytomegalovirus promoter.

Induction of oncogene addiction shift to NF-{kappa}B by camptothecin in solid tumor cells

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

Togano, Tomiteru; Sasaki, Masataka; Watanabe, Mariko

2009-12-04

The biological basis of the resistance of solid tumor cells to chemotherapy is not well understood. While addressing this problem, we found that gastric cancer cell line St-4/CPT, lung cancer cell line A549/CPT, and colon cancer cell line HT-29/CPT, all of which are resistant to camptothecin (CPT), showed strong and constitutive nuclear factor (NF)-{kappa}B activity driven by I{kappa}B kinase compared with their parental cell lines St-4, A549, and HT-29. A new NF-{kappa}B inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), reduced viability and induced apoptosis in St-4/CPT, A549/CPT, and HT-29/CPT cell lines, while their parental cell lines were resistant to DHMEQ. The results in thismore » study present an example of the shift in signals that support the survival of solid tumor cells to NF-{kappa}B during the acquisition of resistance to CPT. The results also indicate that solid tumor cells that become resistant to chemotherapy may be more easily treated by NF-{kappa}B inhibitors.« less

Effects of neratinib and combination with irradiation and chemotherapy in head and neck cancer cells.

PubMed

Schneider, S; Thurnher, D; Kadletz, L; Seemann, R; Brunner, M; Kotowski, U; Schmid, R; Lill, C; Heiduschka, G

2016-11-01

Prognosis of patients with head and neck squamous cell carcinoma (HNSCC) is still poor. Novel therapeutic approaches are of great interest to improve the effects of radiochemotherapy. We evaluated the effects of tyrosine kinase inhibitor neratinib on HNSCC cell lines CAL27, SCC25 and FaDu as a single agent and in combination with irradiation and chemotherapy. Effects of neratinib were evaluated in HNSCC cell lines CAL27, SCC25 and FaDu. Effect on cell viability of neratinib and combination with cisplatin and irradiation was measured using CCK-8 assays and clonogenic assays. Western blot analysis was performed to distinguish the effect on epithelial growth factor receptor and HER2 expression. Apoptosis was evaluated by flow cytometry analysis. Growth inhibition was achieved in all cell lines, whereas combination of cisplatin and neratinib showed greater inhibition than each agent alone. Apoptosis was induced in all cell lines. Combination of neratinib with irradiation or cisplatin showed significantly increased apoptosis. In clonogenic assays, significant growth inhibition was observed in all investigated cell lines. Neratinib, as a single agent or in combination with chemo-irradiation, may be a promising treatment option for patients with head and neck cancer. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

A framework for identification of actionable cancer genome dependencies in small cell lung cancer

PubMed Central

Sos, Martin L.; Dietlein, Felix; Peifer, Martin; Schöttle, Jakob; Balke-Want, Hyatt; Müller, Christian; Koker, Mirjam; Richters, André; Heynck, Stefanie; Malchers, Florian; Heuckmann, Johannes M.; Seidel, Danila; Eyers, Patrick A.; Ullrich, Roland T.; Antonchick, Andrey P.; Vintonyak, Viktor V.; Schneider, Peter M.; Ninomiya, Takashi; Waldmann, Herbert; Büttner, Reinhard; Rauh, Daniel; Heukamp, Lukas C.; Thomas, Roman K.

2012-01-01

Small cell lung cancer (SCLC) accounts for about 15% of all lung cancers. The prognosis of SCLC patients is devastating and no biologically targeted therapeutics are active in this tumor type. To develop a framework for development of specific SCLC-targeted drugs we conducted a combined genomic and pharmacological vulnerability screen in SCLC cell lines. We show that SCLC cell lines capture the genomic landscape of primary SCLC tumors and provide genetic predictors for activity of clinically relevant inhibitors by screening 267 compounds across 44 of these cell lines. We show Aurora kinase inhibitors are effective in SCLC cell lines bearing MYC amplification, which occur in 3–7% of SCLC patients. In MYC-amplified SCLC cells Aurora kinase inhibition associates with G2/M-arrest, inactivation of PI3-kinase (PI3K) signaling, and induction of apoptosis. Aurora dependency in SCLC primarily involved Aurora B, required its kinase activity, and was independent of depletion of cytoplasmic levels of MYC. Our study suggests that a fraction of SCLC patients may benefit from therapeutic inhibition of Aurora B. Thus, thorough chemical and genomic exploration of SCLC cell lines may provide starting points for further development of rational targeted therapeutic intervention in this deadly tumor type. PMID:23035247

Low-dose non-targeted radiation effects in human esophageal adenocarcinoma cell lines.

PubMed

Hanu, Christine; Wong, Raimond; Sur, Ranjan K; Hayward, Joseph E; Seymour, Colin; Mothersill, Carmel

2017-02-01

To investigate non-targeted radiation effects in esophageal adenocarcinoma cell lines (OE19 and OE33) using human keratinocyte and colorectal cancer cell reporters following γ-ray exposure. Both clonogenic assays and ratiometric calcium endpoints were used to check for the occurrence of bystander signals in reporter cells. We report data suggesting that γ-irradiation increases cell killing over the expected linear quadratic (LQ) model levels in the OE19 cell line exposed to doses below 1 Gy, i.e. which may be suggestive to be a low hyper-radiosensitive (HRS) response to direct irradiation. Both EAC cell lines (OE19 and OE33) have the ability to produce bystander signals when irradiated cell conditioned medium (ICCM) is placed onto human keratinocyte reporters, but do not seem to be capable of responding to bystander signals when placed on their autologous reporters. Further work with human keratinocyte reporter models showed statistically significant intracellular calcium fluxes following exposure of the reporters to ICCM harvested from both EAC cell lines exposed to 0.5 Gy. These experiments suggest that the OE19 and OE33 cell lines produce bystander signals in human keratinocyte reporter cells. However, the radiosensitivity of the EAC cell lines used in this study cannot be enhanced by the bystander response since both cell lines could not respond to bystander signals.

Effect of histone deacetylase inhibitors trichostatin A and valproic acid on hair cell regeneration in zebrafish lateral line neuromasts

PubMed Central

He, Yingzi; Cai, Chengfu; Tang, Dongmei; Sun, Shan; Li, Huawei

2014-01-01

In humans, auditory hair cells are not replaced when injured. Thus, cochlear hair cell loss causes progressive and permanent hearing loss. Conversely, non-mammalian vertebrates are capable of regenerating lost sensory hair cells. The zebrafish lateral line has numerous qualities that make it well-suited for studying hair cell development and regeneration. Histone deacetylase (HDAC) activity has been shown to have an important role in regenerative processes in vertebrates, but its function in hair cell regeneration in vivo is not fully understood. Here, we have examined the role of HDAC activity in hair cell regeneration in the zebrafish lateral line. We eliminated lateral line hair cells of 5-day post-fertilization larvae using neomycin and then treated the larvae with HDAC inhibitors. To assess hair cell regeneration, we used 5-bromo-2-deoxyuridine (BrdU) incorporation in zebrafish larvae to label mitotic cells after hair cell loss. We found that pharmacological inhibition of HDACs using trichostatin A (TSA) or valproic acid (VPA) increased histone acetylation in the regenerated neuromasts following neomycin-induced damage. We also showed that treatment with TSA or VPA decreased the number of supporting cells and regenerated hair cells in response to hair cell damage. Additionally, BrdU immunostaining and western blot analysis showed that TSA or VPA treatment caused a significant decrease in the percentage of S-phase cells and induced p21Cip1 and p27Kip1 expression, both of which are likely to explain the decrease in the amount of newly regenerated hair cells in treated embryos. Finally, we showed that HDAC inhibitors induced no observable cell death in neuromasts as measured by cleaved caspase-3 immunohistochemistry and western blot analysis. Taken together, our results demonstrate that HDAC activity has an important role in the regeneration of hair cells in the lateral line. PMID:25431550

Polyketide Derivatives from Annona muricata Linn Leaves as Potencial Anticancer Material by Combination Treatment With Doxorubicin on Hela Cell Line

NASA Astrophysics Data System (ADS)

Artanti, A. N.; Astirin, O. P.; Prayito, A.; Widiyaningsih, R. F.; Prihapsara, F.

2017-02-01

One of the compounds found effication as an anticancer agent on cervical cancer is acetogenin, a polyketide compound that is abundant in Annona muricata L. leaves. This study has been done to examine polyketide derivatives was isolated from Annona muricata L. which has potency to induce apoptosis by p53 expression on hela cell line. An approach recently develop to overcome side effect of chemoterapeutic agent is used of combined chemoterapeutic agent, i.e doxorubicin. The determination of cytotoxic combination activity from polyketide derivative and doxorubicin was evaluated using MTT assay to obtain the value of CI (combination index). The expression of p53 profile was evaluated by immunohistochemistry on hela cell line. Data analysis showed that combination of polyketide derivative from Annona muricata L. (38,5 µg/ml) and doxorubicin with all of concentration performed synergistic effect on hela cell line with CI value from 0,33 - 0,65. The analysis on immucytochemistry showed that polyketide derivative from Annona muricata L. leaves could enhance p53 pathway significantly on hela cell line.

Synthesis of novel lipoamino acid conjugates of sapienic acid and evaluation of their cytotoxicity activities.

PubMed

Gopal, Sanganamoni Chinna; Kaki, Shiva Shanker; Rao, Bhamidipati V S K; Poornachandra, Yedla; Kumar, Chityal Ganesh; Narayana Prasad, Rachapudi Badari

2014-01-01

Novel lipoamino acids were prepared with the coupling of sapienic acid [(Z)-6-hexadecenoic acid] with α - amino group of amino acids and the resulting N-sapienoyl amino acids were tested for their cytotoxicity activities against four cancer based cell lines. Initially, sapienic acid was synthesized by the Wittig coupling of triphenylphosphonium bromide salt of 6-bromohexanoic acid and decanal with a Z specific reagent. The prepared sapienic acid was subsequently converted to its acid chloride which was further coupled with amino acids by the Schotten-Baumann reaction to form N-sapienoyl amino acid conjugates. Structural characterization of the prepared N-sapienoyl amino acid derivatives was done by spectral data (IR, mass spectra and NMR). These lipoamino acid derivatives were screened for in vitro cytotoxicity evaluation. Cytotoxicity evaluation against four cancer cell lines showed that N-sapienoyl isoleucine was active against three cell lines whereas other derivatives either showed activity against only one or two cell lines with very moderate activity and two derivatives were observed to be inactive against the tested cell lines.

Cisplatin resistance induced in germ cell tumour cells is due to reduced susceptibility towards cell death but not to altered DNA damage induction or repair.

PubMed

Fenske, Annabelle E; Glaesener, Stephanie; Bokemeyer, Carsten; Thomale, Juergen; Dahm-Daphi, Jochen; Honecker, Friedemann; Dartsch, Dorothee C

2012-11-28

To identify factors involved in cisplatin (CDDP) resistance of germ cell tumours (GCTs), we exposed NTERA-2 cells, and the platinum-adapted subline NTERA-2R to CDDP and compared their response. While both cell lines showed comparable proliferation, NTERA-2R cells were clearly more resistant to the drug than the parental NTERA-2 cell line. Interestingly, the two lines showed identical extent of DNA adduct formation and elimination, indicating that neither changes in CDDP uptake, nor altered drug efflux, DNA binding, or repair caused the difference in resistance. Similarly, no difference occurred in the time-course of γH2AX formation, which was not linked to 53BP1 accumulation. In contrast, NTERA-2R cells showed a more pronounced dose-dependent S phase delay, a transient G(2)/M-block, and subsequent release into immediate cell death. We thus conclude that the enhanced resistance against CDDP is linked to reduced susceptibility to cell death rather than to an altered DNA adduct formation or adduct removal. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Characterization of resistance to rhabdovirus and retrovirus infection in a human myeloid cell line.

PubMed

Boso, Guney; Somia, Nikunj V

2015-01-01

Viruses interact with various permissive and restrictive factors in host cells throughout their replication cycle. Cell lines that are non-permissive to viral infection have been particularly useful in discovering host cell proteins involved in viral life cycles. Here we describe the characterization of a human myeloid leukemia cell line, KG-1, that is resistant to infection by retroviruses and a Rhabdovirus. We show that KG-1 cells are resistant to infection by Vesicular Stomatits Virus as well as VSV Glycoprotein (VSVG) pseudotyped retroviruses due to a defect in binding. Moreover our results indicate that entry by xenotropic retroviral envelope glycoprotein RD114 is impaired in KG-1 cells. Finally we characterize a post- entry block in the early phase of the retroviral life cycle in KG-1 cells that renders the cell line refractory to infection. This cell line will have utility in discovering proteins involved in infection by VSV and HIV-1.

Establishment of an ASPL-TFE3 renal cell carcinoma cell line (S-TFE).

PubMed

Hirobe, Megumi; Masumori, Naoya; Tanaka, Toshiaki; Kitamura, Hiroshi; Tsukamoto, Taiji

2013-06-01

Xp11 translocation renal cell carcinoma is a rare disease diagnosed in children and adolescents in the advanced stage with an aggressive clinical course. Various gene fusions including the transcription factor E3 (TFE3) gene located on chromosome X cause the tumor. We established an Xp11 translocation renal cell carcinoma cell line from a renal tumor in a 18-y-old Japanese female and named it "S-TFE." The cell line and its xenograft demonstrated definite gene fusion including TFE3. They showed strong nuclear staining for TFE3 in immunohistochemistry, TFE3 gene rearrangement in dual-color, break-apart FISH analysis and ASPL-TFE3 type 1 fusion transcripts detected by RT-PCR and direct DNA sequencing. Although many renal cell carcinoma cell lines have been established and investigated, only a few cell lines are recognized as Xp11.2 translocation carcinoma. S-TFE will be useful to examine the characteristics and drug susceptibility of Xp11 translocation renal cell carcinoma.

Establishment of an ASPL-TFE3 renal cell carcinoma cell line (S-TFE)

PubMed Central

Hirobe, Megumi; Masumori, Naoya; Tanaka, Toshiaki; Kitamura, Hiroshi; Tsukamoto, Taiji

2013-01-01

Xp11 translocation renal cell carcinoma is a rare disease diagnosed in children and adolescents in the advanced stage with an aggressive clinical course. Various gene fusions including the transcription factor E3 (TFE3) gene located on chromosome X cause the tumor. We established an Xp11 translocation renal cell carcinoma cell line from a renal tumor in a 18-y-old Japanese female and named it “S-TFE.” The cell line and its xenograft demonstrated definite gene fusion including TFE3. They showed strong nuclear staining for TFE3 in immunohistochemistry, TFE3 gene rearrangement in dual-color, break-apart FISH analysis and ASPL-TFE3 type 1 fusion transcripts detected by RT-PCR and direct DNA sequencing. Although many renal cell carcinoma cell lines have been established and investigated, only a few cell lines are recognized as Xp11.2 translocation carcinoma. S-TFE will be useful to examine the characteristics and drug susceptibility of Xp11 translocation renal cell carcinoma. PMID:23760492

Host cell reactivation of gamma-irradiated adenovirus 5 in human cell lines of varying radiosensitivity.

PubMed Central

Eady, J. J.; Peacock, J. H.; McMillan, T. J.

1992-01-01

DNA repair processes play an important role in the determination of radiation response in both normal and tumour cells. We have investigated one aspect of DNA repair in a number of human cell lines of varying radiosensitivity using the adenovirus 5 host cell reactivation assay (HCR). In this technique, gamma-irradiated virions are used to infect cells and the ability of the cellular repair systems to process this damage is assayed by a convenient immunoperoxidase method recognising viral structural antigen expression on the cell membrane 48 h after infection. Reduced HCR was exhibited by radioresistant HeLa cells and by a radiosensitive neuroblastoma cell line, HX142. In contrast, an ataxia telangiectasia cell line, AT5 BIVA, did not show reduced HCR. On the basis of these results we can make no general conclusions about the relevance of HCR to cellular radiosensitivity. We have extended these studies to determine whether our cell lines exhibited enhanced viral reactivation (ER) following a small priming dose of gamma-radiation given to the cells before viral infection. No evidence for this phenomenon was found either in normal or tumour cell lines. PMID:1637659

Bioactivity-guided isolation of anticancer agents from Bauhinia kockiana Korth.

PubMed

Chew, Yik Ling; Lim, Yau Yan; Stanslas, Johnson; Ee, Gwendoline Cheng Lian; Goh, Joo Kheng

2014-01-01

Flowers of Bauhinia kockiana were investigated for their anticancer properties. Gallic acid (1), and methyl gallate (2), were isolated via bioassay-directed isolation, and they exhibited anticancer properties towards several cancer cell lines, examined using MTT cell viability assay. Pyrogallol (3) was examined against the same cancer cell lines to deduce the bioactive functional group of the phenolic compounds. The results showed that the phenolic compounds could exhibit moderate to weak cytotoxicity towards certain cell lines (GI50 30 - 86 µM), but were inactive towards DU145 prostate cancer cell (GI50 > 100 µM). It was observed that pyrogallol moiety was one of the essential functional structures of the phenolic compounds in exhibiting anticancer activity. Also, the carboxyl group of compound 1 was also important in anticancer activity. Examination of the PC-3 cells treated with compound 1 using fluorescence microscopy showed that PC-3 cells were killed by apoptosis.

Effect of verteporfin-PDT on the Notch signaling pathway in cholangiocarcinoma (CCA) cell lines

NASA Astrophysics Data System (ADS)

Cerec, Virginie; Andreola, Fausto; Pereira, Stephen P.

2009-06-01

Accumulating preclinical and clinical evidence supports a pro-oncogenic function for Notch signaling in several solid tumors. Therefore, Notch inhibitory agents, such as gamma-secretase inhibitors (GSI), are being investigated as cancer therapeutic agents and a potential adjuvant to conventional chemo/radiotherapy. To date, no in vitro data are available on the cellular response and effect of either photodynamic therapy (PDT) or GSI on human cholangiocarcinoma (CCA). Consequently, we aimed to study the: (i) constitutive expression of Notch signaling pathway in CCA cell lines; (ii) response to Verteporfin-PDT and to GSI, as single agents on CCA cell lines; (iii) effect of Verteporfin-PDT on Notch signaling pathway expression. Expression of Notch signaling components was studied in two cholangiocarcinoma cell lines, HuCCT1 and TFK-1 (intra- and extrahepatic, respectively). No difference in basal expression of Notch1, 2 and Jagged1 was observed in either cell line. In contrast, Notch3 was found to be weakly and highly expressed in HuCCT1 and TFK-1 cells, respectively - supporting our recent microarray data which showed Notch3 overexpression in biliary brushings from patients with extrahepatic CCA. HuCCT1 and TFK-1 differentially responded to Verteporfin-PDT treatment; preliminary data showed no clear effect of GSI on proliferation/apoptosis in either cell line following short exposure (6 and 24h). Following Verteporfin-PDT, Notch1, 2 and Jagged-1 expression was down-regulated in both cell lines, while Notch3 expression was unaffected in HuCCT1 cells and down-regulated in TFK-1 cells. The Notch signaling pathway could represent a potential target for combination therapy in CCA treatment.

Nicotine transport in lung and non-lung epithelial cells.

PubMed

Takano, Mikihisa; Kamei, Hidetaka; Nagahiro, Machi; Kawami, Masashi; Yumoto, Ryoko

2017-11-01

Nicotine is rapidly absorbed from the lung alveoli into systemic circulation during cigarette smoking. However, mechanism underlying nicotine transport in alveolar epithelial cells is not well understood to date. In the present study, we characterized nicotine uptake in lung epithelial cell lines A549 and NCI-H441 and in non-lung epithelial cell lines HepG2 and MCF-7. Characteristics of [ 3 H]nicotine uptake was studied using these cell lines. Nicotine uptake in A549 cells occurred in a time- and temperature-dependent manner and showed saturation kinetics, with a Km value of 0.31mM. Treatment with some organic cations such as diphenhydramine and pyrilamine inhibited nicotine uptake, whereas treatment with organic cations such as carnitine and tetraethylammonium did not affect nicotine uptake. Extracellular pH markedly affected nicotine uptake, with high nicotine uptake being observed at high pH up to 11.0. Modulation of intracellular pH with ammonium chloride also affected nicotine uptake. Treatment with valinomycin, a potassium ionophore, did not significantly affect nicotine uptake, indicating that nicotine uptake is an electroneutral process. For comparison, we assessed the characteristics of nicotine uptake in another lung epithelial cell line NCI-H441 and in non-lung epithelial cell lines HepG2 and MCF-7. Interestingly, these cell lines showed similar characteristics of nicotine uptake with respect to pH dependency and inhibition by various organic cations. The present findings suggest that a similar or the same pH-dependent transport system is involved in nicotine uptake in these cell lines. A novel molecular mechanism of nicotine transport is proposed. Copyright © 2017 Elsevier Inc. All rights reserved.

Synthesis and anti-tumor evaluation of panaxadiol halogen-derivatives.

PubMed

Xiao, Shengnan; Chen, Shuai; Sun, Yuanyuan; Zhou, Wuxi; Piao, Huri; Zhao, Yuqing

2017-09-01

In the current work, 13 novel panaxadiol (PD) derivatives were synthesized by reacting with chloroacetyl chloride and bromoacetyl bromide. Their in vitro antitumor activities were evaluated on three human tumor cell lines (HCT-116, BGC-823, SW-480) and three normal cells (human gastric epithelial cell line-GES-1, hair follicle dermal papilla cell line-HHDPC and rat myocardial cell line-H9C2) by MTT assay. Compared with PD, the results demonstrated that compound 1e, 2d, 2e showed significant anti-tumor activity against three tumor cell lines, the IC50 value of compound 2d against HCT-116 was the lowest (3.836μM). The anti-tumor activity of open-ring compounds are significantly better than the compounds of C-25 cyclization. Compound 1f, 2f, 2g showed the strong anti-tumor activity. The IC50 value of compound 2g against BGC-823 and SW-480 were the lowest (0.6μM and 0.1μM, respectively). Combined with cytotoxicity test, the IC50 value of compound 1e, 2d, 2e are greater than 100. the open-ring compounds (1f, 2f, 2g) showed a strong toxicity. The toxicity of 1f is lower than 2f and 2g. These compounds may be useful for the development of novel antiproliferative agents. Copyright © 2017. Published by Elsevier Ltd.

Cell line specific modulation of connexin43 expression after exposure to ionizing radiation.

PubMed

Banaz-Yaşar, Ferya; Tischka, Rabea; Iliakis, George; Winterhager, Elke; Gellhaus, Alexandra

2005-01-01

Gap junctional intercellular communication plays a significant role in mediating radiation-induced bystander effects. However, the level of Cx43 itself is influenced by ionizing radiation, which could modify the bystander effect. Here we have investigated several cell lines for the modulation of Cx43 expression 24 h after irradiation with 5 Gy X-rays. The mouse endothelial cell line bEnd3 revealed a significantly elevated level of Cx43 already 15 min after exposure to X-rays, whereas human hybrid endothelial cells (EA.hy926) exhibited a transient downregulation of Cx43 mRNA. No obvious changes in the communication properties of the different cell lines could be observed after irradiation. The communication-deficient malignant human trophoblast cell line Jeg3 stably transfected with Cx43 did not reveal any induction of endogenous nor alteration in the exogenous Cx43 transcript level upon exposure to 5 Gy. Taken together, our data show a cell line specific modulation of Cx43 expression after exposure to X-rays.

Development and characterization of a hydrogen peroxide-resistant cholangiocyte cell line: A novel model of oxidative stress-related cholangiocarcinoma genesis

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

Thanan, Raynoo; Liver Fluke and Cholangiocarcinoma Research Center, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002; Techasen, Anchalee

Oxidative stress is a cause of inflammation–related diseases, including cancers. Cholangiocarcinoma is a liver cancer with bile duct epithelial cell phenotypes. Our previous studies in animal and human models indicated that oxidative stress is a major cause of cholangiocarcinoma development. Hydrogen peroxide (H{sub 2}O{sub 2}) can generate hydroxyl radicals, which damage lipids, proteins, and nucleic acids, leading to cell death. However, some cells can survive by adapting to oxidative stress conditions, and selective clonal expansion of these resistant cells would be involved in oxidative stress-related carcinogenesis. The present study aimed to establish H{sub 2}O{sub 2}-resistant cell line from an immortal cholangiocytemore » cell line (MMNK1) by chronic treatment with low-concentration H{sub 2}O{sub 2} (25 μM). After 72 days of induction, H{sub 2}O{sub 2}-resistant cell lines (ox-MMNK1-L) were obtained. The ox-MMNK1-L cell line showed H{sub 2}O{sub 2}-resistant properties, increasing the expression of the anti-oxidant genes catalase (CAT), superoxide dismutase-1 (SOD1), superoxide dismutase-2 (SOD2), and superoxide dismutase-3 (SOD3) and the enzyme activities of CAT and intracellular SODs. Furthermore, the resistant cells showed increased expression levels of an epigenetics-related gene, DNA methyltransferase-1 (DNMT1), when compared to the parental cells. Interestingly, the ox-MMNK1-L cell line had a significantly higher cell proliferation rate than the MMNK1 normal cell line. Moreover, ox-MMNK1-L cells showed pseudopodia formation and the loss of cell-to-cell adhesion (multi-layers) under additional oxidative stress (100 μM H{sub 2}O{sub 2}). These findings suggest that H{sub 2}O{sub 2}-resistant cells can be used as a model of oxidative stress-related cholangiocarcinoma genesis through molecular changes such as alteration of gene expression and epigenetic changes. - Highlights: • An H{sub 2}O{sub 2}-resistant ox-MMNK1-L cells was established from immortalized cholangiocytes. • The resistance was acquired by daily treatment of low H{sub 2}O{sub 2} (25 μM) for 15 passages. • The cells highly expressed catalase, SODs and DNMT1 with rapid cell proliferation. • Pseudopodia and the loss of cell-to-cell adhesion appeared by 100 μM H{sub 2}O{sub 2} treatment. • The resistant cells can be used as a model of oxidative stress-related carcinogenesis.« less

Giant Lysosomes as a Chemotherapy Resistance Mechanism in Hepatocellular Carcinoma Cells.

PubMed

Colombo, Federico; Trombetta, Elena; Cetrangolo, Paola; Maggioni, Marco; Razini, Paola; De Santis, Francesca; Torrente, Yvan; Prati, Daniele; Torresani, Erminio; Porretti, Laura

2014-01-01

Despite continuous improvements in therapeutic protocols, cancer-related mortality is still one of the main problems facing public health. The main cause of treatment failure is multi-drug resistance (MDR: simultaneous insensitivity to different anti-cancer agents), the underlying molecular and biological mechanisms of which include the activity of ATP binding cassette (ABC) proteins and drug compartmentalisation in cell organelles. We investigated the expression of the main ABC proteins and the role of cytoplasmic vacuoles in the MDR of six hepatocellular carcinoma (HCC) cell lines, and confirmed the accumulation of the yellow anti-cancer drug sunitinib in giant (four lines) and small cytoplasmic vacuoles of lysosomal origin (two lines). ABC expression analyses showed that the main ABC protein harboured by all of the cell lines was PGP, whose expression was not limited to the cell membrane but was also found on lysosomes. MTT assays showed that the cell lines with giant lysosomes were more resistant to sorafenib treatment than those with small lysosomes (p<0.01), and that verapamil incubation can revert this resistance, especially if it is administered after drug pre-incubation. The findings of this study demonstrate the involvement of PGP-positive lysosomes in drug sequestration and MDR in HCC cell lines. The possibility of modulating this mechanism using PGP inhibitors could lead to the development of new targeted strategies to enhance HCC treatment.

Establishment and characterization of outer root sheath (ORS) cell line from Jining grey goat.

PubMed

Cui, Zhifeng; Hu, Yanxia; Wang, Hui; Zeng, Yongqing; Dong, Bin; Zhu, Houshun; Dong, Zhongdian; Liu, Zhiyuan

2012-03-01

A new line of outer root sheath (ORS) cells was established from hair follicles of Jining grey goat by using a mechanical separation combined with enzyme digestion. Cell morphology is described at different phases. The chromosome analysis of ORS cells, identification of the ORS cells and morphological reversion test were detected at the 4th and 40th passages. The ORS cells were healthy and the growth characteristics were stable with a population doubling time of 52 h. Chromosome analysis showed that >58% of cells were diploid. Test for ORS cell line CK19 expression was positive. This newly established ORS cell line not only lays the foundation for further studying on the growth, regeneration, development law of goat hair follicle but also provides a mirror for the research of human hair in medical field.

Single Chain Antibodies as Estrogen Receptor Repressors in Breast Cancer

DTIC Science & Technology

2000-06-01

differential display we identified proteinase inhibitor-9 as an mRNA upregulated by estrogen in a human hepatoblastoma cell line (HepG2) stably transfected...antiestrogen ICI 182,780 was a pure antag- human hepatoblastoma cell line (3), contained ER (4), this cell onist. Western blot analysis showed that

Lack of differences in radiation-induced immunogenicity parameters between HPV-positive and HPV-negative human HNSCC cell lines.

PubMed

Schneider, Karolin; Bol, Vanesa; Grégoire, Vincent

2017-09-01

Clinical studies indicate that patients with HPV/p16-associated head & neck squamous cell carcinoma (HNSCC) represent a subgroup with a better prognosis and improved response to conventional radiotherapy. Involvement of immune-based factors has been hypothesized. In the present study, we investigated radiation-induced differences in release of damage associated molecular patterns (DAMPs), cytokines and activation of dendritic cells (DCs) in HPV-positive and negative HNSCC cancer cell lines. Calreticulin (CRT) exposure was detected on cancer cell surface. ATP, HMGB1 and cytokines were measured in culture supernatants. Maturation marker CD83 surface exposure was determined on DCs after co-incubation with irradiated tumor cells. There was no increase in DAMPs and cytokine profiles after radiation treatment and no difference between HPV+ and HPV- cell lines. The HPV/p16-positive SCC90 cells showed a trend for increased total CRT, HMGB1, and number of cytokines compared to all other cell lines. None of the irradiated cancer cell lines could affect DC maturation. Radiation treatment did not increase immunogenicity of HNSCC cell lines assessed by membrane CRT, ATP, HMGB1, cytokines production, and by activation of immature DCs. There was no difference between HPV-positive and HPV-negative cell lines. Copyright © 2017 Elsevier B.V. All rights reserved.

Metabolomics Analysis of Hormone-Responsive and Triple-Negative Breast Cancer Cell Responses to Paclitaxel Identify Key Metabolic Differences.

PubMed

Stewart, Delisha A; Winnike, Jason H; McRitchie, Susan L; Clark, Robert F; Pathmasiri, Wimal W; Sumner, Susan J

2016-09-02

To date, no targeted therapies are available to treat triple negative breast cancer (TNBC), while other breast cancer subtypes are responsive to current therapeutic treatment. Metabolomics was conducted to reveal differences in two hormone receptor-negative TNBC cell lines and two hormone receptor-positive Luminal A cell lines. Studies were conducted in the presence and absence of paclitaxel (Taxol). TNBC cell lines had higher levels of amino acids, branched-chain amino acids, nucleotides, and nucleotide sugars and lower levels of proliferation-related metabolites like choline compared with Luminal A cell lines. In the presence of paclitaxel, each cell line showed unique metabolic responses, with some similarities by type. For example, in the Luminal A cell lines, levels of lactate and creatine decreased while certain choline metabolites and myo-inositol increased with paclitaxel. In the TNBC cell lines levels of glutamine, glutamate, and glutathione increased, whereas lysine, proline, and valine decreased in the presence of drug. Profiling secreted inflammatory cytokines in the conditioned media demonstrated a greater response to paclitaxel in the hormone-positive Luminal cells compared with a secretion profile that suggested greater drug resistance in the TNBC cells. The most significant differences distinguishing the cell types based on pathway enrichment analyses were related to amino acid, lipid and carbohydrate metabolism pathways, whereas several biological pathways were differentiated between the cell lines following treatment.

Human renal adipose tissue induces the invasion and progression of renal cell carcinoma.

PubMed

Campo-Verde-Arbocco, Fiorella; López-Laur, José D; Romeo, Leonardo R; Giorlando, Noelia; Bruna, Flavia A; Contador, David E; López-Fontana, Gastón; Santiano, Flavia E; Sasso, Corina V; Zyla, Leila E; López-Fontana, Constanza M; Calvo, Juan C; Carón, Rubén W; Creydt, Virginia Pistone

2017-11-07

We evaluated the effects of conditioned media (CMs) of human adipose tissue from renal cell carcinoma located near the tumor (hRATnT) or farther away from the tumor (hRATfT), on proliferation, adhesion and migration of tumor (786-O and ACHN) and non-tumor (HK-2) human renal epithelial cell lines. Human adipose tissues were obtained from patients with renal cell carcinoma (RCC) and CMs from hRATnT and hRATfT incubation. Proliferation, adhesion and migration were quantified in 786-O, ACHN and HK-2 cell lines incubated with hRATnT-, hRATfT- or control-CMs. We evaluated versican, adiponectin and leptin expression in CMs from hRATnT and hRATfT. We evaluated AdipoR1/2, ObR, pERK, pAkt y pPI3K expression on cell lines incubated with CMs. No differences in proliferation of cell lines was found after 24 h of treatment with CMs. All cell lines showed a significant decrease in cell adhesion and increase in cell migration after incubation with hRATnT-CMs vs. hRATfT- or control-CMs. hRATnT-CMs showed increased levels of versican and leptin, compared to hRATfT-CMs. AdipoR2 in 786-O and ACHN cells decreased significantly after incubation with hRATfT- and hRATnT-CMs vs. control-CMs. We observed a decrease in the expression of pAkt in HK-2, 786-O and ACHN incubated with hRATnT-CMs. This result could partially explain the observed changes in migration and cell adhesion. We conclude that hRATnT released factors, such as leptin and versican, could enhance the invasive potential of renal epithelial cell lines and could modulate the progression of the disease.

Let-7 miRNA Precursors Co-express with LIN28B in Cervical Cells.

PubMed

Zamora-Contreras, Aida Margarita; Alvarez-Salas, Luis Marat

2018-01-01

The let-7 microRNAs (miRNAs) are frequently dysregulated in carcinogenic processes, including cervical cancer. LIN28 proteins regulate let-7 biogenesis by binding to conserved sequences within the pre-miRNA structure. Nevertheless, recent research has shown that some let-7 miRNAs may escape LIN28 regulation. Correlate pre-let-7 miRNAs and LIN28B levels in cervical cell lines with different malignancy and HPV content. Pre-let-7 levels were determined by RTqPCR. LIN28B and other let-7 targets were analyzed by immunoblot. In silico tools were used to correlate let-7 and LIN28B expression and to analyze prelet- 7 sequences and structures. Lin28B protein was detected in all tested cell lines although it was more expressed in tumor cell lines. High levels of pre-let-7c/f-1 and pre-miR-98 were present in almost all cell lines regardless malignancy and LIN28B expression. Pre-let-7g/i were mainly expressed in tumor cell lines, pre-let-7e and pre-let-7-a3 were absent in all cell lines and pre-let-7a-2 showed indistinct expression. LIN28B showed positive correlation with pre-let-7i/g/f-1 and pre-miR-98 in tumor cell lines, suggesting escape from regulation. Sequence alignment and analysis of pre-let-7 miRNAs showed distinctive structural features within the preE region that may influence the ideal pre-let-7 structuring for LIN28B interaction. Short preE-stems were present in pre-let-7 that may escape LIN28B regulation, but long preEstems were mostly associated with high-level pre-let-7 miRNAs. The observed differences of pre-let-7 levels in cervical cell lines may be the result of alternative preE structuring affecting interaction with LIN28B thus resulting in differential let-7 regulation. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Rewiring carbohydrate catabolism differentially affects survival of pancreatic cancer cell lines with diverse metabolic profiles

PubMed Central

Tataranni, Tiziana; Agriesti, Francesca; Ruggieri, Vitalba; Mazzoccoli, Carmela; Simeon, Vittorio; Laurenzana, Ilaria; Scrima, Rosella; Pazienza, Valerio; Capitanio, Nazzareno; Piccoli, Claudia

2017-01-01

An increasing body of evidence suggests that targeting cellular metabolism represents a promising effective approach to treat pancreatic cancer, overcome chemoresistance and ameliorate patient's prognosis and survival. In this study, following whole-genome expression analysis, we selected two pancreatic cancer cell lines, PANC-1 and BXPC-3, hallmarked by distinct metabolic profiles with specific concern to carbohydrate metabolism. Functional comparative analysis showed that BXPC-3 displayed a marked deficit of the mitochondrial respiratory and oxidative phosphorylation activity and a higher production of reactive oxygen species and a reduced NAD+/NADH ratio, indicating their bioenergetic reliance on glycolysis and a different redox homeostasis as compared to PANC-1. Both cell lines were challenged to rewire their metabolism by substituting glucose with galactose as carbon source, a condition inhibiting the glycolytic flux and fostering full oxidation of the sugar carbons. The obtained data strikingly show that the mitochondrial respiration-impaired-BXPC-3 cell line was unable to sustain the metabolic adaptation required by glucose deprivation/substitution, thereby resulting in a G2\\M cell cycle shift, unbalance of the redox homeostasis, apoptosis induction. Conversely, the mitochondrial respiration-competent-PANC-1 cell line did not show clear evidence of cell sufferance. Our findings provide a strong rationale to candidate metabolism as a promising target for cancer therapy. Defining the metabolic features at time of pancreatic cancer diagnosis and likely of other tumors, appears to be crucial to predict the responsiveness to therapeutic approaches or coadjuvant interventions affecting metabolism. PMID:28476035

Establishment and genetic characterization of ANGM-CSS, a novel, immortal cell line derived from a human glioblastoma multiforme.

PubMed

Notarangelo, Angelantonio; Trombetta, Domenico; D'Angelo, Vincenzo; Parrella, Paola; Palumbo, Orazio; Storlazzi, Clelia Tiziana; Impera, Luciana; Muscarella, Lucia Anna; La Torre, Antonella; Affuso, Andrea; Fazio, Vito Michele; Carella, Massimo; Zelante, Leopoldo

2014-03-01

Glioblastoma multiforme (World Health Organization, grade IV astrocytoma) is the most common and most aggressive malignant primary brain tumor. We report a novel cell line, designated as ANGM-CSS, which was established from a 56-year-old male patient with a surgically removed glioblastoma multiforme. The ANGM-CSS cell line was established in vitro and characterized using histological and immunohistochemical staining, classical and molecular cytogenetic analyses, molecular studies and functional assays using a xenograft model in immunodeficient animals. ANGM-CSS was positive for CD133, nestin and vimentin proteins, whereas GFAP showed staining only in a fraction of the cells. Cytogenetic and molecular cytogenetic analysis revealed a near-tetraploid karyotype, with a modal chromosome number from 88 to 91, and additional cytogenetic abnormalities, such as the t(6;14)(p12;q11.2), t(8;10)(q24.2;q21.1) and t(5;9)(q34;p21) unbalanced translocations. Moreover, ANGM-CSS showed amplification of the MET and EGFR genes whose overexpression was observed at the mRNA level. Interestingly, ANGM-CSS is tumorigenic when implanted in immunodeficient mice, and the cells obtained from the xenografts showed the same morphology and karyotype in vitro as the original cell line. ANGM-CSS represents a biologically relevant cell line to be used to investigate the molecular pathology of glioblastoma multiforme, also to evaluate the efficacy of novel therapeutic drugs in vitro.

Identification of a novel rhabdovirus in Spodoptera frugiperda cell lines.

PubMed

Ma, Hailun; Galvin, Teresa A; Glasner, Dustin R; Shaheduzzaman, Syed; Khan, Arifa S

2014-06-01

The Sf9 cell line, derived from Spodoptera frugiperda, is used as a cell substrate for biological products, and no viruses have been reported in this cell line after extensive testing. We used degenerate PCR assays and massively parallel sequencing (MPS) to identify a novel RNA virus belonging to the order Mononegavirales in Sf9 cells. Sequence analysis of the assembled virus genome showed the presence of five open reading frames (ORFs) corresponding to the genes for the N, P, M, G, and L proteins in other rhabdoviruses and an unknown ORF of 111 amino acids located between the G- and L-protein genes. BLAST searches indicated that the S. frugiperda rhabdovirus (Sf-rhabdovirus) was related in a limited region of the L-protein gene to Taastrup virus, a newly discovered member of the Mononegavirales from a leafhopper (Hemiptera), and also to plant rhabdoviruses, particularly in the genus Cytorhabdovirus. Phylogenetic analysis of sequences in the L-protein gene indicated that Sf-rhabdovirus is a novel virus that branched with Taastrup virus. Rhabdovirus morphology was confirmed by transmission electron microscopy of filtered supernatant samples from Sf9 cells. Infectivity studies indicated potential transient infection by Sf-rhabdovirus in other insect cell lines, but there was no evidence of entry or virus replication in human cell lines. Sf-rhabdovirus sequences were also found in the Sf21 parental cell line of Sf9 cells but not in other insect cell lines, such as BT1-TN-5B1-4 (Tn5; High Five) cells and Schneider's Drosophila line 2 [D.Mel.(2); SL2] cells, indicating a species-specific infection. The results indicate that conventional methods may be complemented by state-of-the-art technologies with extensive bioinformatics analysis for identification of novel viruses. The Spodoptera frugiperda Sf9 cell line is used as a cell substrate for the development and manufacture of biological products. Extensive testing has not previously identified any viruses in this cell line. This paper reports on the identification and characterization of a novel rhabdovirus in Sf9 cells. This was accomplished through the use of next-generation sequencing platforms, de novo assembly tools, and extensive bioinformatics analysis. Rhabdovirus identification was further confirmed by transmission electron microscopy. Infectivity studies showed the lack of replication of Sf-rhabdovirus in human cell lines. The overall study highlights the use of a combinatorial testing approach including conventional methods and new technologies for evaluation of cell lines for unexpected viruses and use of comprehensive bioinformatics strategies for obtaining confident next-generation sequencing results. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Specific down-regulation of XIAP with RNA interference enhances the sensitivity of canine tumor cell-lines to TRAIL and doxorubicin

PubMed Central

Spee, Bart; Jonkers, Martijn DB; Arends, Brigitte; Rutteman, Gerard R; Rothuizen, Jan; Penning, Louis C

2006-01-01

Background Apoptosis resistance occurs in various tumors. The anti-apoptotic XIAP protein is responsible for inhibiting apoptosis by reducing caspase-3 activation. Our aim is to evaluate whether RNA inhibition against XIAP increases the sensitivity of canine cell-lines for chemotherapeutics such as TRAIL and doxorubicin. We used small interfering RNA's (siRNA) directed against XIAP in three cell-lines derived from bile-duct epithelia (BDE), mammary carcinoma (P114), and osteosarcoma (D17). These cell-lines represent frequently occurring canine cancers and are highly comparable to their human counterparts. XIAP down-regulation was measured by means of quantitative PCR (Q-PCR) and Western blotting. The XIAP depleted cells were treated with a serial dilution of TRAIL or doxorubicin and compared to mock- and nonsense-treated controls. Viability was measured with a MTT assay. Results All XIAP siRNA treated cell-lines showed a mRNA down-regulation over 80 percent. Western blot analysis confirmed mRNA measurements. No compensatory effect of IAP family members was seen in XIAP depleted cells. The sensitivity of XIAP depleted cells for TRAIL was highest in BDE cells with an increase in the ED50 of 14-fold, compared to mock- and nonsense-treated controls. The sensitivity of P114 and D17 cell-lines increased six- and five-fold, respectively. Doxorubicin treatment in XIAP depleted cells increased sensitivity in BDE cells more than eight-fold, whereas P114 and D17 cell-lines showed an increase in sensitivity of three- and five-fold, respectively. Conclusion XIAP directed siRNA's have a strong sensitizing effect on TRAIL-reduced cell-viability and a smaller but significant effect with the DNA damaging drug doxorubicin. The increase in efficacy of chemotherapeutics with XIAP depletion provides the rationale for the use of XIAP siRNA's in insensitive canine tumors. PMID:16953886

Novel Mechanisms Revealed in the Trachea Transcriptome of Resistant and Susceptible Chicken Lines following Infection with Newcastle Disease Virus

PubMed Central

Gallardo, Rodrigo A.; Bunn, David A.; Kelly, Terra R.; Dekkers, Jack C. M.; Zhou, Huaijun

2017-01-01

ABSTRACT Newcastle disease virus (NDV) has a devastating impact on poultry production in developing countries. This study examined the transcriptome of tracheal epithelial cells from two inbred chicken lines that differ in NDV susceptibility after challenge with a high-titer inoculum of lentogenic NDV. The Fayoumi line had a significantly lower NDV load postchallenge than the Leghorn line, demonstrating the Fayoumi line's classification as a relatively NDV-resistant breed. Examination of the trachea transcriptome showed a large increase in immune cell infiltration in the trachea in both lines at all times postinfection. The pathways conserved across lines and at all three time points postinfection included iCOS-iCOSL signaling in T helper cells, NF-κB signaling, the role of nuclear factor of activated T cells in the regulation of the immune response, calcium-induced T lymphocyte apoptosis, phospholipase C signaling, and CD28 signaling in T helper cells. Although shared pathways were seen in the Fayoumi and Leghorn lines, each line showed unique responses as well. The downregulation of collagen and the activation of eukaryotic translation initiation factor 2 signaling in the Fayoumis relative to the Leghorns at 2 days postinfection may contribute to the resistance phenotype seen in the Fayoumis. This study provides a further understanding of host-pathogen interactions which could improve vaccine efficacy and, in combination with genome-wide association studies, has the potential to advance strategies for breeding chickens with enhanced resistance to NDV. PMID:28331077

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

PubMed

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

2016-01-01

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

Subtype and pathway specific responses to anticancer compounds in breast cancer.

PubMed

Heiser, Laura M; Sadanandam, Anguraj; Kuo, Wen-Lin; Benz, Stephen C; Goldstein, Theodore C; Ng, Sam; Gibb, William J; Wang, Nicholas J; Ziyad, Safiyyah; Tong, Frances; Bayani, Nora; Hu, Zhi; Billig, Jessica I; Dueregger, Andrea; Lewis, Sophia; Jakkula, Lakshmi; Korkola, James E; Durinck, Steffen; Pepin, François; Guan, Yinghui; Purdom, Elizabeth; Neuvial, Pierre; Bengtsson, Henrik; Wood, Kenneth W; Smith, Peter G; Vassilev, Lyubomir T; Hennessy, Bryan T; Greshock, Joel; Bachman, Kurtis E; Hardwicke, Mary Ann; Park, John W; Marton, Laurence J; Wolf, Denise M; Collisson, Eric A; Neve, Richard M; Mills, Gordon B; Speed, Terence P; Feiler, Heidi S; Wooster, Richard F; Haussler, David; Stuart, Joshua M; Gray, Joe W; Spellman, Paul T

2012-02-21

Breast cancers are comprised of molecularly distinct subtypes that may respond differently to pathway-targeted therapies now under development. Collections of breast cancer cell lines mirror many of the molecular subtypes and pathways found in tumors, suggesting that treatment of cell lines with candidate therapeutic compounds can guide identification of associations between molecular subtypes, pathways, and drug response. In a test of 77 therapeutic compounds, nearly all drugs showed differential responses across these cell lines, and approximately one third showed subtype-, pathway-, and/or genomic aberration-specific responses. These observations suggest mechanisms of response and resistance and may inform efforts to develop molecular assays that predict clinical response.

Cytotoxic cardiac glycosides and other compounds from Asclepias syriaca.

PubMed

Araya, Juan J; Kindscher, Kelly; Timmermann, Barbara N

2012-03-23

Phytochemical investigation of the dried biomass of Asclepias syriaca afforded five new compounds (1-5), along with 19 known structures. Overall, the secondary metabolites isolated and identified from this plant showed a wide structural diversity including pentacyclic triterpenes, cardiac glycosides, flavonoid glycosides, lignans, a phenylethanoid, and a glycosylated megastigmane. In addition, the isolates were tested against the cancer breast cell line Hs578T, and those showing IC(50) values lower than 50 μM (1 and 6-9) were further investigated in three additional breast cancer cell lines (MCF-7, T47D, and Sk-Br-3) and the normal breast cell line Hs578Bst.

A new PDAC mouse model originated from iPSCs-converted pancreatic cancer stem cells (CSCcm)

PubMed Central

Calle, Anna Sanchez; Nair, Neha; Oo, Aung KoKo; Prieto-Vila, Marta; Koga, Megumi; Khayrani, Apriliana Cahya; Hussein, Maram; Hurley, Laura; Vaidyanath, Arun; Seno, Akimasa; Iwasaki, Yoshiaki; Calle, Malu; Kasai, Tomonari; Seno, Masaharu

2016-01-01

Pancreatic ductal adenocarcinoma (PDAC) is the most representative form of pancreatic cancers. PDAC solid tumours are constituted of heterogeneous populations of cells including cancer stem cells (CSCs), differentiated cancer cells, desmoplastic stroma and immune cells. The identification and consequent isolation of pancreatic CSCs facilitated the generation of genetically engineered murine models. Nonetheless, the current models may not be representative for the spontaneous tumour occurrence. In the present study, we show the generation of a novel pancreatic iPSC-converted cancer stem cell lines (CSCcm) as a cutting-edge model for the study of PDAC. The CSCcm lines were achieved only by the influence of pancreatic cancer cell lines conditioned medium and were not subjected to any genetic manipulation. The xenografts tumours from CSCcm lines displayed histopathological features of ADM, PanIN and PDAC lesions. Further molecular characterization from RNA-sequencing analysis highlighted primary culture cell lines (1st CSCcm) as potential candidates to represent the pancreatic CSCs and indicated the establishment of the pancreatic cancer molecular pattern in their subsequent progenies 2nd CSCcm and 3rd CSCcm. In addition, preliminary RNA-seq SNPs analysis showed that the distinct CSCcm lines did not harbour single point mutations for the oncogene Kras codon 12 or 13. Therefore, PDAC-CSCcm model may provide new insights about the actual occurrence of the pancreatic cancer leading to develop different approaches to target CSCs and abrogate the progression of this fatidic disease. PMID:28042501

Establishment and proteomic characterization of a novel synovial sarcoma cell line, NCC-SS2-C1.

PubMed

Oyama, Rieko; Kito, Fusako; Sakumoto, Marimu; Shiozawa, Kumiko; Toki, Shunichi; Endo, Makoto; Yoshida, Akihiko; Kawai, Akira; Kondo, Tadashi

2018-05-01

Synovial sarcoma is an aggressive mesenchymal tumor, characterized by the presence of unique transfusion gene, SS18-SSX. Cell lines enable researchers to investigate the molecular backgrounds of disease and the significance of SS18-SSX in relevant cellular contexts. We report the establishment and proteomic characterization of a novel synovial sarcoma cell line. Primary tissue culture was performed using tumor tissue of synovial sarcoma. The established cell line was authenticated by assessing its DNA microsatellite short tandem repeat analysis and characterized by in vitro assay. Proteomic study was achieved by mass spectrometry, and the results were analyzed by treemap. The cell line NCC-SS2-C1 was established from a primary tumor tissue of a synovial sarcoma patient. The cell line has grown well for 11 mo and has been subcultured more than 15 times. The established cells were authenticated by assessing their short tandem repeat pattern comparing with that of original tumor tissue. The cells showed polygonal in shape and formed spheroid when seeded on the low-attachment dish. Proteomic analysis revealed the molecular pathways which are unique to the original tumor tissue or the established cell line. In conclusion, a novel synovial sarcoma cell line NCC-SS2-C1 was successfully established from the primary tumor tissue. The cell line has characteristic transfusion SS18-SSX and poses aggressive in vitro growth and capability of spheroid formation. Thus, NCC-SS2-C1 cell line will be a useful tool for investigation of the mechanisms of disease and the biological role of fusion gene.

Phenotypes and Karyotypes of Human Malignant Mesothelioma Cell Lines

PubMed Central

Relan, Vandana; Morrison, Leanne; Parsonson, Kylie; Clarke, Belinda E.; Duhig, Edwina E.; Windsor, Morgan N.; Matar, Kevin S.; Naidoo, Rishendran; Passmore, Linda; McCaul, Elizabeth; Courtney, Deborah; Yang, Ian A.; Fong, Kwun M.; Bowman, Rayleen V.

2013-01-01

Background Malignant mesothelioma is an aggressive tumour of serosal surfaces most commonly pleura. Characterised cell lines represent a valuable tool to study the biology of mesothelioma. The aim of this study was to develop and biologically characterise six malignant mesothelioma cell lines to evaluate their potential as models of human malignant mesothelioma. Methods Five lines were initiated from pleural biopsies, and one from pleural effusion of patients with histologically proven malignant mesothelioma. Mesothelial origin was assessed by standard morphology, Transmission Electron Microscopy (TEM) and immunocytochemistry. Growth characteristics were assayed using population doubling times. Spectral karyotyping was performed to assess chromosomal abnormalities. Authentication of donor specific derivation was undertaken by DNA fingerprinting using a panel of SNPs. Results Most of cell lines exhibited spindle cell shape, with some retaining stellate shapes. At passage 2 to 6 all lines stained positively for calretinin and cytokeratin 19, and demonstrated capacity for anchorage-independent growth. At passage 4 to 16, doubling times ranged from 30–72 hours, and on spectral karyotyping all lines exhibited numerical chromosomal abnormalities ranging from 41 to 113. Monosomy of chromosomes 8, 14, 22 or 17 was observed in three lines. One line displayed four different karyotypes at passage 8, but only one karyotype at passage 42, and another displayed polyploidy at passage 40 which was not present at early passages. At passages 5–17, TEM showed characteristic features of mesothelioma ultrastructure in all lines including microvilli and tight intercellular junctions. Conclusion These six cell lines exhibit varying cell morphology, a range of doubling times, and show diverse passage-dependent structural chromosomal changes observed in malignant tumours. However they retain characteristic immunocytochemical protein expression profiles of mesothelioma during maintenance in artificial culture systems. These characteristics support their potential as in vitro model systems for studying cellular, molecular and genetic aspects of mesothelioma. PMID:23516439

Characterisation of adriamycin- and amsacrine-resistant human leukaemic T cell lines.

PubMed Central

Snow, K.; Judd, W.

1991-01-01

Cell lines resistant to adriamycin and amsacrine were derived from cloned sublines of the human T cell line Jurkat. Most of the lines resemble atypical MDR cells (Danks et al., 1987; Beck et al., 1987). Thus, resistant Jurkat sublines were cross resistant to several topoisomerase II inhibiting drugs but had low or no resistance to other classes of drugs, resistance was not reversed by verapamil, Pgp was not overexpressed, and drug accumulation was unaltered in resistant compared to parental (control) sublines. Other findings were that anthracycline metabolism differed between resistant and parental sublines, and that resistant sublines displayed altered expression of small polypeptides (less than 20K MW) and an 85K MW protein. Drug resistant cells showed resistance to the production of drug induced cytogenetic aberrations, DNA breaks, and protein-DNA complexes. Resistance was not mediated by altered binding of drugs to DNA or by increased repair of DNA damage. Indirect evidence suggests that the resistant cells had an altered drug-DNA-topoisomerase II association. The study highlights the complex relationships between DNA breaks, cytogenetic aberrations, protein-DNA complexes and drug cytotoxicity, and shows that the relationships differ for adriamycin and amsacrine, suggesting some differences in the modes of action and/or resistance for the drugs and cell lines. Images Figure 2 Figure 3 PMID:1989661

Fucoidan Does Not Exert Anti-Tumorigenic Effects on Uveal Melanoma Cell Lines

PubMed Central

Dithmer, Michaela; Kirsch, Anna-Maria; Richert, Elisabeth; Fuchs, Sabine; Wang, Fanlu; Schmidt, Harald; Coupland, Sarah E.; Roider, Johann; Klettner, Alexa

2017-01-01

Background. The polysaccharide fucoidan is widely investigated as an anti-cancer agent. Here, we tested the effect of fucoidan on uveal melanoma cell lines. Methods. The effect of 100 µM fucoidan was investigated on five cell lines (92.1, Mel270 OMM1, OMM2.3, OMM2.5) and of 1 µg/mL–1 mg/mL fucoidan in two cell lines (OMM1, OMM2.3). Cell proliferation and viability were investigated with a WST-1 assay, migration in a wound healing (scratch) assay. Vascular Endothelial Growth Factor (VEGF) was measured in ELISA. Angiogenesis was evaluated in co-cultures with endothelial cells. Cell toxicity was induced by hydrogen-peroxide. Protein expression (Akt, ERK1/2, Bcl-2, Bax) was investigated in Western blot. Results. Fucoidan increased proliferation in two and reduced it in one cell line. Migration was reduced in three cell lines. The effect of fucoidan on VEGF was cell type and concentration dependent. In endothelial co-culture with 92.1, fucoidan significantly increased tubular structures. Moreover, fucoidan significantly protected all tested uveal melanoma cell lines from hydrogen-peroxide induced cell death. Under oxidative stress, fucoidan did not alter the expression of Bcl-2, Bax or ERK1/2, while inducing Akt expression in 92.1 cells but not in any other cell line. Conclusion. Fucoidan did not show anti-tumorigenic effects but displayed protective and pro-angiogenic properties, rendering fucoidan unsuitable as a potential new drug for the treatment of uveal melanoma. PMID:28640204

Antiproliferative effect of isopentenylated coumarins on several cancer cell lines.

PubMed

Kawaii, S; Tomono, Y; Ogawa, K; Sugiura, M; Yano, M; Yoshizawa, Y; Ito, C; Furukawa, H

2001-01-01

33 coumarins, mainly the simple isopentenylated coumarins and derived pyrano- and furanocoumarins, were examined for their antiproliferative activity towards several cancer and normal human cell lines. The pyrano- and furanocoumarins showed strong activity against the cancer cell lines, whereas they had weak antiproliferative activity against the normal human cell lines. The decreasing rank order of potency was osthenone (10), clausarin (25), clausenidin (26), dentatin (24), nordentatin (23), imperatorin (29), seselin (27), xanthyletin (21), suberosin (17), phebalosin (8) and osthol (12). The structure-activity relationship established from the results revealed that the 1,1-dimethylallyl and isopentenyl groups have an important role for antiproliferative activity.

Establishment of a new cell line from the snout tissue of golden pompano Trachinotus ovatus, and its application in virus susceptibility.

PubMed

Yu, Y; Wei, S; Wang, Z; Huang, X; Huang, Y; Cai, J; Li, C; Qin, Q

2016-06-01

A new marine-fish cell line, designated GPS, was established from the snout tissue of golden pompano Trachinotus ovatus. GPS cells multiplied well in Leibovitz's L-15 containing 10% foetal bovine serum (FBS) at 28° C and the cells have been subcultured for >60 passages. Polymerase chain reaction (PCR) amplification of 16S ribosomal (r)RNA confirmed the origin of this cell line from T. ovatus. Chromosome analysis showed that GPS cells exhibited chromosomal aneuploidy with a modal chromosome number of 54. Bright green fluorescence signal was observed in enhanced green fluorescent protein (EGFP)-N3 transfected cells, indicating that GPS cells could be used to investigate gene functions in vitro. The GPS cells were highly susceptible to Singapore grouper iridovirus (SGIV), which was demonstrated by the presence of severe cytopathic effect (CPE) and increased viral titres. Real-time quantitative PCR and Western blot analysis showed that the viral gene transcription and protein synthesis occurred during SGIV infection in GPS cells. Thus, this study described the characteristic of a new cell line from the snout tissue of T. ovatus that could be used as a tool for propagation of iridovirus and genetic manipulation to investigate host-pathogen interactions. © 2016 The Fisheries Society of the British Isles.

Recent advances of in vitro culture systems for spermatogonial stem cells in mammals.

PubMed

Sahare, Mahesh G; Suyatno; Imai, Hiroshi

2018-04-01

Spermatogonial stem cells (SSCs) in the mammalian testis are unipotent stem cells for spermatozoa. They show unique cell characteristics as stem cells and germ cells after being isolated from the testis and cultured in vitro. This review introduces recent progress in the development of culture systems for the establishment of SSC lines in mammalian species, including humans. Based on the published reports, the isolation and purification of SSCs, identification and characteristics of SSCs, and culture system for mice, humans, and domestic animals have been summarized. In mice, cell lines from SSCs are established and can be reprogrammed to show pluripotent stem cell potency that is similar to embryonic stem cells. However, it is difficult to establish cell lines for animals other than mice because of the dearth of understanding about species-specific requirements for growth factors and mechanisms supporting the self-renewal of cultured SSCs. Among the factors that are associated with the development of culture systems, the enrichment of SSCs that are isolated from the testis and the combination of growth factors are essential. Providing an example of SSC culture in cattle, a rational consideration was made about how it can be possible to establish cell lines from neonatal and immature testes.

Eco-friendly synthesis of novel cyanopyridine derivatives and their anticancer and PIM-1 kinase inhibitory activities.

PubMed

Abouzid, Khaled A M; Al-Ansary, Ghada H; El-Naggar, Abeer M

2017-07-07

Targeting Pim-1 kinase recently proved to be profitable for conquering cancer proliferation. In the current study, we report the design, synthesis and biological evaluation of two novel series of 2-amino cyanopyridine series (5a-g) and 2-oxocyanopyridine series (6a-g) targeting Pim-1 kinase. All of the newly synthesized compounds were evaluated for their in vitro anticancer activity against a panel of three cell lines, namely, the liver cancer cell line (HepG2), the colon cancer cell line (HCT-116) and the breast cancer cell line (MCF-7). Most of the compounds showed good to moderate anti-proliferative activity against HepG2 and HCT-116 cell lines while only few compounds showed significant cytotoxic activity against MCF-7 cell line. Further, the Pim-1 kinase inhibitory activity for the two series was evaluated where most of the tested compounds showed marked Pim-1 kinase inhibitory activity (26%-89%). Moreover, determination of the IC 50 values unraveled very potent molecules in the submicromolar range where compound 6c possessed an IC 50 value of 0.94 μM. Moreover, apoptosis studies were conducted on the most potent compound 6c to evaluate the proapoptotic potential of our compounds. Interestingly, it induced the level of active caspase 3 and boosted the Bax/Bcl2 ratio 22704 folds in comparison to the control. Finally, a molecular docking study was conducted to reveal the probable interaction with the Pim-1 kinase active site. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Anticancer property of sediment actinomycetes against MCF-7 and MDA-MB-231 cell lines.

PubMed

Ravikumar, S; Fredimoses, M; Gnanadesigan, M

2012-02-01

To investigate the anticancer property of marine sediment actinomycetes against two different breast cancer cell lines. In vitro anticancer activity was carried out against breast (MCF-7 and MDA-MB-231) cancer cell lines. Partial sequences of the 16s rRNA gene, phylogenetic tree construction, multiple sequence analysis and secondary structure analysis were also carried out with the actinomycetes isolates. Of the selected five actinomycete isolates, ACT01 and ACT02 showed the IC50 value with (10.13±0.92) and (22.34±5.82) µg/mL concentrations, respectively for MCF-7 cell line at 48 h, but ACT01 showed the minimum (18.54±2.49 µg/mL) level of IC50 value with MDA-MB-231 cell line. Further, the 16s rRNA partial sequences of ACT01, ACT02, ACT03, ACT04 and ACT05 isolates were also deposited in NCBI data bank with the accession numbers of GQ478246, GQ478247, GQ478248, GQ478249 and GQ478250, respectively. The phylogenetic tree analysis showed that, the isolates of ACT02 and ACT03 were represented in group I and III, respectively, but ACT01 and ACT02 were represented in group II. The multiple sequence alignment of the actinomycete isolates showed that, the maximum identical conserved regions were identified with the nucleotide regions of 125 to 221st base pairs, 65 to 119th base pairs and 55, 48 and 31st base pairs. Secondary structure prediction of the 16s rRNA showed that, the maximum free energy was consumed with ACT03 isolate (-45.4 kkal/mol) and the minimum free energy was consumed with ACT04 isolate (-57.6 kkal/mol). The actinomycete isolates of ACT01 and ACT02 (GQ478246 and GQ478247) which are isolated from sediment sample can be further used as anticancer agents against breast cancer cell lines.

Snake venom causes apoptosis by increasing the reactive oxygen species in colorectal and breast cancer cell lines

PubMed Central

Al-Asmari, Abdulrahman Khazim; Riyasdeen, Anvarbatcha; Al-Shahrani, Mohammad Hamed; Islam, Mozaffarul

2016-01-01

Snake venom possesses various kinds of proteins and neurotoxic polypeptides, which can negatively interfere with the neurotransmitter signaling cascade. This phenomenon occurs mainly due to the blocking of ion channels in the body system. Envenomation prevents or severely interrupts nerve impulses from being transmitted, inhibition of adenosine triphosphate synthesis, and proper functioning of the cardiac muscles. However, some beneficial properties of venoms have also been reported. The aim of this study was to examine the snake venom as an anticancer agent due to its inhibitory effects on cancer progression such as cell motility, cell invasion, and colony formation. In this study, the effect of venoms on phenotypic changes and the change on molecular level in colorectal and breast cancer cell lines were examined. A reduction of 60%–90% in cell motility, colony formation, and cell invasion was observed when these cell lines were treated with different concentrations of snake venom. In addition, the increase in oxidative stress that results in an increase in the number of apoptotic cancer cells was significantly higher in the venom-treated cell lines. Further analysis showed that there was a decrease in the expression of pro-inflammatory cytokines and signaling proteins, strongly suggesting a promising role for snake venom against breast and colorectal cancer cell progression. In conclusion, the snake venoms used in this study showed significant anticancer properties against colorectal and breast cancer cell lines. PMID:27799796

Stem/progenitor cell-like properties of desmoglein 3dim cells in primary and immortalized keratinocyte lines.

PubMed

Wan, Hong; Yuan, Ming; Simpson, Cathy; Allen, Kirsty; Gavins, Felicity N E; Ikram, Mohammed S; Basu, Subham; Baksh, Nuzhat; O'Toole, Edel A; Hart, Ian R

2007-05-01

We showed previously that primary keratinocytes selected for low desmoglein 3 (Dsg3) expression levels exhibited increased colony-forming efficiency and heightened proliferative potential relative to cells with higher Dsg3 expression levels, characteristics consistent with a more "stem/progenitor cell-like" phenotype. Here, we have confirmed that Dsg3(dim) cells derived from cultured primary human adult keratinocytes have comparability with alpha(6)(bri)/CD71(dim) stem cells in terms of colony-forming efficiency. Moreover, these Dsg3(dim) cells exhibit increased reconstituting ability in in vitro organotypic culture on de-epidermalized dermis (DED); they are small, actively cycling cells, and they express elevated levels of various p63 isoforms. In parallel, using the two immortalized keratinocyte cell lines HaCaT and NTERT, we obtained essentially similar though occasionally different findings. Thus, reduced colony-forming efficiency by Dsg3(bri) cells consistently was observed in both cell lines even though the cell cycle profile and levels of p63 isoforms in the bri and dim populations differed between these two cell lines. Dsg3(dim) cells from both immortalized lines produced thicker and better ordered hierarchical structural organization of reconstituted epidermis relative to Dsg3(bri) and sorted control cells. Dsg3(dim) HaCaT cells also show sebocyte-like differentiation in the basal compartment of skin reconstituted after a 4-week organotypic culture. No differences in percentages of side population cells (also a putative marker of stem cells) were detected between Dsg3(dim) and Dsg3(bri) populations. Taken together our data indicate that Dsg3(dim) populations from primary human adult keratinocytes and long-term established keratinocyte lines possess certain stem/progenitor cell-like properties, although the side population characteristic is not one of these features. Disclosure of potential conflicts of interest is found at the end of this article.

Cytotoxicity and genotoxicity of coronaridine from Tabernaemontana catharinensis A.DC in a human laryngeal epithelial carcinoma cell line (Hep-2)

PubMed Central

Rizo, Walace Fraga; Ferreira, Luis Eduardo; Colnaghi, Vanessa; Martins, Juliana Simões; Franchi, Leonardo Pereira; Takahashi, Catarina Satie; Beleboni, Rene Oliveira; Marins, Mozart; Pereira, Paulo Sérgio; Fachin, Ana Lúcia

2013-01-01

Cancer has become a major public health problem worldwide and the number of deaths due to this disease is increasing almost exponentially. In the constant search for new treatments, natural products of plant origin have provided a variety of new compounds to be explored as antitumor agents. Tabernaemontana catharinensis is a medicinal plant that produces alkaloids with expressive antitumor activity, such as heyneanine, coronaridine and voacangine. The aim of present study was firstly to screen the cytotoxic activity of the indole alkaloids heyneanine, coronaridine and voacangine against HeLa (human cervix tumor), 3T3 (normal mouse embryo fibroblasts), Hep-2 (human laryngeal epithelial carcinoma) and B-16 (murine skin) cell lines by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide); and secondly to analyze the apoptotic activity, cell membrane damage and genotoxicity of the compound that showed the best cytotoxic activity against the tumor cell lines tested. Coronaridine was the one that exhibited greater cytotoxic activity in the laryngeal carcinoma cell line Hep-2 (IC50 = 54.47 μg/mL) than the other alkaloids tested (voacangine IC50 = 159.33 g/mL, and heyneanine IC50 = 689.45 μg/mL). Coronaridine induced apoptosis in cell lines 3T3 and Hep-2, even at high concentrations. The evaluation of genotoxicity by comet assay showed further that coronaridine caused minimal DNA damage in the Hep-2 tumor cell line, and the LDH test showed that it did not affect the plasma membrane. These results suggest that further investigation of coronaridine as an antitumor agent has merit. PMID:23569415

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

PubMed

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

2018-04-01

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

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.

Evaluation of Anticancer Activity of Curcumin Analogues Bearing a Heterocyclic Nucleus.

PubMed

Ahsan, Mohamed Jawed; Ahsan, Mohamed Jawed

2016-01-01

We report herein an in vitro anticancer evaluation of a series of seven curcumin analogues (3a-g). The National Cancer Institute (NCI US) Protocol was followed and all the compounds were evaluated for their anticancer activity on nine different panels (leukemia, non small cell lung cancer, colon cancer, CNS cancer, melanoma, ovarian cancer, renal cancer, prostate cancer and breast cancer) represented by 60 NCI human cancer cell lines. All the compounds showed significant anticancer activity in one dose assay (drug concentration 10 μM) and hence were evaluated further in five dose assays (0.01, 0.1, 1, 10 and 100 μM) and three dose related parameters GI50, TGI and LC50 were calculated for each (3a-g) in micro molar drug concentrations (μM). The compound 3d (NSC 757927) showed maximum mean percent growth inhibition (PGI) of 112.2%, while compound 3g (NSC 763374) showed less mean PGI of 40.1% in the one dose assay. The maximum anticancer activity was observed with the SR (leukemia) cell line with a GI50 of 0.03 μM. The calculated average sensitivity of all cell lines of a particular subpanel toward the test agent showed that all the curcumin analogues showed maximum activity on leukemia cell lines with GI50 values between 0.23 and 2.67 μM.

[The characters and specific features of new human embryonic stem cells lines].

PubMed

Krylova, T A; Kol'tsova, A M; Zenin, V V; Gordeeva, O F; Musorina, A S; Goriachaia, T S; Shlykova, S A; Kamenetskaia, Iu K; Pinaev, G P; Polianskaia, G G

2009-01-01

Four continuous human embryonic stem cell lines (SC1, SC2, SC3 and SC4), derived from the blastocysts has been described. The cell lines were cultivated on mitotically inactivated human feeder cells. The cell lines SC1 and SC2 have passed through 150 population doublings and the cell lines SC3 and SC4 -- near 120 populations doublings, which exceeds Hayflick limit sufficiently. These cell lines maintain high activity of alkaline phosphatase, expression of transcription factor OCT-4 and cell surface antigens (SSEA-4, TRA-1-60 and TRA-1-81), confirming their ESC status and human specificity. Immunofluorescent detection of antigens, characteristic of ectoderm, endoderm and mesoderm confirms the ability of these cells to retain their pluripotency under in vitro condition. PCR analysis revealed expression of six genes specific for pluripotent cells (OCT-4, NANOG, DPPA3/STELLA, TDGF/CRIPTO and LEFTYA). Correlation between the level of proliferative activity and the character of DNA-bound fluorescent staining was found. Fluorescent dyes, Hoechst 33342 and PI, produced diffuse staining of the nuclei in slowly proliferating cells of the SC1 and SC2 lines. In contrast, in actively proliferating cells of the SC3 and SC4 lines, the clear staining of the nuclei was observed. Upon changing the cultivation condition, proliferative activity of SC3 and SC4 lines decreased and became similar to that of SC1 and SC2 lines. The character of the fluorescent staining of all these lines was also shown to be similar. These results show that quality of the fluorescent staining with Hoechst 33342 and PI reflects the level of proliferation. Possible causes and mechanisms of this feature of human ESC are discussed.

Nanofork for single cells adhesion measurement via ESEM-nanomanipulator system.

PubMed

Ahmad, Mohd Ridzuan; Nakajima, Masahiro; Kojima, Masaru; Kojima, Seiji; Homma, Michio; Fukuda, Toshio

2012-03-01

In this paper, single cells adhesion force was measured using a nanofork. The nanofork was used to pick up a single cell on a line array substrate inside an environmental scanning electron microscope (ESEM). The line array substrate was used to provide small gaps between the single cells and the substrate. Therefore, the nanofork could be inserted through these gaps in order to successfully pick up a single cell. Adhesion force was measured during the cell pick-up process from the deflection of the cantilever beam. The nanofork was fabricated using focused ion beam (FIB) etching process while the line array substrate was fabricated using nanoimprinting technology. As to investigate the effect of contact area on the strength of the adhesion force, two sizes of gap distance of line array substrate were used, i.e., 1 μm and 2 μm. Results showed that cells attached on the 1 μm gap line array substrate required more force to be released as compared to the cells attached on the 1 μm gap line array substrate.

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

PubMed

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

2014-12-01

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

Antitumor effect of CXCR4 antagonist AMD3100 on the tumorigenic cell line of BHP10-3 papillary thyroid cancer cells.

PubMed

Jung, Young Ho; Lee, Doh Young; Cha, Wonjae; Kim, Bo Hae; Sung, Myung-Whun; Kim, Kwang Hyun; Ahn, Soon-Hyun

2016-10-01

A tumorigenic cell line (BHP10-3M) derived from nontumorigenic papillary thyroid carcinoma (PTC) cells (BHP10-3) having rearranged during transfection (RET)/PTC1 gene rearrangement might have a higher expression of CXCR4, either quantitatively or functionally. The authors also postulated that CXCR4-mediated invasion or tumorigenesis could be blocked by CXCR4 antagonists, including AMD3100. The expression of CXCR4 in BHP10-3 and BHP10-3M cells was assessed using immunoblot analysis, flow cytometry, and quantitative reverse-transcriptase polymerase chain reaction (RT-PCR). The effect of AMD3100 on BHP10-3 and BHP10-3M cell lines was evaluated using cell proliferation assay, invasion assay, and tumor growth experiment in nude mice. Immunoblotting, flow cytometry, and quantitative RT-PCR proved that BHP10-3M cells expressed a higher level of CXCR4 than BHP10-3 cells. Although blocking CXCR4 with AMD3100 did not suppress cell proliferation in both cell lines from 1 ng/mL to 100 ng/mL concentration, AMD3100 suppressed invasion of BHP10-3M cells in vitro in a dose-dependent manner. At higher concentrations from 10(3) ng/mL to 10(5) ng/mL, the proliferation of BHP10-3M cells was inhibited more strongly by AMD3100 than that of BHP10-3 cells. Intraperitoneal injection of AMD3100 inhibited tumor formation by BHP10-3M cells in the thyroid of nude mice. A tumorigenic cell line (BHP10-3M) of PTC showed higher expression of CXCR4 quantitatively and functionally than a nontumorigenic cell line (BHP10-3). The CXCR4 antagonist (AMD3100) showed a significant antitumor effect on the tumorigenic cell line of PTC BHP10-3 cells both in vitro and in vivo. CXCR4 antagonist can be expected to have an adjuvant role in the management of PTC. © 2016 Wiley Periodicals, Inc. Head Neck, 2016 © 2016 Wiley Periodicals, Inc. Head Neck 38: First-1486, 2016. © 2016 Wiley Periodicals, Inc.

The chance of small interfering RNAs as eligible candidates for a personalized treatment of prostate cancer.

PubMed

Pietschke, Katharina; Walker, Tobias; Krajewski, Stefanie; Kurz, Julia; Aufderklamm, Stefan; Schwentner, Christian; Schlensak, Christian; Stenzl, Arnulf; Wendel, Hans P; Nolte, Andrea

2014-01-01

Prostate cancer is one of the leading malignant tumors in men. Current therapies are associated with severe side effects making it problematic for many multi-morbid patients to receive treatment. Prostate specific antigen, serum response factor (SRF), signal transducer and activator of transcription-3 (STAT3), hypoxia-inducible factor-1α (HIF-1α), HIF-2α, E2F1 and Survivin are well known proteins being overexpressed in cancer cells, expediting cell growth and also demonstrated in prostate cancer cells. Targeting these genes using the RNA-Interference pathway could be a new approach for prostate cancer therapy with fewer side effects. Three prostate cancer cell lines were cultured under standard conditions and transfected with three different concentrations (25 nM, 50 nM, 100 nM) of specific small interfering RNAs (siRNAs) targeting SRF, STAT3, HIF1α, HIF2α, E2F1 and Survivin in a non-viral manner. Cells treated with non-specific siRNA (SCR-siRNA) served as control. Changes of messenger RNA (mRNA) levels were determined using quantitative real-time polymerase chain reaction (qRT-PCR). The analysis of the effect of siRNA on the number of cells was detected using CASY cell counter system. Transfections of the PC-3 cell line with specific siRNA especially against Survivin, E2F1, HIF1α- and HIF2α-siRNA resulted in a significant reduction of intracellular mRNA concentration together with a significant decreased number of cells. In the LnCAP and DU-145 cell lines Survivin and E2F1 showed similar effects. The impact of silencing STAT3 or SRF showed little influence on the amount of cells in all three cell lines. This study shows that RNAi succeeds in silencing gene expression and reducing the number of cells in differing dimensions depending on the transfected cell line and used siRNA.

Comparison between effects of free curcumin and curcumin loaded NIPAAm-MAA nanoparticles on telomerase and PinX1 gene expression in lung cancer cells.

PubMed

Badrzadeh, Fariba; Akbarzadeh, Abolfazl; Zarghami, Nosratollah; Yamchi, Mohammad Rahmati; Zeighamian, Vahide; Tabatabae, Fateme Sadate; Taheri, Morteza; Kafil, Hossein Samadi

2014-01-01

Herbal compounds such as curcumin which decrease telomerase and gene expression have been considered as beneficial tools for lung cancer treatment. In this article, we compared the effects of pure curcumin and curcumin-loaded NIPAAm-MAA nanoparticles on telomerase and PinX1 gene expression in a lung cancer cell line. A tetrazolium-based assay was used for determination of cytotoxic effects of curcumin on the Calu-6 lung cancer cell line and telomerase and pinX1 gene expression was measured with real-time PCR. MTT assay showed that Curcumin-loaded NIPAAm-MAA inhibited the growth of the Calu-6 lung cancer cell line in a time and dose-dependent manner. Our q-PCR results showed that the expression of telomerase gene was effectively reduced as the concentration of curcumin-loaded NIPAAm-MAA increased while expression of the PinX1 gene became elevated. The results showed that curcumin- loaded- NIPAAm-MAA exerted cytotoxic effects on the Calu-6 cell line through down-regulation of telomerase and stimulation of pinX1 gene expression. NIPPAm-MAA could be good carrier for such kinds of hydrophobic agent.

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.

Establishment of an immortal cynomolgus macaque fibroblast cell line for propagation of cynomolgus macaque cytomegalovirus (CyCMV).

PubMed

Ambagala, Aruna P; Marsh, Angie K; Chan, Jacqueline K; Mason, Rosemarie; Pilon, Richard; Fournier, Jocelyn; Sandstrom, Paul; Willer, David O; MacDonald, Kelly S

2013-05-01

Cynomolgus macaques are widely used as an animal model in biomedical research. We have established an immortalized cynomolgus macaque fibroblast cell line (MSF-T) by transducing primary dermal fibroblasts isolated from a 13-year-old male cynomolgus macaque with a retrovirus vector expressing human telomerase reverse transcriptase (hTERT). The MSF-T cells showed increased telomerase enzyme activity and reached over 200 in vitro passages compared to the non-transduced dermal fibroblasts, which reached senescence after 43 passages. The MSF-T cell line is free of mycoplasma contamination and is permissive to the newly identified cynomolgus macaque cytomegalovirus (CyCMV). CyCMV productively infects MSF-T cells and induces down-regulation of MHC class I expression. The MSF-T cell line will be extremely useful for the propagation of CyCMV and other cynomolgus herspesviruses in host-derived fibroblast cells, allowing for the retention of host-specific viral genes. Moreover, this cell line will be beneficial for many in vitro experiments related to this animal model.

GAP JUNCTION COMMUNICATON IN A TRANSFECTED HUMAN CELL LINE: ACTION OF MELATONIN AND MAGNETIC FIELDS

EPA Science Inventory

GAP JUNCTION COMMUNICTION IN TRANSFECTED HUMAN CELL LINE: ACTION OF MELATONIN AND MAGNETIC FIELDS.

OBJECTIVE: We previously showed that functional gap junction communication (GJC), as monitored by dye transfer (DT), could be enhanced in mouse C3H 10T112 cells and in mouse...

Genomic instability of osteosarcoma cell lines in culture: impact on the prediction of metastasis relevant genes.

PubMed

Muff, Roman; Rath, Prisni; Ram Kumar, Ram Mohan; Husmann, Knut; Born, Walter; Baudis, Michael; Fuchs, Bruno

2015-01-01

Osteosarcoma is a rare but highly malignant cancer of the bone. As a consequence, the number of established cell lines used for experimental in vitro and in vivo osteosarcoma research is limited and the value of these cell lines relies on their stability during culture. Here we investigated the stability in gene expression by microarray analysis and array genomic hybridization of three low metastatic cell lines and derivatives thereof with increased metastatic potential using cells of different passages. The osteosarcoma cell lines showed altered gene expression during in vitro culture, and it was more pronounced in two metastatic cell lines compared to the respective parental cells. Chromosomal instability contributed in part to the altered gene expression in SAOS and LM5 cells with low and high metastatic potential. To identify metastasis-relevant genes in a background of passage-dependent altered gene expression, genes involved in "Pathways in cancer" that were consistently regulated under all passage comparisons were evaluated. Genes belonging to "Hedgehog signaling pathway" and "Wnt signaling pathway" were significantly up-regulated, and IHH, WNT10B and TCF7 were found up-regulated in all three metastatic compared to the parental cell lines. Considerable instability during culture in terms of gene expression and chromosomal aberrations was observed in osteosarcoma cell lines. The use of cells from different passages and a search for genes consistently regulated in early and late passages allows the analysis of metastasis-relevant genes despite the observed instability in gene expression in osteosarcoma cell lines during culture.

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

PubMed

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

2016-01-01

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

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.

Development and characterization of a cell line TTCF from endangered mahseer Tor tor (Ham.).

PubMed

Yadav, K; Lakra, W S; Sharma, J; Goswami, M; Singh, Akhilesh

2012-08-01

Tor tor is an important game and food fish of India with a distribution throughout Asia from the trans-Himalayan region to the Mekong River basin to Malaysia, Pakistan, Bangladesh and Indonesia. A new cell line named TTCF was developed from the caudal fin of T. tor for the first time. The cell line was optimally maintained at 28°C in Leibovitz-15 (L-15) medium supplemented with 20% fetal bovine serum (FBS). The propagation of TTCF cells showed a high plating efficiency of 63.00%. The cytogenetic analysis revealed a diploid count of 100 chromosomes at passage 15, 30, 45 and 60 passages. The viability of the TTCF cell line was found to be 72% after 6 months of cryopreservation in liquid nitrogen (-196°C). The origin of the cell lines was confirmed by the amplification of 578- and 655-bp sequences of 16S rRNA and cytochrome oxidase subunit I (COI) genes of mitochondrial DNA (mtDNA) respectively. TTCF cells were successfully transfected with green fluorescent protein (GFP) reporter plasmids. Further, immunocytochemistry studies confirm its fibroblastic morphology of cells. Genotoxicity assessment of H₂O₂ in TTCF cell line revealed the utility of TTCF cell line as in vitro model for aquatic toxicological studies.

Identification of triterpene hydroxycinnamates with in vitro antitumor activity from whole cranberry fruit (Vaccinium macrocarpon).

PubMed

Murphy, Brian T; MacKinnon, Shawna L; Yan, Xiaojun; Hammond, Gerald B; Vaisberg, Abraham J; Neto, Catherine C

2003-06-04

Bioactivity-guided fractionation of cranberry fruit was used to determine the identity of triterpenoid esters from Vaccinium macrocarpon, which inhibit tumor cell growth and may play a role in cancer prevention. In our previous study, a fraction from whole fruit exhibited tumor cell growth inhibition in vitro. The major components of this fraction were isolated by chromatographic separation of ethyl acetate extracts, purified by semipreparative HPLC, and identified by NMR as cis- (1) and trans- (2) isomers of 3-O-p-hydroxycinnamoyl ursolic acid. These triterpenoid esters have not been previously reported in Vaccinium fruit. Bioassay of the purified triterpene cinnamates in tumor cell lines in vitro showed slightly greater activity of compound 1 in most cell lines, with GI(50) values of approximately 20 microM in MCF-7 breast, ME180 cervical and PC3 prostate tumor cell lines. Quercetin was slightly less active than 1, while cyanidin-3-galactoside exhibited much lower cytotoxicity, with GI(50) greater than 250 microM in all cell lines. Phenylboronic acid (3) was also isolated from the fruit but showed insignificant antitumor activity.

Fatty acid binding proteins (FABPs) in prostate, bladder and kidney cancer cell lines and the use of IL-FABP as survival predictor in patients with renal cell carcinoma

PubMed Central

2011-01-01

Background Fatty acid binding proteins (FABP) play an important role in carcinogenesis. Modified FABP expression patterns were described for prostate, bladder and for renal cell carcinoma. Studies on metabolic relationships and interactions in permanent cell lines allow a deeper insight into molecular processes. The aim of this study is therefore a systematic overview on mRNA and protein expressions of seven FABPs in frequently used urological cell lines. Methods Nine cell lines of renal carcinomas, seven of urinary bladder carcinomas, and five of prostate carcinomas were investigated. Quantitative RT-qPCR and western blotting were used to determine different FABPs. In addition, 46 paired cancerous and noncancerous tissue samples from nephrectomy specimen with renal cell carcinomas were investigated regarding the ileum FABP mRNA expression level and associated with survival outcome. Results General characteristics of all urological carcinoma cell lines were the expression of E-and IL-FABP on mRNA and protein level, while the expressions differed between the cell lines. The protein expression was not always congruent with the mRNA expression. Renal cell carcinoma cell lines showed expressions of L-, H- and B-FABP mRNA in addition to the general FABP expression in five out of the eight investigated cell lines. In bladder cancer cell lines, we additionally found the expression of A-FABP mRNA in six cell lines, while H-FABP was present only in three cell lines. In prostate cancer cell lines, a strong reduction of A- and E- FABP mRNA was observed. The expression of B-FABP mRNA and protein was observed only in the 22 RV-1 cells. IL-FABP mRNA was over-expressed in renal tumour tissue. The IL-FABP ratio was identified as an independent indicator of survival outcome. Conclusions Distinctly different FABP expression patterns were observed not only between the cell lines derived from the three cancer types, but also between the cell lines from the same cancer. The FABP patterns in the cell lines do not always reflect the real situation in the tumours. These facts have to be considered in functional studies concerning the different FABPs. PMID:21767383

Human T-cell leukemia virus type 1 Tax oncoprotein represses the expression of the BCL11B tumor suppressor in T-cells

PubMed Central

Takachi, Takayuki; Takahashi, Masahiko; Takahashi-Yoshita, Manami; Higuchi, Masaya; Obata, Miki; Mishima, Yukio; Okuda, Shujiro; Tanaka, Yuetsu; Matsuoka, Masao; Saitoh, Akihiko; Green, Patrick L; Fujii, Masahiro

2015-01-01

Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T cell leukemia (ATL), which is an aggressive form of T-cell malignancy. HTLV-1 oncoproteins, Tax and HBZ, play crucial roles in the immortalization of T-cells and/or leukemogenesis by dysregulating the cellular functions in the host. Recent studies show that HTLV-1-infected T-cells have reduced expression of the BCL11B tumor suppressor protein. In the present study, we explored whether Tax and/or HBZ play a role in downregulating BCL11B in HTLV-1-infected T-cells. Lentiviral transduction of Tax in a human T-cell line repressed the expression of BCL11B at both the protein and mRNA levels, whereas the transduction of HBZ had little effect on the expression. Tax mutants with a decreased activity for the NF-κB, CREB or PDZ protein pathways still showed a reduced expression of the BCL11B protein, thereby implicating a different function of Tax in BCL11B downregulation. In addition, the HTLV-2 Tax2 protein reduced the BCL11B protein expression in T-cells. Seven HTLV-1-infected T-cell lines, including three ATL-derived cell lines, showed reduced BCL11B mRNA and protein expression relative to an uninfected T-cell line, and the greatest reductions were in the cells expressing Tax. Collectively, these results indicate that Tax is responsible for suppressing BCL11B protein expression in HTLV-1-infected T-cells; Tax-mediated repression of BCL11B is another mechanism that Tax uses to promote oncogenesis of HTLV-1-infected T-cells. PMID:25613934

Fusion of CCL21 non-migratory active breast epithelial and breast cancer cells give rise to CCL21 migratory active tumor hybrid cell lines.

PubMed

Berndt, Benjamin; Haverkampf, Sonja; Reith, Georg; Keil, Silvia; Niggemann, Bernd; Zänker, Kurt S; Dittmar, Thomas

2013-01-01

The biological phenomenon of cell fusion has been linked to tumor progression because several data provided evidence that fusion of tumor cells and normal cells gave rise to hybrid cell lines exhibiting novel properties, such as increased metastatogenic capacity and an enhanced drug resistance. Here we investigated M13HS hybrid cell lines, derived from spontaneous fusion events between M13SV1-EGFP-Neo breast epithelial cells exhibiting stem cell characteristics and HS578T-Hyg breast cancer cells, concerning CCL21/CCR7 signaling. Western Blot analysis showed that all cell lines varied in their CCR7 expression levels as well as differed in the induction and kinetics of CCR7 specific signal transduction cascades. Flow cytometry-based calcium measurements revealed that a CCL21 induced calcium influx was solely detected in M13HS hybrid cell lines. Cell migration demonstrated that only M13HS hybrid cell lines, but not parental derivatives, responded to CCL21 stimulation with an increased migratory activity. Knockdown of CCR7 expression by siRNA completely abrogated the CCL21 induced migration of hybrid cell lines indicating the necessity of CCL21/CCR7 signaling. Because the CCL21/CCR7 axis has been linked to metastatic spreading of breast cancer to lymph nodes we conclude from our data that cell fusion could be a mechanism explaining the origin of metastatic cancer (hybrid) cells.

Fusion of CCL21 Non-Migratory Active Breast Epithelial and Breast Cancer Cells Give Rise to CCL21 Migratory Active Tumor Hybrid Cell Lines

PubMed Central

Reith, Georg; Keil, Silvia; Niggemann, Bernd; Zänker, Kurt S.; Dittmar, Thomas

2013-01-01

The biological phenomenon of cell fusion has been linked to tumor progression because several data provided evidence that fusion of tumor cells and normal cells gave rise to hybrid cell lines exhibiting novel properties, such as increased metastatogenic capacity and an enhanced drug resistance. Here we investigated M13HS hybrid cell lines, derived from spontaneous fusion events between M13SV1-EGFP-Neo breast epithelial cells exhibiting stem cell characteristics and HS578T-Hyg breast cancer cells, concerning CCL21/CCR7 signaling. Western Blot analysis showed that all cell lines varied in their CCR7 expression levels as well as differed in the induction and kinetics of CCR7 specific signal transduction cascades. Flow cytometry-based calcium measurements revealed that a CCL21 induced calcium influx was solely detected in M13HS hybrid cell lines. Cell migration demonstrated that only M13HS hybrid cell lines, but not parental derivatives, responded to CCL21 stimulation with an increased migratory activity. Knockdown of CCR7 expression by siRNA completely abrogated the CCL21 induced migration of hybrid cell lines indicating the necessity of CCL21/CCR7 signaling. Because the CCL21/CCR7 axis has been linked to metastatic spreading of breast cancer to lymph nodes we conclude from our data that cell fusion could be a mechanism explaining the origin of metastatic cancer (hybrid) cells. PMID:23667660

Human adenovirus serotypes 4p and 11p are efficiently expressed in cell lines of neural tumour origin.

PubMed

Skog, Johan; Mei, Ya-Fang; Wadell, Göran

2002-06-01

Most currently used adenovirus vectors are based upon adenovirus serotypes 2 and 5 (Ad2 and Ad5), which have limited efficiencies for gene transfer to human neural cells. Both serotypes bind to the known adenovirus receptor, CAR (coxsackievirus and adenovirus receptor), and have restricted cell tropism. The purpose of this study was to find vector candidates that are superior to Ad5 in infecting human neural tumours. Using flow cytometry, the vector candidates Ad4p, Ad11p and Ad17p were compared to the commonly used adenovirus vector Ad5v for their binding capacity to neural cell lines derived from glioblastoma, medulloblastoma and neuroblastoma cell lines. The production of viral structural proteins and the CAR-binding properties of the different serotypes were also assessed in these cells. Computer-based models of the fibre knobs of Ad4p and Ad17 were created based upon the crystallized fibre knob structure of adenoviruses and analysed for putative receptor-interacting regions that differed from the fibre knob of Ad5. The non CAR-binding vector candidate Ad11p showed clearly the best binding capacity to all of the neural cell lines, binding more than 90% of cells of all of the neural cell lines tested, in contrast to 20% or less for the commonly used vector Ad5v. Ad4p and Ad11p were also internalized and produced viral proteins more successfully than Ad5. Ad4p showed a low binding ability but a very efficient capacity for infection in cell culture. Ad17p virions neither bound or efficiently infected any of the neural cell lines studied.

Establishment and Biological Characterization of a Panel of Glioblastoma Multiforme (GBM) and GBM Variant Oncosphere Cell Lines.

PubMed

Binder, Zev A; Wilson, Kelli M; Salmasi, Vafi; Orr, Brent A; Eberhart, Charles G; Siu, I-Mei; Lim, Michael; Weingart, Jon D; Quinones-Hinojosa, Alfredo; Bettegowda, Chetan; Kassam, Amin B; Olivi, Alessandro; Brem, Henry; Riggins, Gregory J; Gallia, Gary L

2016-01-01

Human tumor cell lines form the basis of the majority of present day laboratory cancer research. These models are vital to studying the molecular biology of tumors and preclinical testing of new therapies. When compared to traditional adherent cell lines, suspension cell lines recapitulate the genetic profiles and histologic features of glioblastoma multiforme (GBM) with higher fidelity. Using a modified neural stem cell culture technique, here we report the characterization of GBM cell lines including GBM variants. Tumor tissue samples were obtained intra-operatively and cultured in neural stem cell conditions containing growth factors. Tumor lines were characterized in vitro using differentiation assays followed by immunostaining for lineage-specific markers. In vivo tumor formation was assayed by orthotopic injection in nude mice. Genetic uniqueness was confirmed via short tandem repeat (STR) DNA profiling. Thirteen oncosphere lines derived from GBM and GBM variants, including a GBM with PNET features and a GBM with oligodendroglioma component, were established. All unique lines showed distinct genetic profiles by STR profiling. The lines assayed demonstrated a range of in vitro growth rates. Multipotency was confirmed using in vitro differentiation. Tumor formation demonstrated histologic features consistent with high grade gliomas, including invasion, necrosis, abnormal vascularization, and high mitotic rate. Xenografts derived from the GBM variants maintained histopathological features of the primary tumors. We have generated and characterized GBM suspension lines derived from patients with GBMs and GBM variants. These oncosphere cell lines will expand the resources available for preclinical study.

STL-based Analysis of TRAIL-induced Apoptosis Challenges the Notion of Type I/Type II Cell Line Classification

PubMed Central

Bertaux, François; Maler, Oded; Batt, Gregory

2013-01-01

Extrinsic apoptosis is a programmed cell death triggered by external ligands, such as the TNF-related apoptosis inducing ligand (TRAIL). Depending on the cell line, the specific molecular mechanisms leading to cell death may significantly differ. Precise characterization of these differences is crucial for understanding and exploiting extrinsic apoptosis. Cells show distinct behaviors on several aspects of apoptosis, including (i) the relative order of caspases activation, (ii) the necessity of mitochondria outer membrane permeabilization (MOMP) for effector caspase activation, and (iii) the survival of cell lines overexpressing Bcl2. These differences are attributed to the activation of one of two pathways, leading to classification of cell lines into two groups: type I and type II. In this work we challenge this type I/type II cell line classification. We encode the three aforementioned distinguishing behaviors in a formal language, called signal temporal logic (STL), and use it to extensively test the validity of a previously-proposed model of TRAIL-induced apoptosis with respect to experimental observations made on different cell lines. After having solved a few inconsistencies using STL-guided parameter search, we show that these three criteria do not define consistent cell line classifications in type I or type II, and suggest mutants that are predicted to exhibit ambivalent behaviors. In particular, this finding sheds light on the role of a feedback loop between caspases, and reconciliates two apparently-conflicting views regarding the importance of either upstream or downstream processes for cell-type determination. More generally, our work suggests that these three distinguishing behaviors should be merely considered as type I/II features rather than cell-type defining criteria. On the methodological side, this work illustrates the biological relevance of STL-diagrams, STL population data, and STL-guided parameter search implemented in the tool Breach. Such tools are well-adapted to the ever-increasing availability of heterogeneous knowledge on complex signal transduction pathways. PMID:23675292

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

PubMed

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

2018-04-01

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

Cytotoxicity of titanium and silicon dioxide nanoparticles

NASA Astrophysics Data System (ADS)

Wagner, Stefanie; Münzer, Simon; Behrens, Peter; Scheper, Thomas; Bahnemann, Detlef; Kasper, Cornelia

2009-05-01

Different TiO2 and SiO2 nanoparticles have been tested concerning their toxicity on selected mammalian cell lines. Various powders and suspensions, all of which consist of titanium or silicon dioxide nanoparticles have been examined. These particles differ in the crystal structure, the size and the BET-surface area. There was also a classification in fixed particles and in particles easily accessible in solution. With focus on the possible adsorption of the nanoparticles into the human organism, via skin and via respiratory tract, the effects on fibroblasts (NIH-3T3) and on a human lung adenocarcinoma epithelial cell line were examined. Additionally, the particles were tested with HEP-G2 cells, which are often used as model cell line for biocompatibility tests, and PC-12 cells, a rat adrenal pheochromocytoma cell line. The viability of the cells was examined by the MTT-test. The viability results were found to partly depend on the type of cells used. The experimental results show that the adhesion of the cells on the different powders strongly depends on the type of cell lines as well as on the type of powder. It was found that the lower viability of some cells on the powder coatings is not only caused by a cytotoxicity effect of the powders, but is also due to a lower adhesion of the cells on the particle surfaces. Furthermore, it could be shown that the physical properties of the powders cannot be easily correlated to any observed biological effect. While some powders show a significant suppression of the cell growth, others with similar physical properties indicate no toxic effect.

Establishment and genetic characterization of an immortal tumor cell line derived from intestinal-type sinonasal adenocarcinoma.

PubMed

Pérez-Escuredo, Jhudit; García Martínez, Jorge; García-Inclán, Cristina; Vivanco, Blanca; Costales, María; Álvarez Marcos, César; Llorente, José Luis; Hermsen, Mario A

2011-02-01

Intestinal-type sinonasal adenocarcinoma (ITAC) is a rare tumor etiologically related to professional exposure to wood dust. The overall prognosis is poor, mainly due to the difficulty to resect the tumor completely in this anatomically complex region. Therefore, there is great need for alternative treatments. However, the lack of a good tumor model system for ITAC has hampered the development and testing of new therapeutic agents. Here, we report the establishment and characterization of the first human ITAC cell line named ITAC-3. The cell line was initiated from small explants of a T4bN0M0 colonic type ITAC from the ethmoid sinus. Growth and invasion parameters as well as genetic characteristics were analyzed. The population doubling time was 18 h and the cell line was capable of invasion in matrigel. Chromosomal analysis showed a tetraploid karyotype with both numerical and structural aberrations. High resolution microarray CGH analysis identified many copy number alterations, including homozygous deletions. TP53 carried a mutation c.818G>T in exon eight concurring with a strong nuclear protein overexpression. Immunohistochemical analysis showed protein overexpression of EGFR and normal expression of β-catenin and p16. This is the first report of the establishment of a cell line derived from a primary ITAC. The genomic profile of the cell line was the same as the primary tumor from which it was derived. This new cell line will be a useful tool for the development and testing of new therapeutic agents for this tumor type.

Production of coagulation factor VII in human cell lines Sk-Hep-1 and HKB-11.

PubMed

Corrêa de Freitas, Marcela Cristina; Bomfim, Aline de Sousa; Mizukami, Amanda; Picanço-Castro, Virgínia; Swiech, Kamilla; Covas, Dimas Tadeu

2017-09-01

Recombinant factor VII (rFVII) is the main therapeutic choice for hemophilia patients who have developed inhibitory antibodies against conventional treatments (FVIII and FIX). Because of the post-translational modifications, rFVII needs to be produced in mammalian cell lines. In this study, for the first time, we have shown efficient rFVII production in HepG2, Sk-Hep-1, and HKB-11 cell lines. Experiments in static conditions for a period of 96 h showed that HepG2-FVII produced the highest amounts of rhFVII, with an average of 1843 ng/mL. Sk-hep-1-FVII cells reached a maximum protein production of 1432 ng/mL and HKB-11-FVII cells reached 1468 ng/mL. Sk-Hep-1-rFVII and HKB-11-rFVII were selected for the first step of scale-up. Over 10 days of spinner flask culture, HKB-11 and SK-Hep-1 cells showed a cumulative production of rFVII of 152 μg and 202.6 μg in 50 mL, respectively. Thus, these human cell lines can be used for an efficient production of recombinant FVII. With more investment in basic research, human cell lines can be optimized for the commercial production of different bio therapeutic proteins. Copyright © 2017 Elsevier Inc. All rights reserved.

Anti-proliferative activity of 2,6-dichloro-9- or 7-(ethoxycarbonylmethyl)-9H- or 7H-purines against several human solid tumour cell lines.

PubMed

Morales, Fátima; Ramírez, Alberto; Conejo-García, Ana; Morata, Cynthia; Marchal, Juan A; Campos, Joaquín M

2014-04-09

As leads we took several benzo-fused seven- and six-membered scaffolds linked to the pyrimidine or purine moieties with notable anti-proliferative activity against human breast, colon and melanoma cancerous cell lines. We then decided to maintain the double-ringed nitrogenous bases and change the other components to the ethyl acetate moiety. This way six purine and two 5-fluorouracil derivatives were obtained and evaluated against the MCF-7, HCT-116, A-375 and G-361 cancer cell lines. Two QSARs are obtained between the anti-proliferative IC₅₀ values for compounds 26-33 and the clog P against the melanoma cell lines A-375 and G-361. Our results show that two of the analogues [ethyl 2-(2,6-dichloro-9H- or 7H-purine-9- or 7-yl)acetates (30 and 33, respectively)] are potent cytotoxic agents against all the tumour cell lines assayed, showing single-digit micromolar IC₅₀ values. This exemplifies the potential of our previously reported purine compounds to qualify as lead structures for medicinal chemistry campaigns, affording simplified analogues easy to synthesize and with a noteworthy bioactivity. The selective activity of 30 and 33 against the melanoma cell line A-375, via apoptosis, supposes a great advantage for a future therapeutic use. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Heterologous Expression of the Carrot Hsp17.7 gene Increased Growth, Cell Viability, and Protein Solubility in Transformed Yeast (Saccharomyces cerevisiae) under Heat, Cold, Acid, and Osmotic Stress Conditions.

PubMed

Ko, Eunhye; Kim, Minhye; Park, Yunho; Ahn, Yeh-Jin

2017-08-01

In industrial fermentation of yeast (Saccharomyces cerevisiae), culture conditions are often modified from the optimal growth conditions of the cells to maintain large-scale cultures and/or to increase recombinant protein production. However, altered growth conditions can be stressful to yeast cells resulting in reduced cell growth and viability. In this study, a small heat shock protein gene from carrot (Daucus carota L.), Hsp17.7, was inserted into the yeast genome via homologous recombination to increase tolerance to stress conditions that can occur during industrial culture. A DNA construct, Translational elongation factor gene promoter-carrot Hsp17.7 gene-Phosphoribosyl-anthranilate isomerase gene (an auxotrophic marker), was generated by a series of PCRs and introduced into the chromosome IV of the yeast genome. Immunoblot analysis showed that carrot Hsp17.7 accumulated in the transformed yeast cell lines. Growth rates and cell viability of these cell lines were higher than control cell lines under heat, cold, acid, and hyperosmotic stress conditions. Soluble protein levels were higher in the transgenic cell lines than control cell lines under heat and cold conditions, suggesting the molecular chaperone function of the recombinant Hsp17.7. This study showed that a recombinant DNA construct containing a HSP gene from carrot was successfully expressed in yeast by homologous recombination and increased tolerances to abiotic stress conditions.

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.

Selective cytotoxic effect of non-thermal micro-DBD plasma

NASA Astrophysics Data System (ADS)

Kwon, Byung-Su; Choi, Eun Ha; Chang, Boksoon; Choi, Jeong-Hyun; Kim, Kyung Sook; Park, Hun-Kuk

2016-10-01

Non-thermal plasma has been extensively researched as a new cancer treatment technology. We investigated the selective cytotoxic effects of non-thermal micro-dielectric barrier discharge (micro-DBD) plasma in cervical cancer cells. Two human cervical cancer cell lines (HeLa and SiHa) and one human fibroblast (HFB) cell line were treated with micro-DBD plasma. All cells underwent apoptotic death induced by plasma in a dose-dependent manner. The plasma showed selective inhibition of cell proliferation in cervical cancer cells compared to HFBs. The selective effects of the plasma were also observed between the different cervical cancer cell lines. Plasma treatment significantly inhibited the proliferation of SiHa cells in comparison to HeLa cells. The changes in gene expression were significant in the cervical cancer cells in comparison to HFBs. Among the cancer cells, apoptosis-related genes were significantly enriched in SiHa cells. These changes were consistent with the differential cytotoxic effects observed in different cell lines.

Cancer stem cell mediated acquired chemoresistance in head and neck cancer can be abrogated by aldehyde dehydrogenase 1 A1 inhibition.

PubMed

Kulsum, Safeena; Sudheendra, Holalugunda Vittalamurthy; Pandian, Ramanan; Ravindra, Doddathimmasandra Ramanjanappa; Siddappa, Gangotri; R, Nisheena; Chevour, Priyanka; Ramachandran, Balaji; Sagar, Milind; Jayaprakash, Aravindakshan; Mehta, Alka; Kekatpure, Vikram; Hedne, Naveen; Kuriakose, Moni A; Suresh, Amritha

2017-02-01

Chemoresistance leading to disease relapse is one of the major challenges to improve outcome in head and neck cancers. Cancer Stem Cells (CSCs) are increasingly being implicated in chemotherapy resistance, this study investigates the correlation between CSC behavior and acquired drug resistance in in vitro cell line models. Cell lines resistant to Cisplatin (Cal-27 CisR, Hep-2 CisR) and 5FU (Cal-27 5FUR) with high Resistance Indices (RI) were generated (RI ≥ 3) by short-term treatment of head and neck squamous cell carcinoma (HNSCC) cell lines with chemotherapeutic drugs (Cisplatin, Docetaxel, 5FU), using a dose-incremental strategy. The cell lines (Cal-27 DoxR, Hep-2 DoxR, Hep-2 5FUR) that showed low RI, nevertheless had a high cross resistance to Cisplatin/5FU (P < 0.05). Cal-27 CisR and DoxR showed 12-14% enrichment of CD44+ cells, while CisR/5FUR showed 4-6% increase in ALDH1A1+ cells as compared to parental cells (P < 0.05). Increased expression of stem cell markers (CD44, CD133, NOTCH1, ALDH1A1, OCT4, SOX2) in these cell lines, correlated with enhanced spheroid/colony formation, migratory potential, and increased in vivo tumor burden (P < 0.05). Inhibition of ALDH1A1 in Cal-27 CisR led to down regulation of the CSC markers, reduction in migratory, self-renewal and tumorigenic potential (P < 0.05) accompanied by an induction of sensitivity to Cisplatin (P < 0.05). Further, ex vivo treatment of explants (n = 4) from HNSCC patients with the inhibitor (NCT-501) in combination with Cisplatin showed a significant decrease in proliferating cells as compared to individual treatment (P = 0.001). This study hence suggests an ALDH1A1-driven, CSC-mediated mechanism in acquired drug resistance of HNSCC, which may have therapeutic implications. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

ZIP8 expression in human proximal tubule cells, human urothelial cells transformed by Cd+2 and As+3 and in specimens of normal human urothelium and urothelial cancer

PubMed Central

2012-01-01

Background ZIP8 functions endogenously as a Zn+2/HCO3- symporter that can also bring cadmium (Cd+2) into the cell. It has also been proposed that ZIP8 participates in Cd-induced testicular necrosis and renal disease. In this study real-time PCR, western analysis, immunostaining and fluorescent localization were used to define the expression of ZIP8 in human kidney, cultured human proximal tubule (HPT) cells, normal and malignant human urothelium and Cd+2 and arsenite (As+3) transformed urothelial cells. Results It was shown that in the renal system both the non-glycosylated and glycosylated form of ZIP8 was expressed in the proximal tubule cells with localization of ZIP8 to the cytoplasm and cell membrane; findings in line with previous studies on ZIP8. The studies in the bladder were the first to show that ZIP8 was expressed in normal urothelium and that ZIP8 could be localized to the paranuclear region. Studies in the UROtsa cell line confirmed a paranuclear localization of ZIP8, however addition of growth medium to the cells increased the expression of the protein in the UROtsa cells. In archival human samples of the normal urothelium, the expression of ZIP8 was variable in intensity whereas in urothelial cancers ZIP8 was expressed in 13 of 14 samples, with one high grade invasive urothelial cancer showing no expression. The expression of ZIP8 was similar in the Cd+2 and As+3 transformed UROtsa cell lines and their tumor transplants. Conclusion This is the first study which shows that ZIP8 is expressed in the normal urothelium and in bladder cancer. In addition the normal UROtsa cell line and its transformed counterparts show similar expression of ZIP8 compared to the normal urothelium and the urothelial cancers suggesting that the UROtsa cell line could serve as a model system to study the expression of ZIP8 in bladder disease. PMID:22550998

Effects of cholera toxin on human colon carcinoma cell lines.

PubMed

Barkla, D H; Whitehead, R H; Hayward, I P

1992-10-01

This study reports on changes in morphology and membrane transport in 5 human colon carcinoma cell lines treated with cholera toxin (CT). Three of the cell lines that grew as monolayers (LIM 1215, LIM 1899, LIM 2099) and 1 that grew as floating clumps (LIM 2408) did not show morphological changes after CT treatment. However, cell line LIM 1863 that grows as floating "crypt-like" organoids showed rapid and distinctive changes in morphology and membrane transport after CT treatment. At 1 and 6 hrs after CT treatment, light and transmission electron microscopy revealed rapid dilatation of the central lumen of organoids and the appearance of 2 populations of apical vesicular inclusions. The first population was unusual in being non-membrane bound and limited by fuzzy filamentous material. The second population was membrane bound. Scanning electron microscopy at 1-6 hr after CT treatment showed swelling and loss of surface microvilli on some, but not all, cells. At 24 hr after CT treatment the majority of organoids showed evidence of fluid accumulation and small apical vesicles coalesced to form large single vacuoles that obliterated normal cell morphology. By 48 hr, continued swelling produced extreme attenuation of the plasma membrane with cells taking on an "endothelial cell-like" appearance. The response to CT was dose-dependent. Uptake studies using 86Rubidium and blocking studies using ouabain and amiloride indicated that CT is acting on the Na+/K+ ATPase membrane pump to cause the increased fluid uptake by LIM 1863 cells. This study is the first to report specific morphological changes in intestine-derived cells in response to CT.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemical genetic inhibition of Mps1 in stable human cell lines reveals novel aspects of Mps1 function in mitosis.

PubMed

Sliedrecht, Tale; Zhang, Chao; Shokat, Kevan M; Kops, Geert J P L

2010-04-22

Proper execution of chromosome segregation relies on tight control of attachment of chromosomes to spindle microtubules. This is monitored by the mitotic checkpoint that allows chromosome segregation only when all chromosomes are stably attached. Proper functioning of the attachment and checkpoint processes is thus important to prevent chromosomal instability. Both processes rely on the mitotic kinase Mps1. We present here two cell lines in which endogenous Mps1 has been stably replaced with a mutant kinase (Mps1-as) that is specifically inhibited by bulky PP1 analogs. Mps1 inhibition in these cell lines is highly penetrant and reversible. Timed inhibition during bipolar spindle assembly shows that Mps1 is critical for attachment error-correction and confirms its role in Aurora B regulation. We furthermore show that Mps1 has multiple controls over mitotic checkpoint activity. Mps1 inhibition precludes Mad1 localization to unattached kinetochores but also accelerates mitosis. This acceleration correlates with absence of detectable mitotic checkpoint complex after Mps1 inhibition. Finally, we show that short-term inhibition of Mps1 catalytic activity is sufficient to kill cells. Mps1 is involved in the regulation of multiple key processes that ensure correct chromosome segregation and is a promising target for inhibition in anti-cancer strategies. We report here two cell lines that allow specific and highly penetrant inhibition of Mps1 in a reproducible manner through the use of chemical genetics. Using these cell lines we confirm previously suggested roles for Mps1 activity in mitosis, present evidence for novel functions and examine cell viability after short and prolonged Mps1 inhibition. These cell lines present the best cellular model system to date for investigations into Mps1 biology and the effects of penetrance and duration of Mps1 inhibition on cell viability.

Eighteen-Year Cryopreservation Does Not Negatively Affect the Pluripotency of Human Embryos: Evidence from Embryonic Stem Cell Derivation

PubMed Central

Rungsiwiwut, Ruttachuk; Numchaisrika, Pranee; Ahnonkitpanit, Vichuda; Isarasena, Nipan; Virutamasen, Pramuan

2012-01-01

Abstract Human embryonic stem (hES) cells are considered to be a potential source for the therapy of human diseases, drug screening, and the study of developmental biology. In the present study, we successfully derived hES cell lines from blastocysts developed from frozen and fresh embryos. Seventeen- to eighteen-year-old frozen embryos were thawed, cultured to the blastocyst stage, and induced to form hES cells using human foreskin fibroblasts. The Chula2.hES cell line and the Chula4.hES and Chula5.hES cell lines were derived from blastocysts developed from frozen and fresh embryos, respectively. The cell lines expressed pluripotent markers, including alkaline phosphatase (AP), Oct3/4, stage-specific embryonic antigen (SSEA)-4, and tumor recognition antigen (TRA)-1-60 and TRA-1-81 as detected with immunocytochemistry. The real-time polymerase chain reaction (RT-PCR) results showed that the cell lines expressed pluripotent genes, including OCT3/4, SOX2, NANOG, UTF, LIN28, REX1, NODAL, and E-Cadherin. In addition, the telomerase activities of the cell lines were higher than in the fibroblast cells. Moreover, the cell lines differentiated into all three germ layers both in vitro and in vivo. The cell lines had distinct identities, as revealed with DNA fingerprinting, and maintained their normal karyotype after a long-term culture. This study is the first to report the successful derivation of hES cell lines in Thailand and that frozen embryos maintained their pluripotency similar to fresh embryos, as shown by the success of hES cell derivation, even after years of cryopreservation. Therefore, embryos from prolonged cryopreservation could be an alternative source for embryonic stem cell research. PMID:23514952

Human pathogenic Mycoplasma species induced cytokine gene expression in Epstein-Barr virus (EBV)-positive lymphoblastoid cell lines.

PubMed

Schäffner, E; Opitz, O; Pietsch, K; Bauer, G; Ehlers, S; Jacobs, E

1998-04-01

We addressed the question whether the in vitro interaction of two Epstein-Barr virus (EBV)-genome-positive B cell lines (EB-3 and HilB-gamma) with either Mycoplasma pneumoniae or M. hominis, with the mycoplasma species (M. fermentans, M. fermentans subsp. incognitus, M. penetrans, M. genitalium) or with mycoplasma species known to be mere commensals of the respiratory tract (M. orale and M. salivarium) would result in expression of mRNAs for IL-2, IL-2R, IL-4 and IL-6 as determined by reverse transcriptase (RT)-PCR after 4 and 24 h of cocultivation. The pattern of cytokine gene expression observed depended on (i) the origin of the transformed cell line, (ii) the pathogenicity of the Mycoplasma species, and (iii) the length of cocultivation. The EBV-immortalized lymphoblastoid cell line HilB-gamma showed mRNA expression for IL-2, IL-2-receptor, IL-4 and IL-6 peaking 24 h after stimulation with M. pneumoniae and all AIDS-related mycoplasma species tested. The Burkitt lymphoma cell line EB-3 showed a distinct and isolated strong II-2/IL-2 R-mRNA expression within 4 h after contact with the pathogenic and all of the AIDS related mycoplasma species. In neither EBV-containing cell line cytokine was gene expression detectable after stimulation with the commensal mycoplasma species, M. orale and M. salivarium, indicating species differences in the ability of mycoplasmas to interact with and stimulate B-cell lines. Our data suggest that some mcyoplasma species may act as immunomodulatory cofactors by eliciting inappropriate cytokine gene expression in B cells latently infected with EBV. Therefore, this cultivation model may prove useful in evaluating the pathogenetic potential of novel isolated mycoplasma species. Copyright 1998 Academic Press Limited.

Immunomodulation of human B cells following treatment with intravenous immunoglobulins involves increased phosphorylation of extracellular signal-regulated kinases 1 and 2.

PubMed

Dussault, Nathalie; Ducas, Eric; Racine, Claudia; Jacques, Annie; Paré, Isabelle; Côté, Serge; Néron, Sonia

2008-11-01

In the treatment of autoimmune diseases, intravenous Igs (IVIg) are assumed to modulate immune cells through the binding of surface receptors. IVIg act upon definite human B cell populations to modulate Ig repertoire, and such modulation might proceed through intracellular signaling. However, the heterogeneity of human B cell populations complicates investigations of the intracellular pathways involved in IVIg-induced B cell modulation. The aim of this study was to establish a model allowing the screening of IVIg signal transduction in human B cell lines and to attempt transposing observations made in cell lines to normal human B lymphocytes. Nine human B cell lines were treated with IVIg with the goal of selecting the most suitable model for human B lymphocytes. The IgG(+) DB cell line, whose response was similar to that of human B lymphocytes, showed reduced IVIg modulation following addition of PD98059, an inhibitor of extracellular signal-regulated protein kinase 1/2 (ERK1/2). The IVIg-induced ERK1/2 phosphorylation was indeed proportional to the dosage of monomeric IVIg used when tested on DB cells as well as Pfeiffer cells, another IgG(+) cell line. In addition, two other intermediates, Grb2-associated binder 1 (Gab1) and Akt, showed increased phosphorylation in IVIg-treated DB cells. IVIg induction of ERK1/2 phosphorylation was finally observed in peripheral human B lymphocytes, specifically within the IgG(+) B cell population. In conclusion, IVIg immunomodulation of human B cells can thus be linked to intracellular transduction pathways involving the phosphorylation of ERK1/2, which in combination with Gab1 and Akt, may be related to B cell antigen receptor signaling.

Cytotoxic activity of Justicia spicigera is inhibited by bcl-2 proto-oncogene and induces apoptosis in a cell cycle dependent fashion.

PubMed

Cáceres-Cortés, J R; Cantú-Garza, F A; Mendoza-Mata, M T; Chavez-González, M A; Ramos-Mandujano, G; Zambrano-Ramírez, I R

2001-12-01

Identification of organic compounds from plants is of clinical significance because of the effect that they might have in patients with haematopoietic disorders. We studied the effect of the plant extract Justicia spicigera (Acanthaceae) in different haematopoietic cells: human leukaemic cell lines, umbilical cord blood cells, and mouse bone marrow cells. By examining colony formation and performing the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay it was shown that the plant extract of Justicia spicigera contains cytotoxic factors for leukaemic cells and has no proliferative activity on normal haematopoietic progenitor cells. Our results show that this plant extract induces apoptosis in the human leukaemia cell line TF-1, but not in the bcl-2 transfectant cell line TB-1. Similar results were obtained using a haemopoietic cell line 32D and 32DBcl2. The cultures of umbilical cord blood cells and mouse bone marrow that contain granulocyte-macrophage colony-stimulating factor (GM-CSF) do not proliferate or become terminally differentiated in the presence of the infusion of Justicia spicigera. GM-CSF that acts by abrogating programmed cell death is not sufficient to inhibit the apoptotic stimulus in TF-1 and 32D cells. Moreover mouse fibroblasts (3T3) and two cervical carcinoma cell lines CALO and INBL, undergo apoptosis in the presence of different concentrations of an infusion from the plant. Our data show that there is a strong correlation between the cytotoxic effect and cell proliferation. Together, these results indicate that the plant infusion of Justicia spicigera does not contain any haematopoietic activity, induces apoptosis inhibited by bcl-2 and is linked to cell proliferation. Copyright 2001 John Wiley & Sons, Ltd.

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

PubMed

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

2016-06-01

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

Effects of Newcastle Disease Virus Strains AF2240 and V4-UPM on Cytolysis and Apoptosis of Leukemia Cell Lines

PubMed Central

Alabsi, Aied M.; Bakar, Siti Aishah Abu; Ali, Rola; Omar, Abdul Rahman; Bejo, Mohd Hair; Ideris, Aini; Ali, Abdul Manaf

2011-01-01

Newcastle disease virus (NDV) is used as an antineoplastic agent in clinical tumor therapy. It has prompted much interest as an anticancer agent because it can replicate up to 10,000 times better in human cancer cells than in most normal cells. This study was carried out to determine the oncolytic potential of NDV strain AF2240 and V4-UPM on WEHI-3B leukemia cell line. Results from MTT cytotoxicity assay showed that the CD50 values for both strains were 2 and 8 HAU for AF2240 and V4-UPM, respectively. In addition, bromodeoxyuridine (BrdU) and trypan blue dye exclusion assays showed inhibition in cell proliferation after different periods. Increase in the cellular level of caspase-3 and detection of DNA laddering using agarose gel electrophoresis on treated cells with NDV confirmed that the mode of cell death was apoptosis. In addition, flow-cytometry analysis of cellular DNA content showed that the virus caused an increase in the sub-G1 region (apoptosis peaks). In conclusion, NDV strains AF2240 and V4-UPM caused cytolytic effects against WEHI-3B leukemic cell line. PMID:22272097

Human squamous cell carcinoma from skin: establishment and characterization of a new cell line (HSC-5).

PubMed

Hozumi, Y; Kondo, S; Shimoura, T; Aso, K

1990-03-01

A new cell line, designated as HSC-5 and derived from human skin squamous cell carcinoma (SCC), has been established in vitro and maintained proliferative in continuous tissue culture for over two years. The cells grow in a monolayer in vitro and have anaplastic epithelioid features. The doubling time was about 35 hr at the 30th passage. Chromosome analysis showed hypotetraploidy with a modal number of 76. A trial of transplantation of the cultured cells into nude mice was not successful. Analysis of cytokeratins from HSC-5 by two-dimensional gel electrophoresis revealed polypeptides No. 5, 8, 13, 18 and 19. The cell line is available to other investigators.

A silencing suppressor protein (NSs) of a tospovirus enhances baculovirus replication in permissive and semipermissive insect cell lines.

PubMed

Oliveira, Virgínia Carla; Bartasson, Lorrainy; de Castro, Maria Elita Batista; Corrêa, José Raimundo; Ribeiro, Bergmann Morais; Resende, Renato Oliveira

2011-01-01

The nonstructural protein (NSs) of the Tomato spotted wilt virus (TSWV) has been identified as an RNAi suppressor in plant cells. A recombinant Autographa californica multiple nucleopolyhedrovirus (AcMNPV) designated vAcNSs, containing the NSs gene under the control of the viral polyhedrin (polh) gene promoter, was constructed and the effects of NSs in permissive, semipermissive and nonpermissive insect cells to vAcNSs infection were evaluated. vAcNSs produced more budded virus when compared to wild type in semipermissive cells. Co-infection of vAcNSs with wild type baculoviruses clearly enhanced polyhedra production in all host cells. Confocal microscopy analysis showed that NSs accumulated in abundance in the cytoplasm of permissive and semipermissive cells. In contrast, high amounts of NSs were detected in the nuclei of nonpermissive cells. Co-infection of vAcNSs with a recombinant AcMNPV containing the enhanced green fluorescent protein (egfp) gene, significantly increased EGFP expression in semipermissive cells and in Anticarsia gemmatalis-hemocytes. Absence of small RNA molecules of egfp transcripts in this cell line and in a permissive cell line indicates the suppression of gene silencing activity. On the other hand, vAcNSs was not able to suppress RNAi in a nonpermissive cell line. Our data showed that NSs protein of TSWV facilitates baculovirus replication in different lepidopteran cell lines, and these results indicate that NSs could play a similar role during TSWV-infection in its thrips vector. Copyright © 2010 Elsevier B.V. All rights reserved.

Identification of anti-proliferative kinase inhibitors as potential therapeutic agents to treat canine osteosarcoma.

PubMed

Mauchle, Ulrike; Selvarajah, Gayathri T; Mol, Jan A; Kirpensteijn, Jolle; Verheije, Monique H

2015-08-01

Osteosarcoma is the most common primary bone tumour in dogs but various forms of therapy have not significantly improved clinical outcomes. As dysregulation of kinase activity is often present in tumours, kinases represent attractive molecular targets for cancer therapy. The purpose of this study was to identify novel compounds targeting kinases with the potential to induce cell death in a panel of canine osteosarcoma cell lines. The ability of 80 well-characterized kinase inhibitor compounds to inhibit the proliferation of four canine osteosarcoma cell lines was investigated in vitro. For those compounds with activity, the mechanism of action and capability to potentiate the activity of doxorubicin was further evaluated. The screening showed 22 different kinase inhibitors that induced significant anti-proliferative effects across the four canine osteosarcoma cell lines investigated. Four of these compounds (RO 31-8220, 5-iodotubercidin, BAY 11-7082 and an erbstatin analog) showed significant cell growth inhibitory effects across all cell lines in association with variable induction of apoptosis. RO 31-8220 and 5-iodotubercidin showed the highest ability to potentiate the effects of doxorubicin on cell viability. In conclusion, the present study identified several potent kinase inhibitors targeting the PKC, CK1, PKA, ErbB2, mTOR and NF-κB pathways, which may warrant further investigations for the treatment of osteosarcoma in dogs. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

PubMed

Kamal, Ahmed; Pogula, Praveen Kumar; Khan, Mohammed Naseer Ahmed; Seshadri, Bobburi Naga; Sreekanth, Kokkonda

2013-08-01

As a continuation of our efforts to develop the benzimidazole-PBD conjugates as potential anticancer agents, a series of heteroaryl substituted benzimidazole linked PBD conjugates has been synthesized and evaluated for their anticancer potential in 60 human cancer cell lines. Most of the compounds exhibited promising anticancer activity and interestingly, compounds 4c and 4d displayed significant activity in most of the cell lines tested. Whereas, compound 4e showed selectivity in renal cancer cells with GI50 values of <10 and 70 nM against RXF 393 and UO-31 cell lines, respectively. Further, these compounds also showed significant DNA-binding affinity by thermal denaturation study using duplex form of calf thymus (CT) DNA.

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

PubMed

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

2012-05-31

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

Novel Mechanisms Revealed in the Trachea Transcriptome of Resistant and Susceptible Chicken Lines following Infection with Newcastle Disease Virus.

PubMed

Deist, Melissa S; Gallardo, Rodrigo A; Bunn, David A; Kelly, Terra R; Dekkers, Jack C M; Zhou, Huaijun; Lamont, Susan J

2017-05-01

Newcastle disease virus (NDV) has a devastating impact on poultry production in developing countries. This study examined the transcriptome of tracheal epithelial cells from two inbred chicken lines that differ in NDV susceptibility after challenge with a high-titer inoculum of lentogenic NDV. The Fayoumi line had a significantly lower NDV load postchallenge than the Leghorn line, demonstrating the Fayoumi line's classification as a relatively NDV-resistant breed. Examination of the trachea transcriptome showed a large increase in immune cell infiltration in the trachea in both lines at all times postinfection. The pathways conserved across lines and at all three time points postinfection included iCOS-iCOSL signaling in T helper cells, NF-κB signaling, the role of nuclear factor of activated T cells in the regulation of the immune response, calcium-induced T lymphocyte apoptosis, phospholipase C signaling, and CD28 signaling in T helper cells. Although shared pathways were seen in the Fayoumi and Leghorn lines, each line showed unique responses as well. The downregulation of collagen and the activation of eukaryotic translation initiation factor 2 signaling in the Fayoumis relative to the Leghorns at 2 days postinfection may contribute to the resistance phenotype seen in the Fayoumis. This study provides a further understanding of host-pathogen interactions which could improve vaccine efficacy and, in combination with genome-wide association studies, has the potential to advance strategies for breeding chickens with enhanced resistance to NDV. Copyright © 2017 Deist et al.

A flavonoid isolated from Streptomyces sp. (ERINLG-4) induces apoptosis in human lung cancer A549 cells through p53 and cytochrome c release caspase dependant pathway.

PubMed

Balachandran, C; Sangeetha, B; Duraipandiyan, V; Raj, M Karunai; Ignacimuthu, S; Al-Dhabi, N A; Balakrishna, K; Parthasarathy, K; Arulmozhi, N M; Arasu, M Valan

2014-12-05

The aim of this study was to investigate the anticancer activity of a flavonoid type of compound isolated from soil derived filamentous bacterium Streptomyces sp. (ERINLG-4) and to explore the molecular mechanisms of action. Cytotoxic properties of ethyl acetate extract was carried out against A549 lung cancer cell line using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Cytotoxic properties of isolated compound were investigated in A549 lung cancer cell line, COLO320DM cancer cell line and Vero cells. The compound showed potent cytotoxic properties against A549 lung cancer cell line and moderate cytotoxic properties against COLO320DM cancer cell line. Isolated compound showed no toxicity up to 2000 μg/mL in Vero cells. So we have chosen the A549 lung cancer cell line for further anticancer studies. Intracellular visualization was done by using a laser scanning confocal microscope. Apoptosis was measured using DNA fragmentation technique. Treatment of the A549 cancer cells with isolated compound significantly reduced cell proliferation, increased formation of fragmented DNA and apoptotic body. Activation of caspase-9 and caspase-3 indicated that compound may be inducing intrinsic and extrinsic apoptosis pathways. Bcl-2, p53, pro-caspases, caspase-3, caspase-9 and cytochrome c release were detected by western blotting analysis after compound treatment (123 and 164 μM). The activities of pro-caspases-3, caspase-9 cleaved to caspase-3 and caspase-9 gradually increased after the addition of isolated compound. But Bcl-2 protein was down regulated after treatment with isolated compound. Molecular docking studies showed that the compound bound stably to the active sites of caspase-3 and caspase-9. These results strongly suggest that the isolated compound induces apoptosis in A549 cancer cells via caspase activation through cytochrome c release from mitochondria. The present results might provide helpful suggestions for the design of antitumor drugs toward lung cancer treatment. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Combined therapeutic potential of nuclear receptors with receptor tyrosine kinase inhibitors in lung cancer

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

Wairagu, Peninah M.; Institute of Lifestyle Medicine, Wonju College of Medicine, Yonsei University, Wonju, Gangwon-do 220-701; Nuclear Receptor Research Consortium, Wonju College of Medicine, Yonsei University, Wonju, Gangwon-do 220-701

2014-05-09

Highlights: • The 48 NR genes and 48 biological anti-cancer targets are profiled in paired-cells. • Growth inhibition by NR ligands or TKIs is target receptor level-dependent. • T0901317 with gefitinib/PHA665752 shows additive growth inhibition in lung cells. - Abstract: Cancer heterogeneity is a big hurdle in achieving complete cancer treatment, which has led to the emergence of combinational therapy. In this study, we investigated the potential use of nuclear receptor (NR) ligands for combinational therapy with other anti-cancer drugs. We first profiled all 48 NRs and 48 biological anti-cancer targets in four pairs of lung cell lines, where eachmore » pair was obtained from the same patient. Two sets of cell lines were normal and the corresponding tumor cell lines while the other two sets consisted of primary versus metastatic tumor cell lines. Analysis of the expression profile revealed 11 NRs and 15 cancer targets from the two pairs of normal versus tumor cell lines, and 9 NRs and 9 cancer targets from the primary versus metastatic tumor cell lines had distinct expression patterns in each category. Finally, the evaluation of nuclear receptor ligand T0901317 for liver X receptor (LXR) demonstrated its combined therapeutic potential with tyrosine kinase inhibitors. The combined treatment of cMET inhibitor PHA665752 or EGFR inhibitor gefitinib with T0901317 showed additive growth inhibition in both H2073 and H1993 cells. Mechanistically, the combined treatment suppressed cell cycle progression by inhibiting cyclinD1 and cyclinB expression. Taken together, this study provides insight into the potential use of NR ligands in combined therapeutics with other biological anti-cancer drugs.« less

Comparing Apoptosis and Necrosis Effects of Arctium Lappa Root Extract and Doxorubicin on MCF7 and MDA-MB-231 Cell Lines

PubMed Central

Ghafari, Fereshteh; Rajabi, Mohammad Reza; Mazoochi, Tahereh; Taghizadeh, Mohsen; Nikzad, Hossein; Atlasi, Mohammad Ali; Taherian, Aliakbar

2017-01-01

Objective: Breast cancer is a heterogeneous disease and very common malignancy in women worldwide. The efficacy of chemotherapy as an important part of breast cancer treatment is limited due to its side effects. While pharmaceutical companies are looking for better chemicals, research on traditional medicines that generally have fewer side effects is quite interesting. In this study, apoptosis and necrosis effect of Arctium lappa and doxorubicin was compared in MCF7, and MDA-MB-231 cell lines. Materials and Methods: MCF7 and MDA-MB-231 cells were cultured in RPMI 1640 containing 10% FBS and 100 U/ml penicillin/streptomycin. MTT assay and an annexin V/propidium iodide (AV/PI) kit were used respectively to compare the survival rate and apoptotic effects of different concentrations of doxorubicin and Arctium lappa root extract on MDA-MB-231 and MCF7 cells. Results: Arctium lappa root extract was able to reduce cell viability of the two cell lines in a dose and time dependent manner similar to doxorubicin. Flow cytometry results showed that similar to doxorubicin, Arctium Lappa root extract had a dose and time dependent apoptosis effect on both cell lines. 10µg/mL of Arctium lappa root extract and 5 µM of doxorubicin showed the highest anti-proliferative and apoptosis effect in MCF7 and MDA231 cells. Conclusion: The MCF7 (ER/PR-) and MDA-MB-231 (ER/PR+) cell lines represent two major breast cancer subtypes. The similar anti-proliferative and apoptotic effects of Arctium lappa root extract and doxorubicin (which is a conventional chemotherapy drug) on two different breast cancer cell lines strongly suggests its anticancer effects and further studies. PMID:28441789

Comparing Apoptosis and Necrosis Effects of Arctium Lappa Root Extract and Doxorubicin on MCF7 and MDA-MB-231 Cell Lines

PubMed

Ghafari, Fereshteh; Rajabi, Mohammad Reza; Mazoochi, Tahereh; Taghizadeh, Mohsen; Nikzad, Hossein; Atlasi, Mohammad Ali; Taherian, Aliakbar

2017-03-01

Objective: Breast cancer is a heterogeneous disease and very common malignancy in women worldwide. The efficacy of chemotherapy as an important part of breast cancer treatment is limited due to its side effects. While pharmaceutical companies are looking for better chemicals, research on traditional medicines that generally have fewer side effects is quite interesting. In this study, apoptosis and necrosis effect of Arctium lappa and doxorubicin was compared in MCF7, and MDA-MB-231 cell lines. Materials and Methods: MCF7 and MDA-MB-231 cells were cultured in RPMI 1640 containing 10% FBS and 100 U/ml penicillin/streptomycin. MTT assay and an annexin V/propidium iodide (AV/PI) kit were used respectively to compare the survival rate and apoptotic effects of different concentrations of doxorubicin and Arctium lappa root extract on MDA-MB-231 and MCF7 cells. Results: Arctium lappa root extract was able to reduce cell viability of the two cell lines in a dose and time dependent manner similar to doxorubicin. Flow cytometry results showed that similar to doxorubicin, Arctium Lappa root extract had a dose and time dependent apoptosis effect on both cell lines. 10μg/mL of Arctium lappa root extract and 5 μM of doxorubicin showed the highest anti-proliferative and apoptosis effect in MCF7 and MDA231 cells. Conclusion: The MCF7 (ER/PR-) and MDA-MB-231 (ER/PR+) cell lines represent two major breast cancer subtypes. The similar anti-proliferative and apoptotic effects of Arctium lappa root extract and doxorubicin (which is a conventional chemotherapy drug) on two different breast cancer cell lines strongly suggests its anticancer effects and further studies. Creative Commons Attribution License

Proteomic Studies of Cholangiocarcinoma and Hepatocellular Carcinoma Cell Secretomes

PubMed Central

Srisomsap, Chantragan; Sawangareetrakul, Phannee; Subhasitanont, Pantipa; Chokchaichamnankit, Daranee; Chiablaem, Khajeelak; Bhudhisawasdi, Vaharabhongsa; Wongkham, Sopit; Svasti, Jisnuson

2010-01-01

Cholangiocarcinoma (CCA) and hepatocellular carcinoma (HCC) occur with relatively high incidence in Thailand. The secretome, proteins secreted from cancer cells, are potentially useful as biomarkers of the diseases. Proteomic analysis was performed on the secreted proteins of cholangiocarcinoma (HuCCA-1) and hepatocellular carcinoma (HCC-S102, HepG2, SK-Hep-1, and Alexander) cell lines. The secretomes of the five cancer cell lines were analyzed by SDS-PAGE combined with LC/MS/MS. Sixty-eight proteins were found to be expressed only in HuCCA-1. Examples include neutrophil gelatinase-associated lipocalin (lipocalin 2), laminin 5 beta 3, cathepsin D precursor, desmoplakin, annexin IV variant, and annexin A5. Immunoblotting was used to confirm the presence of lipocalin 2 in conditioned media and cell lysate of 5 cell lines. The results showed that lipocalin 2 was a secreted protein which is expressed only in the conditioned media of the cholangiocarcinoma cell line. Study of lipocalin 2 expression in different types of cancer and normal tissues from cholangiocarcinoma patients showed that lipocalin 2 was expressed only in the cancer tissues. We suggest that lipocalin 2 may be a potential biomarker for cholangiocarcinoma. PMID:20069059

Epigenetic inactivation of VGF associated with Urothelial Cell Carcinoma and its potential as a non-invasive biomarker using urine.

PubMed

Hayashi, Masamichi; Bernert, Heike; Kagohara, Luciane Tsukamoto; Maldonado, Leonel; Brait, Mariana; Schoenberg, Mark; Bivalacqua, Trinity; Netto, George J; Koch, Wayne; Sidransky, David; Hoque, Mohammad O

2014-05-30

To identify new epigenetic markers and further characterize Urothelial Cell Carcinoma (UCC), we tested the promoter methylation (PM) status of 19 genes previously identified as cancer specific methylated genes in other solid tumors. We used bisulfite sequencing, methylation specific PCR and quantitative methylation specific PCR (QMSP) to test the PM status of 19 genes in urothelial cancer cell lines. Among the 19 genes tested, VGF was found to be completely methylated in several UCC cell lines. VGF QMSP analysis showed that methylation values of almost all the primary 19 UCC tissues were higher than the paired normal tissues (P=0.009). In another cohort, 12/35 (34.3%) of low grade UCC cases displayed VGF methylation. As a biomarker for non-invasive detection of UCC, VGF showed a significantly higher frequency of methylation in urine from UCC cases (8/20) compared to controls (1/20) (P=0.020). After treatment of cell lines with 5-Aza-2'-deoxycytidine, VGF was robustly re-expressed. Forced expression of VGF in bladder cancer cell lines inhibited cell growth. Selection of candidates from genome-wide screening approach in other solid tumors successfully identified UCC specific methylated genes.

Comparative analysis of different cell systems for Zika virus (ZIKV) propagation and evaluation of anti-ZIKV compounds in vitro.

PubMed

Vicenti, Ilaria; Boccuto, Adele; Giannini, Alessia; Dragoni, Filippo; Saladini, Francesco; Zazzi, Maurizio

2018-01-15

A strong correlation between Zika virus (ZIKV) infection and severe neurological disease in newborns and occasionally adults has emerged in the Brazilian outbreak. Efficient human cell-based assays are required to test candidate inhibitors of ZIKV replication. The aim of this work was to investigate ZIKV propagation and quantification in different cell lines. The human (U87, A549, Huh7), mosquito (C6/36) and monkey (VERO E6) cell lines tested were all permissive to ZIKV infection. When assessed by plaque forming units (PFU) in three different target cell lines, the maximal production of ZIKV was achieved in Huh7 at day 3 post-infection (6.38±0.44 log 10 PFU/ml). The C6/36 cell line showed a low and slow production of virus when compared with other cell lines. A549 readout cells generated a larger number of plaques compared to Huh7 but not to VERO E6 cells. ZIKV PFU and RNA titers showed the highest correlation when Huh7 and A549 were used as the producer and readout cells, respectively. Also, U87 cells produced ZIKV RNA titers which were highly correlated with PFU independently from the readout cell line. Using the best virus-cell system, sofosbuvir and ribavirin EC 50 were 1.2μM and 1.1μM when measured through plaque assay, and 4.2μM and 5.2μM when measured by quantitative real time PCR (qRT-PCR), respectively. In summary, ZIKV can efficiently infect different human cell lines and rapidly reach peak viral titers. Overall, A549 cells appear to be as efficient as the VERO E6 gold standard for plaque assay allowing the use of human, rather than simian, cells for evaluating candidate anti-ZIKV compounds by the reference assay. The possibility to replace the labor-intensive plaque assay with the more rapid and easy-to-perform qRT-PCR is appealing and warrants further investigation. Copyright © 2017 Elsevier B.V. All rights reserved.

Proscillaridin A is cytotoxic for glioblastoma cell lines and controls tumor xenograft growth in vivo.

PubMed

Denicolaï, Emilie; Baeza-Kallee, Nathalie; Tchoghandjian, Aurélie; Carré, Manon; Colin, Carole; Jiglaire, Carine Jiguet; Mercurio, Sandy; Beclin, Christophe; Figarella-Branger, Dominique

2014-11-15

Glioblastoma is the most frequent primary brain tumor in adults. Because of molecular and cellular heterogeneity, high proliferation rate and significant invasive ability, prognosis of patients is poor. Recent therapeutic advances increased median overall survival but tumor recurrence remains inevitable. In this context, we used a high throughput screening approach to bring out novel compounds with anti-proliferative and anti-migratory properties for glioblastoma treatment. Screening of the Prestwick chemical library® of 1120 molecules identified proscillaridin A, a cardiac glycoside inhibitor of the Na(+)/K(+) ATPase pump, with most significant effects on glioblastoma cell lines. In vitro effects of proscillaridin A were evaluated on GBM6 and GBM9 stem-like cell lines and on U87-MG and U251-MG cell lines. We showed that proscillaridin A displayed cytotoxic properties, triggered cell death, induced G2/M phase blockade in all the glioblastoma cell lines and impaired GBM stem self-renewal capacity even at low concentrations. Heterotopic and orthotopic xenotransplantations were used to confirm in vivo anticancer effects of proscillaridin A that both controls xenograft growth and improves mice survival. Altogether, results suggest that proscillaridin A is a promising candidate as cancer therapies in glioblastoma. This sustains previous reports showing that cardiac glycosides act as anticancer drugs in other cancers.

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

PubMed

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

2016-11-29

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

Radiation Response of Cancer Stem-Like Cells From Established Human Cell Lines After Sorting for Surface Markers

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

Al-Assar, Osama; Muschel, Ruth J.; Mantoni, Tine S.

2009-11-15

Purpose: A subpopulation of cancer stem-like cells (CSLC) is hypothesized to exist in different cancer cell lines and to mediate radioresistance in solid tumors. Methods and Materials: Cells were stained for CSLC markers and sorted (fluorescence-activated cell sorter/magnetic beads) to compare foci and radiosensitivity of phosphorylated histone H2AX at Ser 139 (gamma-H2AX) in sorted vs. unsorted populations in eight cell lines from different organs. CSLC properties were examined using anchorage-independent growth and levels of activated Notch1. Validation consisted of testing tumorigenicity and postirradiation enrichment of CSLC in xenograft tumors. Results: The quantity of CSLC was generally in good agreement withmore » primary tumors. CSLC from MDA-MB-231 (breast) and Panc-1 and PSN-1 (both pancreatic) cells had fewer residual gamma-H2AX foci than unsorted cells, pointing to radioresistance of CSLC. However, only MDA-MB-231 CSLC were more radioresistant than unsorted cells. Furthermore, MDA-MB-231 CSLC showed enhanced anchorage-independent growth and overexpression of activated Notch1 protein. The expression of cancer stem cell surface markers in the MDA-MB-231 xenograft model was increased after exposure to fractionated radiation. In contrast to PSN-1 cells, a growth advantage for MDA-MB-231 CSLC xenograft tumors was found compared to tumors arising from unsorted cells. Conclusions: CSLC subpopulations showed no general radioresistant phenotype, despite the quantities of CSLC subpopulations shown to correspond relatively well in other reports. Likewise, CSLC characteristics were found in some but not all of the tested cell lines. The reported problems in testing for CSLC in cell lines may be overcome by additional techniques, beyond sorting for markers.« less

Induction of morphological changes in death-induced cancer cells monitored by holographic microscopy.

PubMed

El-Schich, Zahra; Mölder, Anna; Tassidis, Helena; Härkönen, Pirkko; Falck Miniotis, Maria; Gjörloff Wingren, Anette

2015-03-01

We are using the label-free technique of holographic microscopy to analyze cellular parameters including cell number, confluence, cellular volume and area directly in the cell culture environment. We show that death-induced cells can be distinguished from untreated counterparts by the use of holographic microscopy, and we demonstrate its capability for cell death assessment. Morphological analysis of two representative cell lines (L929 and DU145) was performed in the culture flasks without any prior cell detachment. The two cell lines were treated with the anti-tumour agent etoposide for 1-3days. Measurements by holographic microscopy showed significant differences in average cell number, confluence, volume and area when comparing etoposide-treated with untreated cells. The cell volume of the treated cell lines was initially increased at early time-points. By time, cells decreased in volume, especially when treated with high doses of etoposide. In conclusion, we have shown that holographic microscopy allows label-free and completely non-invasive morphological measurements of cell growth, viability and death. Future applications could include real-time monitoring of these holographic microscopy parameters in cells in response to clinically relevant compounds. Copyright © 2015 Elsevier Inc. All rights reserved.

Biomechanics of Tetrahymena escaping from a dead end

PubMed Central

Kikuchi, Kenji

2018-01-01

Understanding the behaviours of swimming microorganisms in various environments is important for understanding cell distribution and growth in nature and industry. However, cell behaviour in complex geometries is largely unknown. In this study, we used Tetrahymena thermophila as a model microorganism and experimentally investigated cell behaviour between two flat plates with a small angle. In this configuration, the geometry provided a ‘dead end' line where the two flat plates made contact. The results showed that cells tended to escape from the dead end line more by hydrodynamics than by a biological reaction. In the case of hydrodynamic escape, the cell trajectories were symmetric as they swam to and from the dead end line. Near the dead end line, T. thermophila cells were compressed between the two flat plates while cilia kept beating with reduced frequency; those cells again showed symmetric trajectories, although the swimming velocity decreased. These behaviours were well reproduced by our computational model based on biomechanics. The mechanism of hydrodynamic escape can be understood in terms of the torque balance induced by lubrication flow. We therefore conclude that a cell's escape from the dead end was assisted by hydrodynamics. These findings pave the way for understanding cell behaviour and distribution in complex geometries. PMID:29491169

LINE-1 couples EMT programming with acquisition of oncogenic phenotypes in human bronchial epithelial cells.

PubMed

Reyes-Reyes, Elsa M; Aispuro, Ivan; Tavera-Garcia, Marco A; Field, Matthew; Moore, Sara; Ramos, Irma; Ramos, Kenneth S

2017-11-28

Although several lines of evidence have established the central role of epithelial-to-mesenchymal-transition (EMT) in malignant progression of non-small cell lung cancers (NSCLCs), the molecular events connecting EMT to malignancy remain poorly understood. This study presents evidence that Long Interspersed Nuclear Element-1 (LINE-1) retrotransposon couples EMT programming with malignancy in human bronchial epithelial cells (BEAS-2B). This conclusion is supported by studies showing that: 1) activation of EMT programming by TGF-β1 increases LINE-1 mRNAs and protein; 2) the lung carcinogen benzo(a)pyrene coregulates TGF-β1 and LINE-1 mRNAs, with LINE-1 positioned downstream of TGF-β1 signaling; and, 3) forced expression of LINE-1 in BEAS-2B cells recapitulates EMT programming and induces malignant phenotypes and tumorigenesis in vivo . These findings identify a TGFβ1-LINE-1 axis as a critical effector pathway that can be targeted for the development of precision therapies during malignant progression of intractable NSCLCs.

Minimum line width of ion beam-modified polystyrene by negative carbon ions for nerve-cell attachment and neurite extension

NASA Astrophysics Data System (ADS)

Sommani, P.; Tsuji, H.; Sato, H.; Kitamura, T.; Hattori, M.; Gotoh, Y.; Ishikawa, J.

2007-04-01

The minimum line width of the negative-ion-modified polystyrene (PS) for guidance and immobilizations of nerve-cell body and neurite extension have been investigated. Carbon negative ions were implanted into PS at fluence of 3 × 1015 ions/cm2 and energy of 5-20 keV through the various triangle apertures of the micro-pattern mask. After in vitro culture of the nerve-like cells of rat adrenal pheochromocytoma (PC12h), results showed that the minimum line widths for a single cell attachment and for neurite extension were 5-7 and 3-5 μm, respectively. While the minimum line width for attachment of cell group with long neurite was about 20 μm. The suitable widths for a large number of cells and for neurite extension were 20 and 5 μm, respectively. Therefore, the guidance for a clear separation of the attachment size of cell body and neurite extension could be achieved by the different modified line widths.

Comparison of Cultivars and Seasonal Variation in Blueberry (Vaccinium Species) Leaf Extract on Adult T-Cell Leukemia Cell Line Growth Suppression.

PubMed

Kai, Hisahiro; Fuse, Takuichi; Kunitake, Hisato; Morishita, Kazuhiro; Matsuno, Koji

2014-06-30

The inhibitory effects of blueberry leaves on the proliferation of adult T-cell leukemia (ATL) cell lines have previously been reported. A comparison of blueberry leaf extracts from different cultivars and seasonal variation were investigated regarding their effects on ATL cell line proliferation. The inhibitory effects of 80% ethanol leaf extracts from different blueberry cultivars collected from April to December in 2006 or 2008 were evaluated using two ATL cell lines. The bioactivities of leaf extracts of rabbit-eye blueberry ( Vaccinium virgatum Aiton; RB species), southern highbush blueberry ( V. spp.; SB species), northern highbush blueberry ( V. corymbosum L.; NB species), and wild blueberry ( V. bracteatum Thunb.; WB species) were compared. Of these, leaves of the RB species collected in December showed a significantly stronger inhibitory effect in both cell lines than the SB, NB, or WB species. These results suggest elevated biosynthesis of ATL-preventative bioactive compounds in the leaves of the RB species before the defoliation season.

Comparison of Cultivars and Seasonal Variation in Blueberry (Vaccinium Species) Leaf Extract on Adult T-Cell Leukemia Cell Line Growth Suppression

PubMed Central

Kai, Hisahiro; Fuse, Takuichi; Kunitake, Hisato; Morishita, Kazuhiro; Matsuno, Koji

2014-01-01

The inhibitory effects of blueberry leaves on the proliferation of adult T-cell leukemia (ATL) cell lines have previously been reported. A comparison of blueberry leaf extracts from different cultivars and seasonal variation were investigated regarding their effects on ATL cell line proliferation. The inhibitory effects of 80% ethanol leaf extracts from different blueberry cultivars collected from April to December in 2006 or 2008 were evaluated using two ATL cell lines. The bioactivities of leaf extracts of rabbit-eye blueberry (Vaccinium virgatum Aiton; RB species), southern highbush blueberry (V. spp.; SB species), northern highbush blueberry (V. corymbosum L.; NB species), and wild blueberry (V. bracteatum Thunb.; WB species) were compared. Of these, leaves of the RB species collected in December showed a significantly stronger inhibitory effect in both cell lines than the SB, NB, or WB species. These results suggest elevated biosynthesis of ATL-preventative bioactive compounds in the leaves of the RB species before the defoliation season. PMID:28933373

Establishment of a rat hepatoma-derived cell line proliferating in D-phenylalanine medium and expressing D-amino-acid oxidase.

PubMed

Yoda, N; Konno, R; Nagashima, S

2001-01-01

A cell line (R-Y121B.DF) has been established from a cell line (R-Y121B) derived from a rat hepatoma line (H4-II-E). The R-Y121B.DF cells have been continuously cultured in a serum-free modified Eagle's minimum essential medium in which L-phenylalanine was replaced by D-phenylalanine. They had D-amino-acid oxidase (DAO) activity which is essential for the growth in the medium containing D-amino acids. The enzyme activity of the R-Y121B.DF cells was approximately one-fourth of that of the rat liver. Northern hybridization using a DAO cDNA probe detected a hybridizing signal in the R-Y121B.DF cells and the rat liver but not in the parental R-Y121B and H4-II-E cells. Reverse transcription-polymerase chain reaction using DAO-specific primers amplified a DNA fragment of the expected size in the R-Y121B.DF cells but not in the R-Y121B and H4-II-E cells. This fragment was confirmed to be DAO cDNA by nucleotide sequencing. Western blotting showed that DAO protein was present in the R-Y121B.DF cells and the rat liver but not in the R-Y121B and H4-II-E cells. Southern hybridization showed that the DAO gene structure was not different among the R-Y121B.DF cells, R-Y121B cells, H4-II-E cells, and the rat liver. These results indicate that the R-Y121B.DF is a unique cell line which proliferates in the medium containing D-phenylalanine and explicitly expresses DAO. This line is useful for the study of DAO in vitro.

Cellular characteristics of primary and immortal canine embryonic fibroblast cells.

PubMed

You, Seungkwon; Moon, Jai-Hee; Kim, Tae-Kyung; Kim, Sung-Chan; Kim, Jai-Woo; Yoon, Du-Hak; Kwak, Sungwook; Hong, Ki-Chang; Choi, Yun-Jaie; Kim, Hyunggee

2004-08-31

Using normal canine embryonic fibroblasts (CaEF) that were shown to be senescent at passages 7th-9th, we established two spontaneously immortalized CaEF cell lines (designated CGFR-Ca-1 and -2) from normal senescent CaEF cells, and an immortal CaEF cell line by exogenous introduction of a catalytic telomerase subunit (designated CGFR-Ca-3). Immortal CGFR- Ca-1, -2 and -3 cell lines grew faster than primary CaEF counterpart in the presence of either 0.1% or 10% FBS. Cell cycle analysis demonstrated that all three immortal CaEF cell lines contained a significantly high proportion of S-phase cells compared to primary CaEF cells. CGFR-Ca-1 and -3 cell lines showed a loss of p53 mRNA and protein expression leading to inactivation of p53 regulatory function, while the CGFR-Ca-2 cell line was found to have the inactive mutant p53. Unlike the CGFR-Ca-3 cell line that down-regulated p16INK4a mRNA due to its promoter methylation but had an intact p16INK4a regulatory function, CGFR-Ca-1 and -2 cell lines expressed p16INK4a mRNA but had a functionally inactive p16INK4a regulatory pathway as judged by the lack of obvious differences in cell growth and phenotype when reconstituted with wild-type p16INK4a. All CGFR-Ca-1, -2 and -3 cell lines were shown to be untransformed but immortal as determined by anchorage-dependent assay, while these cell lines were fully transformed when overexpressed oncogenic H-rasG12V. Taken together, similar to the nature of murine embryo fibroblasts, the present study suggests that normal primary CaEF cells have relatively short in vitro lifespans and should be spontaneously immortalized at high frequency.

Antiproliferative activity of flavonoids on several cancer cell lines.

PubMed

Kawaii, S; Tomono, Y; Katase, E; Ogawa, K; Yano, M

1999-05-01

Twenty-seven Citrus flavonoids were examined for their antiproliferative activities against several tumor and normal human cell lines. As a result, 7 flavonoids were judged to be active against the tumor cell lines, while they had weak antiproliferative activity against the normal human cell lines. The rank order of potency was luteolin, natsudaidain, quercetin, tangeretin, eriodictyol, nobiletin, and 3,3',4',5,6,7,8-heptamethoxyflavone. The structure-activity relationship established from comparison among these flavones and flavanones showed that the ortho-catechol moiety in ring B and a C2-C3 double bond were important for the antiproliferative activity. As to polymethoxylated flavones, C-3 hydroxyl and C-8 methoxyl groups were essential for high activity.

MiRNA-Target Interaction Reveals Cell-Specific Post-Transcriptional Regulation in Mammalian Cell Lines

PubMed Central

Kulkarni, Varun; Naqvi, Afsar Raza; Uttamani, Juhi Raju; Nares, Salvador

2016-01-01

MicroRNAs are 18–22 nucleotides long, non-coding RNAs that bind transcripts with complementary sequences leading to either mRNA degradation or translational suppression. However, the inherent differences in preferred mode of miRNA regulation among cells of different origin have not been examined. In our previous transcriptome profiling studies, we observed that post-transcriptional regulation can differ substantially depending on the cell in context. Here we examined mechanistic differences in the regulation of a let-7a targeted (wild type) or resistant (mutant) engineered renilla transcript across various mammalian cell lines of diverse origin. Dual luciferase assays show that compared to mutant (mut), the reporter gene containing wild type (wt) let-7a binding sites was efficiently suppressed upon transfection in various cell lines. Importantly, the strength of miRNA regulation varied across the cell lines. Total RNA analysis demonstrates that wt renilla mRNA was expressed to similar or higher levels compared to mut suggesting that translation repression is a predominant mode of miRNA regulation. Nonetheless, transcript degradation was observed in some cell lines. Ago-2 immunoprecipitation show that miRNA repressed renilla mRNA are associated with functional mi-RISC (miRNA-RNA induced silencing complex). Given the immense potential of miRNA as a therapeutic option, these findings highlight the necessity to thoroughly examine the mode of mRNA regulation in order to achieve the beneficial effects in targeting cells. PMID:26761000

Escherichia coli STb toxin induces apoptosis in intestinal epithelial cell lines.

PubMed

Syed, H Claudia; Dubreuil, J Daniel

2012-09-01

A previous study conducted in our laboratory demonstrated that cells having internalized Escherichia coli STb toxin display apoptotic-like morphology. We therefore investigated if STb could induce programmed cell death in both a human and an animal intestinal epithelial cell lines. HRT-18 (Human Colon Tumor) and IEC-18 (Rat Ileum Epithelial Cells) cell lines were used. As STb is frequently tested in a rat model, the IEC-18 cell line was most relevant to our work. The cell lines were treated with various amounts of purified STb (nanomole range) for a period of 24 h after which cells were harvested and examined for apoptotic characteristics. Caspase-9, the initiator of mitochondrion-mediated apoptosis, and caspase-3, an effector of caspase-9, were both activated following STb intoxication of HRT-18 and IEC-18 cells whereas caspase-8, the initiator caspase of the extrinsic pathway, was not activated. For both cell lines, agarose gel electrophoresis of the cell DNA content reveals laddering of DNA, resulting from DNA fragmentation, a characteristic of apoptosis. Hoechst 33342-stained DNA of STb-treated cell lines, observed using fluorescence microscopy, revealed condensation and fragmentation of the nuclei. Apoptotic indexes calculated from fragmented nuclei of Hoechst 33342-stained DNA for HRT-18 and IEC-18 cells showed an STb dose-dependent response. Overall, these data indicate that STb toxin induces a mitochondrion-mediated caspase-dependent apoptotic pathway. Copyright © 2012 Elsevier Ltd. All rights reserved.

Synthesis, characterization and anticancer studies of Ni(II), Pd(II) and Pt(II) complexes with Schiff base derived from N-methylhydrazinecarbothioamide and 2-hydroxy-5-methoxy-3-nitrobenzaldehyde

NASA Astrophysics Data System (ADS)

Arafath, Md. Azharul; Adam, Farook; Razali, Mohd. R.; Ahmed Hassan, Loiy E.; Ahamed, Mohamed B. Khadeer; Majid, Amin Malik S. A.

2017-02-01

A carbothioamide NSO tridentate Schiff base ligand (HL) and its square planar complexes Na[NiLOAc], Na[PdLOAc] and [PtLdmso] have been synthesized and characterized on the basis of melting point, elemental analysis, FT-IR, 1H NMR, 13C NMR, UV-Vis spectra. The structure of HL was elucidated with X-ray diffraction analysis. In the present study, the synthesized compounds were evaluated for their anticancer properties against three human cancer cell lines breast cancer (MCF-7), cervical (Hela), and colon (HCT-116). In addition, the cytotoxicity of the synthesized compounds was tested on a normal human cell line (human endothelial cell line EA.hy926). Among the tested compounds, the complex [NiLOAc] excelled in halting proliferation of the cervical and colon cancer cells with median inhibitory concentration (IC50) values of 28.33 and 34.4 μM, respectively. The complex, [PdLOAc] demonstrated selective cytotoxicity against breast cancer line MCF-7 with IC50 = 47.5 μM, while HL showed inhibitory effect against colon cancer cell line (HCT-116) with IC50 = 55.66 μM. The complex, [PtLdmso] showed mild activity against breast cancer (MCF-7) and cervical cancer (Hela) cells with IC50 = 64.44 and 68.3 μM, respectively, whereas, it displayed insignificant cytotoxicity against human endothelial cells (EA.hy926) with IC50 > 200 μM. Cancer cells treated with [NiLOAc] showed apoptotic features such as membrane blebbing and DNA condensation. Thus, the findings of the present study demonstrated that the series of metal complexes of HL could form the new lead for development of cancer chemotherapies to treat human cervical, breast and colon malignancies.

Pharmacological importance, characterization and applications of gold and silver nanoparticles synthesized by Panax ginseng fresh leaves.

PubMed

Singh, Priyanka; Singh, Hina; Ahn, Sungeun; Castro-Aceituno, Verónica; Jiménez, Zuly; Simu, Shakina Yesmin; Kim, Yeon Ju; Yang, Deok Chun

2017-11-01

Previously, we showed the rapid and eco-friendly synthesis of gold and silver nanoparticles within 3 and 45 min by fresh leaves extract of herbal medicinal plant Panax ginseng. In addition, we characterized the nanoparticles in terms of shape, size, morphology and stability by FE-TEM, EDX, elemental mapping, SEAD, XRD and particles size analysis. In addition of this, we showed their antimicrobial, anti-coagulant, and biofilm inhibition activity of nanoparticles. Continuing our previous study, here we highlight the further characterization and biomedical applications of P. ginseng leaf-mediated gold and silver nanoparticles. We characterized the nanoparticles further in terms of active functional group and capping layer, surface charge, and temperature stability. Based on these factors, we explored the nanoparticles for antioxidant efficacy, biocompatibility in HaCaT cells, 3T3-L1 pre-adipocytes cells, for anticancer efficacy in A549 lung cancer and B16BL6 skin melenoma cancer cell lines and for anti-inflammation efficacy in RAW 264.7 cell lines. Based on our findings, we suggest that the P. ginseng-mediated gold nanoparticles have high antioxidant activity and highly biocompatibility in HaCaT cells, 3T3-L1 pre-adipocytes cells, RAW 264.7 cells lines and could be considered for future drug delivery carriers. The silver nanoparticles also showed high potent antioxidant efficacy, additionally it showed high anticancer effect in A549 lung cancer and B16BL6 skin melenoma cancer cell lines as compared to precursor salts. Moreover, both gold and silver nanoparticles have anti-inflammatory efficacies in RAW 264.7 cells. Thus, the study may provide useful insights of P. ginseng leaves extract-mediated biocompatible gold and silver nanoparticles and improving their applicability in designing nanoparticles carrier systems for drug delivery applications.

Growth Inhibitory Effect of Palatine Tonsil-derived Mesenchymal Stem Cells on Head and Neck Squamous Cell Carcinoma Cells

PubMed Central

Lim, Yun-Sung; Lee, Jin-Choon; Lee, Yoon Se; Wang, Soo-Geun

2012-01-01

Objectives Mesenchymal stem cells (MSCs) play an important role in the development and growth of tumor cells. However, the effect of human MSCs on the growth of human tumors is not well understood. The purpose of this study is to confirm the growth effect of palatine tonsil-derived MSCs (TD-MSCs) on head and neck squamous cell carcinoma (HNSCC) cell lines and to elucidate the mechanism of their action. Methods TD-MSCs were isolated from patient with chronic tonsillitis and tonsillar hypertrophy. Two human HNSCC cell lines (PNUH-12 and SNU-899) were studied and cocultured with isolated palatine tonsil-derived MSC. The growth inhibitory effect of MSCs on HNSCC cell lines was tested through methylthiazolyldiphenyl-tetrazolium (MTT) assay. The apoptosis induction effect of MSCs on cell lines was assessed with flow cytometry and reverse transcriptase (RT)-PCR. Results Palatine tonsil-derived MSCs exhibited a growth inhibitory effect on both cell lines. Cell cycle analysis showed an accumulation of tumor cells predominantly in G0/G1 phase with an increase in concentration of TD-MSCs, which was confirmed by increased mRNA expression of cell cycle negative regulator p21. Apoptosis of tumor cells increased significantly as concentration of cocultured TD-MSCs increased. Additionally, mRNA expression of caspase 3 was upregulated with increased concentration of TD-MSCs. Conclusion TD-MSCs have a potential growth inhibitory effect on HNSCC cell lines in vitro by inducing apoptotic cell death and G1 phase arrest of cell lines. PMID:22737289

Hepatocyte growth factor/scatter factor enhances the invasion of mesothelioma cell lines and the expression of matrix metalloproteinases

PubMed Central

Harvey, P; Clark, I M; Jaurand, M-C; Warn, R M; Edwards, D R

2000-01-01

Hepatocyte growth factor/scatter factor (HGF/SF) is a multifunctional factor involved both in development and tissue repair, as well as pathological processes such as cancer and metastasis. It has been identified in vivo in many types of tumours together with its tyrosine kinase receptor, Met. We show here that exogenous HGF/SF acts as a strong chemoattractant for human mesothelioma cell lines. The factor also enhanced cell adhesion to and invasion into Matrigel. The mesothelioma cell lines synthesized a panel of matrix metalloproteinases critical for tumour progression such as MMP-1, 2, 3, 9 and membrane-bound MT1-MMP. HGF/SF stimulated the expression of MMP-1, 9 and MT1-MMP and had a slight effect on expression of the MMP inhibitor TIMP-1 but not TIMP-2. However, there was no simple correlation between the levels of MMPs and TIMPs of the cell lines and their different invasion properties or between HGF/SF stimulatory effects on MMP expression and invasion. In addition, effects of protease inhibitors on invasion suggested that serine proteases were also expressed in human mesothelioma cell lines and were involved in HGF/SF-induced invasion. The results show a predominant role for HGF/SF in mesothelioma cell invasion, stimulating simultaneously adhesion, motility, invasion and regulation of MMP and TIMP levels. © 2000 Cancer Research Campaign PMID:11027427

Synthesis, crystal structure and spectroscopy of bioactive Cd(II) polymeric complex of the non-steroidal anti-inflammatory drug diclofenac sodium: Antiproliferative and biological activity

NASA Astrophysics Data System (ADS)

Tabrizi, Leila; Chiniforoshan, Hossein; McArdle, Patrick

2015-02-01

The interaction of Cd(II) with the non-steroidal anti-inflammatory drug diclofenac sodium (Dic) leads to the formation of the complex [Cd2(L)41.5(MeOH)2(H2O)]n(L = Dic), 1, which has been isolated and structurally characterized by X-ray crystallography. Diclofenac sodium and its metal complex 1 have also been evaluated for antiproliferative activity in vitro against the cells of three human cancer cell lines, MCF-7 (breast cancer cell line), T24 (bladder cancer cell line), A-549 (non-small cell lung carcinoma), and a mouse fibroblast L-929 cell line. The results of cytotoxic activity in vitro expressed as IC50 values indicated the diclofenac sodium and cadmium chloride are non active or less active than the metal complex of diclofenac (1). Complex 1 was also found to be a more potent cytotoxic agent against T-24 and MCF-7 cancer cell lines than the prevalent benchmark metallodrug, cisplatin, under the same experimental conditions. The superoxide dismutase activity was measured by Fridovich test which showed that complex 1 shows a low value in comparison with Cu complexes. The binding properties of this complex to biomolecules, bovine or human serum albumin, are presented and evaluated. Antibacterial and growth inhibitory activity is also higher than that of the parent ligand compound.

Protein alterations associated with temozolomide resistance in subclones of human glioblastoma cell lines.

PubMed

Sun, Stella; Wong, T S; Zhang, X Q; Pu, Jenny K S; Lee, Nikki P; Day, Philip J R; Ng, Gloria K B; Lui, W M; Leung, Gilberto K K

2012-03-01

Temozolomide (TMZ) is the standard chemotherapeutic agent for human malignant glioma, but intrinsic or acquired chemoresistance represents a major obstacle to successful treatment of this highly lethal group of tumours. Obtaining better understanding of the molecular mechanisms underlying TMZ resistance in malignant glioma is important for the development of better treatment strategies. We have successfully established a passage control line (D54-C10) and resistant variants (D54-P5 and D54-P10) from the parental TMZ-sensitive malignant glioma cell line D54-C0. The resistant sub-cell lines showed alterations in cell morphology, enhanced cell adhesion, increased migration capacities, and cell cycle arrests. Proteomic analysis identified a set of proteins that showed gradual changes in expression according to their 50% inhibitory concentration (IC(50)). Successful validation was provided by transcript profiling in another malignant glioma cell line U87-MG and its resistant counterparts. Moreover, three of the identified proteins (vimentin, cathepsin D and prolyl 4-hydroxylase, beta polypeptide) were confirmed to be upregulated in high-grade glioma. Our data suggest that acquired TMZ resistance in human malignant glioma is associated with promotion of malignant phenotypes, and our reported molecular candidates may serve not only as markers of chemoresistance but also as potential therapeutic targets in the treatment of TMZ-resistant human malignant glioma, providing a platform for future investigations.

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

PubMed Central

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

2015-01-01

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

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.

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

Establishment and characterization of a new human acinar cell carcinoma cell line, Faraz-ICR, from pancreas.

PubMed

Rezaei, Marzieh; Hosseini, Ahmad; Nikeghbalian, Saman; Ghaderi, Abbas

Basic research in the field of acinar cell carcinoma (ACC) as a rare neoplasm of the pancreas is dependent on the availability of pragmatic model such as new pancreatic cancer cell lines. Thus, establishment and characterization of new pancreatic cancer cell lines from ACC origin are deemed important. Faraz-ICR cell line was derived from a 58-years old woman with pancreatic acinar cell carcinoma by the collagenase digestion protocol. We characterized the cell line by examining its morphology and cytostructural and functional profile. Faraz-ICR has a doubling time of 35 hours and grows in soft agar with a colony-forming efficiency of 25%. The cell had nearly normal pattern of chromosomes in karyotype analysis and Comparative Genomic Hybridization (CGH) array analysis. Evaluation of cells by flowcytometry showed that Faraz-ICR is negative for EpCAM and mesenchymal markers in different passages, and has epithelial nature. Immunofluorescence staining revealed that cells were strongly positive for vimentin, desmin, ezrin, S100, nestin and they were negative for pan-cytokeratins, chromogranin and alpha smooth muscle actin. We were able to establish a new pancreatic carcinoma cell line with partial aspects of Epithelial-mesenchymal transition and aggressiveness. This cell line might be suitable for studying various anticancer drugs and protein profile aiming to see any possible tumor associated marker for ACC. Copyright © 2017 IAP and EPC. Published by Elsevier B.V. All rights reserved.

Establishment and characterization of a cell line from human adenoid cystic carcinoma of the lacrimal glands and a nude mouse transplantable model.

PubMed

Lin, Tingting; Zhu, Limin; Zhou, Beiqing; Xie, Lianfeng; Lv, Jianmei; Dong, Lijie; He, Yanjin

2015-06-01

Using tissue block culture techniques, we established a new human tumor cell line derived from adenoid cystic carcinoma of the lacrimal glands (LACC-1). The LACC-1 cell line was successfully subcultured for more than 100 passages during the last two years. The outgrowth of cells was observed by day 5 after seeding, and then the cells were generated slowly. The first passage proceeded by day 32, and the classical epithelioid cell colonies formed by day 69 after inoculation. After eight passages, homogeneous epithelioid tumor cells appeared when we combined continuous passage, mechanical scraping, repeated adherence, and dissociation methods to remove the fibroblast cells. LACC-1 cells appeared as a histologically solid pattern and continuous passage culture. The population doubling time was approximately 37.1 h. LACC-1 cells appeared as an epithelioid monolayer culture on the cell culture flask and presented with a cobblestone-like appearance when they reached confluency. The nucleus was large and round with many abnormal mitoses. The nucleoplasm ratio was high. Multinucleated tumor giant cells appeared. LACC-1 cells showed a tendency to have overlapping growth without contact inhibition when the cell density continued to increase. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) showed that the LACC-1 cells were malignant tumor cells that were poorly differentiated. The surface of the LACC-1 cells exhibited affluent microvilli, protuberances and filopodia under SEM. The no. 84 generation LACC-1 cell line was inoculated subcutaneously into the subaxillary of nude mice and the tumorigenic potential was evident. The formation rate of the transplanted tumors was 100% at day 7 after inoculation. This finding showed that the LACC-1 cell line was malignant with tumorigenic ability. The xenograft tumors retained the same histological characteristics of a solid pattern as the LACC-1 original tumor after inoculation for 49 days. Under TEM observation, the xenograft tumor cells had the same ultrastructure as the LACC-1 cells. Immunohistochemical examination revealed the similarity of both cytoskeletal proteins (e.g., cytokeratin, vimentin, desmin and α-SMA) and S-100 expression in the original tumor, LACC-1 cells and xenograft tumors. Immunoreactivity of these proteins was gradually decreased in these three tissues. Reverse transcription-polymerase chain reaction demonstrated that the xenograft tumors originated from the human. Based on these results, the LACC-1 cell line provides a useful model for studying the biological characteristics of human ACC of the lacrimal glands.

Newly established human retinoblastoma cell lines exhibit an "immortalized" but not an invasive phenotype in vitro.

PubMed

Griegel, S; Hong, C; Frötschl, R; Hülser, D F; Greger, V; Horsthemke, B; Rajewsky, M F

1990-07-15

Retinoblastoma (RB), an intraocular childhood tumor occurring in a hereditary (mostly bilateral) or non-hereditary (unilateral) form, is associated with the inactivation of both alleles of a putative tumor suppressor gene (RB-I) located on chromosome 13q14. Both the process of RB development and the biological characteristics of RB cells are as yet poorly understood. We have established 7 new RBL lines (RBL13, RBL14, RBL18 and RBL30, derived from unilateral RB; and RBL7, RBL15 and RBL20, derived from bilateral RB). Southern blot analyses of restriction fragment length polymorphisms in DNA samples from 6 cell lines revealed loss of constitutional heterozygosity at one or several polymorphic loci on chromosome 13 in 4 cases. Gross deletions involving the RB-I locus and amplification of the N-myc gene were not detected in any of the RBL lines. The phenotypic properties of the RBL lines were analyzed in comparison with cells from the original RB tumors, with 4 RB lines established by others (RB383, RB355, RB247C3 and Y79) and with the adenovirus-EIA-transformed human retinoblast line HER-Xhol-CC2. It was found that RB tumors consist of phenotypically heterogeneous cell subpopulations with varying nutrient requirements and differentiation potential in vitro. All cell lines showed the typical characteristics of established ("immortalized") cells. In some cases, cells from original RB tumors or cell lines were able to form colonies when cell aggregates of 2-10 cells were suspended in semi-solid agar medium; however, anchorage-independent colonies never developed from single cells. Cell lines RBL13, RBL18, RB247C3, RB355, RB383 and Y79 were tested for invasion into embryonic chick heart fragments in vitro and found to be non-invasive. None of the RBL or RB lines were tumorigenic in nu/nu (T-) mice. Y79 cells (propagated in culture for many years) exhibited properties distinctly different from those of the other cell lines, and thus cannot be considered phenotypically representative of RB cells.

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.

Differential repair of radiation-induced DNA damage in cells of human squamous cell carcinoma and the effect of caffeine and cysteamine on induction and repair of DNA double-strand breaks

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

Smeets, M.F.M.A.; Mooren, E.H.M.; Abdel-Wahab, A.H.A.

1994-11-01

The goal of these experiments was to investigate further the relationship between DNA double-strand breaks and cell killing in human tumor cells, first by comparing different cell lines, and second by radiomodification studies. Field-inversion gel electrophoresis was used to quantify double-strand breaks. Two subclones of the radioresistant human squamous cell carcinoma line SQ20B (SQD9 and SQG6) were compared. These subclones differed in DNA index by a factor of 1.7 but showed the same resistance to radiation as cells of the parental cell line. It was found that, although induction of DSBs was not significantly different in the two cell lines,more » the t{sub 1/2} of the fast component of repair was significantly shorter for SQD9 cells, leading to greater overall repair which was not reflected in increased survival. Caffeine and cysteamine were tested as modifiers of radiosensitivity, using the radioresistant SQ20B line and the radiosensitive SCC61 cell line. No effect of caffeine was seen when the drug was present only during irradiation. Postirradiation incubations with caffeine, however, resulted in a dose reduction factor greater than 2.0 in cell survival for both cell lines. In contrast, induction of DSBs was reduced by caffeine, and no effect on DSB repair was observed. Cysteamine led to a dose protection factor greater than 1.8 in cell survival in both cell lines. A reduction in induced DSBs was found at high doses corresponding approximately with the increase in cell survival. Over the same (low) dose range, however, the correlation between DSB induction and cell killing was poor. These data indicate that DSB induction does not correlate well with cell killing either for different cell lines, for radiochemical modification (cysteamine) or for some other types of modification (caffeine). 31 refs., 8 figs.« less

Establishment and characterization of SUIT-58 pancreas cancer cell line and its subline S58-SF adapted to serum-free condition derived from metastatic liver tumor.

PubMed

Takahashi, Nobuyasu; Aoyama, Fumiyo; Ohuchida, Jiro; Sameshima, Naoki; Asada, Yujiro; Sawaguchi, Akira

2015-10-01

A new pancreas cancer cell line, SUIT-58, was established from metastatic liver tumor. The cultured cells exhibited polygonal shape, and proliferated in a form of sheet-structure showing prominent nucleoli and frequent mitotic features. Chromosome count ranged from 54 to 73 with modal chromosome numbers 72 and 73. It was noteworthy that this cell line grew in the serum-free media and maintained in this condition for 30 passages (designated as S58-SF). Both SUIT-58 and S58-SF cell lines were successfully transplanted into nude mice, and their tumor doubling times in xenografts were calculated as 5.4 and 2.8 days, respectively. Histopathologically, the xenografts formed glandular structure that resembled the original tumor. In culture media, the doubling time of SUIT-58 and S58-SF cell lines was calculated as 32 and 35.7 h, respectively. Although the cellular arrangements of SUIT-58 and S58-SF cell lines are different to some extent, their subcellular structures under electron microscope were similar with a large number of lysosomes and distinct desmosomes at cell-cell adhesion sites. The present SUIT-58 and its derivative cell line S58-SF will be applicable for biological studies to develop a new clinical treatment of refractory pancreatic cancer.

Ligand activation of peroxisome proliferator-activated receptor-β/δ (PPAR β/δ) inhibits cell growth in a mouse mammary gland cancer cell line

PubMed Central

Foreman, Jennifer E.; Sharma, Arun K.; Amin, Shantu; Gonzalez, Frank J.; Peters, Jeffrey M.

2009-01-01

The effects of ligand activation of PPARβ/δ were examined in the mouse mammary tumor cell line (C20). Expression of PPARβ/δ was markedly lower in C20 cells as compared to the human non-tumorigenic mammary gland derived cell line (MCF10A) and mouse keratinocytes. Ligand activation of PPARβ/δ in C20 cells caused upregulation of the PPARβ/δ target gene angiopoietin-like 4 (Angptl4). Inhibition of C20 cell proliferation and clonogenicity was observed following treatment with GW0742 or GW501516, two highly specific PPARβ/δ ligands. In addition, an increase in apoptosis was observed in C20 cells cultured with 10 µM GW501516 that preceded the observed inhibition of cell proliferation. Results from this study show that proliferation of the C20 mouse mammary gland cancer cell line is inhibited by ligand activation of PPARβ/δ due in part to increased apoptosis. PMID:19660859

Cytotoxic activity of carotenoid rich fractions from Haematococcus pluvialis and Dunaliella salina microalgae and the identification of the phytoconstituents using LC-DAD/ESI-MS.

PubMed

El-Baz, Farouk K; Hussein, Rehab A; Mahmoud, Khaled; Abdo, Sayeda M

2018-02-01

Microalgae represent a rich source that satisfies the growing need for novel ingredients of nutriceuticals, pharmaceuticals, and food supplements. Haematococcus pluvialis and Dunaliella salina microalgae are isolated from the Egyptian hydro-flora and are reported for their potent antioxidant activities. The cytotoxic activity of different fractions of both microalgae was investigated on 4 cell lines HePG2, MCF7, HCT116, and A549. The carotenoid rich fraction of H. pluvialis showed potent cytotoxic activity against colon cancer cell line and moderate activity against both liver and breast cancer cell lines. On the other hand, the carotenoid rich fraction of D. salina showed mild cytotoxic activity on breast and liver cancer cell lines. The carotenoid rich fraction of H. pluvialis was analysed using LC-DAD/ESI-MS and the major carotenoids were identified either free as well as bounded to fatty acids. Copyright © 2017 John Wiley & Sons, Ltd.

Radiation-Induced Dedifferentiation of Head and Neck Cancer Cells Into Cancer Stem Cells Depends on Human Papillomavirus Status

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

Vlashi, Erina, E-mail: evlashi@mednet.ucla.edu; Jonsson Comprehensive Cancer Center at UCLA, Los Angeles, California; Chen, Allen M.

Purpose: To test the hypothesis that the radiation response of cancer stem cells (CSCs) in human papillomavirus (HPV)-positive and HPV-negative head and neck squamous cell carcinoma (HNSCC) differs and is not reflected in the radiation response of the bulk tumor populations, that radiation therapy (RT) can dedifferentiate non-stem HNSCC cells into CSCs, and that radiation-induced dedifferentiation depends on the HPV status. Methods and Materials: Records of a cohort of 162 HNSCC patients were reviewed, and their outcomes were correlated with their HPV status. Using a panel of HPV-positive and HPV-negative HNSCC cell lines expressing a reporter for CSCs, we characterized HPV-positivemore » and HPV-negative lines via flow cytometry, sphere-forming capacity assays in vitro, and limiting dilution assays in vivo. Non-CSCs were treated with different doses of radiation, and the dedifferentiation of non-CSCs into CSCs was investigated via flow cytometry and quantitative reverse transcription–polymerase chain reaction for re-expression of reprogramming factors. Results: Patients with HPV-positive tumors have superior overall survival and local–regional control. Human papillomavirus–positive HNSCC cell lines have lower numbers of CSCs, which inversely correlates with radiosensitivity. Human papillomavirus–negative HNSCC cell lines lack hierarchy owing to enhanced spontaneous dedifferentiation. Non-CSCs from HPV-negative lines show enhanced radiation-induced dedifferentiation compared with HPV-positive lines, and RT induced re-expression of Yamanaka reprogramming factors. Conclusions: Supporting the favorable prognosis of HPV-positive HNSCCs, we show that (1) HPV-positive HNSCCs have a lower frequency of CSCs; (2) RT can dedifferentiate HNSCC cells into CSCs; and (3) radiation-induced dedifferentiation depends on the HPV status of the tumor.« less

Comparison of a teratogenic transcriptome-based predictive test based on human embryonic versus inducible pluripotent stem cells.

PubMed

Shinde, Vaibhav; Perumal Srinivasan, Sureshkumar; Henry, Margit; Rotshteyn, Tamara; Hescheler, Jürgen; Rahnenführer, Jörg; Grinberg, Marianna; Meisig, Johannes; Blüthgen, Nils; Waldmann, Tanja; Leist, Marcel; Hengstler, Jan Georg; Sachinidis, Agapios

2016-12-30

Human embryonic stem cells (hESCs) partially recapitulate early embryonic three germ layer development, allowing testing of potential teratogenic hazards. Because use of hESCs is ethically debated, we investigated the potential for human induced pluripotent stem cells (hiPSCs) to replace hESCs in such tests. Three cell lines, comprising hiPSCs (foreskin and IMR90) and hESCs (H9) were differentiated for 14 days. Their transcriptome profiles were obtained on day 0 and day 14 and analyzed by comprehensive bioinformatics tools. The transcriptomes on day 14 showed that more than 70% of the "developmental genes" (regulated genes with > 2-fold change on day 14 compared to day 0) exhibited variability among cell lines. The developmental genes belonging to all three cell lines captured biological processes and KEGG pathways related to all three germ layer embryonic development. In addition, transcriptome profiles were obtained after 14 days of exposure to teratogenic valproic acid (VPA) during differentiation. Although the differentially regulated genes between treated and untreated samples showed more than 90% variability among cell lines, VPA clearly antagonized the expression of developmental genes in all cell lines: suppressing upregulated developmental genes, while inducing downregulated ones. To quantify VPA-disturbed development based on developmental genes, we estimated the "developmental potency" (D p ) and "developmental index" (D i ). Despite differences in genes deregulated by VPA, uniform D i values were obtained for all three cell lines. Given that the D i values for VPA were similar for hESCs and hiPSCs, D i can be used for robust hazard identification, irrespective of whether hESCs or hiPSCs are used in the test systems.

Radiation-Induced Dedifferentiation of Head and Neck Cancer Cells Into Cancer Stem Cells Depends on Human Papillomavirus Status.

PubMed

Vlashi, Erina; Chen, Allen M; Boyrie, Sabrina; Yu, Garrett; Nguyen, Andrea; Brower, Philip A; Hess, Clayton B; Pajonk, Frank

2016-04-01

To test the hypothesis that the radiation response of cancer stem cells (CSCs) in human papillomavirus (HPV)-positive and HPV-negative head and neck squamous cell carcinoma (HNSCC) differs and is not reflected in the radiation response of the bulk tumor populations, that radiation therapy (RT) can dedifferentiate non-stem HNSCC cells into CSCs, and that radiation-induced dedifferentiation depends on the HPV status. Records of a cohort of 162 HNSCC patients were reviewed, and their outcomes were correlated with their HPV status. Using a panel of HPV-positive and HPV-negative HNSCC cell lines expressing a reporter for CSCs, we characterized HPV-positive and HPV-negative lines via flow cytometry, sphere-forming capacity assays in vitro, and limiting dilution assays in vivo. Non-CSCs were treated with different doses of radiation, and the dedifferentiation of non-CSCs into CSCs was investigated via flow cytometry and quantitative reverse transcription-polymerase chain reaction for re-expression of reprogramming factors. Patients with HPV-positive tumors have superior overall survival and local-regional control. Human papillomavirus-positive HNSCC cell lines have lower numbers of CSCs, which inversely correlates with radiosensitivity. Human papillomavirus-negative HNSCC cell lines lack hierarchy owing to enhanced spontaneous dedifferentiation. Non-CSCs from HPV-negative lines show enhanced radiation-induced dedifferentiation compared with HPV-positive lines, and RT induced re-expression of Yamanaka reprogramming factors. Supporting the favorable prognosis of HPV-positive HNSCCs, we show that (1) HPV-positive HNSCCs have a lower frequency of CSCs; (2) RT can dedifferentiate HNSCC cells into CSCs; and (3) radiation-induced dedifferentiation depends on the HPV status of the tumor. Copyright © 2016 Elsevier Inc. All rights reserved.

Boric acid inhibits human prostate cancer cell proliferation.

PubMed

Barranco, Wade T; Eckhert, Curtis D

2004-12-08

The role of boron in biology includes coordinated regulation of gene expression in mixed bacterial populations and the growth and proliferation of higher plants and lower animals. Here we report that boric acid, the dominant form of boron in plasma, inhibits the proliferation of prostate cancer cell lines, DU-145 and LNCaP, in a dose-dependent manner. Non-tumorigenic prostate cell lines, PWR-1E and RWPE-1, and the cancer line PC-3 were also inhibited, but required concentrations higher than observed human blood levels. Studies using DU-145 cells showed that boric acid induced a cell death-independent proliferative inhibition, with little effect on cell cycle stage distribution and mitochondrial function.

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.

[Overexpression of liver kinase B1 inhibits the proliferation of lung cancer cells].

PubMed

Li, Yang; Zhang, Libin; Wang, Ping

2017-01-01

Objective To explore the effect of overexpressed liver kinase B1(LKB1) on the proliferation of lung cancer cell lines. Methods The expression levels of LKB1 and PTEN in A549, NCI-H23, NCI-H157, XWLC-05, NCI-H446 lung cancer cells were detected by immunocytochemistry (ICC) and Western blotting. Plasmid pcDNA3.1 + -LKB1 and empty vector pcDNA3.1 + -null were separately transfected into the above five cell lines, and then the expression of LKB1 mRNA and protein were determined by quantitative real-time PCR and Western blotting, respectively. Finally, CCK-8 assay was used to analyze the proliferation ability of the transfected cells. Results LKB1 and PTEN were positive in NCI-H23 cells; LKB1 was negative while PTEN was positive in A549 and NCI-H446 cells; both LKB1 and PTEN were negative in NCI-H157 and XWLC-05 cells. Quantitative real-time PCR and Western blotting showed that the expression level of LKB1 significantly increased in the above cell lines transfected with plasmid pcDNA3.1 + -LKB1 compared with the ones with empty vector pcDNA3.1 + -null. Besides, CCK-8 assay showed that the overexpression of LKB1 in the lung cancer cells transfected with pcDNA3.1 + -LKB1 had an obvious inhibitory effect on cell proliferation. Conclusion The expression of LKB1 is down-regulated in most of the lung cell lines to different extent and the over-expression of LKB1 can remarkably inhibit the proliferation ability of lung cancer cell lines.

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

PubMed

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

2014-07-01

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

The human-induced pluripotent stem cell initiative—data resources for cellular genetics

PubMed Central

Streeter, Ian; Harrison, Peter W.; Faulconbridge, Adam; Flicek, Paul; Parkinson, Helen; Clarke, Laura

2017-01-01

The Human Induced Pluripotent Stem Cell Initiative (HipSci) isf establishing a large catalogue of human iPSC lines, arguably the most well characterized collection to date. The HipSci portal enables researchers to choose the right cell line for their experiment, and makes HipSci's rich catalogue of assay data easy to discover and reuse. Each cell line has genomic, transcriptomic, proteomic and cellular phenotyping data. Data are deposited in the appropriate EMBL-EBI archives, including the European Nucleotide Archive (ENA), European Genome-phenome Archive (EGA), ArrayExpress and PRoteomics IDEntifications (PRIDE) databases. The project will make 500 cell lines from healthy individuals, and from 150 patients with rare genetic diseases; these will be available through the European Collection of Authenticated Cell Cultures (ECACC). As of August 2016, 238 cell lines are available for purchase. Project data is presented through the HipSci data portal (http://www.hipsci.org/lines) and is downloadable from the associated FTP site (ftp://ftp.hipsci.ebi.ac.uk/vol1/ftp). The data portal presents a summary matrix of the HipSci cell lines, showing available data types. Each line has its own page containing descriptive metadata, quality information, and links to archived assay data. Analysis results are also available in a Track Hub, allowing visualization in the context of public genomic annotations (http://www.hipsci.org/data/trackhubs). PMID:27733501

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.

Human embryonic stem cell-derived neural crest cells capable of expressing markers of osteochondral or meningeal-choroid plexus differentiation.

PubMed

Sternberg, Hal; Jiang, Jianjie; Sim, Pamela; Kidd, Jennifer; Janus, Jeffrey; Rinon, Ariel; Edgar, Ron; Shitrit, Alina; Larocca, David; Chapman, Karen B; Binette, Francois; West, Michael D

2014-01-01

The transcriptome and fate potential of three diverse human embryonic stem cell-derived clonal embryonic progenitor cell lines with markers of cephalic neural crest are compared when differentiated in the presence of combinations of TGFβ3, BMP4, SCF and HyStem-C matrices. The cell lines E69 and T42 were compared with MEL2, using gene expression microarrays, immunocytochemistry and ELISA. In the undifferentiated progenitor state, each line displayed unique markers of cranial neural crest including TFAP2A and CD24; however, none expressed distal HOX genes including HOXA2 or HOXB2, or the mesenchymal stem cell marker CD74. The lines also showed diverse responses when differentiated in the presence of exogenous BMP4, BMP4 and TGFβ3, SCF, and SCF and TGFβ3. The clones E69 and T42 showed a profound capacity for expression of endochondral ossification markers when differentiated in the presence of BMP4 and TGFβ3, choroid plexus markers in the presence of BMP4 alone, and leptomeningeal markers when differentiated in SCF without TGFβ3. The clones E69 and T42 may represent a scalable source of primitive cranial neural crest cells useful in the study of cranial embryology, and potentially cell-based therapy.

Establishment and Characterization of a Telomerase-Immortalized Sheep Trophoblast Cell Line.

PubMed

Zhang, Yufei; Shi, Jing; Liu, Shuying

2016-01-01

The primary sheep trophoblast cells (STCs) have a finite lifespan in culture. This feature limits the scope for long-term in vitro studies with STCs. This study was an attempt to establish and characterize a telomerase-immortalized sheep trophoblast cell line. STCs were isolated and purified by using Percoll and specific immunoaffinity purification, respectively. The purified STCs were transfected with a plasmid carrying sequences of human telomerase reverse transcriptase (hTERT) to create immortalized sheep trophoblast cell line (hTERT-STCs). hTERT-STCs showed a stable expression of hTERT gene, serially passaged for a year, and showed active proliferation without signs of senescence. Cytokeratin 7 (CK-7), secreted human chorionic gonadotrophin subunit β (CG-β), placental lactogen (PL), and endogenous jaagsiekte sheep retrovirus (enJSRV) envelope genes were expressed in hTERT-STCs. Transwell cell invasion assay indicated that hTERT-STCs still possessed the same invasive characteristics as normal primary sheep trophoblast cells. hTERT-STCs could not grow in soft agar and did not develop into tumors in nude mice. In this study, we established a strain of immortalized sheep trophoblast cell line which could be gainfully employed in the future as an experimental model to study trophoblast cells with secretory function, invasive features, and probable biological function of enJSRV envelope genes.

Tonic B-cell receptor signaling in diffuse large B-cell lymphoma.

PubMed

Havranek, Ondrej; Xu, Jingda; Köhrer, Stefan; Wang, Zhiqiang; Becker, Lisa; Comer, Justin M; Henderson, Jared; Ma, Wencai; Man Chun Ma, John; Westin, Jason R; Ghosh, Dipanjan; Shinners, Nicholas; Sun, Luhong; Yi, Allen F; Karri, Anusha R; Burger, Jan A; Zal, Tomasz; Davis, R Eric

2017-08-24

We used clustered regularly interspaced short palindromic repeats/Cas9-mediated genomic modification to investigate B-cell receptor (BCR) signaling in cell lines of diffuse large B-cell lymphoma (DLBCL). Three manipulations that altered BCR genes without affecting surface BCR levels showed that BCR signaling differs between the germinal center B-cell (GCB) subtype, which is insensitive to Bruton tyrosine kinase inhibition by ibrutinib, and the activated B-cell (ABC) subtype. Replacing antigen-binding BCR regions had no effect on BCR signaling in GCB-DLBCL lines, reflecting this subtype's exclusive use of tonic BCR signaling. Conversely, Y188F mutation in the immunoreceptor tyrosine-based activation motif of CD79A inhibited tonic BCR signaling in GCB-DLBCL lines but did not affect their calcium flux after BCR cross-linking or the proliferation of otherwise-unmodified ABC-DLBCL lines. CD79A-GFP fusion showed BCR clustering or diffuse distribution, respectively, in lines of ABC and GCB subtypes. Tonic BCR signaling acts principally to activate AKT, and forced activation of AKT rescued GCB-DLBCL lines from knockout (KO) of the BCR or 2 mediators of tonic BCR signaling, SYK and CD19. The magnitude and importance of tonic BCR signaling to proliferation and size of GCB-DLBCL lines, shown by the effect of BCR KO, was highly variable; in contrast, pan-AKT KO was uniformly toxic. This discrepancy was explained by finding that BCR KO-induced changes in AKT activity (measured by gene expression, CXCR4 level, and a fluorescent reporter) correlated with changes in proliferation and with baseline BCR surface density. PTEN protein expression and BCR surface density may influence clinical response to therapeutic inhibition of tonic BCR signaling in DLBCL. © 2017 by The American Society of Hematology.

Withaferin A modulates the Spindle assembly checkpoint by degradation of Mad2-Cdc20 complex in colorectal cancer cell lines.

PubMed

Das, Tania; Roy, Kumar Singha; Chakrabarti, Tulika; Mukhopadhyay, Sibabrata; Roychoudhury, Susanta

2014-09-01

Withania somnifera L. Dunal (Ashwagandha) is used over centuries in the ayurvedic medicines in India. Withaferin A, a withanolide, is the major compound present in leaf extract of the plant which shows anticancer activity against leukemia, breast cancer and colorectal cancer. It arrests the ovarian cancer cells in the G2/M phase in dose dependent manner. In the current study we show the effect of Withaferin A on cell cycle regulation of colorectal cancer cell lines HCT116 and SW480 and its effect on cell fate. Treatment of these cells with this compound leads to apoptosis in a dose dependent manner. It causes the G2/M arrest in both the cell lines. We show that Withaferin A (WA) causes mitotic delay by blocking Spindle assembly checkpoint (SAC) function. Apoptosis induced by Withaferin A is associated with proteasomal degradation of Mad2 and Cdc20, an important constituent of the Spindle Checkpoint Complex. Further overexpression of Mad2 partially rescues the deleterious effect of WA by restoring proper anaphase initiation and keeping more number of cells viable. We hypothesize that Withaferin A kills cancer cells by delaying the mitotic exit followed by inducing chromosome instability. Copyright © 2014 Elsevier Inc. All rights reserved.

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

Human renal adipose tissue induces the invasion and progression of renal cell carcinoma

PubMed Central

Campo-Verde-Arbocco, Fiorella; López-Laur, José D.; Romeo, Leonardo R.; Giorlando, Noelia; Bruna, Flavia A.; Contador, David E.; López-Fontana, Gastón; Santiano, Flavia E.; Sasso, Corina V.; Zyla, Leila E.; López-Fontana, Constanza M.; Calvo, Juan C.; Carón, Rubén W.; Creydt, Virginia Pistone

2017-01-01

We evaluated the effects of conditioned media (CMs) of human adipose tissue from renal cell carcinoma located near the tumor (hRATnT) or farther away from the tumor (hRATfT), on proliferation, adhesion and migration of tumor (786-O and ACHN) and non-tumor (HK-2) human renal epithelial cell lines. Human adipose tissues were obtained from patients with renal cell carcinoma (RCC) and CMs from hRATnT and hRATfT incubation. Proliferation, adhesion and migration were quantified in 786-O, ACHN and HK-2 cell lines incubated with hRATnT-, hRATfT- or control-CMs. We evaluated versican, adiponectin and leptin expression in CMs from hRATnT and hRATfT. We evaluated AdipoR1/2, ObR, pERK, pAkt y pPI3K expression on cell lines incubated with CMs. No differences in proliferation of cell lines was found after 24 h of treatment with CMs. All cell lines showed a significant decrease in cell adhesion and increase in cell migration after incubation with hRATnT-CMs vs. hRATfT- or control-CMs. hRATnT-CMs showed increased levels of versican and leptin, compared to hRATfT-CMs. AdipoR2 in 786-O and ACHN cells decreased significantly after incubation with hRATfT- and hRATnT-CMs vs. control-CMs. We observed a decrease in the expression of pAkt in HK-2, 786-O and ACHN incubated with hRATnT-CMs. This result could partially explain the observed changes in migration and cell adhesion. We conclude that hRATnT released factors, such as leptin and versican, could enhance the invasive potential of renal epithelial cell lines and could modulate the progression of the disease. PMID:29212223

Generation and characterization of human iPSC line generated from mesenchymal stem cells derived from adipose tissue.

PubMed

Zapata-Linares, Natalia; Rodriguez, Saray; Mazo, Manuel; Abizanda, Gloria; Andreu, Enrique J; Barajas, Miguel; Prosper, Felipe; Rodriguez-Madoz, Juan R

2016-01-01

In this work, mesenchymal stem cells derived from adipose tissue (ADSCs) were used for the generation of the human-induced pluripotent stem cell line G15.AO. Cell reprogramming was performed using retroviral vectors containing the Yamanaka factors, and the generated G15.AO hiPSC line showed normal karyotype, silencing of the exogenous reprogramming factors, induction of the typical pluripotency-associated markers, alkaline phosphatase enzymatic activity, and in vivo and in vitro differentiation ability to the three germ layers. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Radiation sensitivity of Merkel cell carcinoma cell lines

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

Leonard, J.H.; Ramsay, J.R.; Birrell, G.W.

1995-07-30

Merkel cell carcinoma (MCC), being a small cell carcinoma, would be expected to be sensitive to radiation. Clinical analysis of patients at our center, especially those with macroscopic disease, would suggest the response is quite variable. We have recently established a number of MCC cell lines from patients prior to radiotherapy, and for the first time are in a position to determine their sensitivity under controlled conditions. Some of the MCC lines grew as suspension cultures and could not be single cell cloned; therefore, it was not possible to use clonogenic survival for all cell lines. A tetrazolium based (MTT)more » assay was used for these lines, to estimate cell growth after {gamma} irradiation. Control experiments were conducted on lymphoblastoid cell lines (LCL) and the adherent MCC line, MCC13, to demonstrate that the two assays were comparable under the conditions used. We have examined cell lines from MCC, small cell lung cancer (SCLC), malignant melanomas, Epstein Barr virus (EBV) transformed lymphocytes (LCL), and skin fibroblasts for their sensitivity to {gamma} irradiation using both clonogenic cell survival and MTT assays. The results show that the tumor cell lines have a range of sensitivities, with melanoma being more resistant (surviving fraction at 2 Gy (SF2) 0.57 and 0.56) than the small cell carcinoma lines, MCC (SF2 range 0.21-0.45, mean SF2 0.30, n = 8) and SCLC (SF2 0.31). Fibroblasts were the most sensitive (SF2 0.13-0.20, mean 0.16, n = 5). The MTT assay, when compared to clonogenic assay for the MCC13 adherent line and the LCL, gave comparable results under the conditions used. Both assays gave a range of SF2 values for the MCC cell lines, suggesting that these cancers would give a heterogeneous response in vivo. The results with the two derivative clones of MCC14 (SF2 for MCC14/1 0.38, MCC14/2 0.45) would further suggest that some of them may develop resistance during clonogenic evolution. 25 refs., 3 figs., 1 tab.« less

Global gene expression analyses of hematopoietic stem cell-like cell lines with inducible Lhx2 expression

PubMed Central

Richter, Karin; Wirta, Valtteri; Dahl, Lina; Bruce, Sara; Lundeberg, Joakim; Carlsson, Leif; Williams, Cecilia

2006-01-01

Background Expression of the LIM-homeobox gene Lhx2 in murine hematopoietic cells allows for the generation of hematopoietic stem cell (HSC)-like cell lines. To address the molecular basis of Lhx2 function, we generated HSC-like cell lines where Lhx2 expression is regulated by a tet-on system and hence dependent on the presence of doxycyclin (dox). These cell lines efficiently down-regulate Lhx2 expression upon dox withdrawal leading to a rapid differentiation into various myeloid cell types. Results Global gene expression of these cell lines cultured in dox was compared to different time points after dox withdrawal using microarray technology. We identified 267 differentially expressed genes. The majority of the genes overlapping with HSC-specific databases were those down-regulated after turning off Lhx2 expression and a majority of the genes overlapping with those defined as late progenitor-specific genes were the up-regulated genes, suggesting that these cell lines represent a relevant model system for normal HSCs also at the level of global gene expression. Moreover, in situ hybridisations of several genes down-regulated after dox withdrawal showed overlapping expression patterns with Lhx2 in various tissues during embryonic development. Conclusion Global gene expression analysis of HSC-like cell lines with inducible Lhx2 expression has identified genes putatively linked to self-renewal / differentiation of HSCs, and function of Lhx2 in organ development and stem / progenitor cells of non-hematopoietic origin. PMID:16600034

Antiapoptotic property of human alpha-synuclein in neuronal cell lines is associated with the inhibition of caspase-3 but not caspase-9 activity.

PubMed

Li, Wenxue; Lee, Michael K

2005-06-01

Abnormalities of alpha-synuclein (alpha-Syn) are mechanistically linked to Parkinson's disease (PD) and other alpha-synucleinopathies. To gain additional insights into the relationships between alpha-Syn expression and cell death, we examined the effects of expressing human alpha-Syn (Hualpha-Syn) variants on the cellular vulnerability to apoptotic stimuli. We show that the expression of wild-type (WT) and A30P mutant, but not A53T mutant, Hualpha-Syn leads to the protection of neuronal cell lines from apoptosis but not necrosis. Significantly, Hualpha-Syn did not protect non-neuronal cell lines from apoptosis. We also show that A53T mutant is a loss of function in regards to the antiapoptotic property since the expression of WT Hualpha-Syn with an excess of A53T mutant Hualpha-Syn leads to protection of the cells from apoptosis. The antiapoptotic property is specific to human alpha-Syn as neither beta-Syn nor mouse alpha-Syn protected cells from apoptosis, and the carboxy-terminal 20 amino acids are required for the antiapoptotic property. Analyses of capase-3 and caspase-9 activation reveal that the antiapoptotic property of Hualpha-Syn in neuronal cell lines is associated with the attenuation of caspase-3 activity without affecting the caspase-9 activity or the levels of cleaved, active caspase-3. We conclude that Hualpha-Syn modulates the activity of cleaved caspase-3 product in neuronal cell lines.

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.

DNA Repair in Human Pluripotent Stem Cells Is Distinct from That in Non-Pluripotent Human Cells

PubMed Central

Luo, Li Z.; Park, Sang-Won; Bates, Steven E.; Zeng, Xianmin; Iverson, Linda E.; O'Connor, Timothy R.

2012-01-01

The potential for human disease treatment using human pluripotent stem cells, including embryonic stem cells and induced pluripotent stem cells (iPSCs), also carries the risk of added genomic instability. Genomic instability is most often linked to DNA repair deficiencies, which indicates that screening/characterization of possible repair deficiencies in pluripotent human stem cells should be a necessary step prior to their clinical and research use. In this study, a comparison of DNA repair pathways in pluripotent cells, as compared to those in non-pluripotent cells, demonstrated that DNA repair capacities of pluripotent cell lines were more heterogeneous than those of differentiated lines examined and were generally greater. Although pluripotent cells had high DNA repair capacities for nucleotide excision repair, we show that ultraviolet radiation at low fluxes induced an apoptotic response in these cells, while differentiated cells lacked response to this stimulus, and note that pluripotent cells had a similar apoptotic response to alkylating agent damage. This sensitivity of pluripotent cells to damage is notable since viable pluripotent cells exhibit less ultraviolet light-induced DNA damage than do differentiated cells that receive the same flux. In addition, the importance of screening pluripotent cells for DNA repair defects was highlighted by an iPSC line that demonstrated a normal spectral karyotype, but showed both microsatellite instability and reduced DNA repair capacities in three out of four DNA repair pathways examined. Together, these results demonstrate a need to evaluate DNA repair capacities in pluripotent cell lines, in order to characterize their genomic stability, prior to their pre-clinical and clinical use. PMID:22412831

Gene Expression in Single Cells Isolated from the CWR-R1 Prostate Cancer Cell Line and Human Prostate Tissue Based on the Side Population Phenotype.

PubMed

Gangavarapu, Kalyan J; Miller, Austin; Huss, Wendy J

2016-09-01

Defining biological signals at the single cell level can identify cancer initiating driver mutations. Techniques to isolate single cells such as microfluidics sorting and magnetic capturing systems have limitations such as: high cost, labor intense, and the requirement of a large number of cells. Therefore, the goal of our current study is to identify a cost and labor effective, reliable, and reproducible technique that allows single cell isolation for analysis to promote regular laboratory use, including standard reverse transcription PCR (RT-PCR). In the current study, we utilized single prostate cells isolated from the CWR-R1 prostate cancer cell line and human prostate clinical specimens, based on the ATP binding cassette (ABC) transporter efflux of dye cycle violet (DCV), side population assay. Expression of four genes: ABCG2; Aldehyde dehydrogenase1A1 (ALDH1A1); androgen receptor (AR); and embryonic stem cell marker, Oct-4, were determined. Results from the current study in the CWR-R1 cell line showed ABCG2 and ALDH1A1 gene expression in 67% of single side population cells and in 17% or 100% of non-side population cells respectively. Studies using single cells isolated from clinical specimens showed that the Oct-4 gene is detected in only 22% of single side population cells and in 78% of single non-side population cells. Whereas, AR gene expression is in 100% single side population and non-side population cells isolated from the same human prostate clinical specimen. These studies show that performing RT-PCR on single cells isolated by FACS can be successfully conducted to determine gene expression in single cells from cell lines and enzymatically digested tissue. While these studies provide a simple yes/no expression readout, the more sensitive quantitative RT-PCR would be able to provide even more information if necessary.

Gene Expression in Single Cells Isolated from the CWR-R1 Prostate Cancer Cell Line and Human Prostate Tissue Based on the Side Population Phenotype

PubMed Central

Gangavarapu, Kalyan J; Miller, Austin; Huss, Wendy J

2016-01-01

Defining biological signals at the single cell level can identify cancer initiating driver mutations. Techniques to isolate single cells such as microfluidics sorting and magnetic capturing systems have limitations such as: high cost, labor intense, and the requirement of a large number of cells. Therefore, the goal of our current study is to identify a cost and labor effective, reliable, and reproducible technique that allows single cell isolation for analysis to promote regular laboratory use, including standard reverse transcription PCR (RT-PCR). In the current study, we utilized single prostate cells isolated from the CWR-R1 prostate cancer cell line and human prostate clinical specimens, based on the ATP binding cassette (ABC) transporter efflux of dye cycle violet (DCV), side population assay. Expression of four genes: ABCG2; Aldehyde dehydrogenase1A1 (ALDH1A1); androgen receptor (AR); and embryonic stem cell marker, Oct-4, were determined. Results from the current study in the CWR-R1 cell line showed ABCG2 and ALDH1A1 gene expression in 67% of single side population cells and in 17% or 100% of non-side population cells respectively. Studies using single cells isolated from clinical specimens showed that the Oct-4 gene is detected in only 22% of single side population cells and in 78% of single non-side population cells. Whereas, AR gene expression is in 100% single side population and non-side population cells isolated from the same human prostate clinical specimen. These studies show that performing RT-PCR on single cells isolated by FACS can be successfully conducted to determine gene expression in single cells from cell lines and enzymatically digested tissue. While these studies provide a simple yes/no expression readout, the more sensitive quantitative RT-PCR would be able to provide even more information if necessary. PMID:27785389

Characterization of immortalized human mammary epithelial cell line HMEC 2.6.

PubMed

Joshi, Pooja S; Modur, Vishnu; Cheng, JiMing; Robinson, Kathy; Rao, Krishna

2017-10-01

Primary human mammary epithelial cells have a limited life span which makes it difficult to study them in vitro for most purposes. To overcome this problem, we have developed a cell line that was immortalized using defined genetic elements, and we have characterized this immortalized non-tumorigenic human mammary epithelial cell line to establish it as a potential model system. human mammary epithelial cells were obtained from a healthy individual undergoing reduction mammoplasty at SIU School of Medicine. The cells were transduced with CDK4R24C followed by transduction with human telomerase reverse transcriptase. Post all manipulation, the cells displayed a normal cell cycle phase distribution and were near diploid in nature, which was confirmed by flow cytometry and karyotyping. In vitro studies showed that the cells were anchorage dependent and were non-invasive in nature. The cell line expressed basal epithelial markers such as cytokeratin 7, CD10, and p63 and was negative for the expression of estrogen receptor and progesterone receptor. Upon G-band karyotyping, the cell line displayed the presence of a few cytogenic abnormalities, including trisomy 20 and trisomy 7, which are also commonly present in other immortalized mammary cell lines. Furthermore, the benign nature of these cells was confirmed by multiple in vitro and in vivo experiments. Therefore, we think that this cell line could serve as a good model to understand the molecular mechanisms involved in the development and progression of breast cancer and to also assess the effect of novel therapeutics on human mammary epithelial cells.

Antiproliferative Activity of Xanthones Isolated from Artocarpus obtusus

PubMed Central

Hashim, Najihah Mohd; Rahmani, Mawardi; Ee, Gwendoline Cheng Lian; Sukari, Mohd Aspollah; Yahayu, Maizatulakmal; Oktima, Winda; Ali, Abd Manaf; Go, Rusea

2012-01-01

An investigation of the chemical constituents in Artocarpus obtusus species led to the isolation of three new xanthones, pyranocycloartobiloxanthone A (1), dihydroartoindonesianin C (2), and pyranocycloartobiloxanthone B (3). The compounds were subjected to antiproliferative assay against human promyelocytic leukemia (HL60), human chronic myeloid leukemia (K562), and human estrogen receptor (ER+) positive breast cancer (MCF7) cell lines. Pyranocycloartobiloxanthone A (1) consistently showed strong cytotoxic activity against the three cell lines compared to the other two with IC50 values of 0.5, 2.0 and 5.0 μg/mL, respectively. Compound (1) was also observed to exert antiproliferative activity and apoptotic promoter towards HL60 and MCF7 cell lines at respective IC50 values. The compound (1) was not toxic towards normal cell lines human nontumorigenic breast cell line (MCF10A) and human peripheral blood mononuclear cells (PBMCs) with IC50 values of more than 30 μg/mL. PMID:21960741

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.

Establishment and characterization of a new cell line (SSP-9) derived from Atlantic salmon Salmo salar that expresses type I ifn.

PubMed

Rodriguez Saint-Jean, S; González, C; Monrás, M; Romero, A; Ballesteros, N; Enríquez, R; Perez-Prieto, S

2014-11-01

In the present work, the establishment and biological characterization of a new cell line, SSP-9, derived from the pronephros of the Atlantic salmon Salmo salar, are reported. These cells grew well in Leibovitz's (L15) medium supplemented with 10% foetal calf serum at temperatures from 15 to 25° C, and they have been sub-cultured over 100 passages to produce a continuous cell line with an epithelial-like morphology. The SSP-9 cells attached and spread efficiently at different plating densities, retaining 80% of cell viability after storage in liquid nitrogen. When karyotyped, the cells had 40-52 chromosomes, with a modal number of 48. Viral susceptibility tests showed that SSP-9 cells were susceptible to infectious pancreatic necrosis virus and infectious haematopoietic necrosis virus, producing infectious virus and regular cytopathic effects. Moreover, these cells could be stimulated by poly I:C, showing significant up-regulation in the expression of the genes that regulate immune responses, such as ifn and mx-1. SSP-9 cells constitutively express genes characteristic of macrophages, such as major histocompatibility complex (mhc-II) and interleukin 12b (il-12b), and flow cytometry assays confirmed that SSP-9 cells can be permanently transfected with plasmids expressing a reporter gene. Accordingly, this new cell line is apparently suitable for transgenic manipulation, and to study host cell-virus interactions and immune processes. © 2014 The Fisheries Society of the British Isles.

Anticancer and enhanced antimicrobial activity of biosynthesizd silver nanoparticles against clinical pathogens

NASA Astrophysics Data System (ADS)

Rajeshkumar, Shanmugam; Malarkodi, Chelladurai; Vanaja, Mahendran; Annadurai, Gurusamy

2016-07-01

The present investigation shows the biosynthesis of eco-friendly silver nanoparticles using culture supernatant of Enterococcus sp. and study the effect of enhanced antimicrobial activity, anticancer activity against pathogenic bacteria, fungi and cancer cell lines. Silver nanoparticles was synthesized by adding 1 mM silver nitrate into the 100 ml of 24 h freshly prepared culture supernatant of Enterococcus sp. and were characterized by UV-vis spectroscopy, X-ray diffraction (XRD), Transmission Electron Microscope (TEM), Selected Area Diffraction X-Ray (SAED), Energy Dispersive X Ray (EDX) and Fourier Transform Infra red Spectroscopy (FT-IR). The synthesized silver nanoparticles were impregnated with commercial antibiotics for evaluation of enhanced antimicrobial activity. Further these synthesized silver nanoparticles were assessed for its anticancer activity against cancer cell lines. In this study crystalline structured nanoparticles with spherical in the size ranges from 10 to 80 nm and it shows excellent enhanced antimicrobial activity than the commercial antibiotics. The in vitro assay of silver nanoparticles on anticancer have great potential to inhibit the cell viability. Amide linkages and carboxylate groups of proteins from Enterococcus sp. may bind with silver ions and convert into nanoparticles. The activities of commercial antibiotics were enhanced by coating silver nanoparticles shows significant improved antimicrobial activity. Silver nanoparticles have the great potential to inhibit the cell viability of liver cancer cells lines (HepG2) and lung cancer cell lines (A549).

Expression of Tac antigen component of bovine interleukin-2 receptor in different leukocyte populations infected with Theileria parva or Theileria annulata.

PubMed Central

Dobbelaere, D A; Prospero, T D; Roditi, I J; Kelke, C; Baumann, I; Eichhorn, M; Williams, R O; Ahmed, J S; Baldwin, C L; Clevers, H

1990-01-01

The Tac antigen component of the bovine interleukin-2 receptor was expressed as a Cro-beta-galactosidase fusion protein in Escherichia coli and used to raise antibodies in rabbits. These antibodies were used for flow cytofluorimetric analysis to investigate the expression of Tac antigen in a variety of Theileria parva-infected cell lines and also in three Theileria annulata-infected cell lines. Cells expressing Tac antigen on their surface were found in all T. parva-infected cell lines tested whether these were of T- or B-cell origin. T cells expressing Tac antigen could be CD4- CD8-, CD4+ CD8-, CD4- CD8+, or CD4+ CD8+. Tac antigen expression was observed both in cultures which had been maintained in the laboratory for several years and in transformed cell lines which had recently been established by infection of lymphocytes in vitro with T. parva. Northern (RNA) blot analysis demonstrated Tac antigen transcripts in RNA isolated from all T. parva-infected cell lines. Three T. annulata-infected cell lines which were not of T-cell origin were also tested. Two of them expressed Tac antigen on their surface. Abundant Tac antigen mRNA was detected in these T. annulata-infected cell lines, but only trace amounts were demonstrated in the third cell line, which contained very few Tac antigen-expressing cells. In all cell lines tested, whether cloned or uncloned, a proportion of the cells did not express detectable levels of Tac antigen on their surface. This was also the case for a number of other leukocyte surface markers. In addition, we showed that the interleukin-2 receptors were biologically functional, because addition of recombinant interleukin-2 to cultures stimulated cell proliferation. Recombinant interleukin-2 treatment also resulted in increased amounts of steady-state Tac antigen mRNA. The relevance of interleukin-2 receptor expression on Theileria-infected cells is discussed. Images PMID:1979317

Aberrant Apoptotic Response of Colorectal Cancer Cells to Novel Nucleoside Analogues.

PubMed

Harmse, Leonie; Dahan-Farkas, Nurit; Panayides, Jenny-Lee; van Otterlo, Willem; Penny, Clement

2015-01-01

Despite the increased understanding of colorectal cancer and the introduction of targeted drug therapy, the metastatic phase of the disease remains refractory to treatment. Since the deregulation of normal apoptosis contributes to the pathogenesis of colorectal cancer, novel nucleoside analogues were synthesized here and evaluated for their ability to induce apoptosis and cause cell death in two colorectal adeno-carcinoma cell lines, Caco-2 and HT-29. Three novel nucleoside analogues assessed here showed cytotoxic activity, as measured by the MTT assay against both cell lines: the IC50 values ranged between 3 and 37 μM, with Caco-2 cells being more sensitive than HT-29 cells. Compared to camptothecin, the positive control, the nucleoside analogues were significantly less toxic to normal unstimulated leukocytes (p>0.05). Moreover, the nucleosides were able to induce apoptosis as measured by an increase in caspase 8 and caspase 3 activity above that of the control. This was additionally supported by data derived from Annexin V-FITC assays. Despite marginal changes to the mitochondrial membrane potential, all three nucleosides caused a significant increase in cytosolic cytochrome c (p>0.05), with a corresponding decrease in mitochondrial cytochrome c. Morphological analysis of both cell lines showed the rapid appearance of vacuoles following exposure to two of the nucleosides, while a third caused cellular detachment, delayed cytoplasmic vacuolisation and nuclear abnormalities. Preliminary investigations, using the autophagic indicator monodansylcadaverine and chloroquine as positive control, showed that two of the nucleosides induced the formation of autophagic vacuoles. In summary, the novel nucleoside analogues showed selective cytotoxicity towards both cancer cell lines and are effective initiators of an unusual apoptotic response, demonstrating their potential to serve as structural scaffolds for more potent analogues.

CXCL2 synthesized by oral squamous cell carcinoma is involved in cancer-associated bone destruction

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

Oue, Erika; Section of Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University; Global Center of Excellence

Highlights: Black-Right-Pointing-Pointer Oral cancer cells synthesize CXCL2. Black-Right-Pointing-Pointer CXCL2 synthesized by oral cancer is involved in osteoclastogenesis. Black-Right-Pointing-Pointer CXCL2-neutralizing antibody inhibited osteoclastogenesis induced by oral cancer cells. Black-Right-Pointing-Pointer We first report the role of CXCL2 in cancer-associated bone destruction. -- Abstract: To explore the mechanism of bone destruction associated with oral cancer, we identified factors that stimulate osteoclastic bone resorption in oral squamous cell carcinoma. Two clonal cell lines, HSC3-C13 and HSC3-C17, were isolated from the maternal oral cancer cell line, HSC3. The conditioned medium from HSC3-C13 cells showed the highest induction of Rankl expression in the mouse stromal cellmore » lines ST2 and UAMS-32 as compared to that in maternal HSC3 cells and HSC3-C17 cells, which showed similar activity. The conditioned medium from HSC3-C13 cells significantly increased the number of osteoclasts in a co-culture with mouse bone marrow cells and UAMS-32 cells. Xenograft tumors generated from these clonal cell lines into the periosteal region of the parietal bone in athymic mice showed that HSC3-C13 cells caused extensive bone destruction and a significant increase in osteoclast numbers as compared to HSC3-C17 cells. Gene expression was compared between HSC3-C13 and HSC3-C17 cells by using microarray analysis, which showed that CXCL2 gene was highly expressed in HSC3-C13 cells as compared to HSC3-C17 cells. Immunohistochemical staining revealed the localization of CXCL2 in human oral squamous cell carcinomas. The increase in osteoclast numbers induced by the HSC3-C13-conditioned medium was dose-dependently inhibited by addition of anti-human CXCL2-neutralizing antibody in a co-culture system. Recombinant CXCL2 increased the expression of Rankl in UAMS-32 cells. These results indicate that CXCL2 is involved in bone destruction induced by oral cancer. This is the first report showing the role of CXCL2 in cancer-associated bone destruction.« less

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

In vivo and in vitro biophysical properties of hair cells from the lateral line and inner ear of developing and adult zebrafish.

PubMed

Olt, Jennifer; Johnson, Stuart L; Marcotti, Walter

2014-05-15

Hair cells detect and process sound and movement information, and transmit this with remarkable precision and efficiency to afferent neurons via specialized ribbon synapses. The zebrafish is emerging as a powerful model for genetic analysis of hair cell development and function both in vitro and in vivo. However, the full exploitation of the zebrafish is currently limited by the difficulty in obtaining systematic electrophysiological recordings from hair cells under physiological recording conditions. Thus, the biophysical properties of developing and adult zebrafish hair cells are largely unknown. We investigated potassium and calcium currents, voltage responses and synaptic activity in hair cells from the lateral line and inner ear in vivo and using near-physiological in vitro recordings. We found that the basolateral current profile of hair cells from the lateral line becomes more segregated with age, and that cells positioned in the centre of the neuromast show more mature characteristics and those towards the edge retain a more immature phenotype. The proportion of mature-like hair cells within a given neuromast increased with zebrafish development. Hair cells from the inner ear showed a developmental change in current profile between the juvenile and adult stages. In lateral line hair cells from juvenile zebrafish, exocytosis also became more efficient and required less calcium for vesicle fusion. In hair cells from mature zebrafish, the biophysical characteristics of ion channels and exocytosis resembled those of hair cells from other lower vertebrates and, to some extent, those in the immature mammalian vestibular and auditory systems. We show that although the zebrafish provides a suitable animal model for studies on hair cell physiology, it is advisable to consider that the age at which the majority of hair cells acquire a mature-type configuration is reached only in the juvenile lateral line and in the inner ear from >2 months after hatching. © 2014 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.

The Glycoproteins of All Filovirus Species Use the Same Host Factors for Entry into Bat and Human Cells but Entry Efficiency Is Species Dependent.

PubMed

Hoffmann, Markus; González Hernández, Mariana; Berger, Elisabeth; Marzi, Andrea; Pöhlmann, Stefan

2016-01-01

Ebola and marburgviruses, members of the family Filoviridae, can cause severe hemorrhagic fever in humans. The ongoing Ebola virus (EBOV) disease epidemic in Western Africa claimed more than 11,300 lives and was associated with secondary cases outside Africa, demonstrating that filoviruses pose a global health threat. Bats constitute an important natural reservoir of filoviruses, including viruses of the recently identified Cuevavirus genus within the Filoviridae family. However, the interactions of filoviruses with bat cells are incompletely understood. Here, we investigated whether filoviruses employ different strategies to enter human and bat cells. For this, we examined host cell entry driven by glycoproteins (GP) from all filovirus species into cell lines of human and fruit bat origin. We show that all GPs were able to mediate entry into human and most fruit bat cell lines with roughly comparable efficiency. In contrast, the efficiency of entry into the cell line EidNi/41 derived from a straw-colored fruit bat varied markedly between the GPs of different filovirus species. Furthermore, inhibition studies demonstrated that filoviruses employ the same host cell factors for entry into human, non-human primate and fruit bat cell lines, including cysteine proteases, two pore channels and NPC1 (Niemann-Pick C1 molecule). Finally, processing of GP by furin and the presence of the mucin-like domain in GP were dispensable for entry into both human and bat cell lines. Collectively, these results show that filoviruses rely on the same host cell factors for entry into human and fruit bat cells, although the efficiency of the usage of these factors might differ between filovirus species.

The synthetic ligand of peroxisome proliferator-activated receptor-gamma ciglitazone affects human glioblastoma cell lines.

PubMed

Strakova, Nicol; Ehrmann, Jiri; Dzubak, Petr; Bouchal, Jan; Kolar, Zdenek

2004-06-01

Glioblastoma multiforme is the most common malignant brain tumor in adults, and it is among the most lethal of all cancers. Recent studies have shown that ligand activation of peroxisome proliferator-activated receptor (PPAR)-gamma can induce differentiation and inhibit proliferation of several cancer cells. In this study, we have investigated whether one PPARgamma ligand in particular, ciglitazone, inhibits cell viability and, additionally, whether it affects the cell cycle and apoptosis of human glioblastoma cell lines T98G, U-87 MG, A172, and U-118 MG. All glioblastoma cell lines were found to express PPARgamma protein, and following treatment with ciglitazone, localization was unchanged. Ciglitazone inhibited viability in a dose-dependent manner in all four tested glioblastoma cells after 24 h of treatment. Analysis of the cell cycle showed arrest in the G(1) phase and partial block in G(2)/M phase of the cell cycle. Cyclin D1 and cyclin B expression was decreased. Phosphorylation of Rb protein dropped as well. We found that ciglitazone was followed by increased expression of p27(Kip1) and p21(Waf1/Cip1). It also led to apoptosis induction: bax expression in T98G was elevated. Expression of the antiapoptotic protein bcl-2 was reduced in U-118 MG and U-87 MG and showed a slight decrease in A172 cells. Flow cytometry confirmed the induction of apoptosis. Moreover, PPARgamma ligand decreased telomerase activity in U-87 MG and U-118 MG cell lines. Our results demonstrate that ciglitazone inhibits the viability of human glioblastoma cell lines via induction of apoptosis; as a result, this ligand may offer potential new therapy for the treatment of central nervous system neoplasms.

The Glycoproteins of All Filovirus Species Use the Same Host Factors for Entry into Bat and Human Cells but Entry Efficiency Is Species Dependent

PubMed Central

Hoffmann, Markus; González Hernández, Mariana; Berger, Elisabeth; Marzi, Andrea; Pöhlmann, Stefan

2016-01-01

Ebola and marburgviruses, members of the family Filoviridae, can cause severe hemorrhagic fever in humans. The ongoing Ebola virus (EBOV) disease epidemic in Western Africa claimed more than 11,300 lives and was associated with secondary cases outside Africa, demonstrating that filoviruses pose a global health threat. Bats constitute an important natural reservoir of filoviruses, including viruses of the recently identified Cuevavirus genus within the Filoviridae family. However, the interactions of filoviruses with bat cells are incompletely understood. Here, we investigated whether filoviruses employ different strategies to enter human and bat cells. For this, we examined host cell entry driven by glycoproteins (GP) from all filovirus species into cell lines of human and fruit bat origin. We show that all GPs were able to mediate entry into human and most fruit bat cell lines with roughly comparable efficiency. In contrast, the efficiency of entry into the cell line EidNi/41 derived from a straw-colored fruit bat varied markedly between the GPs of different filovirus species. Furthermore, inhibition studies demonstrated that filoviruses employ the same host cell factors for entry into human, non-human primate and fruit bat cell lines, including cysteine proteases, two pore channels and NPC1 (Niemann-Pick C1 molecule). Finally, processing of GP by furin and the presence of the mucin-like domain in GP were dispensable for entry into both human and bat cell lines. Collectively, these results show that filoviruses rely on the same host cell factors for entry into human and fruit bat cells, although the efficiency of the usage of these factors might differ between filovirus species. PMID:26901159

In-silico and in-vitro anti-cancer potential of a curcumin analogue (1E, 6E)-1, 7-di (1H-indol-3-yl) hepta-1, 6-diene-3, 5-dione.

PubMed

Sufi, Shamim Akhtar; Adigopula, Lakshmi Narayana; Syed, Safiulla Basha; Mukherjee, Victor; Coumar, Mohane S; Rao, H Surya Prakash; Rajagopalan, Rukkumani

2017-01-01

Previously we showed that BDMC, an analogue of curcumin suppresses growth of human breast and laryngeal cancer cell line by causing apoptosis. Here, we demonstrate the enhanced anti-cancer activity of a heterocyclic ring (indole) incorporated curcumin analogue ((1E, 6E)-1, 7-di (1H-indol-3-yl) hepta-1, 6-diene-3, 5-Dione), ICA in short, in comparison to curcumin. ICA was synthesized by a one pot condensation reaction. Anti-cancer potential of ICA was assessed in three human cancer cell lines of different origin (Lung adenocarcinoma (A549), leukemia (K562) and colon cancer (SW480)) by MTT assay. Mode of cell death was determined by acridine orange-ethidium bromide (Ao-Eb) staining. Putative cellular targets of ICA were investigated by molecular docking studies. Cell cycle analysis following curcumin or ICA treatment in SW480 cell line was carried out by flow cytometry. Expression levels of Cyclin D1 and apoptotic markers, such as Caspase 3, 8 and 9 were studied by western blot analysis in SW480 cell line treated with or without ICA and curcumin. The yield of ICA synthesis was found to be 69% with a purity of 98%. ICA demonstrated promising anti-cancer activity compared to curcumin alone, as discerned by MTT assay. ICA was non-toxic to the cell line of normal origin. We further observed that ICA is ∼2 fold more potent than curcumin in inhibiting the growth of SW480 cells. Ao-Eb staining revealed that ICA could induce apoptosis in all the cell lines tested. Molecular docking studies suggest that ICA may possibly exhibit its anticancer effect by inhibiting EGFR in A549, Bcr-Abl in K562 and GSK-3β kinase in SW480 cell line. Moreover, ICA showed strong binding avidity for Bcl-2 protein in silico, which could result in induction of apoptosis. Cell cycle analysis revealed that both curcumin and ICA induced concomitant cell cycle arrest at G0/G1 and G2/M phase. Western blot shows that ICA could effectively down regulate the expression of cell cycle protein cyclin D1, while promoting the activation of Caspase 3, 8 and 9 when compared to curcumin in human colon cancer cell line SW480. The result of this study indicates that ICA could hold promise to be a potential anti-cancer agent. Since ICA has shown encouraging results in terms of its anti-cancer activity compared to curcumin, further research is necessary to fully delineate the underlying molecular mechanism of its anticancer potential. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Establishment and characterization of a new conditionally immortalized human astrocyte cell line.

PubMed

Furihata, Tomomi; Ito, Ryo; Kamiichi, Atsuko; Saito, Kosuke; Chiba, Kan

2016-01-01

Astrocytes are the most abundant cell types in mammalian brains, within which they participate in various neuronal activities, partly by utilizing the numerous transporters expressed at their plasma membranes. Accordingly, detailed characterization of astrocytic functions, including transporters, are essential for understanding of mechanistic basis of normal brain functions, as well as the pathogenesis and treatment of various brain diseases. As a part of overall efforts to facilitate such studies, this study reports on the establishment of a new human astrocyte cell line, which is hereafter referred to as human astrocyte/conditionally immortalized, clone 35 (HASTR/ci35). This line, which was developed utilizing a cell immortalization method, showed excellent proliferative ability and expressed various astrocyte markers, including glial fibrillary acidic protein. When co-cultured with neuronal cells, HASTR/ci35 cells could facilitate their dendritic network formation. Furthermore, HASTR/ci35 cells not only possessed significant glutamate and adenosine transporter activities but also exhibited organic ion transporter activities. To summarize, HASTR/ci35 cells possess several key astrocytic characteristics, including various transporter functions, while simultaneously showing infinite proliferation and scalability. Based on these findings, HASTR/ci35 cells can be expected to contribute significantly to various human astrocyte study fields. In vitro astrocyte models are valuable experimental tools in various astrocyte studies. Here, we report the establishment of a new human astrocyte cell line, HASTR/ci35, which show various key astrocyte properties, including astrocytic transporter activities, glycogen storage and facilitation of neuronal cell differentiation. Thus, HASTR/ci35 is expected to significantly contribute to advances toward detailed understanding of human astrocyte functions. © 2015 International Society for Neurochemistry.

Neoplastic transformation of human thyroid epithelial cells by ionizing radiation

NASA Astrophysics Data System (ADS)

Herceg, Zdenko

Neoplastic transformation of human thyroid epithelial cells has been investigated following exposure to ionizing radiation in vitro. The effects of radiation type, irradiation regime, and postirradiation passaging were examined using a human thyroid epithelial cell line, designated HToriS, which was previously immortalized with SV40 genome. Exponentially growing HToriS cells were irradiated with graded doses of 137 Cs gamma- and 238pu alpha-irradiation. Cells were irradiated with either a single or multiple doses of 0.5, 1, 2, 3, or 4 Gy gamma-radiation, or single doses of 0.125, 0.25, 0.5, 1, or 1.5 Gy gamma-radiation. Following passaging, the cells were transplanted into the athymic nude mice, and the animals were screened for tumour formation. Statistically significant increases in tumour incidence were obtained with both gamma- and alpha-irradiation and with both single and multiple irradiation regimes as compared with the un-irradiated group. Regardless of radiation type and or radiation regime there appears to be a trend, with increasing doses of radiation, in which tumour incidence increases and reaches a maximum, after which the tumour incidence decreases. Tumours were characterized by histopathological examination as undifferentiated carcinomas. Investigation of expression time following irradiation demonstrated that post-irradiation passaging, generally regarded as a critical step for expression of radiation-induced DNA damage, was not a prerequisite for the neoplastic conversion of irradiated cells with this system. Cell lines were established from the tumours and their identification and characterization carried out. All cell lines established were determined to be derived from the parent HTori3 cells by DNA fingerprinting, karyotype analysis, cytokeratin staining, and SV40 large T-antigen staining. Tumorigenicity of the cell lines was confirmed by retransplantation. Comparison of the morphology in vitro showed that the tumour cell lines retained the basic epithelial morphology of the parent HToriS cells. Investigation of radiosensitivity showed that none of the 6 tumour cell lines examined had a higher radiosensitivity compared to the parent HToriS cells. This excludes the possibility that the observed transformation was the result of the selection of a pre-existing transformed subpopulation of the parent cells but that radiation-induced transformants were being induced de novo. The tumour cell lines were screened for mutations in H- and K-ras oncogenes using restriction enzyme analysis of PCR amplified DNA. No mutations were detected in 26 tumour cell lines suggesting that mutations in these two genes do not appear to be involved in radiation- induced neoplastic transformation in human thyroid epithelial cells. Screening for mutations in p53 protein using immunoprecipitation method detected no mutations in 6 tumour cell lines. This human thyroid epithelial cell line may thus be useful for the in vitro study of cellular and molecular mechanisms that are involved in human epithelial cell carcinogenesis.

Unsuccessful derivation of human embryonic stem cell lines from pairs of human blastomeres.

PubMed

Fong, Chui-Yee; Richards, Mark; Bongso, Ariff

2006-08-01

Human embryonic stem cells (hESC) that differentiate into all three primordial germ layers have been established. Differentiation of these cells into desirable lineages offers hope for future transplantation therapies. Currently, hESC lines are derived from the inner cell mass (ICM) of blastocysts, leading to destruction of the embryo, and thus the process is ethically controversial. Successful attempts at deriving hESC lines from blastomeres without destruction of the ensuing embryo have not been reported. One or two blastomeres are routinely biopsied from 8-cell embryos for preimplantation genetic diagnosis. In this study it was therefore attempted to derive hESC lines from paired blastomeres. Of 66 pairs of 8-cell stage blastomeres, four pairs produced two morula and two blastocyst-like structures. When plated on mitomycin-C-treated mouse embryonic fibroblasts, one morula and one blastocyst-like structure separately produced small colonies containing hESC-like cells with prominent nucleoli and high nuclear-cytoplasmic ratios. When these colonies were detached and plated onto fresh feeders, there was no further colony formation or ensuing hESC lines. The results showed that it might not be possible to derive hESC lines directly from paired blastomeres. A minimum number of blastomeres in close contact with one another may be required to successfully generate an hESC line as blastomeres, like ICM and hESC cells, may be 'social' cells.

Moringa oleifera as an Anti-Cancer Agent against Breast and Colorectal Cancer Cell Lines.

PubMed

Al-Asmari, Abdulrahman Khazim; Albalawi, Sulaiman Mansour; Athar, Md Tanwir; Khan, Abdul Quaiyoom; Al-Shahrani, Hamoud; Islam, Mozaffarul

2015-01-01

In this study we investigated the anti-cancer effect of Moringa oleifera leaves, bark and seed extracts. When tested against MDA-MB-231 and HCT-8 cancer cell lines, the extracts of leaves and bark showed remarkable anti-cancer properties while surprisingly, seed extracts exhibited hardly any such properties. Cell survival was significantly low in both cells lines when treated with leaves and bark extracts. Furthermore, a striking reduction (about 70-90%) in colony formation as well as cell motility was observed upon treatment with leaves and bark. Additionally, apoptosis assay performed on these treated breast and colorectal cancer lines showed a remarkable increase in the number of apoptotic cells; with a 7 fold increase in MD-MB-231 to an increase of several fold in colorectal cancer cell lines. However, no significant apoptotic cells were detected upon seeds extract treatment. Moreover, the cell cycle distribution showed a G2/M enrichment (about 2-3 fold) indicating that these extracts effectively arrest the cell progression at the G2/M phase. The GC-MS analyses of these extracts revealed numerous known anti-cancer compounds, namely eugenol, isopropyl isothiocynate, D-allose, and hexadeconoic acid ethyl ester, all of which possess long chain hydrocarbons, sugar moiety and an aromatic ring. This suggests that the anti-cancer properties of Moringa oleifera could be attributed to the bioactive compounds present in the extracts from this plant. This is a novel study because no report has yet been cited on the effectiveness of Moringa extracts obtained in the locally grown environment as an anti-cancer agent against breast and colorectal cancers. Our study is the first of its kind to evaluate the anti-malignant properties of Moringa not only in leaves but also in bark. These findings suggest that both the leaf and bark extracts of Moringa collected from the Saudi Arabian region possess anti-cancer activity that can be used to develop new drugs for treatment of breast and colorectal cancers.

Cyclopentenone derivatives and polyhydroxylated steroids from the soft coral Sinularia acuta.

PubMed

Zhang, Nai-Xia; Tang, Xu-Li; van Ofwegen, Leen; Xue, Lei; Song, Wen-Juan; Li, Ping-Lin; Li, Guo-Qiang

2015-02-01

Four new polyhydroxylated steroids, 1-4, and the racemic form of cyclopentenone 9, together with four known steroids, 5-8, one known cyclopentenone derivative, 10, and one known butenolide derivative, 11, were isolated from the soft coral Sinularia acuta collected from Weizhou Island of Guangxi Province, P. R. China. Their structures were elucidated on the basis of spectroscopic analyses and by comparison of the corresponding data with those previously reported. The cytotoxicities of the isolates 1-11 in vitro against the selected tumor cell lines HL-60, HeLa, and K562 were evaluated. Compounds 2 and 5 showed potent cytotoxicities against HL-60 cell lines with IC50 values of 7.3 and 9.9 μM, respectively. Compounds 5 and 6 showed moderate activities against K562 cell lines with IC50 values of 10.9 and 11.7 μM, respectively, while compounds 1, 2, and 6 showed weak activities against HeLa cell lines with respective IC50 values of 44.8, 27.1, and 18.2 μM. This is the first report on chemical and bioactivity research of S. acuta. Copyright © 2015 Verlag Helvetica Chimica Acta AG, Zürich.

Normal neutrophil differentiation and secondary granule gene expression in the EML and MPRO cell lines.

PubMed

Lawson, N D; Krause, D S; Berliner, N

1998-11-01

The EML and MPRO cell lines express a dominant negative retinoic acid receptor alpha that causes a block at specific stages of myelopoiesis. The EML cell line is multipotent and gives rise to erythroid, lymphoid, and myeloid lineages depending on the presence of appropriate cytokines. The MPRO cell line is promyelocytic and undergoes neutrophilic differentiation when induced with all-trans retinoic acid in the presence of granulocyte/macrophage colony-stimulating factor. Previous studies have shown that both of these cell lines undergo morphological differentiation into neutrophils. In this study, we show that unlike other models of neutrophil differentiation such as NB4 and HL60, both EML and MPRO cell lines undergo complete, normal granulocytic differentiation programs. Similar to HL60, MPRO and EML induce expression of CD11b/CD18 and also exhibit downregulation of CD34 on differentiation. In contrast to HL60 and NB4, EML and MPRO cell lines coordinately upregulate secondary granule transcripts for lactoferrin and neutrophil gelatinase. Furthermore, we have confirmed previous observations that serum can induce a low level of differentiation in MPRO cells and that it is possible to grow these cells in serum-free medium, thereby eliminating this effect. Based on these studies, it appears that these lines can serve as a model for normal retinoic acid-induced neutrophil differentiation and provide insight into the role of the retinoic acid-responsive pathway in normal and leukemic myelopoiesis.

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

PubMed

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

2006-05-01

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

Influence of oxygen partial pressure on the characteristics of human hepatocarcinoma cells.

PubMed

Trepiana, Jenifer; Meijide, Susana; Navarro, Rosaura; Hernández, M Luisa; Ruiz-Sanz, José Ignacio; Ruiz-Larrea, M Begoña

2017-08-01

Most of the in vitro studies using liver cell lines have been performed under atmospheric oxygen partial pressure (21% O 2 ). However, the oxygen concentrations in the liver and cancer cells are far from this value. In the present study, we have evaluated the influence of oxygen on 1) the tumor cell lines features (growth, steady-state ROS levels, GSH content, activities of antioxidant enzymes, p66 Shc and SOD expressions, metalloproteinases secretion, migration, invasion, and adhesion) of human hepatocellular carcinoma cell lines, and b) the response of the cells to an oxidant stimulus (aqueous leaf extract of the V. baccifera plant species). For this purpose, three hepatocarcinoma cell lines with different p53 status, HepG2 (wild-type), Huh7 (mutated), and Hep3B (deleted), were cultured (6-30 days) under atmospheric (21%) and more physiological (8%) pO 2 . Results showed that after long-term culturing at 8% versus 21% O 2 , the cellular proliferation rate and the steady-state levels of mitochondrial O 2 - were unaffected. However, the intracellular basal ROS levels were higher independently of the characteristics of the cell line. Moreover, the lower pO 2 was associated with lower glutathione content, the induction of p66 Shc and Mn-SOD proteins, and increased SOD activity only in HepG2. This cell line also showed a higher migration rate, secretion of active metalloproteinases, and a faster invasion. HepG2 cells were more resistant to the oxidative stress induced by V. baccifera. Results suggest that the long-term culturing of human hepatoma cells at a low, more physiological pO 2 induces antioxidant adaptations that could be mediated by p53, and may alter the cellular response to a subsequent oxidant challenge. Data support the necessity of validating outcomes from studies performed with hepatoma cell cultures under ambient O 2 . Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Isolation of a novel chronic lymphocytic leukemic (CLL) cell line and development of an in vivo mouse model of CLL.

PubMed

Kellner, Joshua; Wierda, William; Shpall, Elizabeth; Keating, Michael; McNiece, Ian

2016-01-01

Leukemic cell lines have become important tools for studies of disease providing a monoclonal cell population that can be extensively expanded in vitro while preserving leukemic cellular characteristics. However, studies of chronic lymphocytic leukemia (CLL) have been impeded in part by the lack of continuous human cell lines. CLL cells have a high spontaneous apoptosis rate in vitro and exhibit minimal proliferation in xenograft models. Therefore, there is a need for development of primary CLL cell lines and we describe the isolation of such a line from the bone marrow of a CLL patient (17p deletion and TP53 mutation) which has been in long term culture for more than 12 months with continuous proliferation. The CLL cell line (termed MDA-BM5) which was generated in vitro with continuous co-culture on autologous stromal cells is CD19+CD5+ and shows an identical pattern of somatic hypermutation as determined in the patient's bone marrow (BM), confirming the origin of the cells from the original CLL clone. MDA-BM5 cells were readily transplantable in NOD/SCID gamma null mice (NSG) with disease developing in the BM, liver and spleen. BM cells from quaternary serial transplantation in NSG mice demonstrated the presence of CD19+CD5+ cells with Ig restricted to lambda which is consistent with the original patient cells. These studies describe a new CLL cell line from a patient with del(17p) that provides a unique model for in vitro and in vivo studies. Copyright © 2015 Elsevier Ltd. All rights reserved.

Establishment of an immortal chicken embryo liver-derived cell line.

PubMed

Lee, Jeongyoon; Foster, Douglas N; Bottje, Walter G; Jang, Hyeon-Min; Chandra, Yohanna G; Gentles, Lauren E; Kong, Byung-Whi

2013-06-01

A continuously growing immortal cell substrate can be used for virus propagation, diagnostic purposes, and vaccine production. The aim of this study was to develop an immortal chicken cell line for efficient propagation of avian infectious viruses. From the various chicken embryo cells that were tested for life span extension, an immortalized chicken embryo liver (CEL) cell line, named CEL-im, was derived spontaneously without either oncogenic viruses or carcinogenic chemical treatment. Currently, CEL-im cells are growing 0.8 to 1.1 population doublings per day and have reached 120 passages. The CEL-im cell line is permissive for poultry infectious viruses, including avian metapneumovirus (AMPV), Marek's disease virus serotype 1 (MDV-1), and infectious laryngotracheitis virus. The CEL-im cells produced high AMPV titer (>10(5) pfu/mL), whereas very low titers (~10 pfu/mL) for MDV-1 and infectious laryngotracheitis virus were produced. To identify genetic alterations in the immortal CEL-im cell line, telomerase activity and mRNA expression for major cell cycle regulatory genes were determined during the immortalizing process. The CEL-im cell line has negative telomerase activity, and when compared with the primary passage 2 CEL cell counterpart, mRNA expression of tumor suppressor protein p53, mouse double minute 2 (Mdm2), cyclin dependent kinase (CDK) inhibitor p21 (p21(WAF)), and CDK inhibitor p16 (p16(INK4)) were downregulated in the CEL-im cell line, whereas retinoblastoma (Rb), transcription factor E2F, member 1 (E2F-1), and alternative reading frame of p16(INK4) (ARF) were upregulated. These results are similar to genetic alterations found previously in immortal chicken embryo fibroblast (CEF) cell lines that showed efficient propagation of MDV-1. Therefore, this newly established CEL-im cell line can serve as an alternative cell substrate for the propagation of poultry viruses, such as AMPV.

Characterization of the Murine Myeloid Precursor Cell Line MuMac-E8

PubMed Central

Fricke, Stephan; Riemschneider, Sina; Kohlschmidt, Janine; Hilger, Nadja; Fueldner, Christiane; Knauer, Jens; Sack, Ulrich; Emmrich, Frank; Lehmann, Jörg

2014-01-01

Starting point for the present work was the assumption that the cell line MuMac-E8 represents a murine cell population with stem cell properties. Preliminary studies already pointed to the expression of stem-cell associated markers and a self-regenerative potential of the cells. The cell line MuMac-E8 should be examined for their differential stage within stem cell hierarchy. MuMac-E8 cells were derived from a chimeric mouse model of arthritis. It could be shown that MuMac-E8 cells express mRNA of some genes associated with pluripotent stem cells (Nanog, Nucleostemin), of genes for hematopoietic markers (EPCR, Sca-1, CD11b, CD45), for the mesenchymal marker CD105 and of genes for the neural markers Pax-6 and Ezrin. In methylcellulose and May-Grünwald-Giemsa staining, hematopoietic colonies were obtained but the hematopoietic system of lethally irradiated mice could not be rescued. Osteogenic differentiation was not detectable. Thus, it became evident that MuMac-E8 represents not a stem cell line. However, MuMac-E8 cells expressed several myeloid surface markers (i.e. CD11b, F4/80, CD14, CD64), showed phagocytosis and is capable of producing nitric oxide. Thus, this cell line seems to be arrested an advanced stage of myeloid differentiation. Adherence data measured by impedance-based real-time cell analysis together with cell morphology data suggested that MuMac-E8 represents a new macrophage precursor cell line exhibiting weak adherence. This cell line is suitable as an in-vitro model for testing of macrophage functions. Moreover, it might be also useful for differentiation or reprogramming studies. PMID:25546418

Heterogeneity of osteosarcoma cell lines led to variable responses in reprogramming.

PubMed

Choong, Pei Feng; Teh, Hui Xin; Teoh, Hoon Koon; Ong, Han Kiat; Choo, Kong Bung; Sugii, Shigeki; Cheong, Soon Keng; Kamarul, Tunku

2014-01-01

Four osteosarcoma cell lines, Saos-2, MG-63, G-292 and U-2 OS, were reprogrammed to pluripotent state using Yamanaka factors retroviral transduction method. Embryonic stem cell (ESC)-like clusters started to appear between 15 to 20 days post transduction. Morphology of the colonies resembled that of ESC colonies with defined border and tightly-packed cells. The reprogrammed sarcomas expressed alkaline phosphatase and pluripotency markers, OCT4, SSEA4, TRA-1-60 and TRA-1-81, as in ESC up to Passage 15. All reprogrammed sarcomas could form embryoid body-like spheres when cultured in suspension in a low attachment dish for up to 10 days. Further testing on the directed differentiation capacity of the reprogrammed sarcomas showed all four reprogrammed sarcoma lines could differentiate into adipocytes while reprogrammed Saos-2-REP, MG-63-REP and G-292-REP could differentiate into osteocytes. Among the 4 osteosarcoma cell lines, U-2 OS reported the highest transduction efficiency but recorded the lowest reprogramming stability under long term culture. Thus, there may be intrinsic differences governing the variable responses of osteosarcoma cell lines towards reprogramming and long term culture effect of the reprogrammed cells. This is a first report to associate intrinsic factors in different osteosarcoma cell lines with variable reprogramming responses and effects on the reprogrammed cells after prolonged culture.

Isolation of Canine parvovirus with a view to identify the prevalent serotype on the basis of partial sequence analysis.

PubMed

Kaur, Gurpreet; Chandra, Mudit; Dwivedi, P N; Sharma, N S

2015-01-01

The aim of this study was to isolate Canine parvovirus (CPV) from suspected dogs on madin darby canine kidney (MDCK) cell line and its confirmation by polymerase chain reaction (PCR) and nested PCR (NPCR). Further, VP2 gene of the CPV isolates was amplified and sequenced to determine prevailing antigenic type. A total of 60 rectal swabs were collected from dogs showing signs of gastroenteritis, processed and subjected to isolation in MDCK cell line. The samples showing cytopathic effects (CPE) were confirmed by PCR and NPCR. These samples were subjected to PCR for amplification of VP2 gene of CPV, sequenced and analyzed to study the prevailing antigenic types of CPV. Out of the 60 samples subjected to isolation in MDCK cell line five samples showed CPE in the form of rounding of cells, clumping of cells and finally detachment of the cells. When these samples and the two commercially available vaccines were subjected to PCR for amplification of VP2 gene, a 1710 bp product was amplified. The sequence analysis revealed that the vaccines belonged to the CPV-2 type and the samples were of CPV-2b type. It can be concluded from the present study that out of a total of 60 samples 5 samples exhibited CPE as observed in MDCK cell line. Sequence analysis of the VP2 gene among the samples and vaccine strains revealed that samples belonged to CPV-2b type and vaccines belonging to CPV-2.

Establishment and characterization of a fin cell line from blunt snout bream, Megalobrama amblycephala.

PubMed

Zhu, Dong-Mei; Yang, Kun; Wang, Wei-Min; Song, Wen

2013-12-01

This study established and characterized a new cell line (MAF) from the fin of blunt snout bream (Megalobrama amblycephala), a freshwater fish cultivated in China. MAF cells proliferated well in medium 199 supplemented with 10 % fetal bovine serum at 28 °C and have been subcultured more than 95 times in almost a year. MAF cells were revived at 90-95 % viability after 3-6 months of storage in liquid nitrogen. Karyotyping indicated that the modal chromosome number of MAF cells was 48. The MAF cell line consisted predominantly of fibroblastic and epithelial-like cells from M. amblycephala, which was confirmed by immunofluorescence and mitochondrial 12s rRNA sequencing. Viral susceptibility tests showed that MAF cells were susceptible to infection by snakehead rhabdovirus, spring viremia carp virus, and channel catfish virus, which was demonstrated by the presence of cytopathic effect, high viral titers, and PCR products. Bacterial cytotoxicity studies showed that extracellular products from Aeromonas hydrophila were toxic to MAF cells. Cu²⁺ was also cytotoxic to MAF cells, and the 24-h IC₅₀ value was 144.48 μmol/l. When MAF cells were transfected with pEGFP-N1 plasmid, bright fluorescent signals were observed, and the transfection efficiency reached up to 5 %. These results suggest that the MAF cell line may provide a valuable tool for studying virus pathogenesis, as well as cytotoxicity testing and genetic manipulation studies.

12-Chloracetyl-PPD, a novel dammarane derivative, shows anti-cancer activity via delay the progression of cell cycle G2/M phase and reactive oxygen species-mediate cell apoptosis.

PubMed

Wang, Xu De; Sun, Yuan Yuan; Zhao, Chen; Qu, Fan Zhi; Zhao, Yu Qing

2017-03-05

(20R)-Dammarane-3β, 12β, 20, 25-tetrol (25-OH-PPD) is a ginsenoside isolated from Panax ginseng (C. A. Meyer). This compound exhibits anti-cancer activities on many human cancer cell lines. In this study, we investigated anti-cancer mechanisms of 12β-O-( L -Chloracetyl)-dammar-20(22)-ene-3β,25-diol(12-Chloracetyl-PPD), a modified 25-OH-PPD. We found that compound 12-Chloracetyl-PPD resulted in a concentration-dependent inhibition of viability in prostate, breast, and gastric cancer cells, without affecting the viability of normal cell (human gastric epithelial cell line-GES-1, hair follicle dermal papilla cell line-HHDPC and rat myocardial cell line-H9C2). In MDA-MB-435 and C4-2B cancer cells, 12-Chloracetyl-PPD induced G2/M cell cycle arrest, down-regulated mouse double minute 2 (MDM2) expression, up-regulated p53 expression, triggered apoptosis, and stimulated reactive oxygen species production. Apoptosis can be attenuated by the reactive oxygen species scavenger N-acetylcysteine. Our results suggested that compound 12-Chloracetyl-PPD showed obvious anti-cancer activity based on delaying cell cycle arrest and inducing cell apoptosis by reactive oxygen species production, which supported development of 12-Chloracetyl-PPD as a potential agent for cancer chemotherapy. Copyright © 2017 Elsevier B.V. All rights reserved.

Generation and characteristics of human Sertoli cell line immortalized by overexpression of human telomerase

PubMed Central

Wen, Liping; Yuan, Qingqing; Sun, Min; Niu, Minghui; Wang, Hong; Fu, Hongyong; Zhou, Fan; Yao, Chencheng; Wang, Xiaobo; Li, Zheng; He, Zuping

2017-01-01

Sertoli cells are required for normal spermatogenesis and they can be reprogrammed to other types of functional cells. However, the number of primary Sertoli cells is rare and human Sertoli cell line is unavailable. In this study, we have for the first time reported a stable human Sertoli cell line, namely hS1 cells, by overexpression of human telomerase. The hS1 cells expressed a number of hallmarks for human Sertoli cells, including SOX9, WT1, GDNF, SCF, BMP4, BMP6, GATA4, and VIM, and they were negative for 3β-HSD, SMA, and VASA. Higher levels of AR and FSHR were observed in hS1 cells compared to primary human Sertoli cells. Microarray analysis showed that 70.4% of global gene profiles of hS1 cells were similar to primary human Sertoli cells. Proliferation assay demonstrated that hS1 cells proliferated rapidly and they could be passaged for more than 30 times in 6 months. Neither Y chromosome microdeletion nor tumorgenesis was detected in this cell line and 90% normal karyotypes existed in hS1 cells. Collectively, we have established the first human Sertoli cell line with phenotype of primary human Sertoli cells, an unlimited proliferation potential and high safety, which could offer sufficient human Sertoli cells for basic research as well as reproductive and regenerative medicine. PMID:28152522

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

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

PubMed

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

2015-02-01

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

Failure in activation of the canonical NF-κB pathway by human T-cell leukemia virus type 1 Tax in non-hematopoietic cell lines

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

Mizukoshi, Terumi; Komori, Hideyuki; Mizuguchi, Mariko

2013-09-01

Human T-cell leukemia virus type 1 (HTLV-1) Tax (Tax1) plays crucial roles in leukemogenesis in part through activation of NF-κB. In this study, we demonstrated that Tax1 activated an NF-κB binding (gpκB) site of the gp34/OX40 ligand gene in a cell type-dependent manner. Our examination showed that the gpκΒ site and authentic NF-κB (IgκB) site were activated by Tax1 in hematopoietic cell lines. Non-hematopoietic cell lines including hepatoma and fibroblast cell lines were not permissive to Tax1-mediated activation of the gpκB site, while the IgκB site was activated in those cells in association with binding of RelB. However RelA bindingmore » was not observed in the gpκB and IgκB sites. Our results suggest that HTLV-1 Tax1 fails to activate the canonical pathway of NF-κB in non-hematopoietic cell lines. Cell type-dependent activation of NF-κB by Tax1 could be associated with pathogenesis by HTLV-1 infection. - Highlights: • HTLV-1 Tax1 does not activate RelA of NF-κB in non-hematopoietic cell lines. • Tax1 activates the NF-κB non-canonical pathway in non-hematopoietic cell lines. • Tax1 does not induce RelA nuclear translocation in those cell lines, unlike TNFα. • The OX40L promoter κB site is activated by ectopic, but not endogenous, RelA.« less

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.

Establishment and characterization of a platinum- and paclitaxel-resistant high grade serous ovarian carcinoma cell line.

PubMed

Teng, Pang-Ning; Bateman, Nicholas W; Wang, Guisong; Litzi, Tracy; Blanton, Brian E; Hood, Brian L; Conrads, Kelly A; Ao, Wei; Oliver, Kate E; Darcy, Kathleen M; McGuire, William P; Paz, Keren; Sidransky, David; Hamilton, Chad A; Maxwell, G Larry; Conrads, Thomas P

2017-07-01

High grade serous ovarian cancer (HGSOC) patients have a high recurrence rate after surgery and adjuvant chemotherapy due to inherent or acquired drug resistance. Cell lines derived from HGSOC tumors that are resistant to chemotherapeutic agents represent useful pre-clinical models for drug discovery. Here, we describe establishment of a human ovarian carcinoma cell line, which we term WHIRC01, from a patient-derived mouse xenograft established from a chemorefractory HGSOC patient who did not respond to carboplatin and paclitaxel therapy. This newly derived cell line is platinum- and paclitaxel-resistant with cisplatin, carboplatin, and paclitaxel half-maximal lethal doses of 15, 130, and 20 µM, respectively. Molecular characterization of this cell line was performed using targeted DNA exome sequencing, transcriptomics (RNA-seq), and mass spectrometry-based proteomic analyses. Results from exomic sequencing revealed mutations in TP53 consistent with HGSOC. Transcriptomic and proteomic analyses of WHIRC01 showed high level of alpha-enolase and vimentin, which are associated with cell migration and epithelial-mesenchymal transition. WHIRC01 represents a chemorefractory human HGSOC cell line model with a comprehensive molecular profile to aid future investigations of drug resistance mechanisms and screening of chemotherapeutic agents.

Development and characterization of an embryonic cell line from endangered endemic cyprinid Honmoroko Gnathopogon caerulescens (Sauvage, 1883).

PubMed

Higaki, Shogo; Shimada, Manami; Koyama, Yoshie; Fujioka, Yasuhiro; Sakai, Noriyoshi; Takada, Tatsuyuki

2015-09-01

Establishing a cell line from endemic species facilitates the cell biological research of these species in the laboratory. In this study, an epithelium-like cell line RME1 was established from the blastula-stage embryos of the critically endangered cyprinid Honmoroko Gnathopogon caerulescens, which is endemic to ancient Lake Biwa in Japan. To the best of our knowledge, this is the first embryonic cell line from an endangered fish species. This cell line is well adapted to grow at 28°C in the culture medium, which was successfully used for establishing testicular and ovarian cell lines of G. caerulescens, and has displayed stable growth over 60 passages since its initiation in June 2011. Although RME1 did not express the genes detected in blastula-stage embryos, such as oct4, sox2, nanog, and klf4, it showed a high euploidy rate (2n = 50; 67.2%) with normal diploid karyotype morphology, suggesting that RME1 retains the genomic organization of G. caerulescens and can prove to be a useful tool to investigate the unique properties of endangered endemic fishes at cellular level.

Changes in cell wall properties coincide with overexpression of extensin fusion proteins in suspension cultured tobacco cells.

PubMed

Tan, Li; Pu, Yunqiao; Pattathil, Sivakumar; Avci, Utku; Qian, Jin; Arter, Allison; Chen, Liwei; Hahn, Michael G; Ragauskas, Arthur J; Kieliszewski, Marcia J

2014-01-01

Extensins are one subfamily of the cell wall hydroxyproline-rich glycoproteins, containing characteristic SerHyp4 glycosylation motifs and intermolecular cross-linking motifs such as the TyrXaaTyr sequence. Extensins are believed to form a cross-linked network in the plant cell wall through the tyrosine-derivatives isodityrosine, pulcherosine, and di-isodityrosine. Overexpression of three synthetic genes encoding different elastin-arabinogalactan protein-extensin hybrids in tobacco suspension cultured cells yielded novel cross-linking glycoproteins that shared features of the extensins, arabinogalactan proteins and elastin. The cell wall properties of the three transgenic cell lines were all changed, but in different ways. One transgenic cell line showed decreased cellulose crystallinity and increased wall xyloglucan content; the second transgenic cell line contained dramatically increased hydration capacity and notably increased cell wall biomass, increased di-isodityrosine, and increased protein content; the third transgenic cell line displayed wall phenotypes similar to wild type cells, except changed xyloglucan epitope extractability. These data indicate that overexpression of modified extensins may be a route to engineer plants for bioenergy and biomaterial production.

Xeroderma pigmentosum cells contain low levels of photoreactivating enzyme.

PubMed Central

Sutherland, B M; Rice, M; Wagner, E K

1975-01-01

Fibroblasts from patients with xeroderma pigmentosum contain low levels of photoreactivating enzyme in comparison to normal cells. Levels vary from 0 (line 1199) to 50 (line 1259) percent of normal. The depressed enzyme levels are not an artifact of low growth rate, age of cell donor, cell culture conditions, assay conditions, the presence of inhibitors, or mycoplasma contamination. We show that human fibroblasts can monomerize pyrimidine dimers in vivo. PMID:1054487

Effect of HSP27 on Human Breast Tumor Cell Growth and Motility

DTIC Science & Technology

1999-08-01

the small stress protein, HSP27 , on growth and motility characteristics of normal and tumor-derived human mammary cell lines. We hypothesized that...cells overexpressing HSP27 would show increased motility, altered chemotactic properties, increased resistance to heat killing and to certain drugs...Donna has prepared and studied 19 clonal MDA23 1 breast tumor cell lines that overexpress human HSP27 , and determined that, while heat resistance is

Heat shock protein 72 expression allows permissive replication of oncolytic adenovirus dl1520 (ONYX-015) in rat glioblastoma cells

PubMed Central

Madara, Jonathan; Krewet, James A; Shah, Maulik

2005-01-01

In this study we have made novel observations with regards to potentiation of the tumoricidal activity of the oncolytic adenovirus, dl1520 (ONYX-015) in rat glioblastoma cell lines expressing heat shock protein 72 (HSP72) due to permissive virus replication. ONYX-015 is a conditionally replicating adenovirus that is deleted for the E1B 55 kDA gene product whose normal function is to interact with cell-cycle regulatory proteins to permit virus replication. However, many murine and rodent cell lines are not permissive for adenovirus replication. Previously, it has been reported that the heat shock response is necessary for adenovirus replication and that induction of heat shock proteins is mediated by E1 region gene products. Therefore, we hypothesized that HSP72 expression may allow for permissive replication of ONYX-015 in previously non-permissive cells. Rat glioma cell lines 9L and RT2 were transfected with a plasmids expressing HSP72 or GFP. After infection with ONYX-015, no tumoricidal activity is observed in GFP expressing cell lines despite adequate transduction. In contrast, HSP72 transfected cells show cytopathic effects by 72 hours and greater than 75% loss of viability by 96 hours. Burst assays show active virus replication in the HSP72 expressing cell lines. Therefore, 9L-HSP72 and RT2-HSP72 are ideal models to evaluate the efficacy of ONYX-015 in an immunocompetent rat model. Our study has implications for creating rodent tumor models for pre-clinical studies with E1 region deleted conditionally replicating adenovirus. PMID:15762988

In Vitro Co-Delivery Evaluation of Novel Pegylated Nano-Liposomal Herbal Drugs of Silibinin and Glycyrrhizic Acid (Nano-Phytosome) to Hepatocellular Carcinoma Cells

PubMed Central

Ochi, Mohammad Mahdi; Amoabediny, Ghasem; Rezayat, Seyed Mahdi; Akbarzadeh, Azim; Ebrahimi, Bahman

2016-01-01

Objective This study aimed to evaluate a co-encapsulated pegylated nano-liposome system based on two herbal anti-tumor drugs, silibinin and glycyrrhizic acid, for delivery to a hepatocellular carcinoma (HCC) cell line (HepG2). Materials and Methods In this experimental study, co-encapsulated nano-liposomes by the thin layer film hydration method with HEPES buffer and sonication at 60% amplitude. Liposomes that co-encapsulated silibinin and glycyrrhizic acid were prepared with a specified molar ratio of dipalmitoylphosphatidylcholine (DPPC), cholesterol (CHOL), and methoxy-polyethylene glycol 2000 (PEG2000)–derived distearoyl phosphatidylethanolamine (mPEG2000-DSPE). We used the MTT technique to assess cytotoxicity for various concentrations of co-encapsulated nano-liposomes, free silibinin (25% w/v) and glycyrrhizic acid (75% w/v) on HepG2 and fibroblast cell lines over a 48-hour period. Results Formulation of pegylated nano-liposomes showed a narrow size distribution with an average diameter of 46.3 nm. The encapsulation efficiency (EE) for silibinin was 24.37%, whereas for glycyrrhizic acid it was 68.78%. Results of in vitro cytotoxicity showed significantly greater co-encapsulated nano-liposomes on the HepG2 cell line compared to the fibroblast cell line. The half maximal inhibitory concentration (IC50) for co-encapsulated pegylated nanoliposomal herbal drugs was 48.68 µg/ml and free silibinin with glycyrrhizic acid was 485.45 µg/ml on the HepG2 cell line. Conclusion This in vitro study showed that nano-liposome encapsulation of silibinin with glycyrrhizic acid increased the biological activity of free drugs, increased the stability of silibinin, and synergized the therapeutic effect of silibinin with glycyrrhizic acid. The IC50 of the co-encapsulated nano-liposomes was lower than the combination of free silibinin and glycyrrhizic acid on the HepG2 cell line. PMID:27540518

Cloning of transgenic tobacco BY-2 cells; an efficient method to analyse and reduce high natural heterogeneity of transgene expression.

PubMed

Nocarova, Eva; Fischer, Lukas

2009-04-22

Phenotypic characterization of transgenic cell lines, frequently used in plant biology studies, is complicated because transgene expression in individual cells is often heterogeneous and unstable. To identify the sources and to reduce this heterogeneity, we transformed tobacco (Nicotiana tabacum L.) BY-2 cells with a gene encoding green fluorescent protein (GFP) using Agrobacterium tumefaciens, and then introduced a simple cloning procedure to generate cell lines derived from the individual transformed cells. Expression of the transgene was monitored by analysing GFP fluorescence in the cloned lines and also in lines obtained directly after transformation. The majority ( approximately 90%) of suspension culture lines derived from calli that were obtained directly from transformation consisted of cells with various levels of GFP fluorescence. In contrast, nearly 50% of lines generated by cloning cells from the primary heterogeneous suspensions consisted of cells with homogenous GFP fluorescence. The rest of the lines exhibited "permanent heterogeneity" that could not be resolved by cloning. The extent of fluorescence heterogeneity often varied, even among genetically identical clones derived from the primary transformed lines. In contrast, the offspring of subsequent cloning of the cloned lines was uniform, showing GFP fluorescence intensity and heterogeneity that corresponded to the original clone. The results demonstrate that, besides genetic heterogeneity detected in some lines, the primary lines often contained a mixture of epigenetically different cells that could be separated by cloning. This indicates that a single integration event frequently results in various heritable expression patterns, which are probably accidental and become stabilized in the offspring of the primary transformed cells early after the integration event. Because heterogeneity in transgene expression has proven to be a serious problem, it is highly advisable to use transgenes tagged with a visual marker for BY-2 transformation. The cloning procedure can be used not only for efficient reduction of expression heterogeneity of such transgenes, but also as a useful tool for studies of transgene expression and other purposes.

Biomedical applications of SPION@APTES@PEG-folic acid@carboxylated quercetin nanodrug on various cancer cells

NASA Astrophysics Data System (ADS)

Akal, Z. Ü.; Alpsoy, L.; Baykal, A.

2016-08-01

In this study, carboxylated quercetin (CQ) was conjugated to superparamagnetic iron oxide nanoparticles (SPIONs) which were modified by (3-aminopropyl) triethoxysilane (APTES), Folic acid (FA) and carboxylated Polyethylene glycol (PEG); (SPION@APTES@FA-PEG@CQ), nanodrug has been synthesized via polyol and accompanying by various chemical synthesis routes. The characterization of the final product was done via X-ray powder diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), Thermal gravimetric analysis (TGA), Transmission electron spectroscopy (TEM) and Vibrating sample magnetometer (VSM). Its cytotoxic and apoptotic activities on over expressed folic acid receptor (FR +) (MCF-7, HeLa) and none expressed folic acid receptor (FR-) (A549) cancer cell lines were determined by using MTT assay, Real-Time Cell Analysis, TUNEL assay, Annexin assay and RT-PCR analysis for Caspase3/7 respectively. SPION@APTES@FA-PEG@CQ nanodrug showed higher cytotoxicity against HeLa and MCF-7 cell lines as compared with A549 cell line. Moreover, SPION@APTES@FA-PEG@CQ nanodrug also caused higher apoptotic and necrotic effects in 100 μg/mL HeLa and MCF-7 cells than A549 cells. The findings showed that SPION@APTES@FA-PEG@CQ nanodrug has cytotoxic, apoptotic and necrotic effects on HeLa and MCF-7 which are FR over expressed cell lines and can be potentially used for the delivery of quercetin to cervical and breast cancer cells.

Cytotoxic outcomes of orthodontic bands with and without silver solder in different cell lineages.

PubMed

Jacoby, Letícia Spinelli; Rodrigues Junior, Valnês da Silva; Campos, Maria Martha; Macedo de Menezes, Luciane

2017-05-01

The safety of orthodontic materials is a matter of high interest. In this study, we aimed to assess the in-vitro cytotoxicity of orthodontic band extracts, with and without silver solder, by comparing the viability outcomes of the HaCat keratinocytes, the fibroblastic cell lineages HGF and MRC-5, and the kidney epithelial Vero cells. Sterilized orthodontic bands with and without silver solder joints were added to culture media (6 cm 2 /mL) and incubated for 24 hours at 37°C under continuous agitation. Subsequently, the cell cultures were exposed to the obtained extracts for 24 hours, and an assay was performed to evaluate the cell viability. Copper strip extracts were used as positive control devices. The extracts from orthodontic bands with silver solder joints significantly reduced the viability of the HaCat, MRC-5, and Vero cell lines, whereas the viability of HGF was not altered by this material. Conversely, the extracts of orthodontic bands without silver solder did not significantly modify the viability index of all evaluated cell lines. Except for HGF fibroblasts, all tested cell lines showed decreased viability percentages after exposure to extracts of orthodontic bands containing silver solder joints. These data show the relevance of testing the toxicity of orthodontic devices in different cell lines. Copyright © 2017 American Association of Orthodontists. Published by Elsevier Inc. All rights reserved.

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.

Dual effects of N-acetyl-L-cysteine dependent on NQO1 activity: Suppressive or promotive of 9,10-phenanthrenequinone-induced toxicity

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

Toyooka, Tatsushi; Shinmen, Takuya; Aarts, Jac M.M.J.G.

2012-11-01

A typical antioxidant, N-acetyl-L-cysteine (NAC) generally protects cells from oxidative damage induced by reactive oxygen species (ROS). 9,10-Phenanthrenequinone (9,10-PQ), a major quinone in diesel exhaust particles, produces ROS in redox cycling following two-electron reduction by NAD(P)H:quinone oxidoreductase 1 (NQO1), which has been considered as a cause of its cyto- and genotoxicity. In this study, we show that NAC unexpectedly augments the toxicity of 9,10-PQ in cells with low NQO1 activity. In four human skin cell lines, the expression and the activity of NQO1 were lower than in human adenocarcinoma cell lines, A549 and MCF7. In the skin cells, the cytotoxicitymore » of 9,10-PQ was significantly enhanced by addition of NAC. The formation of DNA double strand breaks accompanying phosphorylation of histone H2AX, was also remarkably augmented. On the other hand, the cyto- and genotoxicity were suppressed by addition of NAC in the adenocarcinoma cells. Two contrasting experiments: overexpression of NQO1 in CHO-K1 cells which originally expressed low NQO1 levels, and knock‐down of NQO1 in the adenocarcinoma cell line A549 by transfection of RNAi, also showed that NAC suppressed 9,10-PQ-induced toxicity in cell lines expressing high NQO1 activity and enhanced it in cell lines with low NQO1 activity. The results suggested that dual effects of NAC on the cyto- and genotoxicity of 9,10-PQ were dependent on tissue-specific NQO1 activity. -- Highlights: ► NAC augmented the cytotoxicity of 9,10-PQ in skin cell lines. ► 9,10-PQ-induced DSBs accompanying γ-H2AX were also augmented by NAC. ► NAC suppressed the cyto- and genotoxicity of 9,10-PQ in adenocarcinoma cell lines. ► The dual effects of NAC on toxicity of 9,10-PQ were dependent on NQO1 activity.« less

B7-1 (CD80) as target for immunotoxin therapy for Hodgkin's disease.

PubMed Central

Vooijs, W. C.; Otten, H. G.; van Vliet, M.; van Dijk, A. J.; de Weger, R. A.; de Boer, M.; Bohlen, H.; Bolognesi, A.; Polito, L.; de Gast, G. C.

1997-01-01

In this preclinical study, the potential applicability of an anti-B7-1 immunotoxin (IT) for the treatment of Hodgkin's disease (HD) was investigated. Immunohistochemical analysis demonstrated strong expression of B7-1 on Hodgkin and Reed-Sternberg (R-S) cells and clear expression on dendritic cells, macrophages and some B-cells in tissues, but not on other tissue cells. Flow cytometric analysis demonstrated that B7-1 was expressed on a few monocytes, but not on CD34+ cells from bone marrow, resting T- or B-cells from peripheral blood or epithelial and endothelial cell lines. An anti-B7-1 immunotoxin containing the anti-B7-1 monoclonal antibody (MAb) B7-24 and saporin as toxin moiety was constructed and showed an affinity similar to that shown by the native MAb. It exhibited strong cytotoxicity against the B7-1+ B-cell line Raji (IC50 10(-11) M), R-S cell lines HDLM2, KM/H2 and L428 and also against a B7-1-transfected epithelial cell line, A431, whose parental line lacks expression of B7-1. In clonogenic assays with Raji cells or KM/H2 cells, a 3- or 4-log kill, respectively, was observed. No cytotoxicity was found against the B7-1- epithelial and endothelial cell lines or against haematopoietic progenitor cells. In conclusion, an anti-B7-1 immunotoxin was developed that had good cytotoxicity against R-S cell lines and that may be used in the elimination of R-S cells in vivo. A concomitant elimination of activated antigen-presenting cells may avoid development of antitoxin and anti-mouse Ig responses and allow repeated administration. Images Figure 1 PMID:9365164

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

PubMed

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

2003-01-03

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

Epigenetic inactivation of VGF associated with Urothelial Cell Carcinoma and its potential as a non-invasive biomarker using urine

PubMed Central

Kagohara, Luciane Tsukamoto; Maldonado, Leonel; Brait, Mariana; Schoenberg, Mark; Bivalacqua, Trinity; Netto, George J; Koch, Wayne; Sidransky, David; Hoque, Mohammad O.

2014-01-01

Background: To identify new epigenetic markers and further characterize Urothelial Cell Carcinoma (UCC), we tested the promoter methylation (PM) status of 19 genes previously identified as cancer specific methylated genes in other solid tumors. Methods: We used bisulfite sequencing, methylation specific PCR and quantitative methylation specific PCR (QMSP) to test the PM status of 19 genes in urothelial cancer cell lines. Results: Among the 19 genes tested, VGF was found to be completely methylated in several UCC cell lines. VGF QMSP analysis showed that methylation values of almost all the primary 19 UCC tissues were higher than the paired normal tissues (P=0.009). In another cohort, 12/35 (34.3%) of low grade UCC cases displayed VGF methylation. As a biomarker for non-invasive detection of UCC, VGF showed a significantly higher frequency of methylation in urine from UCC cases (8/20) compared to controls (1/20) (P=0.020). After treatment of cell lines with 5-Aza-2'-deoxycytidine, VGF was robustly re-expressed. Forced expression of VGF in bladder cancer cell lines inhibited cell growth. Conclusion: Selection of candidates from genome-wide screening approach in other solid tumors successfully identified UCC specific methylated genes. PMID:24830820

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.

Leptin reverts pro-apoptotic and antiproliferative effects of α-linolenic acids in BCR-ABL positive leukemic cells: involvement of PI3K pathway.

PubMed

Beaulieu, Aurore; Poncin, Géraldine; Belaid-Choucair, Zakia; Humblet, Chantal; Bogdanovic, Gordana; Lognay, Georges; Boniver, Jacques; Defresne, Marie-Paule

2011-01-01

It is suspected that bone marrow (BM) microenvironmental factors may influence the evolution of chronic myeloid leukaemia (CML). In this study, we postulated that adipocytes and lipids could be involved in the progression of CML. To test this hypothesis, adipocytes were co-cultured with two BCR-ABL positive cell lines (PCMDS and K562). T cell (Jurkat) and stroma cell (HS-5) lines were used as controls. In the second set of experiments, leukemic cell lines were treated with stearic, oleic, linoleic or α-linolenic acids in presence or absence of leptin. Survival, proliferation, leptin production, OB-R isoforms (OB-Ra and OB-Rb), phosphoinositide 3-kinase (PI3k) and BCL-2 expression have been tested after 24h, 48h and 72h of treatment. Our results showed that adipocytes induced a decrease of CML proliferation and an increase in lipid accumulation in leukemic cells. In addition, CML cell lines induced adipocytes cell death. Chromatography analysis showed that BM microenvironment cells were full of saturated (SFA) and monounsaturated (MUFA) fatty acids, fatty acids that protect tumor cells against external agents. Stearic acid increased Bcl-2 expression in PCMDS, whereas oleic and linoleic acids had no effects. In contrast, α-linolenic acid decreased the proliferation and the survival of CML cell lines as well as BCL-2 and OB-R expression. The effect of α-linolenic acids seemed to be due to PI3K pathway and Bcl-2 inhibition. Leptin production was detected in the co-culture medium. In the presence of leptin, the effect of α-linolenic acid on proliferation, survival, OB-R and BCl-2 expression was reduced.

Cytoarchitecture of Zika virus infection in human neuroblastoma and Aedes albopictus cell lines.

PubMed

Offerdahl, Danielle K; Dorward, David W; Hansen, Bryan T; Bloom, Marshall E

2017-01-15

The Zika virus (ZIKV) pandemic is a global concern due to its role in the development of congenital anomalies of the central nervous system. This mosquito-borne flavivirus alternates between mammalian and mosquito hosts, but information about the biogenesis of ZIKV is limited. Using a human neuroblastoma cell line (SK-N-SH) and an Aedes albopictus mosquito cell line (C6/36), we characterized ZIKV infection by immunofluorescence, transmission electron microscopy (TEM), and electron tomography (ET) to better understand infection in these disparate host cells. ZIKV replicated well in both cell lines, but infected SK-N-SH cells suffered a lytic crisis. Flaviviruses scavenge host cell membranes to serve as replication platforms and ZIKV showed the hallmarks of this process. Via TEM, we identified virus particles and 60-100nm spherular vesicles. ET revealed these vesicular replication compartments contain smaller 20-30nm spherular structures. Our studies indicate that SK-N-SH and C6/36 cells are relevant models for viral cytoarchitecture study. Published by Elsevier Inc.

Integrative genomic and functional analysis of human oral squamous cell carcinoma cell lines reveals synergistic effects of FAT1 and CASP8 inactivation.

PubMed

Hayes, Tyler F; Benaich, Nathan; Goldie, Stephen J; Sipilä, Kalle; Ames-Draycott, Ashley; Cai, Wenjun; Yin, Guangliang; Watt, Fiona M

2016-12-01

Oral squamous cell carcinoma (OSCC) is genetically highly heterogeneous, which contributes to the challenges of treatment. To create an in vitro model that accurately reflects this heterogeneity, we generated a panel of HPV-negative OSCC cell lines. By whole exome sequencing of the lines and matched patient blood samples, we demonstrate that the mutational spectrum of the lines is representative of primary OSCC in The Cancer Genome Atlas. We show that loss of function mutations in FAT1 (an atypical cadherin) and CASP8 (Caspase 8) frequently occur in the same tumour. OSCC cells with inactivating FAT1 mutations exhibited reduced intercellular adhesion. Knockdown of FAT1 and CASP8 individually or in combination in OSCC cells led to increased cell migration and clonal growth, resistance to Staurosporine-induced apoptosis and, in some cases, increased terminal differentiation. The OSCC lines thus represent a valuable resource for elucidating the impact of different mutations on tumour behaviour. Copyright © 2016 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

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

PubMed

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

2009-02-01

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

In vitro evaluation of (99m)Tc-EDDA/tricine-HYNIC-Q-Litorin in gastrin-releasing peptide receptor positive tumor cell lines.

PubMed

Yurt Lambrecht, Fatma; Durkan, Kübra; Ozgür, Aykut; Gündüz, Cumhur; Avcı, Cığır Biray; Susluer, Sunde Yılmaz

2013-05-01

Bombesin and its derivatives exhibit a high affinity for gastrin-releasing peptide receptor (GRPr), which is over-expressed in a variety of human cancers (prostate, pancreatic, lung, etc.). The aim of this study was to investigate the in vitro potential of the hydrazinonicotinamide (HYNIC)-Q-Litorin. (99m)Tc labeling was performed by using different co-ligands: tricine and ethylenediamine diacetic acid (EDDA). The radiochemical stability of radiolabeled peptide conjugates was checked at room temperature and in cysteine solution up to 24 h. The in vitro cell uptake of (99m)Tc-EDDA-HYNIC-Q-Litorin and (99m)Tc-tricine-HYNIC-Q-Litorin were evaluated on pancreatic tumor and control cell lines. Optimum specific activity and incubation time were determined for all the cell lines. The results showed that the cell uptake of the radiolabeled peptide conjugates in tumor cell lines were higher than in the control cell line. The findings of this study indicated the need for further development of in vivo study as a radiopharmaceutical for pancreatic tumor imaging.

Human T-cell leukemia virus type 1 Tax oncoprotein represses the expression of the BCL11B tumor suppressor in T-cells.

PubMed

Takachi, Takayuki; Takahashi, Masahiko; Takahashi-Yoshita, Manami; Higuchi, Masaya; Obata, Miki; Mishima, Yukio; Okuda, Shujiro; Tanaka, Yuetsu; Matsuoka, Masao; Saitoh, Akihiko; Green, Patrick L; Fujii, Masahiro

2015-04-01

Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T cell leukemia (ATL), which is an aggressive form of T-cell malignancy. HTLV-1 oncoproteins, Tax and HBZ, play crucial roles in the immortalization of T-cells and/or leukemogenesis by dysregulating the cellular functions in the host. Recent studies show that HTLV-1-infected T-cells have reduced expression of the BCL11B tumor suppressor protein. In the present study, we explored whether Tax and/or HBZ play a role in downregulating BCL11B in HTLV-1-infected T-cells. Lentiviral transduction of Tax in a human T-cell line repressed the expression of BCL11B at both the protein and mRNA levels, whereas the transduction of HBZ had little effect on the expression. Tax mutants with a decreased activity for the NF-κB, CREB or PDZ protein pathways still showed a reduced expression of the BCL11B protein, thereby implicating a different function of Tax in BCL11B downregulation. In addition, the HTLV-2 Tax2 protein reduced the BCL11B protein expression in T-cells. Seven HTLV-1-infected T-cell lines, including three ATL-derived cell lines, showed reduced BCL11B mRNA and protein expression relative to an uninfected T-cell line, and the greatest reductions were in the cells expressing Tax. Collectively, these results indicate that Tax is responsible for suppressing BCL11B protein expression in HTLV-1-infected T-cells; Tax-mediated repression of BCL11B is another mechanism that Tax uses to promote oncogenesis of HTLV-1-infected T-cells. © 2015 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.

Induction of Apoptosis of 2,4′,6-Trihydroxybenzophenone in HT-29 Colon Carcinoma Cell Line

PubMed Central

Lay, Ma Ma; Karsani, Saiful Anuar

2014-01-01

2,4′,6-Trihydroxy-4-methoxybenzophenone was isolated from the ethyl acetate fraction of Phaleria macrocarpa (Scheff.) Boerl. fruits. It was found to inhibit cell proliferation in HT-29 human colon carcinoma cell line but caused little damage to WRL-68 normal human liver and MRC-5 normal human fibroblast lung cell lines. The compound was found to sharply affect the viability of HT-29 cells in a dose- and time-dependent manner. HT-29 cells treated with the compound showed morphological changes under microscopic examination such as cell shrinkage, membrane blebbing, DNA fragmentation, and the occurrence of apoptotic nuclei. The percentage of early apoptotic, late apoptotic, and dead or necrotic cells was determined by flow cytometry using annexin V-FTIC/PI staining. In addition, flow cytometry showed that, when the HT-29 cells were treated with 115 µM of the compound, it resulted in G0/G1 phase arrest in a time-dependent manner. Western blot revealed an upregulation of PUMA, Bak, Bcl-2, and Mcl-1 proteins suggesting that the compound induced apoptosis in HT-29 cells by regulating these proteins. PMID:24579081

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

PubMed Central

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

2013-01-01

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

Overexpressing Ferredoxins in Chlamydomonas reinhardtii Increase Starch and Oil Yields and Enhance Electric Power Production in a Photo Microbial Fuel Cell

PubMed Central

Huang, Li-Fen; Lin, Ji-Yu; Pan, Kui-You; Huang, Chun-Kai; Chu, Ying-Kai

2015-01-01

Ferredoxins (FDX) are final electron carrier proteins in the plant photosynthetic pathway, and function as major electron donors in diverse redox-driven metabolic pathways. We previously showed that overexpression of a major constitutively expressed ferredoxin gene PETF in Chlamydomonas decreased the reactive oxygen species (ROS) level and enhanced tolerance to heat stress. In addition to PETF, an endogenous anaerobic induced FDX5 was overexpressed in transgenic Chlamydomonas lines here to address the possible functions of FDX5. All the independent FDX transgenic lines showed decreased cellular ROS levels and enhanced tolerance to heat and salt stresses. The transgenic Chlamydomonas lines accumulated more starch than the wild-type line and this effect increased almost three-fold in conditions of nitrogen depletion. Furthermore, the lipid content was higher in the transgenic lines than in the wild-type line, both with and without nitrogen depletion. Two FDX-overexpressing Chlamydomonas lines were assessed in a photo microbial fuel cell (PMFC); power density production by the transgenic lines was higher than that of the wild-type cells. These findings suggest that overexpression of either PETF or FDX5 can confer tolerance against heat and salt stresses, increase starch and oil production, and raise electric power density in a PMFC. PMID:26287179

Inhibin/activin-betaC and -betaE subunits in the Ishikawa human endometrial adenocarcinoma cell line.

PubMed

Kimmich, Tanja; Brüning, Ansgar; Käufl, Stephanie D; Makovitzky, Josef; Kuhn, Christina; Jeschke, Udo; Friese, Klaus; Mylonas, Ioannis

2010-08-01

Inhibins and activins are important regulators of the female reproductive system. Recently, two novel inhibin subunits, named betaC and betaE, have been identified and shown to be expressed in several human tissues. However, only limited data on the expression of these novel inhibin subunits in normal human endometrial tissue and endometrial adenocarcinoma cell lines exist. Samples of proliferative and secretory human endometrium were obtained from five premenopausal, non-pregnant patients undergoing gynecological surgery for benign diseases. Normal endometrial tissue and Ishikawa endometrial adenocarcinoma cell lines were analyzed by immunohistochemistry, immunofluorescence and RT-PCR. Expression of the inhibin betaC and betaE subunits could be demonstrated at the protein level by means of immunohistochemical evaluation and at the transcriptional level by establishing a betaC- and betaE-specific RT-PCR analysis in normal human endometrial tissue and the parental Ishikawa cell line. Interestingly, in a highly de-differentiated subclone of the Ishikawa cell line lacking estrogen receptor expression, the expression of the inhibin-betaC subunit appeared strongly reduced. Here, we show for the first time that the novel inhibin/activin-betaC and -betaE subunits are expressed in normal human endometrium and the estrogen receptor positive human endometrial carcinoma cell line Ishikawa using RT-PCR and immunohistochemical detection methods. Interestingly, the Ishikawa minus cell line (lacking estrogen receptor expression) demonstrated no to minimal expression of the betaC subunit as observed with immunofluorescence and RT-PCR, suggesting a possible hormone- dependency of this subunit in human endometrial cancer cells. Moreover, because the Ishikawa cell line minus is thought to be a more malignant endometrial cell line than its estrogen receptor positive counterpart, inhibin-betaC subunit might be substantially involved in the pathogenesis and malignant transformation in human endometrium.

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.

Establishment and characterization of immortalized gingival epithelial and fibroblastic cell lines for the development of organotypic cultures.

PubMed

Bao, Kai; Akguel, Baki; Bostanci, Nagihan

2014-01-01

In vitro studies using 3D co-cultures of gingival cells can resemble their in vivo counterparts much better than 2D models that typically only utilize monolayer cultures with short-living primary cells. However, the use of 3D gingival models is still limited through lack of appropriate cell lines. We aimed to establish immortalized cell line models of primary human gingival epithelium keratinocytes (HGEK) and gingival fibroblasts (GFB). Immortalized cell lines (HGEK-16 and GFB-16) were induced by E6 and E7 oncoproteins of human papillomavirus. In addition, 3D multilayered organotypic cultures were formed by embedding GFB-16 cells within a collagen (Col) matrix and seeding of HGEK-16 cells on the upper surfaces. Cell growth was analyzed in both immortalized cell lines and their parental primary cells. The expression levels of cell type-specific markers, i.e. cytokeratin (CK) 10, CK13, CK16, CK18, CK19 for HGEK-16 and Col I and Col II for GFB-16, were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). Expansion of the primary cultures was impeded at early passages, while the transformed immortalized cell lines could be expanded for more than 30 passages. In 3D cultures, immortalized HGEK formed a multilayer of epithelial cells. qRT-PCR showed that cell-specific marker expression in the 3D cultures was qualitatively and quantitatively closer to that in human gingival tissue than to monolayer cultures. These results indicate that immortalized gingival fibroblastic and epithelial cell lines can successfully form organotypic multilayered cultures and, therefore, may be useful tools for studying gingival tissue in vitro. © 2014 S. Karger AG, Basel.

A p53-dependent response limits the viability of mammalian haploid cells

PubMed Central

Olbrich, Teresa; Mayor-Ruiz, Cristina; Vega-Sendino, Maria; Gomez, Carmen; Ortega, Sagrario; Ruiz, Sergio; Fernandez-Capetillo, Oscar

2017-01-01

The recent development of haploid cell lines has facilitated forward genetic screenings in mammalian cells. These lines include near-haploid human cell lines isolated from a patient with chronic myelogenous leukemia (KBM7 and HAP1), as well as haploid embryonic stem cells derived from several organisms. In all cases, haploidy was shown to be an unstable state, so that cultures of mammalian haploid cells rapidly become enriched in diploids. Here we show that the observed diploidization is due to a proliferative disadvantage of haploid cells compared with diploid cells. Accordingly, single-cell–sorted haploid mammalian cells maintain the haploid state for prolonged periods, owing to the absence of competing diploids. Although the duration of interphase is similar in haploid and diploid cells, haploid cells spend longer in mitosis, indicative of problems in chromosome segregation. In agreement with this, a substantial proportion of the haploids die at or shortly after the last mitosis through activation of a p53-dependent cytotoxic response. Finally, we show that p53 deletion stabilizes haploidy in human HAP1 cells and haploid mouse embryonic stem cells. We propose that, similar to aneuploidy or tetraploidy, haploidy triggers a p53-dependent response that limits the fitness of mammalian cells. PMID:28808015

Discovery of a drug targeting microenvironmental support for lymphoma cells by screening using patient-derived xenograft cells

PubMed Central

Sugimoto, Keiki; Hayakawa, Fumihiko; Shimada, Satoko; Morishita, Takanobu; Shimada, Kazuyuki; Katakai, Tomoya; Tomita, Akihiro; Kiyoi, Hitoshi; Naoe, Tomoki

2015-01-01

Cell lines have been used for drug discovery as useful models of cancers; however, they do not recapitulate cancers faithfully, especially in the points of rapid growth rate and microenvironment independency. Consequently, the majority of conventional anti-cancer drugs are less sensitive to slow growing cells and do not target microenvironmental support, although most primary cancer cells grow slower than cell lines and depend on microenvironmental support. Here, we developed a novel high throughput drug screening system using patient-derived xenograft (PDX) cells of lymphoma that maintained primary cancer cell phenotype more than cell lines. The library containing 2613 known pharmacologically active substance and off-patent drugs were screened by this system. We could find many compounds showing higher cytotoxicity than conventional anti-tumor drugs. Especially, pyruvinium pamoate showed the highest activity and its strong anti-tumor effect was confirmed also in vivo. We extensively investigated its mechanism of action and found that it inhibited glutathione supply from stromal cells to lymphoma cells, implying the importance of the stromal protection from oxidative stress for lymphoma cell survival and a new therapeutic strategy for lymphoma. Our system introduces a primary cancer cell phenotype into cell-based phenotype screening and sheds new light on anti-cancer drug development. PMID:26278963

Urtica dioica inhibits cell growth and induces apoptosis by targeting Ornithine decarboxylase and Adenosine deaminase as key regulatory enzymes in adenosine and polyamines homeostasis in human breast cancer cell lines.

PubMed

Fattahi, Sadegh; Ghadami, Elham; Asouri, Mohsen; Motevalizadeh Ardekanid, Ali; Akhavan-Niaki, Haleh

2018-02-28

Breast cancer is a heterogeneous and multifactorial disease with variable disease progression risk, and treatment response. Urtica dioica is a traditional herb used as an adjuvant therapeutic agent in cancer. In the present study, we have evaluated the effects of the aqueous extract of Urtica dioica on Adenosine deaminase (ADA) and Ornithine decarboxylase (ODC1) gene expression in MCF-7, MDA-MB-231, two breast cancer cell lines being estrogen receptor positive and estrogen receptor negative, respectively.  Cell lines were cultured in suitable media. After 24 h, different concentrations of the extract were added and after 72 h, ADA and ODC1 gene expression as well as BCL2 and BAX apoptotic genes were assessed by Taqman real time PCR assay. Cells viability was assessed by MTT assay, and apoptosis was also evaluated at cellular level. The intra and extracellular levels of ODC1 and ADA enzymes were evaluated by ELISA. Results showed differential expression of ADA and ODC1 genes in cancer cell lines. In MCF-7 cell line, the expression level of ADA was upregulated in a dose-dependent manner but its expression did not change in MDA-MB cell line. ODC1 expression was increased in both examined cell lines. Also, increased level of the apoptotic BAX/BCL-2 ratio was detected in MCF-7 cells. These results demonstrated that Urtica dioica induces apoptosis in breast cancer cells by influencing ODC1 and ADA genes expression, and estrogen receptors. The different responses observed with these cell lines could be due to the interaction of Urtica dioica as a phytoestrogen with the estrogen receptor.

Similarities and differences between helminth parasites and cancer cell lines in shaping human monocytes: Insights into parallel mechanisms of immune evasion.

PubMed

Narasimhan, Prakash Babu; Akabas, Leor; Tariq, Sameha; Huda, Naureen; Bennuru, Sasisekhar; Sabzevari, Helen; Hofmeister, Robert; Nutman, Thomas B; Tolouei Semnani, Roshanak

2018-04-01

A number of features at the host-parasite interface are reminiscent of those that are also observed at the host-tumor interface. Both cancer cells and parasites establish a tissue microenvironment that allows for immune evasion and may reflect functional alterations of various innate cells. Here, we investigated how the phenotype and function of human monocytes is altered by exposure to cancer cell lines and if these functional and phenotypic alterations parallel those induced by exposure to helminth parasites. Thus, human monocytes were exposed to three different cancer cell lines (breast, ovarian, or glioblastoma) or to live microfilariae (mf) of Brugia malayi-a causative agent of lymphatic filariasis. After 2 days of co-culture, monocytes exposed to cancer cell lines showed markedly upregulated expression of M1-associated (TNF-α, IL-1β), M2-associated (CCL13, CD206), Mreg-associated (IL-10, TGF-β), and angiogenesis associated (MMP9, VEGF) genes. Similar to cancer cell lines, but less dramatically, mf altered the mRNA expression of IL-1β, CCL13, TGM2 and MMP9. When surface expression of the inhibitory ligands PDL1 and PDL2 was assessed, monocytes exposed to both cancer cell lines and to live mf significantly upregulated PDL1 and PDL2 expression. In contrast to exposure to mf, exposure to cancer cell lines increased the phagocytic ability of monocytes and reduced their ability to induce T cell proliferation and to expand Granzyme A+ CD8+ T cells. Our data suggest that despite the fact that helminth parasites and cancer cell lines are extraordinarily disparate, they share the ability to alter the phenotype of human monocytes.

The molecular basis of cytotoxicity of α-spinasterol from Ganoderma resinaceum: Induction of apoptosis and overexpression of p53 in breast and ovarian cancer cell lines.

PubMed

Sedky, Nada K; El Gammal, Zaynab H; Wahba, Amir E; Mosad, Eman; Waly, Zahraa Y; El-Fallal, Amira Ali; Arafa, Reem K; El-Badri, Nagwa

2018-05-01

Despite advances in therapy of breast and ovarian cancers, they still remain among the most imperative causes of cancer death in women. The first can be considered one of the most widespread diseases among females, while the latter is more lethal and needs prompt treatment. Thus, the research field can still benefit from discovery of new compounds that can be of potential use in management of these grave illnesses. We hereby aimed to assess the antitumor activity of the phytosterol α-spinasterol isolated from Ganoderma resinaceum mushroom on human breast cancer cell lines (MCF-7, MDA-MB-231), as well as, on human ovarian cancer cell line (SKOV-3). The anti-tumor activity of α-spinasterol, isolated from the mycelial extract of the Egyptian G. resinaceum, on human breast and ovarian cancer cell lines was evaluated by MTT cell viability assay and AnnexinV/propidium iodide apoptosis assay. The molecular mechanism underlying this effect was assessed by the relative expression of the following markers; tumor suppressor (p53, BRCA1, BRCA2), apoptotic marker (Bax) and cell cycle progression markers (cyclin dependent kinases cdk4/6) using real-time PCR. Cell cycle analysis was performed for the three investigated cancer cell lines to explore the effect on cell cycle progression. Our findings showed that α-spinasterol exhibited a higher antitumor activity on MCF-7 cells relative to SKOV-3 cells, while its lowest antitumor activity was against MDA-MB-231 cells. A significant increase in the expression of p53 and Bax was observed in cells treated with α-spinasterol, while cdk4/6 were significantly down-regulated upon exposure to α-spinasterol. Cell cycle analysis of α-spinasterol treated cells showed a G 0 -G 1 arrest. In conclusion, α-spinasterol isolated from G. resinaceum mushroom exerts a potent inhibitory activity on breast and ovarian cancer cell lines in a time- and dose-dependent manner. This can be reasonified in lights of the compound's ability to increase p53 and Bax expressions, and to lower the expression of cdk4/6. © 2017 Wiley Periodicals, Inc.

Re-evaluation of tumor-specific cytotoxicity of mitomycin C, bleomycin and peplomycin.

PubMed

Sasaki, Masahiro; Okamura, Masahiko; Ideo, Atsushi; Shimada, Jun; Suzuki, Fumika; Ishihara, Mariko; Kikuchi, Hirotaka; Kanda, Yumiko; Kunii, Shiro; Sakagami, Hiroshi

2006-01-01

Three antitumor antibiotics, mitomycin C, bleomycin sulfate and peplomycin sulfate, were compared for their tumor-specific cytotoxicity, using human oral squamous cell lines (HSC-2, HSC-3, HSC-4, Ca9-22 and NA), human promyelocytic leukemic cell line HL-60 and human normal oral cell types (gingival fibroblast HGF, pulp cell HPC and periodontal ligament fibroblast HPLF). Among these three compounds, mitomycin C showed the highest tumor-specificity, due to its higher cytotoxic activity against human oral tumor cell lines than bleomycin and peplomycin. However, there was considerable variation of drug sensitivity among the six tumor cell lines. Mitomycin C induced internucleosomal DNA fragmentation and caspase-3, -8 and -9 activation in HL-60 cells only after 24 h. On the other hand, mitomycin C induced no clear-cut DNA fragmentation in HCS-2 cells, although it activated caspase-3, -8 and -9 to a slightly higher extent. Western blot analysis demonstrated that mitomycin C did not induce any apparent change in the intracellular concentration of anti-apoptotic protein (Bcl-2) and pro-apoptotic proteins (Bax, Bad). Electron microscopy of mitomycin C-treated HL-60 cells showed intact mitochondria (as regards to integrity and size) and cell surface microvilli, without production of an apoptotic body or autophagosome, at an early stage after treatment. The present study suggests the incomplete induction of apoptosis or the induction of another type of cell death by mitomycin C treatment.

Xylose-rich polysaccharides from the primary walls of embryogenic cell line of Pinus caribaea.

PubMed

Mollard, A; Domon, J M; David, H; Joseleau, J P

1997-08-01

Embryogenic cell lines of Pinus caribaea were isolated from somatic embryogenesis from zygotic embryos. Previous studies showed that the proteins and glycoproteins were characteristic of the embryogenic state. In the present work we were seeking typical feature in the polysaccharide from the cell walls of embryogenic calli at nine days of culture. Sequential extraction with water, ammonium oxalate, dimethyl sulfoxide, sodium borohydride and 4.3 M potassium hydroxide revealed that the extracted polysaccharides contained high proportions of arabinose and significant amounts of xylose. Fractionation of the hydrosoluble polymers on DEAE cellulose afforded a xylose-rich fraction (80% xylose, 24% glucose and lower properties of fucose and mannose). Methylation analysis and 13C-NMR spectra showed that the glycan backbone consisted of beta 1 --> 4 linked xylosyl residues Similar study of the fractions extracted respectively with DMSO and 4.3 M KOH showed the presence of polydisperse glycoxylans but excluded the presence of xyloglucan in significant amount. This could be a characteristic feature of embryogenic cells walls of Pinus caribaea or could be typical of cells grown as calluses. In the various fractions obtained from DEAE cellulose chromatography of the alkaline extract the infrequent occurrence of fucoxylans beside an arabinogalactan showed again the unusual nature of the cell wall polymers of this embryogenic lines, which seems to differ greatly from those found in the primary wall of cells from suspension cultures.

The human-induced pluripotent stem cell initiative-data resources for cellular genetics.

PubMed

Streeter, Ian; Harrison, Peter W; Faulconbridge, Adam; Flicek, Paul; Parkinson, Helen; Clarke, Laura

2017-01-04

The Human Induced Pluripotent Stem Cell Initiative (HipSci) isf establishing a large catalogue of human iPSC lines, arguably the most well characterized collection to date. The HipSci portal enables researchers to choose the right cell line for their experiment, and makes HipSci's rich catalogue of assay data easy to discover and reuse. Each cell line has genomic, transcriptomic, proteomic and cellular phenotyping data. Data are deposited in the appropriate EMBL-EBI archives, including the European Nucleotide Archive (ENA), European Genome-phenome Archive (EGA), ArrayExpress and PRoteomics IDEntifications (PRIDE) databases. The project will make 500 cell lines from healthy individuals, and from 150 patients with rare genetic diseases; these will be available through the European Collection of Authenticated Cell Cultures (ECACC). As of August 2016, 238 cell lines are available for purchase. Project data is presented through the HipSci data portal (http://www.hipsci.org/lines) and is downloadable from the associated FTP site (ftp://ftp.hipsci.ebi.ac.uk/vol1/ftp). The data portal presents a summary matrix of the HipSci cell lines, showing available data types. Each line has its own page containing descriptive metadata, quality information, and links to archived assay data. Analysis results are also available in a Track Hub, allowing visualization in the context of public genomic annotations (http://www.hipsci.org/data/trackhubs). © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Mycoplasma pneumonia of swine (MPS) resistance and immune characteristics of pig lines generated by crossing an MPS pulmonary lesion selected Landrace line and a highly immune capacity selected Large White line.

PubMed

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

2016-08-01

To understand the influence of crossbreeding on Mycoplasma pneumonia of swine (MPS) resistance and immune characteristics, two crossbred lines were characterized. One crossbred line, LaWa, was generated by crossing the MPS pulmonary lesion selected Landrace line (La) and the highly immune-selected Large White line (Wa). The second crossbred line, LaWb, was generated by crossing the La line and the nonselected Large White line (Wb). The crossbred LbWb line (nonselected Landrace line × nonselected Large White line) and the La line were used as controls. The LaWa and LaWb lines had an intermediate level of MPS lung lesions between La and LbWb lines, although the difference was not statistically significant. After stimulation with sheep red blood cells (SRBCs), the LaWb and LaWa lines showed immune characteristics similar to that of the La line; the number of monocytes in peripheral blood increased, while B cells, T cells, secretion of SRBC-specific immunoglobulin G, and interleukin (IL)-13 decreased. Additionally, the number of natural killer (NK) cells and the expression of IL-4 and IL-17 were significantly higher in the LaWb and LaWa lines, respectively. These data suggested that crossbreeding of La and Wa lines resulted in the inheritance of some of the selected immune responses. © 2015 Japanese Society of Animal Science.

Low concentrations of alendronate increase the local invasive potential of osteoblastic sarcoma cell lines via connexin 43 activation.

PubMed

Yoshitani, Kazuhiro; Kido, Akira; Honoki, Kanya; Akahane, Manabu; Fujii, Hiromasa; Tanaka, Yasuhito

2011-07-15

Bisphosphonates (BPs) are agents used for treating disorders of excessive bone resorption. In addition, due to their cell-killing activity, BPs were potent candidates for adjuvant cancer therapy. On the other hand, low-concentrations of BPs have been reported to increase cellular viability in several types of tumor cells. Therefore, we focused on the effect of BPs on cellular aggressiveness of malignant bone tumors at low concentrations. MTS assay was performed using osteosarcoma cell lines MG63 and HOS, fibrosarcoma cell line HT1080, and prostate cancer cell line PC3. All the cell lines showed toxicity at high concentrations. On the other hand, at lower concentrations, the cellular viabilities of HOS and MG63 were rather higher than those of untreated controls. Since this tendency was most evident, HOS was used for further assays, including cellular motility, bone resorption activity, and cathepsin K activity. The low-concentration of alendronate enhanced cellular viability and motility, which correlated with the expression of connexin 43 at the mRNA and protein levels. Interestingly, oleamide, a potent connexin 43 inhibitor, had an inhibitory effect on the enhanced proliferation. Our data suggest that alendronate may enhance the proliferation of osteoblastic cell line through connexin 43 activation. Copyright © 2011 Elsevier GmbH. All rights reserved.

Significance of CD133 positive cells in four novel HPV-16 positive cervical cancer-derived cell lines and biopsies of invasive cervical cancer.

PubMed

Javed, Shifa; Sharma, Bal Krishan; Sood, Swati; Sharma, Sanjeev; Bagga, Rashmi; Bhattacharyya, Shalmoli; Rayat, Charan Singh; Dhaliwal, Lakhbir; Srinivasan, Radhika

2018-04-02

Cervical cancer is a major cause of cancer-related mortality in women in the developing world. Cancer Stem cells (CSC) have been implicated in treatment resistance and metastases development; hence understanding their significance is important. Primary culture from tissue biopsies of invasive cervical cancer and serial passaging was performed for establishing cell lines. Variable Number Tandem Repeat (VNTR) assay was performed for comparison of cell lines with their parental tissue. Tumorsphere and Aldefluor assays enabled isolation of cancer stem cells (CSC); immunofluorescence and flow cytometry were performed for their surface phenotypic expression in cell lines and in 28 tissue samples. Quantitative real-time PCR for stemness and epithelial-mesenchymal transition (EMT) markers, MTT cytotoxicity assay, cell cycle analysis and cell kinetic studies were performed. Four low-passage novel cell lines designated RSBS-9, - 14 and - 23 from squamous cell carcinoma and RSBS-43 from adenocarcinoma of the uterine cervix were established. All were HPV16+. VNTR assay confirmed their uniqueness and derivation from respective parental tissue. CSC isolated from these cell lines showed CD133 + phenotype. In tissue samples of untreated invasive cervical cancer, CD133 + CSCs ranged from 1.3-23% of the total population which increased 2.8-fold in radiation-resistant cases. Comparison of CD133 + with CD133 - bulk population cells revealed increased tumorsphere formation and upregulation of stemness and epithelial-mesenchymal transition (EMT) markers with no significant difference in cisplatin sensitivity. Low-passage cell lines developed would serve as models for studying tumor biology. Cancer Stem Cells in cervical cancer display CD133 + phenotype and are increased in relapsed cases and hence should be targeted for achieving remission.

Oncolytic Activity of Avian Influenza Virus in Human Pancreatic Ductal Adenocarcinoma Cell Lines

PubMed Central

Pizzuto, Matteo S.; Silic-Benussi, Micol; Pavone, Silvia; Ciminale, Vincenzo; Capua, Ilaria

2014-01-01

ABSTRACT Pancreatic ductal adenocarcinoma (PDA) is the most lethal form of human cancer, with dismal survival rates due to late-stage diagnoses and a lack of efficacious therapies. Building on the observation that avian influenza A viruses (IAVs) have a tropism for the pancreas in vivo, the present study was aimed at testing the efficacy of IAVs as oncolytic agents for killing human PDA cell lines. Receptor characterization confirmed that human PDA cell lines express the alpha-2,3- and the alpha-2,6-linked glycan receptor for avian and human IAVs, respectively. PDA cell lines were sensitive to infection by human and avian IAV isolates, which is consistent with this finding. Growth kinetic experiments showed preferential virus replication in PDA cells over that in a nontransformed pancreatic ductal cell line. Finally, at early time points posttreatment, infection with IAVs caused higher levels of apoptosis in PDA cells than gemcitabine and cisplatin, which are the cornerstone of current therapies for PDA. In the BxPC-3 PDA cell line, apoptosis resulted from the engagement of the intrinsic mitochondrial pathway. Importantly, IAVs did not induce apoptosis in nontransformed pancreatic ductal HPDE6 cells. Using a model based on the growth of a PDA cell line as a xenograft in SCID mice, we also show that a slightly pathogenic avian IAV significantly inhibited tumor growth following intratumoral injection. Taken together, these results are the first to suggest that IAVs may hold promise as future agents of oncolytic virotherapy against pancreatic ductal adenocarcinomas. IMPORTANCE Despite intensive studies aimed at designing new therapeutic approaches, PDA still retains the most dismal prognosis among human cancers. In the present study, we provide the first evidence indicating that avian IAVs of low pathogenicity display a tropism for human PDA cells, resulting in viral RNA replication and a potent induction of apoptosis in vitro and antitumor effects in vivo. These results suggest that slightly pathogenic IAVs may prove to be effective for oncolytic virotherapy of PDA and provide grounds for further studies to develop specific and targeted viruses, with the aim of testing their efficacy in clinical contexts. PMID:24899201

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.

Anticancer Effects of Extracts from the Fruit of Morinda Citrifolia (Noni) in Breast Cancer Cell Lines.

PubMed

Sharma, K; Pachauri, S D; Khandelwal, K; Ahmad, H; Arya, A; Biala, P; Agrawal, S; Pandey, R R; Srivastava, A; Srivastav, A; Saxena, J K; Dwivedi, A K

2016-03-01

Morinda citrifolia L. (NONI) fruits have been used for thousands of years for the treatment of many health problems including cancer, cold, diabetes, flu, hypertension, and pain. Plant extracts have reported several therapeutic benefits, but extraction of individual compound from the extract often exhibits limited clinical utility as the synergistic effect of various natural ingredients gets lost. They generally constitute polyphenols and flavonoids. Studies have suggested that these phytochemicals, especially polyphenols, display high antioxidant properties, which help to reduce the risk of degenerative diseases, such as cancer and cardiovascular diseases. Several in-vitro and in-vivo studies have shown that Noni fruits have antioxidant, anti-inflammatory, anti-dementia, liver-protective, anticancer, analgesic, and immunomodulatory effects. Till date about 7 in vitro cancer studies have been done, but a detailed in vitro study including cell cycle and caspase activation assay on breast cancer cell line has not been done. In the present study different Noni fruit fractions have tested on cancer cell lines MCF-7, MDA-MB-231 (breast adenocarcinoma) and one non-cancer cell line HEK-293 (Human embryonic kidney). Out of which ethylacetate extract showed a higher order of in vitro anticancer activity profile. The ethylacetate extract strongly inhibited the proliferation of MCF-7, MDA-MB-231 and HEK-293 cell lines with IC50 values of 25, 35, 60 µg/ml respectively. The extract showed increase in apoptotic cells in MCF-7 and MDA-MB-231 cells and arrested the cell cycle in the G1/S phase in MCF-7 and G0/G1 phase in MDA-MB-231 cells. Noni extract also decreases the intracellular ROS generation and mitochondrial membrane potential. © Georg Thieme Verlag KG Stuttgart · New York.

Human Brain Microvascular Endothelial Cells Derived from the BC1 iPS Cell Line Exhibit a Blood-Brain Barrier Phenotype.

PubMed

Katt, Moriah E; Xu, Zinnia S; Gerecht, Sharon; Searson, Peter C

2016-01-01

The endothelial cells that form capillaries in the brain are highly specialized, with tight junctions that minimize paracellular transport and an array of broad-spectrum efflux pumps that make drug delivery to the brain extremely challenging. One of the major limitations in blood-brain barrier research and the development of drugs to treat central nervous system diseases is the lack of appropriate cell lines. Recent reports indicate that the derivation of human brain microvascular endothelial cells (hBMECs) from human induced pluripotent stem cells (iPSCs) may provide a solution to this problem. Here we demonstrate the derivation of hBMECs extended to two new human iPSC lines: BC1 and GFP-labeled BC1. These hBMECs highly express adherens and tight junction proteins VE-cadherin, ZO-1, occludin, and claudin-5. The addition of retinoic acid upregulates VE-cadherin expression, and results in a significant increase in transendothelial electrical resistance to physiological values. The permeabilities of tacrine, rhodamine 123, and Lucifer yellow are similar to values obtained for MDCK cells. The efflux ratio for rhodamine 123 across hBMECs is in the range 2-4 indicating polarization of efflux transporters. Using the rod assay to assess cell organization in small vessels and capillaries, we show that hBMECs resist elongation with decreasing diameter but show progressive axial alignment. The derivation of hBMECs with a blood-brain barrier phenotype from the BC1 cell line highlights that the protocol is robust. The expression of GFP in hBMECs derived from the BC1-GFP cell line provides an important new resource for BBB research.

Cytotoxic consequences of Halloysite nanotube/iron oxide nanocomposite and iron oxide nanoparticles upon interaction with bacterial, non-cancerous and cancerous cells.

PubMed

Abhinayaa, R; Jeevitha, G; Mangalaraj, D; Ponpandian, N; Vidhya, Kalieswaran; Angayarkanni, Jayaraman

2018-05-19

Cytotoxic effects of iron oxide (Fe 3 O 4 ) nanoparticles and Halloysite nanotube/iron oxide (HNT/Fe 3 O 4 ) nanocomposite are compared based on their interaction with Gram-negative bacteria Escherichia coli and Gram-positive bacteria Bacillus subtilis. Similarly, the action of these two nanomaterials on non-cancerous Vero cell lines and human lung cancerous (A-549) cell lines are compared. The cytotoxicity studies on Fe 3 O 4 nanoparticles and HNT/Fe 3 O 4 nanocomposite showed difference in the rate of killing of bacterial cells. This is reflected in differential cell growth, cell membrane integrity loss, lactate dehydrogenase (LDH) release and reactive oxygen species (ROS) production. These factors are measured over a range of concentrations of Fe 3 O 4 nanoparticles and HNT/Fe 3 O 4 nanocomposite and at specified time intervals, to test if there is any statistically significant difference between the toxicity of the two nanomaterials. Between the two nanomaterials, HNT/Fe 3 O 4 nanocomposite is found to be less toxic to bacterial cells than Fe 3 O 4 nanoparticles. HNT, when attached to the Fe 3 O 4 nanoparticles, changes their surface characteristics and suppresses their inherent toxicity on bacteria. In the study on the effect on cell lines, Fe 3 O 4 nanoparticles and HNT/Fe 3 O 4 nanocomposite are both seen to be biocompatible with Vero cell lines. However, HNT/Fe 3 O 4 nanocomposite showed more cytotoxicity than Fe 3 O 4 nanoparticles on A-549 cell lines. Copyright © 2018 Elsevier B.V. All rights reserved.

In vivo and in vitro invasion in relation to phenotypic characteristics of human colorectal carcinoma cells.

PubMed Central

de Vries, J. E.; Dinjens, W. N.; De Bruyne, G. K.; Verspaget, H. W.; van der Linden, E. P.; de Bruïne, A. P.; Mareel, M. M.; Bosman, F. T.; ten Kate, J.

1995-01-01

In this study we investigated the tumorigenicity, growth pattern and spontaneous metastatic ability of a series of nine human colorectal carcinoma cell lines after subcutaneous and intracaecal xenografting in nude mice. CaCo2 cells were found to be poorly tumorigenic to non-tumorigenic in either site; the other cell lines were tumorigenic in both sites. SW1116, SW480 and SW620 did not show local invasive in the NCI-H716 and LS174T cells were both invasive in the caecum, but only NCI-H716 was invasive in the subcutis. HT29 and 5583 (S and E) cells were invasive in the caecum and from that site metastatic to the lungs and/or the liver. HT29 and 5583S cells were both invasive in the subcutis, but 5583E cells were not. Of each category of in vivo behaviour in the caecum, one cell line was further investigated with regard to invasion in vitro (into embryonic chick heart fragments), E-cadherin expression in vivo and in vitro and in vitro production of u-PA and t-PA. These parameters were chosen in view of their purported role in extracellular matrix degradation and intercellular adhesion, which are all involved in the invasive and metastatic cascade. Invasion in vitro was not predictive for invasion or metastasis in vivo. In the cell line which showed invasion in embryonic chick heart tissue, heterogeneous E-cadherin expression was observed in vitro together with a relatively high production of u-PA. The non-invasive cell lines showed in vitro homogeneous expression of E-cadherin with a relatively low production of u-PA. In vivo expression of E-cadherin was either absent or heterogeneous. We conclude that: (1) colorectal carcinoma xenografts show site-specific modification of in vivo invasive and metastatic behaviour; (2) invasion in vitro does not correlate with invasion and metastasis in vivo; (3) in vitro non-invasion might be associated with homogeneous E-cadherin expression and low production of u-PA; (4) E-cadherin expression in vitro differs from E-cadherin expression in vivo. The results support the notion that the microenvironment in which cancer cells grow is one of the factors involved in the regulation of invasive and metastatic behaviour. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:7841040

The first total synthesis and biological evaluation of marine natural products ma'edamines A and B.

PubMed

Saha, Sanjay; Venkata Ramana Reddy, Ch; Chiranjeevi, T; Addepally, Uma; Chinta Rao, T S; Patro, Balaram

2013-02-15

We have developed the first total syntheses of marine natural products ma'edamines A (18) and B (20). Structurally, they contain a pyrazine-2-(1H)-one core and were screened for antiproliferative activity on several cancer cell lines. Out of the six cell lines tested, ma'edamines A and B showed significant cytotoxicity against human colon cancer line COLO 205 (IC(50) 7.9 and 10.3 μM, respectively), breast cancer cell line MCF-7 (IC(50): 6.9 and 10.5 μM, respectively) and human lung adenocarcinoma cell line A549 (IC(50): 12.2 and 15.4 μM, respectively). The apoptotic effect of ma'edamines was confirmed by comet assay. Hence ma'edamines are likely to be useful as leads for development of a new class of anti-cancer agents. Copyright © 2012 Elsevier Ltd. All rights reserved.

Non-Small Cell Lung Cancer Cells Expressing CD44 Are Enriched for Stem Cell-Like Properties

PubMed Central

Leung, Elaine Lai-Han; Fiscus, Ronald R.; Tung, James W.; Tin, Vicky Pui-Chi; Cheng, Lik Cheung; Sihoe, Alan Dart-Loon; Fink, Louis M.; Ma, Yupo; Wong, Maria Pik

2010-01-01

Background The cancer stem cell theory hypothesizes that cancers are perpetuated by cancer stem cells (CSC) or tumor initiating cells (TIC) possessing self-renewal and other stem cell-like properties while differentiated non-stem/initiating cells have a finite life span. To investigate whether the hypothesis is applicable to lung cancer, identification of lung CSC and demonstration of these capacities is essential. Methodology/Principal Finding The expression profiles of five stem cell markers (CD34, CD44, CD133, BMI1 and OCT4) were screened by flow cytometry in 10 lung cancer cell lines. CD44 was further investigated by testing for in vitro and in vivo tumorigenecity. Formation of spheroid bodies and in vivo tumor initiation ability were demonstrated in CD44+ cells of 4 cell lines. Serial in vivo tumor transplantability in nude mice was demonstrated using H1299 cell line. The primary xenografts initiated from CD44+ cells consisted of mixed CD44+ and CD44− cells in similar ratio as the parental H1299 cell line, supporting in vivo differentiation. Semi-quantitative Real-Time PCR (RT-PCR) showed that both freshly sorted CD44+ and CD44+ cells derived from CD44+-initiated tumors expressed the pluripotency genes OCT4/POU5F1, NANOG, SOX2. These stemness markers were not expressed by CD44− cells. Furthermore, freshly sorted CD44+ cells were more resistant to cisplatin treatment with lower apoptosis levels than CD44− cells. Immunohistochemical analysis of 141 resected non-small cell lung cancers showed tumor cell expression of CD44 in 50.4% of tumors while no CD34, and CD133 expression was observed in tumor cells. CD44 expression was associated with squamous cell carcinoma but unexpectedly, a longer survival was observed in CD44-expressing adenocarcinomas. Conclusion/Significance Overall, our results demonstrated that stem cell-like properties are enriched in CD44-expressing subpopulations of some lung cancer cell lines. Further investigation is required to clarify the role of CD44 in tumor cell renewal and cancer propagation in the in vivo environment. PMID:21124918

New isopimarane diterpenes and nortriterpene with cytotoxic activity from Ephorbia alatavica Boiss.

PubMed

Rozimamat, Rushangul; Hu, Rui; Aisa, Haji Akber

2018-06-01

Three new isopimarane diterpenes and one new nor-triterpenes, along with five known diterpenes were isolated from the whole areal part of Ephorbia alatavica Boiss. The structures of the new compounds (1-4) were determined based on extensive spectroscopic analysis, including HR-ESIMS, 1D and 2D NMR data. A plausible biosynthetic pathway for new compounds (1-4) were hypothesized. All isolated compounds were screen for cytotoxicity activity against MCF-8, HeLa and A549 cell lines in vitro by MTT assay. New compound 1 and known 9 showed potential cytotoxic activities with IC 50 values of 15.327 μg/mL, 23.066 μg/mL against MCF-8 cell lines, compound1 showed noteworthy cytotoxic activity with IC 50 13.033 μg/mL against A549 cancer cell line. New compounds 2, 4 and 4 showed moderate cytotoxic activities three human cancer lines with IC 50 value around 50 μg/mL, which compared with positive control doxorubicin (DOX). Copyright © 2018 Elsevier B.V. All rights reserved.

HDAC gene expression in pancreatic tumor cell lines following treatment with the HDAC inhibitors panobinostat (LBH589) and trichostatine (TSA).

PubMed

Mehdi, Ouaïssi; Françoise, Silvy; Sofia, Costa Lima; Urs, Giger; Kevin, Zemmour; Bernard, Sastre; Igor, Sielezneff; Anabela, Cordeiro-da-Silva; Dominique, Lombardo; Eric, Mas; Ali, Ouaïssi

2012-01-01

In this study, the effect of LBH589 and trichostatin (TSA), a standard histone deacetylase inhibitor (HDACi) toward the growth of pancreatic cancer cell lines was studied. Thus, we examined for the first time, the HDAC family gene expression levels before and after drug treatment. Several human pancreatic cancer cell lines (Panc-1, BxPC-3, SOJ-6) and a normal human pancreatic duct immortalized epithelial cell line (HPDE/E6E7) were used as target cells. The cell growth was measured by MTT assay, cell cycle alteration, membrane phosphatidylserine exposure, DNA fragmentation, mitochondrial membrane potential loss, RT-PCR and Western blots were done using standard methods. The effect of drugs on tumor growth in vivo was studied using subcutaneous xenograft model. Except in the case of certain HDAC gene/tumor cell line couples: (SIRT1/HPDE-SOJ6/TSA- or LBH589-treated cells; LBH589-treated Panc-1 Cells; HDAC2/BxPC-3/LBH589-treated cells or TSA-treated SOJ-6-1 cells), there were no major significant changes of HDACs genes transcription in cells upon drug treatment. However, significant variation in HDACs and SIRTs protein expression levels could be seen among individual cell samples. The in vivo results showed that LBH589 formulation exhibited similar tumor reduction efficacy as the commercial drug gemcitabine. Our data demonstrate that LBH589 induced the death of pancreatic tumor cell by apoptosis. In line with its in vitro activity, LBH589 achieved a significant reduction in tumor growth in BxPC-3 pancreatic tumor cell line subcutaneous xenograft mouse model. Furthermore, exploring the impact of LBH589 on HDACs encoding genes expression revealed for the first time that some of them, depending on the cell line considered, seem to be regulated during translation. Copyright © 2012 IAP and EPC. Published by Elsevier B.V. All rights reserved.

Cytotoxic effects in 3T3-L1 mouse and WI-38 human fibroblasts following 72 hour and 7 day exposures to commercial silica nanoparticles

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

Stępnik, Maciej, E-mail: mstep@imp.lodz.pl; Arkusz, Joanna; Smok-Pieniążek, Anna

The potential toxic effects in murine (3T3-L1) and human (WI-38) fibroblast cell lines of commercially available silica nanoparticles (NPs), Ludox CL (nominal size 21 nm) and CL-X (nominal size of 30 nm) were investigated with particular attention to the effect over long exposure times (the tests were run after 72 h exposure up to 7 days). These two formulations differed in physico-chemical properties and showed different stabilities in the cell culture medium used for the experiments. Ludox CL silica NPs were found to be cytotoxic only at the higher concentrations to the WI-38 cells (WST-1 and LDH assays) but notmore » to the 3T3-L1 cells, whereas the Ludox CL-X silica NPs, which were less stable over the 72 h exposure, were cytotoxic to both cell lines in both assays. In the clonogenic assay both silica NPs induced a concentration dependent decrease in the surviving fraction of 3T3-L1 cells, with the Ludox CL-X silica NPs being more cytotoxic. Cell cycle analysis showed a trend indicating alterations in both cell lines at different phases with both silica NPs tested. Buthionine sulfoximine (γ-glutamylcysteine synthetase inhibitor) combined with Ludox CL-X was found to induce a strong decrease in 3T3-L1 cell viability which was not observed for the WI-38 cell line. This study clearly indicates that longer exposure studies may give important insights on the impact of nanomaterials on cells. However, and especially when investigating nanoparticle effects after such long exposure, it is fundamental to include a detailed physico-chemical characterization of the nanoparticles and their dispersions over the time scale of the experiment, in order to be able to interpret eventual impacts on cells. -- Highlights: ► Ludox CL silica NPs are cytotoxic to WI-38 fibroblasts but not to 3T3-L1 fibroblasts. ► Ludox CL-X silica NPs are cytotoxic to both cell lines. ► In clonogenic assay both silica NPs induce cytotoxicity, higher for CL-X silica. ► Cell cycle analysis shows alterations in both cell lines with both silica NP tested. ► Buthionine sulfoximine enhances cytotoxicity of Ludox CL-X in 3T3-L1 cells.« less

An AKT activity threshold regulates androgen-dependent and androgen-independent PSA expression in prostate cancer cell lines.

PubMed

Paliouras, Miltiadis; Diamandis, Eleftherios P

2008-06-01

The androgen receptor (AR) plays an important role in early prostate cancer by activating transcription of a number of genes participating in cell proliferation and growth and cancer progression. However, as the cancer progresses, prostate cancer cells transform from an androgen-dependent to an androgen-independent state. Androgen-independent prostate cancer can manifest itself in several forms, including a percentage of cancers that show reduced levels of prostate-specific antigen (PSA) and can progress without the need for the ligand or active receptor. Therefore, our goal was to examine the role of intracellular signaling pathways in an androgen-independent prostate cancer in vitro model. Using the cell line PC3(AR)(2), we stimulated cells with 5-alpha-dihydrotestosterone (DHT) and epidermal growth factor (EGF) and then analyzed PSA expression. We observed lower PSA expression when cells were jointly stimulated with DHT and EGF, and this was associated with an increase in AKT activity. We examined the role of AKT in AR activity and PSA expression by creating stable PC3(AR)(2) cell lines transfected with a PI3K-Ras-effector loop mutant. These cell lines showed lower DHT-stimulated PSA expression that correlated to changes in the phosphorylated state of AR. Therefore, we propose an in vitro androgen-independent model in which a PI3K/AKT activity threshold and subsequent AR transactivation regulate PSA expression.

Effect of UVC light on growth, incorporation of thymidine, and DNA chain elongation in cells derived from the Indian meal moth and the cabbage looper.

PubMed

Styer, S C; Griffiths, T D

1992-04-01

After exposure to 10 or 20 J/m2 UVC light, cells of the UMN-PIE-1181 line, an embryonic cell line derived from the Indian meal moth, Plodia interpunctella, exhibited a rapid and prolonged depression in the rate of incorporation of [3H]thymidine, whereas cells of the TN-368 line, an ovarian cell line derived from Trichoplusia ni, the cabbage looper, showed only a slight drop in incorporation and a rapid recovery after exposure to 10 or 40 J/m2 UVC light. The extent of this depression was not correlated to the amount of cell killing by UVC light in these cell lines or in IAL-PID2 cells. Blockage of fork progression was correlated to the depression in thymidine incorporation. TN-368 cells exhibited little blockage after exposure to 10 or 20 J/m2 UVC light, whereas UMN-PIE-1181 cells exhibited significant blockage at these fluences. Photoreactivation did not entirely relieve blockage, depression in thymidine incorporation, or cell killing, indicating that, although the (5-6) dimer appears to be the major lesion responsible for these effects, other lesions such as the (6-4) photoproduct may play a role.

Impedance Analysis of Ovarian Cancer Cells upon Challenge with C-terminal Clostridium Perfringens Enterotoxin

NASA Astrophysics Data System (ADS)

Gordon, Geoffrey; Lo, Chun-Min

2007-03-01

Both in vitro and animal studies in breast, prostate, and ovarian cancers have shown that clostridium perfringens enterotoxin (CPE), which binds to CLDN4, may have an important therapeutic benefit, as it is rapidly cytotoxic in tissues overexpressing CLDN4. This study sought to evaluate the ability of C-terminal clostridium perfringens enterotoxin (C-CPE), a CLDN4-targetting molecule, to disrupt tight junction barrier function. Electric cell-substrate impedance sensing (ECIS) was used to measure both junctional resistance and average cell-substrate separation of ovarian cancer cell lines after exposure to C-CPE. A total of 14 ovarian cancer cell lines were used, and included cell lines derived from serous, mucinous, and clear cells. Our results showed that junctional resistance increases as CLDN4 expression increases. In addition, C-CPE is non-cytotoxic in ovarian cancer cells expressing CLDN4. However, exposure to C-CPE results in a significant (p<0.05) dose- and CLDN4-dependent decrease in junctional resistance and an increase in cell-substrate separation. Treatment of ovarian cancer cell lines with C-CPE disrupts tight junction barrier function.

Anticancer activity of Sargassum oligocystum water extract against human cancer cell lines.

PubMed

Zandi, K; Ahmadzadeh, S; Tajbakhsh, S; Rastian, Z; Yousefi, F; Farshadpour, F; Sartavi, K

2010-08-01

Antitumor drug resistance and side effects of antitumor compounds are the most common problems in medicine. Therefore, finding new antitumor agents with low side effects could be interesting. This study was designed to assay antitumor activity of the extract from brown alga Sargassum oligocystum, gathered from Persian Gulf seashore, against K562 and Daudi human cancer cell lines. The research was performed as an in vitro study. The effect of the alga extract on proliferation of cell lines were measured by two methods: MTT assay and trypan blue exclusion test. The most effective antitumor activity has been shown at concentrations 500 microg/ml and 400 microg/ml of the alga extract against Daudi and K562 cell lines, respectively. The results showed that the extracts of brown alga Sargassum oligocystum have remarkable antitumor activity against K562 and Daudi cell lines. It is justified to be suggested for further research such as algal extract fractionation and purification and in vivo studies in order to formulate natural compounds with antitumor activities.

Enhanced electrochemical sensing of leukemia cells using drug/lipid co-immobilized on the conducting polymer layer.

PubMed

Gurudatt, N G; Naveen, M Halappa; Ban, Changill; Shim, Yoon-Bo

2016-12-15

Electrochemical biosensors using five anticancer drug and lipid molecules attached on the conducting polymer layer to obtain the orientation of drug molecules toward cancer cells, were evaluated as sensing materials and their performances were compared. Conjugation of the drug molecules with a lipid, phosphatidylcholine (PC) has enhanced the sensitivity towards leukemia cells and differentiates cancer cells from normal cells. The composition of each layer of sensor probe was confirmed by electrochemical and surface characterization experiments. Both impedance spectroscopy and voltammetry show the enhanced interaction of leukemia cells using the drug/lipid modified sensor probe. As the number of leukemia cells increased, the charge transfer resistance (Rct) in impedance spectra increased and the amine oxidation peak current of drug molecules in voltammograms decreased at around 0.7-1.0V. Of test drug molecules, raltitrexed (Rtx) showed the best performance for the cancer cells detection. Cancer and normal cell lines from different origins were examined to evaluate the degree of expression of folate receptors (FR) on cells surface, where cervical HeLa cell line was found to be shown the highest expression of the receptor. Impedance and chronoamperometric experiments for leukemia cell line (Jurkat E6-1) showed linear dynamic ranges of 1.0×10(3)-2.5×10(5) cells/mL and 1.0×10(3)-8.0×10(3) cells/mL with detection limits of 68±5 cells/mL and 21±3 cells/mL, respectively. Copyright © 2016 Elsevier B.V. All rights reserved.

Apoptotic effects on cultured cells of atmospheric-pressure plasma produced using various gases

NASA Astrophysics Data System (ADS)

Tominami, Kanako; Kanetaka, Hiroyasu; Kudo, Tada-aki; Sasaki, Shota; Kaneko, Toshiro

2016-01-01

This study investigated the effects of low-temperature atmospheric-pressure plasma on various cells such as rat fibroblastic Rat-1 cell line, rat neuroblastoma-like PC12 cell line, and rat macrophage-like NR8383 cell line. The plasma was irradiated directly to a culture medium containing plated cells for 0-20 s. The applied voltage, excitation frequency, and argon or helium gas flow were, respectively, 3-6 kV, 10 kHz, and 3 L/min. Cell viability and apoptotic activity were evaluated using annexin-V/propidium iodide staining. Results showed that the low-temperature atmospheric-pressure plasma irradiation promoted cell death in a discharge-voltage-dependent and irradiation-time-dependent manner. Furthermore, different effects are produced depending on the cell type. Moreover, entirely different mechanisms might be responsible for the induction of apoptosis in cells by helium and argon plasma.

Enterovirus Species B Bias of RD Cell Line and Its Influence on Enterovirus Diversity Landscape.

PubMed

Faleye, Temitope Oluwasegun Cephas; Adeniji, Johnson Adekunle

2015-12-01

Despite its widespread use in poliovirus isolation, studies show that most RD cell line isolates are species B enteroviruses (EB), it was therefore employed to further catalogue the EB diversity in two different regions of Nigeria. Concentrates of 18 environmental samples were inoculated into RD cell line. Isolates were subjected to PCR assays to detect enteroviruses, species C and B members and partial VP1 gene which was subsequently sequenced and used for identification and phylogenetic analysis. Isolates were further passaged in L20B cell line to detect polioviruses. Sixty-eight isolates were recovered from the 18 concentrates, all of which were positive for the enterovirus 5'-UTR screen. Thirteen of the 68 isolates were positive for the species C screen and replicated in L20B cell line, eleven of which also contained species B enteroviruses. Some of the mixed isolates were successfully typed, but as species B members. In all, isolates recovered in this study were identified as CVB5, E6, E7, E11, E13, E19, E20, E33, EVB75 and WPV3, while some could not be typed. Alongside the ten different enterovirus serotypes confirmed, results of this study document for the first time in Nigeria, EVB75. It showed the EB bias of RD cell line might indicate something much more fundamental in its biology. Finally, the finding of WPV3 in a region considered low risk for poliovirus emphasizes the need to expand poliovirus environmental surveillance to enable early detection of poliovirus silent circulation before occurrence of clinical manifestations.

Alpha-2 Heremans Schmid Glycoprotein (AHSG) Modulates Signaling Pathways in Head and Neck Squamous Cell Carcinoma Cell Line SQ20B

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

Thompson, Pamela D.; Sakwe, Amos; Koumangoye, Rainelli

2014-02-15

This study was performed to identify the potential role of Alpha-2 Heremans Schmid Glycoprotein (AHSG) in Head and Neck Squamous Cell Carcinoma (HNSCC) tumorigenesis using an HNSCC cell line model. HNSCC cell lines are unique among cancer cell lines, in that they produce endogenous AHSG and do not rely, solely, on AHSG derived from serum. To produce our model, we performed a stable transfection to down-regulate AHSG in the HNSCC cell line SQ20B, resulting in three SQ20B sublines, AH50 with 50% AHSG production, AH20 with 20% AHSG production and EV which is the empty vector control expressing wild-type levels ofmore » AHSG. Utilizing these sublines, we examined the effect of AHSG depletion on cellular adhesion, proliferation, migration and invasion in a serum-free environment. We demonstrated that sublines EV and AH50 adhered to plastic and laminin significantly faster than the AH20 cell line, supporting the previously reported role of exogenous AHSG in cell adhesion. As for proliferative potential, EV had the greatest amount of proliferation with AH50 proliferation significantly diminished. AH20 cells did not proliferate at all. Depletion of AHSG also diminished cellular migration and invasion. TGF-β was examined to determine whether levels of the TGF-β binding AHSG influenced the effect of TGF-β on cell signaling and proliferation. Whereas higher levels of AHSG blunted TGF-β influenced SMAD and ERK signaling, it did not clearly affect proliferation, suggesting that AHSG influences on adhesion, proliferation, invasion and migration are primarily due to its role in adhesion and cell spreading. The previously reported role of AHSG in potentiating metastasis via protecting MMP-9 from autolysis was also supported in this cell line based model system of endogenous AHSG production in HNSCC. Together, these data show that endogenously produced AHSG in an HNSCC cell line, promotes in vitro cellular properties identified as having a role in tumorigenesis. Highlights: • Head and neck squamous cell carcinoma cell lines synthesize and secret AHSG. • AHSG depleted cell lines are significantly inhibited in their ability to proliferate, adhere, migrate, invade and protect MMP-9. • Human AHSG and bovine fetuin-A are functionally equivalent in regards to growth promotion of cancer cell lines.« less

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.

Performance characteristics of qualified cell lines for isolation and propagation of influenza viruses for vaccine manufacturing

PubMed Central

Donis, Ruben O.; Chen, i-Mei; Davis, C Todd; Foust, Angie; Hossain, M. Jaber; Johnson, Adam; Klimov, Alexander; Loughlin, Rosette; Xu, Xiyan; Tsai, Theodore; Blayer, Simone; Trusheim, Heidi; Colegate, Tony; Fox, John; Taylor, Beverly; Hussain, Althaf; Barr, Ian; Baas, Chantal; Louwerens, Jaap; Geuns, Ed; Lee, Min-Shi; Venhuizen, odewijk; Neumeier, Elisabeth; Ziegler, Thedi

2018-01-01

Cell culture is now available as a method for the production of influenza vaccines in addition to eggs. In accordance with currently accepted practice, viruses recommended as candidates for vaccine manufacture are isolated and propagated exclusively in hens' eggs prior to distribution to manufacturers. Candidate vaccine viruses isolated in cell culture are not available to support vaccine manufacturing in mammalian cell bioreactors so egg-derived viruses have to be used. Recently influenza A (H3N2) viruses have been difficult to isolate directly in eggs. As mitigation against this difficulty, and the possibility of no suitable egg-isolated candidate viruses being available, it is proposed to consider using mammalian cell lines for primary isolation of influenza viruses as candidates for vaccine production in egg and cell platforms. To investigate this possibility, we tested the antigenic stability of viruses isolated and propagated in cell lines qualified for influenza vaccine manufacture and subsequently investigated antigen yields of such viruses in these cell lines at pilot-scale. Twenty influenza A and B-positive, original clinical specimens were inoculated in three MDCK cell lines. The antigenicity of recovered viruses was tested by hemagglutination inhibition using ferret sera against contemporary vaccine viruses and the amino acid sequences of the hemagglutinin and neuraminidase were determined. MDCK cell lines proved to be highly sensitive for virus isolation. Compared to the virus sequenced from the original specimen, viruses passaged three times in the MDCK lines showed up to 2 amino acid changes in the hemagglutinin. Antigenic stability was also established by hemagglutination inhibition titers comparable to those of the corresponding reference virus. Viruses isolated in any of the three MDCK lines grew reasonably well but variably in three MDCK cells and in VERO cells at pilot-scale. These results indicate that influenza viruses isolated in vaccine certified cell lines may well qualify for use in vaccine production. PMID:24975811

Performance characteristics of qualified cell lines for isolation and propagation of influenza viruses for vaccine manufacturing.

PubMed

Donis, Ruben O; Davis, C Todd; Foust, Angie; Hossain, M Jaber; Johnson, Adam; Klimov, Alexander; Loughlin, Rosette; Xu, Xiyan; Tsai, Theodore; Blayer, Simone; Trusheim, Heidi; Colegate, Tony; Fox, John; Taylor, Beverly; Hussain, Althaf; Barr, Ian; Baas, Chantal; Louwerens, Jaap; Geuns, Ed; Lee, Min-Shi; Venhuizen, Odewijk; Neumeier, Elisabeth; Ziegler, Thedi

2014-11-12

Cell culture is now available as a method for the production of influenza vaccines in addition to eggs. In accordance with currently accepted practice, viruses recommended as candidates for vaccine manufacture are isolated and propagated exclusively in hens' eggs prior to distribution to manufacturers. Candidate vaccine viruses isolated in cell culture are not available to support vaccine manufacturing in mammalian cell bioreactors so egg-derived viruses have to be used. Recently influenza A (H3N2) viruses have been difficult to isolate directly in eggs. As mitigation against this difficulty, and the possibility of no suitable egg-isolated candidate viruses being available, it is proposed to consider using mammalian cell lines for primary isolation of influenza viruses as candidates for vaccine production in egg and cell platforms. To investigate this possibility, we tested the antigenic stability of viruses isolated and propagated in cell lines qualified for influenza vaccine manufacture and subsequently investigated antigen yields of such viruses in these cell lines at pilot-scale. Twenty influenza A and B-positive, original clinical specimens were inoculated in three MDCK cell lines. The antigenicity of recovered viruses was tested by hemagglutination inhibition using ferret sera against contemporary vaccine viruses and the amino acid sequences of the hemagglutinin and neuraminidase were determined. MDCK cell lines proved to be highly sensitive for virus isolation. Compared to the virus sequenced from the original specimen, viruses passaged three times in the MDCK lines showed up to 2 amino acid changes in the hemagglutinin. Antigenic stability was also established by hemagglutination inhibition titers comparable to those of the corresponding reference virus. Viruses isolated in any of the three MDCK lines grew reasonably well but variably in three MDCK cells and in VERO cells at pilot-scale. These results indicate that influenza viruses isolated in vaccine certified cell lines may well qualify for use in vaccine production. Published by Elsevier Ltd.

Antiproliferative Cardenolide Glycosides of Elaeodendron alluaudianum from the Madagascar Rainforest1

PubMed Central

Hou, Yanpeng; Cao, Shugeng; Brodie, Peggy; Callmander, Martin; Ratovoson, Fidisoa; Randrianaivo, Richard; Rakotobe, Etienne; Rasamison, Vincent E.; Rakotonandrasana, Stephan; TenDyke, Karen; Suh, Edward M.; Kingston, David G. I.

2010-01-01

Bioassay-guided fractionation of an ethanol extract of a Madagascar collection of Elaeodendron alluaudianum led to the isolation of two new cardenolide glycosides (1 and 2). The 1H and 13C NMR spectra of both compounds were fully assigned using a combination of 2D NMR experiments, including 1H-1H COSY, HSQC, HMBC, and ROESY sequences. Both compounds 1 and 2 were tested against the A2780 human ovarian cancer cell line and the U937 human histiocytic lymphoma cell line assays, and showed significant antiproliferative activity with IC50 values of 0.12 and 0.07 μM against the A2780 human ovarian cancer cell line, and 0.15 and 0.08 μM against the U937 human histiocytic lymphoma cell line, respectively. PMID:19058971

New cytotoxic diarylheptanoids from the rhizomes of Alpinia officinarum Hance.

PubMed

Liu, Dan; Liu, Yan-Wen; Guan, Fu-Qin; Liang, Jing-Yu

2014-07-01

Two new dimeric diarylheptanoids, named Alpinin C (1) and D (2), a new natural product of diarylheptanoid (3) along with three known diarylheptanoids (4-6) were isolated from the rhizomes of Alpinia officinarum Hance. Their structures were elucidated based on extensive spectroscopic analyses (1D and 2D NMR, HRTOFMS, IR). The isolated compounds were evaluated for their cytotoxicity against human tumor cell lines HepG2, MCF-7, T98G and B16-F10. Compound 1 showed selective cytotoxicity against cell lines of MCF-7 and T98G, while compound 6 showed significant cytotoxicity to the all tested tumor cell lines with IC50 in the range from 8.46 to 22.68 μmol/L. Copyright © 2014 Elsevier B.V. All rights reserved.

Human monoclonal antibodies reactive with human myelomonocytic leukemia cells.

PubMed

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

1989-04-01

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

Characterization of an Sf-rhabdovirus-negative Spodoptera frugiperda cell line as an alternative host for recombinant protein production in the baculovirus-insect cell system.

PubMed

Maghodia, Ajay B; Geisler, Christoph; Jarvis, Donald L

2016-06-01

Cell lines derived from the fall armyworm, Spodoptera frugiperda (Sf), are widely used as hosts for recombinant protein production in the baculovirus-insect cell system (BICS). However, it was recently discovered that these cell lines are contaminated with a virus, now known as Sf-rhabdovirus [1]. The detection of this adventitious agent raised a potential safety issue that could adversely impact the BICS as a commercial recombinant protein production platform. Thus, we examined the properties of Sf-RVN, an Sf-rhabdovirus-negative Sf cell line, as a potential alternative host. Nested RT-PCR assays showed Sf-RVN cells had no detectable Sf-rhabdovirus over the course of 60 passages in continuous culture. The general properties of Sf-RVN cells, including their average growth rates, diameters, morphologies, and viabilities after baculovirus infection, were virtually identical to those of Sf9 cells. Baculovirus-infected Sf-RVN and Sf9 cells produced equivalent levels of three recombinant proteins, including an intracellular prokaryotic protein and two secreted eukaryotic glycoproteins, and provided similar N-glycosylation patterns. In fact, except for the absence of Sf-rhabdovirus, the only difference between Sf-RVN and Sf9 cells was SF-RVN produced higher levels of infectious baculovirus progeny. These results show Sf-RVN cells can be used as improved, alternative hosts to circumvent the potential safety hazard associated with the use of Sf-rhabdovirus-contaminated Sf cells for recombinant protein manufacturing with the BICS. Copyright © 2016 Elsevier Inc. All rights reserved.

Inhibiting the NF-kappaB pathway to assess its function in the cellular response to space radiation

NASA Astrophysics Data System (ADS)

Koch, Kristina; Baumstark-Khan, Christa; Hellweg, Christine; Testard, Isabelle; Reitz, Guenther

2012-07-01

Radiation is regarded as one of the limiting factors for space missions. Therefore the cellular radiation response needs to be studied in order to estimate risks and to develop appropriate countermeasures. Exposure of human cells to ionizing radiation can provoke cell cycle arrest, leading to cellular senescence or premature differentiation, and different types of cell death. Previous heavy ion experiments have shown that the Nuclear Factor κB (NF-κB) pathway is activated by fluences that can be reached during long-term missions and thereby NF-κB was identified as an important modulating factor in the cellular radiation response. It could improve cellular survival after exposure to high radiation doses and influence the cancer risk of astronauts. The classical and the genotoxic stress induced NF-κB pathway result in nuclear translocation of the p65/p50 dimer. Both pathways might contribute to the cellular radiation response. Chemical inhibitors were tested to suppress the NF-κB pathway in recombinant HEK-pNF-κB-d2EGFP/Neo cells. The efficacy and cytotoxicity of the inhibitors targeting different elements of the NF-κB pathway were analyzed and found mostly inappropriate as inhibitors were partly cytotoxic or unspecific. Alternatively a functional knock-out of RelA (p65) was used to identify the contribution of the NF-κB pathway to different cellular outcomes. Small hairpin RNA constructs (shRNA) were transfected into the HEK-pNF-κB-d2EGFP/Neo cell line. Their functionality was assessed by quantitative Reverse Transcriptase real-time PCR (qRT-PCR) to verify that the RelA mRNA amount was reduced by more than 80% in the knock-down cells The original cell line had been stably transfected with a reporter system to monitor NF-κB activation by measuring destabilized Enhanced Green Fluorescent Protein (d2EGFP)-expression. It was shown that after 18 hours d2EGFP reaches its highest expression level after activation of NF-κB and can be measured by FACS analysis. Results of measuring d2EGFP showed a suppressed level of EGFP(+) cells in the knock-down cell line, indicating a decreased NF-κB level. Growth behavior of the original and the knock-down cell line was investigated, showing that the decreased RelA level leads to an elongated lag phase while the doubling time during the exponential growth phase remained unaltered. Further the colony forming ability of both cell lines was compared. Both cell lines were irradiated with X-Rays. The RelA-knock-down cell line showed an increased radiosensitivity towards X-Rays, proving that NF-κB plays an important role in the survival ability of the cell. The knock-down cell line will now be used to study the involvement of NF-κB pathway in the cellular response to heavy ion exposure and other space relevant radiation qualities.

Isolation and characterization of conditionally immortalized mouse glomerular endothelial cell lines.

PubMed

Rops, Angelique L; van der Vlag, Johan; Jacobs, Cor W; Dijkman, Henry B; Lensen, Joost F; Wijnhoven, Tessa J; van den Heuvel, Lambert P; van Kuppevelt, Toin H; Berden, Jo H

2004-12-01

The culture and establishment of glomerular cell lines has proven to be an important tool for the understanding of glomerular cell functions in glomerular physiology and pathology. Especially, the recent establishment of a conditionally immortalized visceral epithelial cell line has greatly boosted the research on podocyte biology. Glomeruli were isolated from H-2Kb-tsA58 transgenic mice that contain a gene encoding a temperature-sensitive variant of the SV40 large tumor antigen, facilitating proliferative growth at 33 degrees C and differentiation at 37 degrees C. Glomerular endothelial cells were isolated from glomerular outgrowth by magnetic beads loaded with CD31, CD105, GSL I-B4, and ULEX. Clonal cell lines were characterized by immunofluorescence staining with antibodies/lectins specific for markers of endothelial cells, podocytes, and mesangial cells. Putative glomerular endothelial cell lines were analyzed for (1) cytokine-induced expression of adhesion molecules; (2) tube formation on Matrigel coating; and (3) the presence of fenestrae. As judged by immunostaining for Wilms tumor-1, smooth muscle actin (SMA), podocalyxin, and von Willebrand factor (vWF), we obtained putative endothelial, podocyte and mesangial cell lines. The mouse glomerular endothelial cell clone #1 (mGEnC-1) was positive for vWF, podocalyxin, CD31, CD105, VE-cadherin, GSL I-B4, and ULEX, internalized acetylated-low-density lipoprotein (LDL), and showed increased expression of adhesion molecules after activation with proinflammatory cytokines. Furthermore, mGEnC-1 formed tubes and contained nondiaphragmed fenestrae. The mGEnC-1 represents a conditionally immortalized cell line with various characteristics of differentiated glomerular endothelial cells when cultured at 37 degrees C. Most important, mGEnC-1 contains nondiaphragmed fenestrae, which is a unique feature of glomerular endothelial cells.

Actin cytoskeleton organization, cell surface modification and invasion rate of 5 glioblastoma cell lines differing in PTEN and p53 status

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

Djuzenova, Cholpon S., E-mail: djuzenova_t@ukw.de; Fiedler, Vanessa; Memmel, Simon

Glioblastoma cells exhibit highly invasive behavior whose mechanisms are not yet fully understood. The present study explores the relationship between the invasion capacity of 5 glioblastoma cell lines differing in p53 and PTEN status, expression of mTOR and several other marker proteins involved in cell invasion, actin cytoskeleton organization and cell morphology. We found that two glioblastoma lines mutated in both p53 and PTEN genes (U373-MG and SNB19) exhibited the highest invasion rates through the Matrigel or collagen matrix. In DK-MG (p53wt/PTENwt) and GaMG (p53mut/PTENwt) cells, F-actin mainly occurred in the numerous stress fibers spanning the cytoplasm, whereas U87-MG (p53wt/PTENmut),more » U373-MG and SNB19 (both p53mut/PTENmut) cells preferentially expressed F-actin in filopodia and lamellipodia. Scanning electron microscopy confirmed the abundant filopodia and lamellipodia in the PTEN mutated cell lines. Interestingly, the gene profiling analysis revealed two clusters of cell lines, corresponding to the most (U373-MG and SNB19, i.e. p53 and PTEN mutated cells) and less invasive phenotypes. The results of this study might shed new light on the mechanisms of glioblastoma invasion. - Highlights: • We examine 5 glioblastoma lines on the invasion capacity and actin cytoskeleton. • Glioblastoma cell lines mutated in both p53 and PTEN were the most invasive. • Less invasive cells showed much less lamellipodia, but more actin stress fibers. • A mechanism for the differences in tumor cell invasion is proposed.« less

Long term storage in liquid nitrogen leads to only minor phenotypic and gene expression changes in the mammary carcinoma model cell line BT474.

PubMed

Fazekas, Judit; Grunt, Thomas W; Jensen-Jarolim, Erika; Singer, Josef

2017-05-23

Cancer cell lines are indispensible surrogate models in cancer research, as they can be used off-the-shelf, expanded to the desired extent, easily modified and exchanged between research groups for affirmation, reproduction or follow-up experiments.As malignant cells are prone to genomic instability, phenotypical changes may occur after certain passages in culture. Thus, cell lines have to be regularly authenticated to ensure data quality. In between experiments these cell lines are often stored in liquid nitrogen for extended time periods.Although freezing of cells is a necessary evil, little research is performed on how long-term storage affects cancer cell lines. Therefore, this study investigated the effects of a 28-year long liquid nitrogen storage period on BT474 cells with regard to phenotypical changes, differences in cell-surface receptor expression as well as cytokine and gene expressional variations. Two batches of BT474 cells, one frozen in 1986, the other directly purchased from ATCC were investigated by light microscopy, cell growth analysis, flow cytometry and cytokine as well as whole-transcriptome expression profiling. The cell lines were morphologically indifferent and showed similar growth rates and similar cell-surface receptor expression. Transcriptome analysis revealed significant differences in only 26 of 40,716 investigated RefSeq transcripts with 4 of them being up-regulated and 22 down-regulated. This study demonstrates that even after very long periods of storage in liquid nitrogen, cancer cell lines display only minimal changes in their gene expression profiles. However, also such minor changes should be carefully assessed before continuation of experiments, especially if phenotypic alterations can be additionally observed.

6-shogaol induces apoptosis and enhances radiosensitivity in head and neck squamous cell carcinoma cell lines.

PubMed

Kotowski, Ulana; Kadletz, Lorenz; Schneider, Sven; Foki, Elisabeth; Schmid, Rainer; Seemann, Rudolf; Thurnher, Dietmar; Heiduschka, Gregor

2018-02-01

Ginger (Zingiber officinale Roscoe) is used for a wide array of conditions in traditional medicine in Asia, but little is known about the effect on head and neck cancer. In this study, the effect of two major pharmacologically active compounds of ginger, 6-gingerol and 6-shogaol, were studied on head and neck cancer cell lines. Furthermore, experiments in combination with established treatment methods for head and neck cancer were performed. Proliferation assays showed a dose-dependent reduction of cell viability. Flow cytometry analysis revealed the induction of apoptosis. Western blot analysis indicated that the antiapoptotic protein survivin was suppressed after treatment. Although a combination of 6-shogaol with cisplatin exhibited no synergistic effect, the combination with irradiation showed a synergistic reduction of clonogenic survival. In conclusion, ginger compounds have many noteworthy effects on head and neck cancer cell lines. In particular, the enhancement of radiosensitivity is remarkable. Copyright © 2017 John Wiley & Sons, Ltd.

Irradiated KHYG-1 retains cytotoxicity: potential for adoptive immunotherapy with a natural killer cell line.

PubMed

Suck, G; Branch, D R; Keating, A

2006-05-01

To evaluate gamma-irradiation on KHYG-1, a highly cytotoxic natural killer (NK) cell line and potential candidate for cancer immunotherapy. The NK cell line KHYG-1 was irradiated at 1 gray (Gy) to 50 Gy with gamma-irradiation, and evaluated for cell proliferation, cell survival, and cytotoxicity against tumor targets. We showed that a dose of at least 10 Gy was sufficient to inhibit proliferation of KHYG-1 within the first day but not its cytolytic activity. While 50 Gy had an apoptotic effect in the first hours after irradiation, the killing of K562 and HL60 targets was not different from non-irradiated cells but was reduced for the Ph + myeloid leukemia lines, EM-2 and EM-3. gamma-irradiation (at least 10 Gy) of KHYG-1 inhibits cell proliferation but does not diminish its enhanced cytolytic activity against several tumor targets. This study suggests that KHYG-1 may be a feasible immunotherapeutic agent in the treatment of cancers.

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).

Generation and characterization of p53 null transformed hepatic progenitor cells: oval cells give rise to hepatocellular carcinoma.

PubMed

Dumble, Melissa L; Croager, Emma J; Yeoh, George C T; Quail, Elizabeth A

2002-03-01

Oval cells are bipotential liver stem cells able to differentiate into hepatocytes and bile duct epithelia. In normal adult liver oval cells are quiescent, existing in low numbers around the periportal region, and proliferate following severe, prolonged liver trauma. There is evidence implicating oval cells in the development of hepatocellular carcinoma, and hence the availability of an immortalized oval cell line would be invaluable for the study of liver cell lineage differentiation and carcinogenesis. A novel approach in the generation of cell lines is the use of the p53 knockout mouse. Absence of p53 allows a cell to cycle past the normal Hayflick limit, rendering it immortalized, although subsequent genetic alterations are thought necessary for transformation. p53 knockout mice were fed a choline-deficient, ethionine-supplemented diet, previously shown to increase oval cell numbers in wild-type mice. The oval cells were isolated by centrifugal elutriation and maintained in culture. Colonies of hepatic cells were isolated and characterized with respect to phenotype, growth characteristics and tumorigenicity. Analysis of gene expression by Northern blotting and immunocytochemistry suggests they are oval-like cells by virtue of albumin and transferrin expression, as well as the oval cell markers alpha fetoprotein, M(2)-pyruvate kinase and A6. Injection into athymic nude mice shows the cell lines are capable of forming tumors which phenotypically resemble hepatocellular carcinoma. Thus, the use of p53 null hepatic cells successfully generated immortalized and tumorigenic hepatic stem cell lines. The results presented support the idea that deleting p53 allows immortalization and contributes to the transformation of the oval-like cell lines. Further, the tumorigenic status of the cell lines is direct evidence for the participation of oval cells in the formation of hepatocellular carcinoma.

Study of phytochemical, anti-microbial, anti-oxidant, and anti-cancer properties of Allium wallichii.

PubMed

Bhandari, Jaya; Muhammad, BushraTaj; Thapa, Pratiksha; Shrestha, Bhupal Govinda

2017-02-08

There is growing interest in the use of plants for the treatment and prevention of cancer. Medicinal plants are currently being evaluated as source of promising anticancer agents. In this paper, we have investigated the anticancer potential of plant Allium wallichii, a plant native to Nepal and growing at elevations of 2300-4800 m. This is the first study of its kind for the plant mentioned. The dried plant was extracted in aqueous ethanol. Phytochemical screening, anti-microbial assay, anti-oxidant assay, cytotoxicity assay and the flow-cytometric analysis were done for analyzing different phytochemicals present, anti-microbial activity, anti-oxidant activity and anti-cancer properties of Allium wallichii. We observed the presence of steroids, terpenoids, flavonoids, reducing sugars and glycosides in the plant extract and the plant showed moderate anti-microbial and anti-oxidant activity. The IC 50 values of Allium wallichii in different cancer cell lines are 69.69 μg/ml for Prostate cancer (PC3) cell line, 55.29 μg/ml for Breast Cancer (MCF-7) cell line and 46.51 μg/ml for cervical cancer (HeLa) cell line as compared to Doxorubicin (0.85 μg/ml). The cell viability assay using FACS showed that the IC 50 value of Allium wallichii for Burkitt's lymphoma (B-Lymphoma) cell line was 3.817 ± 1.99 mg/ml. Allium wallichii can be an important candidate to be used as an anticancer agent. Separation of pure compounds with bioassay guided extraction, spectrometric analysis and subsequent cytotoxicity assay of the pure bioactive compounds from Allium wallichii is highly recommended as the crude extract itself showed promising cytotoxicity.

Human somatic cells subjected to genetic induction with six germ line-related factors display meiotic germ cell-like features

PubMed Central

Medrano, Jose V.; Martínez-Arroyo, Ana M.; Míguez, Jose M.; Moreno, Inmaculada; Martínez, Sebastián; Quiñonero, Alicia; Díaz-Gimeno, Patricia; Marqués-Marí, Ana I.; Pellicer, Antonio; Remohí, Jose; Simón, Carlos

2016-01-01

The in vitro derivation of human germ cells has attracted interest in the last years, but their direct conversion from human somatic cells has not yet been reported. Here we tested the ability of human male somatic cells to directly convert into a meiotic germ cell-like phenotype by inducing them with a combination of selected key germ cell developmental factors. We started with a pool of 12 candidates that were reduced to 6, demonstrating that ectopic expression of the germ line-related genes PRDM1, PRDM14, LIN28A, DAZL, VASA and SYCP3 induced direct conversion of somatic cells (hFSK (46, XY), and hMSC (46, XY)) into a germ cell-like phenotype in vitro. Induced germ cell-like cells showed a marked switch in their transcriptomic profile and expressed several post-meiotic germ line related markers, showed meiotic progression, evidence of epigenetic reprogramming, and approximately 1% were able to complete meiosis as demonstrated by their haploid status and the expression of several post-meiotic markers. Furthermore, xenotransplantation assays demonstrated that a subset of induced cells properly colonize the spermatogonial niche. Knowledge obtained from this work can be used to create in vitro models to study gamete-related diseases in humans. PMID:27112843

[Inhibitory effects of 11 coumarin compounds against growth of human bladder carcinoma cell line E-J in vitro].

PubMed

Yang, Xiu-wei; Xu, Bo; Ran, Fu-xiang; Wang, Rui-qing; Wu, Jun; Cui, Jing-rong

2007-01-01

To screen antitumor active compounds, drug-like or leading compounds from Chinese traditional and herbal drugs. Eleven coumarin compounds isolated from the Chinese traditional and herbal drugs were studied for their antitumor activities in vitro by determining the inhibition rates against growth of human bladder carcinoma cell line E-J. It showed that umbelliferone, scoparone, demethylfuropinarine, isopimpinellin, forbesoside, columbianadin, decursin and glycycoumarin inhibited the growth of human bladder carcinoma cell line E-J in vitro and their activities showed a concentration-effect relationship. The inhibitory effects of forbesoside, columbianadin, decursin and umbelliferone, with IC50 values of 7.50x10(-7), 2.30x10(-6), 6.00x10(-6) and 1.30x10(-6) mol/L, respectively, were stronger than those of the other tested compounds. However, xanthotoxin, esculin and sphondin did not inhibit the growth of human bladder carcinoma cell line E-J in this assay condition. These findings indicate that forbesoside, columbianadin, esculin, decursin and umbelliferone would be effective or regarded as potent drug-like or leading compounds against human bladder carcinoma.

Synthesis, characterization and cytotoxic studies of water soluble [(η5-C5H5)2Mo(thionucleobase/thionucleoside)]Cl complexes in breast and colon cancer cell lines

PubMed Central

Acevedo-Acevedo, Débora; Matta, Jaime; Meléndez, Enrique

2010-01-01

Four new water soluble molybdenocene complexes were synthesized in aqueous solution at pH 7.0. The new species, [(η5-C5H5)2Mo(L)]Cl (L= 6-mercaptopurine, 2-amino-6-mercaptopurine, (-)-2-amino-6-mercaptopurine ribose and 6-mercaptopurine ribose), were characterized by spectroscopic methods. NMR spectroscopic data showed the presence of two coordination isomers, S(6), N(7) and S(6), N(1), in aqueous solution, being S(6), N(7) the most stable. The antiproliferative activities of the new species were investigated in HT-29 colon and MCF-7 breast cancer cell lines. The incorporation of molybdenocene (Cp2Mo2+) into the thionucleobases/thionucleosides decreases their cytotoxic activities in HT-29 colon cancer cell line. In contrast, in the MCF-7 cell line, [Cp2Mo(2-amino-6-mercaptopurine)]Cl showed a high cytotoxic activity. This is most likely a consequence of the enhanced lipophilic character on the thionucleobase combined with synergism between Cp2Mo2+ and the thionucleobase ligand. PMID:21399723

New 15-membered tetraaza (N4) macrocyclic ligand and its transition metal complexes: Spectral, magnetic, thermal and anticancer activity

NASA Astrophysics Data System (ADS)

El-Boraey, Hanaa A.; EL-Gammal, Ohyla A.

2015-03-01

Novel tetraamidemacrocyclic 15-membered ligand [L] i.e. naphthyl-dibenzo[1,5,9,12]tetraazacyclopentadecine-6,10,11,15-tetraoneand its transition metal complexes with Fe(II), Co(II), Ni(II), Cu(II), Ru(III) and Pd(II) have been synthesized and characterized by elemental analysis, spectral, thermal as well as magnetic and molar conductivity measurements. On the basis of analytical, spectral (IR, MS, UV-Vis, 1H NMR and EPR) and thermal studies distorted octahedral or square planar geometry has been proposed for the complexes. The antitumor activity of the synthesized ligand and some complexes against human breast cancer cell lines (MCF-7) and human hepatocarcinoma cell lines (HepG2) has been studied. The complexes (IC50 = 2.27-2.7, 8.33-31.1 μg/mL, respectively) showed potent antitumor activity, towards the former cell lines comparable with their ligand (IC50 = 13, 26 μg/mL, respectively). The results show that the activity of the ligand towards breast cancer cell line becomes more pronounced and significant when coordinated to the metal ion.

Cytotoxic and antimicrobial activities of endophytic fungi isolated from Bacopa monnieri (L.) Pennell (Scrophulariaceae).

PubMed

Katoch, Meenu; Singh, Gurpreet; Sharma, Sadhna; Gupta, Nidhi; Sangwan, Payare Lal; Saxena, Ajit Kumar

2014-02-11

Endophytes, which reside in plant tissues, have the potential to produce novel metabolites with immense benefits for health industry. Cytotoxic and antimicrobial activities of endophytic fungi isolated from Bacopa monnieri (L.) Pennell were investigated. Endophytic fungi were isolated from the Bacopa monnieri. Extracts from liquid cultures were tested for cytotoxicity against a number of cancer cell lines using the MTT assay. Antimicrobial activity was determined using the micro dilution method. 22% of the examined extracts showed potent (IC50 of <20 μg/ml) cytotoxic activity against HCT-116 cell line. 5.5%, 11%, 11% of the extracts were found to be cytotoxic for MCF-7, PC-3, and A-549 cell lines respectively. 33% extracts displayed antimicrobial activity against at least one test organism with MIC value 10-100 μg/ml. The isolate B9_Pink showed the most potent cytotoxic activity for all the cell lines examined and maximum antimicrobial activity against the four pathogens examined which was followed by B19. Results indicated the potential for production of bioactive agents from endophytes of Bacopa monnieri.

Variation in cell wall composition among forage maize (Zea mays L.) inbred lines and its impact on digestibility: analysis of neutral detergent fiber composition by pyrolysis-gas chromatography-mass spectrometry.

PubMed

Fontaine, Anne-Sophie; Bout, Siobhán; Barrière, Yves; Vermerris, Wilfred

2003-12-31

Cell wall digestibility is an important determinant of forage quality, but the relationship between cell wall composition and digestibility is poorly understood. We analyzed the neutral detergent fiber (NDF) fraction of nine maize inbred lines and one brown midrib3 mutant with pyrolysis-gas chromatography-mass spectrometry (Py-GC-MS). Among 29 pyrolysis fragments that were quantified, two carbohydrate-derived and six lignin-derived fragments showed statistically significant genetic variation. The pyrolysis products 4-vinyl phenol and 2,6-dimethoxy-4-vinyl phenol were negatively correlated with digestibility, whereas furfural and 3-(4-hydroxyphenyl)-3-oxopropanal showed a positive correlation with digestibility. Linear discriminant analysis of the pyrolysis data resulted in the resolution of groups of inbred lines with different digestibility properties based on their chemical composition. These analyses reveal that digestibility is governed by complex interactions between different cell wall compounds, but that several pyrolysis fragments can be used as markers to distinguish between maize lines with different digestibility.

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.

Monocrotophos Induces the Expression of Xenobiotic Metabolizing Cytochrome P450s (CYP2C8 and CYP3A4) and Neurotoxicity in Human Brain Cells.

PubMed

Tripathi, Vinay Kumar; Kumar, Vivek; Pandey, Ankita; Vatsa, Pankhi; Dhasmana, Anupam; Singh, Rajat Pratap; Appikonda, Sri Hari Chandan; Hwang, Inho; Lohani, Mohtashim

2017-07-01

Expression of various cytochrome P450s (CYPs) in mammalian brain cells is well documented. However, such studies are hampered in neural/glial cells of human origin due to nonavailability of human brain cells. To address this issue, we investigated the expression and inducibility of CYP2C8 and CYP3A4 and their responsiveness against cyclophosphamide (CPA) and organophosphorus pesticide monocrotophos (MCP), a known developmental neurotoxicant in human neural (SH-SY5Y) and glial (U373-MG) cell lines. CPA induced significant expression of CYP2C8 and CYP3A4 in both types of cells in a time-dependent manner. Neural cell line exhibited relatively higher constitutive and inducible expression of CYPs than the glial cell line. MCP exposure alone could not induce the significant expression of CYPs, whereas the cells preexposed to CPA showed a significant response to MCP. Similar to the case of CPA induced expressions, neural cells were found to be more vulnerable than glial cells. Our data indicate differential expressions of CYPs in cultured human neural and glial cell lines. The findings were synchronized with protein ligand docking studies, which showed a significant modulatory capacity of MCP by strong interaction with CYP regulators-CAR and PXR. Similarly, the known CYP inducer CPA has also shown significant high docking scores with the two studied CYP regulators. We also observed a significant induction in reactive oxygen species (ROS), lipid peroxides (LPO), micronucleus (MN), chromosomal aberration (CA), and reduction in reduced glutathione (GSH) and catalase following the exposure of MCP. Moreover, the expressions of apoptotic markers such as caspase-3, caspase-9, Bax, and p53 were significantly upregulated, whereas the levels of antiapoptotic marker, Bcl2, was downregulated after the exposure of MCP in both cell lines. These findings confirm the involvement of ROS-mediated oxidative stress, which subsequently triggers apoptosis pathways in both human neural (SH-SY5Y) and glial (U373-MG) cell lines following the exposure of MCP.

Relationship between DNA ploidy level and tumor sociology behavior in 12 nervous cell lines

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

Kiss, R.; Camby, I.; Salmon, I.

1995-06-01

Cell population sociology was studied in two medulloblastomas and 10 astrocytic human tumor cell lines by means of the characterization of the structure of neoplastic cell colonies growing on histological slides. This was carried out via digital cell image analysis of Feulgen-stained nuclei, to which the Delaunay triangulation and Voronoi paving mathematical techniques were applied. Such assessments were compared to the DNA ploidy level (assessed by means of DNA histogram typing). The results show that the cell colony architecture characteristics differed markedly according to whether the cell lines were euploid (diploid or tetraploid) or aneuploid (hyperdiploid, triploid, hypertriploid, or polymorphic).more » In fact, the cell colonies from the euploid cell nuclei populations were larger and more dense than those from the aneuploid ones. Furthermore, for an identical period of culture, the cell lines from high-grade malignant astrocytic tumors (glioblastomas) exhibited cell colonies that were larger and more dense than those in cell lines from low-grade astrocytic tumors (astrocytomas). In each of these two groups, the diploid cell nuclei populations exhibited cell colonies larger and more dense than the nondiploid colonies. The present methodology is now being applied in vivo to histological sections of surgically removed human brain tumors in order to distinguish between high-risk clinical subgroups and medium-risk subgroups in clearly circumscribed histopathological groups. 38 refs., 5 figs., 2 tabs.« less

Snail regulates cell survival and inhibits cellular senescence in human metastatic prostate cancer cell lines.

PubMed

Emadi Baygi, Modjtaba; Soheili, Zahra Soheila; Schmitz, Ingo; Sameie, Shahram; Schulz, Wolfgang A

2010-12-01

The epithelial-mesenchymal transition (EMT) is regarded as an important step in cancer metastasis. Snail, a master regulator of EMT, has been recently proposed to act additionally as a cell survival factor and inducer of motility. We have investigated the function of Snail (SNAI1) in prostate cancer cells by downregulating its expression via short (21-mer) interfering RNA (siRNA) and measuring the consequences on EMT markers, cell viability, death, cell cycle, senescence, attachment, and invasivity. Of eight carcinoma cell lines, the prostate carcinoma cell lines LNCaP and PC-3 showed the highest and moderate expression of SNAI1 mRNA, respectively, as measured by quantitative RT-PCR. Long-term knockdown of Snail induced a severe decline in cell numbers in LNCaP and PC-3 and caspase activity was accordingly enhanced in both cell lines. In addition, suppression of Snail expression induced senescence in LNCaP cells. SNAI1-siRNA-treated cells did not tolerate detachment from the extracellular matrix, probably due to downregulation of integrin α6. Expression of E-cadherin, vimentin, and fibronectin was also affected. Invasiveness of PC-3 cells was not significantly diminished by Snail knockdown. Our data suggest that Snail acts primarily as a survival factor and inhibitor of cellular senescence in prostate cancer cell lines. We therefore propose that Snail can act as early driver of prostate cancer progression.

The Microprocessor controls the activity of mammalian retrotransposons

PubMed Central

Heras, Sara R.; Macias, Sara; Plass, Mireya; Fernandez, Noemí; Cano, David; Eyras, Eduardo; Garcia-Perez, José L.; Cáceres, Javier F.

2013-01-01

More than half of the human genome is made of Transposable Elements. Their ongoing mobilization is a driving force in genetic diversity; however, little is known about how the host regulates their activity. Here, we show that the Microprocessor (Drosha-DGCR8), which is required for microRNA biogenesis, also recognizes and binds RNAs derived from human LINE-1 (Long INterspersed Element 1), Alu and SVA retrotransposons. Expression analyses demonstrate that cells lacking a functional Microprocessor accumulate LINE-1 mRNA and encoded proteins. Furthermore, we show that structured regions of the LINE-1 mRNA can be cleaved in vitro by Drosha. Additionally, we used a cell culture-based assay to show that the Microprocessor negatively regulates LINE-1 and Alu retrotransposition in vivo. Altogether, these data reveal a new role for the Microprocessor as a post-transcriptional repressor of mammalian retrotransposons acting as a defender of human genome integrity. PMID:23995758

The Microprocessor controls the activity of mammalian retrotransposons.

PubMed

Heras, Sara R; Macias, Sara; Plass, Mireya; Fernandez, Noemí; Cano, David; Eyras, Eduardo; Garcia-Perez, José L; Cáceres, Javier F

2013-10-01

More than half of the human genome is made of transposable elements whose ongoing mobilization is a driving force in genetic diversity; however, little is known about how the host regulates their activity. Here, we show that the Microprocessor (Drosha-DGCR8), which is required for microRNA biogenesis, also recognizes and binds RNAs derived from human long interspersed element 1 (LINE-1), Alu and SVA retrotransposons. Expression analyses demonstrate that cells lacking a functional Microprocessor accumulate LINE-1 mRNA and encoded proteins. Furthermore, we show that structured regions of the LINE-1 mRNA can be cleaved in vitro by Drosha. Additionally, we used a cell culture-based assay to show that the Microprocessor negatively regulates LINE-1 and Alu retrotransposition in vivo. Altogether, these data reveal a new role for the Microprocessor as a post-transcriptional repressor of mammalian retrotransposons and a defender of human genome integrity.

Cellular and Tumor Radiosensitivity is Correlated to Epidermal Growth Factor Receptor Protein Expression Level in Tumors Without EGFR Amplification;Epidermal growth factor receptor; Radiotherapy; Squamous cell carcinoma; Biomarker; Local tumor control

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

Kasten-Pisula, Ulla; Saker, Jarob; Eicheler, Wolfgang

2011-07-15

Purpose: There is conflicting evidence for whether the expression of epidermal growth factor receptor in human tumors can be used as a marker of radioresponse. Therefore, this association was studied in a systematic manner using squamous cell carcinoma (SCC) cell lines grown as cell cultures and xenografts. Methods and Materials: The study was performed with 24 tumor cell lines of different tumor types, including 10 SCC lines, which were also investigated as xenografts on nude mice. Egfr gene dose and the length of CA-repeats in intron 1 were determined by polymerase chain reaction, protein expression in vitro by Western blotmore » and in vivo by enzyme-linked immunosorbent assay, and radiosensitivity in vitro by colony formation. Data were correlated with previously published tumor control dose 50% data after fractionated irradiation of xenografts of the 10 SCC. Results: EGFR protein expression varies considerably, with most tumor cell lines showing moderate and only few showing pronounced upregulation. EGFR upregulation could only be attributed to massive gene amplification in the latter. In the case of little or no amplification, in vitro EGFR expression correlated with both cellular and tumor radioresponse. In vivo EGFR expression did not show this correlation. Conclusions: Local tumor control after the fractionated irradiation of tumors with little or no gene amplification seems to be dependent on in vitro EGFR via its effect on cellular radiosensitivity.« less

Properties of solid polymer electrolyte fluorocarbon film. [used in hydrogen/oxygen fuel cells

NASA Technical Reports Server (NTRS)

Alston, W. B.

1973-01-01

The ionic fluorocarbon film used as the solid polymer electrolyte in hydrogen/oxygen fuel cells was found to exhibit delamination failures. Polarized light microscopy of as-received film showed a lined region at the center of the film thickness. It is shown that these lines were not caused by incomplete saponification but probably resulted from the film extrusion process. The film lines could be removed by an annealing process. Chemical, physical, and tensile tests showed that annealing improved or sustained the water contents, spectral properties, thermo-oxidative stability, and tensile properties of the film. The resistivity of the film was significantly decreased by the annealing process.

Characterization of Acyl-CoA Synthetase Isoforms In Pancreatic Beta Cells: Gene Silencing Shows Participation of ACSL3 and ACSL4 In Insulin Secretion

PubMed Central

Ansari, Israr-ul H.; Longacre, Melissa J.; Stoker, Scott W.; Kendrick, Mindy A.; O’Neill, Lucas M.; Zitur, Laura J.; Fernandez, Luis A.; Ntambi, James M.; MacDonald, Michael J.

2017-01-01

Long-chain acyl-CoA synthetases (ACSLs) convert fatty acids to fatty acyl-CoAs to regulate various physiologic processes. We characterized the ACSL isoforms in a cell line of homogeneous rat beta cells (INS-1 832/13 cells) and human pancreatic islets. ACSL4 and ACSL3 proteins were present in the beta cells and human and rat pancreatic islets and concentrated in insulin secretory granules and less in mitochondria and negligible in other intracellular organelles. ACSL1 and ACSL6 proteins were not seen in INS-1 832/13 cells or pancreatic islets. ACSL5 protein was seen only in INS-1 832/13 cells. With shRNA-mediated gene silencing we developed stable ACSL knockdown cell lines from INS-1 832/13 cells. Glucose-stimulated insulin release was inhibited ~ 50% with ACSL4 and ACSL3 knockdown and unaffected in cell lines with knockdown of ACSL5, ACLS6 and ACSL1. Lentivirus shRNA-mediated gene silencing of ACSL4 and ACSL3 in human pancreatic islets inhibited glucose-stimulated insulin release. ACSL4 and ACSL3 knockdown cells showed inhibition of ACSL enzyme activity more with arachidonate than with palmitate as a substrate, consistent with their preference for unsaturated fatty acids as substrates. ACSL4 knockdown changed the patterns of fatty acids in phosphatidylserines and phosphatidylethanolamines. The results show the involvement of ACLS4 and ACLS3 in insulin secretion. PMID:28193492

Isolation and characterization of an anticancer catechol compound from Semecarpus anacardium.

PubMed

Nair, P K Raveedran; Melnick, Steven J; Wnuk, Stanislaw F; Rapp, Magdalena; Escalon, Enrique; Ramachandran, Cheppail

2009-04-21

The fruits and seeds of Semecarpus anacardium are used widely for the treatment of human cancers and other diseases in the Ayurvedic and Sidda systems of medicine in India. The principal aim of this investigation was to isolate and characterize the anticancer compound from the kernel of Semecarpus anacardium nut. The bioactivity-tailored isolation and detailed chemical characterization were used to identify the active compound. Cytotoxicity, apoptosis, cell cycle arrest as well as synergism between the identified anticancer compound and doxorubicin in human tumor cell lines were analyzed. GC/MS, IR, proton NMR, carbon NMR and collisionally induced dissociation (CID) spectra analysis showed that the isolated active compound is 3-(8'(Z),11'(Z)-pentadecadienyl) catechol (SA-3C). SA-3C is cytotoxic to tumor cell lines with IC(50) values lower than doxorubicin and even multidrug resistant tumor cell lines were equally sensitive to SA-3C. SA-3C induced apoptosis in human leukemia cell lines in a dose-dependent manner and showed synergistic cytotoxicity with doxorubicin. The cell cycle arrest induced by SA-3C at S- and G(2)/M-phases correlated with inhibition of checkpoint kinases. SA-3C isolated from the kernel of Semecarpus anacardium can be developed as an important anticancer agent for single agent and/or multiagent cancer therapy.

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

PubMed

Hong, Y; Winkler, C; Schartl, M

1998-03-31

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

Production of medakafish chimeras from a stable embryonic stem cell line

PubMed Central

Hong, Yunhan; Winkler, Christoph; Schartl, Manfred

1998-01-01

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

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.

Participation of an abnormality in the transforming growth factor-beta signaling pathway in resistance of malignant glioma cells to growth inhibition induced by that factor.

PubMed

Zhang, Lei; Sato, Eiji; Amagasaki, Kenichi; Nakao, Atsuhito; Naganuma, Hirofumi

2006-07-01

Malignant glioma cells secrete and activate transforming growth factor-beta (TGFbeta) and are resistant to growth inhibition by that factor. Nevertheless, the mechanism underlying this effect remains poorly understood. In this study, the mechanism of the resistance to growth inhibition induced by TGFbeta was investigated. The authors examined the expression of downstream components of the TGFbeta receptor, including Smad2, Smad3, Smad4, and Smad7, and the effect of TGFbeta1 treatment on the phosphorylation of Smad2 and the nuclear translocation of Smad2 and Smad3 by using 10 glioma cell lines and the A549 cell line, which is sensitive to TGFbeta-mediated growth inhibition. The expression of two transcriptional corepressor proteins, SnoN and Ski, and the effect of TGFbeta1 treatment on the expression of the SnoN protein and the cell cycle regulators p21, p15, cyclin-dependent kinase-4 (CDK4), and cyclin D1 were also examined. Expression of the Smad2 and Smad3 proteins was lower in the glioma cell lines than in the A549 cell line and in normal astrocytes. In particular, Smad3 expression was low or very low in nine of the 10 malignant glioma cell lines. Expression of Smad4 was low in four glioma cell lines, and expression of the Smad7 protein was similar when compared with protein expression in the A549 cell line and in normal astrocytes. The levels of Smad2 phosphorylation after TGFbeta1 treatment were lower in glioma cell lines than in the A549 cell line, except for one glioma cell line. Seven of the 10 glioma cell lines exhibited lower levels of nuclear translocation of Smad2 and Smad3, and two cell lines that expressed very low levels of Smad3 protein showed no nuclear translocation. All glioma cell lines expressed the SnoN protein and its expression was unaltered by treatment with TGFbeta1. Three glioma cell lines expressed high levels of the Ski protein. The expression of the p21(cip1), p15(INK4B), CDK4, and cyclin D1 proteins was not altered by TGFbeta1, treatment, except in one cell line that displayed a slight increase in p21 protein. Overall, the expression of the Smad2 and Smad3 proteins was low in the glioma cell lines, the phosphorylation and nuclear translocation of Smad2 and Smad3 were impaired, and the TGFbeta receptor signal did not affect the expression of the SnoN, p21, p15, cyclin D1, and CDK4 proteins. These results suggest that the ability to resist TGFbeta-mediated growth inhibition in malignant glioma cells is due to abnormalities in the TGFbeta signaling pathway.

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.

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

PubMed

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

2017-10-10

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

[Multiplication of Brucella abortus and production of nitric oxide in two macrophage cell lines of different origin].

PubMed

Serafino, J; Conde, S; Zabal, O; Samartino, L

2007-01-01

Brucella abortus is a bacterium which causes abortions and infertility in cattle and undulant fever in humans. It multiplies intracellularly, evading the mechanisms of cellular death. Nitric oxide (NO) is important in the regulation of the immune response. In the present work, we studied the ability of three B. abortus strains to survive intracellularly in two macrophage cell lines. The bacterial multiplication in both cell lines was determined at two different times in UFC/ ml units. Moreover the inoculated cells were also observed under light-field and fluorescence microscopy stained with Giemsa and acridine orange, respectively. The stain of both cellular lines showed similar results with respect to the UFC/ml determination. The presence of B. abortus was confirmed by electronic microscopy. In both macrophage cell lines inoculated with the rough strain RB51, the multiplication diminished and the level of NO was higher, compared with cells inoculated with smooth strains (S19 and 2308). These results suggest that the absence of O-chain of LPS probably affects the intracellular growth of B. abortus.

[Effect of Recombinant Adenovirus AdE-SH2-Caspase 8 on the Apoptosis of Imatinib-resistant K562/G01 Cell Line].

PubMed

Wang, Lin; Fei, Chang; Huang, Zheng-Lan; Li, Hui; Liu, Zhang-Lin; Feng, Wen-Li

2015-08-01

To investigate the effect of SH2-Caspase 8 fusion protein expressed by recombinant adenovirus AdE-SH2-Caspase8-HA-GFP (SC) on the apoptosis of K562/G01 cell line, which is a BCR/ABL positive chronic myeloid leukemia cell line and resistant to imatinib. The K562/G01 cell line was infected with AdE-SH2-Caspase 8-HA-GFP adenovirus (SC), then the cells were divided into 3 groups: AdE-SH2m-Caspase 8-HA-GFP (SmC) group, AdE-GFP (CMV) group and PBS group as control. The infection efficiency was observed under fluorescent microscopy and by flow cytometry. The expression of fusion protein SH2-Caspase 8-HA was measured by Western blot. The morphology of the cells detected by Wright's staining. The apoptosis of the cells were detected by flow cytometry and DNA ladder. The expression of Caspase 3 and PARP were detected by Western blot. The infection efficiency of SC on K562/G01 cells was high which was confirmed by fluorescent microscopy and FCM. SH2-Caspase 8-HA fusion protein were expressed correctly in K562/G01 cells. After treatment with SC the apoptosis of K562/G01 cells could be observed by microscopy. The result of FCM showed that early apoptosis of K562/G01 cells increased significantly as compared with control groups (P < 0.05). DNA ladder showed that the classic DNA ladders appeared in K562/G01 cells after treatment with SC. The wester blot detection showed that the expression level of apoptosis-related protein Caspase 3 and PARP increased. The recombinant adenovirus SC expressing SH2-Caspase 8 fusion protein can induces the apoptosis of K562/G01 cells.

Induction of apoptosis in human choriocarcinoma cell lines by treatment with 3,4-dihydro-6-[4-(3,4-dimethoxybenzoyl)-1-piperazinyl]-2(1H)-quinolinone (vesnarinone).

PubMed

Isaka, Keiichi; Fujito, Atsuya; Sagawa, Yasukazu; Yudate, Tamaki; Nishi, Hirotaka; Ito, Hiroe; Takayama, Masaomi

2002-01-01

Induction of apoptosis is an attractive strategy in cancer therapy but it clinical practice is not yet sufficient in choriocarcinoma. The quinolinone derivative, vesnarinone, is a novel inotropic agent used for treating congestive heart failure and may also have a potential anticancer activity. It induces apoptosis and differentiation in some tumor cell lines. We examined the antitumor effect of vesnarinone in eight cell lines established from human choriocarcinoma and hydatidiform mole using MTT assay and also analyzed the nuclear fragmentation of tumor cells by DNA electrophoresis assay. Vesnarinone inhibited the proliferation of choriocarcinoma cell lines in a dose-dependent manner and induced DNA fragmentation in cells. However, the BM cell line prepared by subcultivation from hydatidiform mole showed no growth suppression or DNA fragmentation in response to vesnarinone. On the other hand, PCR-SSCP analysis and direct DNA sequencing have shown that a human choriocarcinoma cell line, SCH, has a mutant p53 gene at codon 249. When SCH cells were treated with vesnarinone cellular proliferation was significantly inhibited. Vesnarinone suppressed the proliferation of all choriocarcinoma cell lines and induced apoptosis, regardless of the existence of p53 mutation. In addition, it has been found by RT-PCR that expression of c-Myc mRNA is upregulated by treating choriocarcinoma cells with vesnarinone. The finding suggests that vesnarinone might induce expression of c-Myc gene in choriocarcinoma cells, the product of which may be associated with the inhibition of cell growth and induce apoptosis. These results suggest that vesnarinone is a useful reagent for the treatment of choriocarcinoma.

Nuclear donor cell lines considerably influence cloning efficiency and the incidence of large offspring syndrome in bovine somatic cell nuclear transfer.

PubMed

Liu, J; Wang, Y; Su, J; Luo, Y; Quan, F; Zhang, Y

2013-08-01

Total five ear skin fibroblast lines (named F1, F2, F3, F4 and F5) from different newborn Holstein cows have been used as nuclear donor cells for producing cloned cows by somatic cell nuclear transfer (SCNT). The effects of these cell lines on both in vitro and in vivo developmental rates of cloned embryos, post-natal survivability and incidence of large offspring syndrome (LOS) were examined in this study. We found that the different cell lines possessed the same capacity to support pre-implantation development of cloned embryos, the cleavage and blastocyst formation rates ranged from 80.2 ± 0.9 to 84.5 ± 2.5% and 28.5 ± 0.9 to 33.3 ± 1.4%, respectively. However, their capacities to support the in vivo development of SCNT embryos showed significant differences (p < 0.05). The pregnancy rates at 90 and 240 day were significantly lower in groups F2 (4.9% and 3.3%) and F3 (5.4% and 5.4%) compared to groups F1 (23.3% and 16.3%), F4 (25.7% and 18.6%) and F5 (25.9% and 19.8%) (p < 0.05). The cloning efficiency was significantly higher in group F5 than those in group F1, F2, F3 and F4 (9.3% vs 4.1%, 1.2%, 2.0% and 5.0%, respectively, p < 0.05). Moreover, large offspring syndrome (LOS) incidence in group F5 was significantly lower than those in other groups (p < 0.05). All cloned offspring from cell line F1, F2, F3 and F4 showed LOS and gestation length delay, while all cloned offspring from F5 showed normal birthweight and gestation length. We concluded that the nuclear donor cell lines have significant impact on the in vivo development of cloned embryos and the incidence of LOS in cloned calves. © 2013 Blackwell Verlag GmbH.

Effect of Temporal Pattern of Radiation in Intensity Modulated Radiotherapy on Cell Cycle Progression and Apoptosis of ACHN Renal Cell Carcinoma Cell Line.

PubMed

Khorramizadeh, Maryam; Saberi, Alihossein; Tahmasebi-Birgani, Mohammadjavad; Shokrani, Parvaneh; Amouhedari, Alireza

The existence of a hypersensitive radiation response to doses below 1 Gy is well established for many normal and tumor cell lines. The aim of this study was to ascertain the impact of temporal pattern modeling IMRT on survival, cell cycle and apoptosis of human RCC cell line ACHN, so as to provide radiobiological basis for optimizing IMRT plans for this disease. The ACHN renal cell carcinoma cell line was used in this study. Impact of the triangle, V, small-large or large-small temporal patterns in the presence and absence of threshold dose of hyper-radiosensitivity at the beginning of patterns were studied using soft agarclonogenic assays. Cell cycle and apoptosis analysis were performed after irradiation with the temporal patterns. For triangle and small-large dose sequences, survival fraction was significantly reduced after irradiation with or without threshold dose of hyper-radiosensitivity at the beginning of the patterns. In all of the dose patterns, cell cycle distributions and the percentage of apoptotic cells at 24 h after irradiation with or without priming dose of hyper-radiosensitivity showed no significant difference. However, apoptotic cells were increased when beams with the smallest dose applied at the beginning of dose pattern like triangle and small-large dose sequence. These data show that the biologic effects of single fraction may differ in clinical settings depending on the size and sequence of the partial fractions. Doses at the beginning but not at the end of sequences may change cytotoxicity effects of radiation.

Antiproliferative and cytotoxic effects of green coffee and yerba mate extracts, their main hydroxycinnamic acids, methylxanthine and metabolites in different human cell lines.

PubMed

Amigo-Benavent, M; Wang, S; Mateos, R; Sarriá, B; Bravo, L

2017-08-01

This work aimed at studying the effects of green coffee bean (GCBE) and yerba mate (YME) extracts, their main phenolic components (5-caffeoylquinic acid, 5-CQA; 3,5-dicaffeoylquinic acid, 3,5-DCQA) and metabolites (ferulic acid, FA; caffeic acid, CA; dihydrocaffeic acid, DHCA; and dihydroferulic acid, DHFA) along with caffeine (CAF) on the viability and proliferation of different human cell lines. Extracts (10-1000 μg/mL) and standards (10-1000 μM) were assayed in colon (Caco-2), lung (A549), oesophageal (OE-33), urinary bladder (T24) human carcinoma cells, and a non-cancer cell line (CCD-18Co). YME significantly reduced viability of cancer cells at all assayed concentrations, the higher doses also reducing cell proliferation. GCBE effects on cell viability were more effective at 100 and 1000 μg/mL, showing modest effects on cell proliferation. The highest doses of 5-CQA and 3,5-DCQA reduced cell viability and proliferation in all cell lines, whereas FA, DHCA and DHFA had lower and variable effects. Caffeine had no effect. Dietary-attainable concentrations (0.1, 1 and 10 μg/mL) of YME were tested for cytotoxicity and reactive oxygen species generation, showing no cytotoxic effect. Low concentrations of all tested compounds were non-cytotoxic to CCD-18Co cells. YME and to a lower degree GCBE, their phenolic components and metabolites may decrease cancer cell viability and proliferation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Inhibitory effect of emodin on human hepatoma cell line SMMC-7721 and its mechanism.

PubMed

Zhang, Xia; Chen, Yingping; Zhang, Ting; Zhang, Yaming

2015-03-01

Da Huang (Radix et Rhizoma Rhei) is the dried root or rhizome of Rheum palmatum L., Rheum tanguticum Maxim ex Balf. or Rheum officinale Braill of family Polygonaceae. It has heat clearing, damp drying, fire purging and toxin removing effects. Because of its definite curative efficacy, it has been widely applied in clinical settings. To study the inhibitory effect of emodin on human hepatoma cell line SMMC-7721 and its mechanism. MTT assay, flow cytometry and electron microscopy were used to investigate the inhibitory effect of different concentrations of emodin on human hepatoma cell line SMMC-7721. 12 h, 24 h and 48 h after the action of 20, 40 and 80 umol/L emodin on SMMC-7721 cells, the proliferation of human hepatoma SMMC-7721 cells was inhibited; the inhibitory effects showed time-and concentration-dependence. 48 h after the action of different concentrations of emodin on SMMC-7721 cells, cells in G2/M phase increased significantly, while the proportion of S phase cells gradually declined. Emodin can inhibit human hepatoma cell line SMMC-7721.

In vitro culture of various typed meningiomas and characterization of a human malignant meningioma cell line (HKBMM).

PubMed

Ishiwata, Isamu; Ishiwata, Chieko; Ishiwata, Emiko; Sato, Yoshiro; Kiguchi, Kazushige; Tachibana, Toshiaki; Ishikawa, Hiroshi

2004-12-01

We placed on culture the 13 cases of meningiomas, succeeded in making a primary culture of 10 cases and maintained 5 cases in vitro over considerable period of time (over three month), and one cell line derived from a malignant meningioma were established. In the early period of the primary culture, meningioma cells were spindle- or round-shaped cells. In the case of psammomatous type, the cultured cells were characterized as forming psammoma bodies. A cell line designated "HKBMM" was established from a human malignant meningioma occurred from frontal lobe. This line grew well without interruption for 5 years and was subcultivated over 120 times. The cells were spindle and fibrous in shape, and neoplastic and pleomorphic features, and multilayering without contact inhibition. The cells proliferated rapidly, and the population doubling time was about 29 hours. The chromosome number showed a wide distribution of aneuploidy. The mode was in the diploid range. The culture cells were easily transplanted into the subcutis of nude mice and produced the tumor resembling the original tumor.

A Glucosamine-Specific Lectin from Green Dragon No. 8 Beans (Phaseolus vulgaris) Induced Apoptosis on Nasopharyngeal Carcinoma Cells

PubMed Central

Chan, Yau Sang; Xia, Lixin; Ng, Tzi Bun

2015-01-01

A lectin exhibiting antiproliferative activity on tumor cell lines but devoid of antifungal activity has been purified from Phaseolus vulgaris cv. Green Dragon no. 8 seeds. The lectin was a 60 kDa dimeric protein with two 30 kDa subunits. It was a glucosamine-specific lectin as implied from the inhibitory effect of glucosamine on hemagglutinating activity of the lectin. The steps for isolation of the lectin involved Affi-gel blue gel (affinity gel), Mono Q (anion exchanger), and Superdex 75 column (size exclusion). The lectin was purified 20.8-fold from the crude extract of the beans. The purified lectin showed antiproliferative activity on breast cancer MCF7 cell line and nasopharyngeal cancer HONE1 and CNE2 cell lines, but a low activity on normal skin fibroblast HSF98 cell line. The lectin was shown to induce apoptosis on HONE1 cells, as indicated by increased phosphatidylserine externalization and mitochondrial depolarization. It also blocked HONE1 cell division and kept the cells at the G2/M phase of the cell cycle. PMID:26290674

Isolation of Canine parvovirus with a view to identify the prevalent serotype on the basis of partial sequence analysis

PubMed Central

Kaur, Gurpreet; Chandra, Mudit; Dwivedi, P. N.; Sharma, N. S.

2015-01-01

Aim: The aim of this study was to isolate Canine parvovirus (CPV) from suspected dogs on madin darby canine kidney (MDCK) cell line and its confirmation by polymerase chain reaction (PCR) and nested PCR (NPCR). Further, VP2 gene of the CPV isolates was amplified and sequenced to determine prevailing antigenic type. Materials and Methods: A total of 60 rectal swabs were collected from dogs showing signs of gastroenteritis, processed and subjected to isolation in MDCK cell line. The samples showing cytopathic effects (CPE) were confirmed by PCR and NPCR. These samples were subjected to PCR for amplification of VP2 gene of CPV, sequenced and analyzed to study the prevailing antigenic types of CPV. Results: Out of the 60 samples subjected to isolation in MDCK cell line five samples showed CPE in the form of rounding of cells, clumping of cells and finally detachment of the cells. When these samples and the two commercially available vaccines were subjected to PCR for amplification of VP2 gene, a 1710 bp product was amplified. The sequence analysis revealed that the vaccines belonged to the CPV-2 type and the samples were of CPV-2b type. Conclusion: It can be concluded from the present study that out of a total of 60 samples 5 samples exhibited CPE as observed in MDCK cell line. Sequence analysis of the VP2 gene among the samples and vaccine strains revealed that samples belonged to CPV-2b type and vaccines belonging to CPV-2. PMID:27046996

The anticancer effect of saffron in two p53 isogenic colorectal cancer cell lines

PubMed Central

2012-01-01

Background Saffron extract, a natural product, has been shown to induce apoptosis in several tumor cell lines. Nevertheless, the p53-dependency of saffron’s mechanism of action in colon cancer remains unexplored. Material and methods In order to examine saffron’s anti-proliferative and pro-apoptotic effects in colorectal cancer cells, we treated two p53 isogenic HCT116 cell lines (HCT wildtype and HCT p53−/−) with different doses of the drug and analyzed cell proliferation and apoptosis in a time-dependent manner. MTT viability and crystal violet assays were performed in order to determine the effective dose of saffron on both cell lines. The cell cycle progress was examined by Flow cytometric analysis. Apoptosis was assessed using Annexin-PI-staining and Western Blotting for caspase 3 and PARP cleavage. Autophagy was determined by Western Blotting of the light chain 3 (LC3)-II and Beclin 1 proteins. The protein content of phospho-H2AX (γH2AX), a sensor of DNA double strand breaks, was also analyzed by Western Blotting. Results Saffron extract induced a p53-dependent pattern of cell cycle distribution with a full G2/M stop in HCT116 p53 wildtype cells. However, it induced a remarkable delay in S/G2 phase transit with entry into mitosis in HCT116 p53 −/− cells. The apoptotic Pre-G1 cell fraction as well as Annexin V staining and caspase 3 cleavage showed a more pronounced apoptosis induction in HCT116 p53 wildtype cells. Obviously, the significantly higher DNA-damage, reflected by ɣH2AX protein levels in cells lacking p53, was coped by up-regulation of autophagy. The saffron-induced LC3-II protein level was a remarkable indication of the accumulation of autophagosomes, a response to the cellular stress condition of drug treatment. Conclusions This is the first study showing the effect of saffron in HCT116 colorectal cancer cells with different p53 status. Saffron induced DNA-damage and apoptosis in both cell lines. However, autophagy has delayed the induction of apoptosis in HCT116 p53 −/− cells. Considering the fact that most tumors show a functional p53 inactivation, further research is needed to elucidate the long-term effects of saffron in p53 −/− tumors. PMID:22640402

Synthesis, Antitumor Activity, and Mechanism of Action of 6-Acrylic Phenethyl Ester-2-pyranone Derivatives

PubMed Central

Fang, Sai; Chen, Lei; Yu, Miao; Cheng, Bao; Lin, Yongsheng; Morris-Natschke, Susan L.; Lee, Kuo-Hsiung; Gu, Qiong; Xu, Jun

2015-01-01

Based on the scaffolds of caffeic acid phenethyl ester (CAPE) as well as bioactive lactone-containing compounds, 6-acrylic phenethyl ester-2-pyranone derivatives were synthesized and evaluated against five tumor cell lines (HeLa, C6, MCF-7, A549, and HSC-2). Most of the new derivatives exhibited moderate to potent cytotoxic activity. Moreover, HeLa cell lines showed higher sensitivity to these compounds. Particularly, compound 5o showed potent cytotoxic activity (IC50 = 0.50 – 3.45 μM) against the five cell lines. Further investigation on the mechanism of action showed that 5o induced apoptosis, arrested the cell cycle at G2/M phases in HeLa cells, and inhibited migration through disruption of the actin cytoskeleton. In addition, ADME properties were also calculated in silico, and compound 5o showed good ADMET properties with good absorption, low hepatotoxicity, and good solubility, and thus, could easily be bound to carrier proteins, without inhibition of CYP2D6. A structure-activity relationship (SAR) analysis indicated that compounds with ortho-substitution on the benzene ring exhibited obviously increased cytotoxic potency. This study indicated that compound 5o is a promising compound as an antitumor agent. PMID:25800703

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

PubMed

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

1989-07-01

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

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

PubMed

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

2017-11-01

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

Effect of incubation temperature on neuropeptide Y and neuropeptide Y receptors in turkey and chicken satellite cells.

PubMed

Clark, Daniel L; McCormick, Janet L; Velleman, Sandra G

2018-05-01

Neuropeptide Y (NPY) is an appetite stimulating peptide released from the central nervous system and impacts the function of many different cell types. A recent transcriptome study showed that NPY expression was altered when turkey breast muscle satellite cells were incubated at low or high temperatures, suggesting NPY may mediate temperature effects on satellite cells. However, to date minimal information exists describing the expression and function of NPY in satellite cells. The objective of this study was to determine how temperature impacts NPY and NPY receptor gene expression in satellite cells isolated from turkeys and chickens with differing genetic lineages. Two broiler and two turkey breast muscle satellite cell lines were incubated at 35, 38 or 41 °C during proliferation and differentiation. In both turkey lines, NPY, and receptors NPY2R and NPY5R expression increased at elevated temperatures after 72 h of proliferation. During differentiation NPY and NPY5R expression increased in both turkey lines with higher temperatures, whereas NPY2R was minimally affected by temperature. In contrast, in both chicken cell lines there were few significant differences for NPY and NPY receptor expression across temperature during proliferation. During differentiation, the temperature effect was different in the two chicken cell lines. In the BPM8 chicken line, there were few differences in NPY and NPY receptors across temperature; whereas elevated temperatures increased NPY, NPY2R, and NPY5R expression in the 708 line. The differences between turkey and chicken lines suggest NPY has species specific satellite cell functions in response to heat stress. Copyright © 2018 Elsevier Inc. All rights reserved.

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

PubMed

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

2017-06-07

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

An Efficient Electroporation Protocol for the Genetic Modification of Mammalian Cells

PubMed Central

Chicaybam, Leonardo; Barcelos, Camila; Peixoto, Barbara; Carneiro, Mayra; Limia, Cintia Gomez; Redondo, Patrícia; Lira, Carla; Paraguassú-Braga, Flávio; Vasconcelos, Zilton Farias Meira De; Barros, Luciana; Bonamino, Martin Hernán

2017-01-01

Genetic modification of cell lines and primary cells is an expensive and cumbersome approach, often involving the use of viral vectors. Electroporation using square-wave generating devices, like Lonza’s Nucleofector, is a widely used option, but the costs associated with the acquisition of electroporation kits and the transient transgene expression might hamper the utility of this methodology. In the present work, we show that our in-house developed buffers, termed Chicabuffers, can be efficiently used to electroporate cell lines and primary cells from murine and human origin. Using the Nucleofector II device, we electroporated 14 different cell lines and also primary cells, like mesenchymal stem cells and cord blood CD34+, providing optimized protocols for each of them. Moreover, when combined with sleeping beauty-based transposon system, long-term transgene expression could be achieved in all types of cells tested. Transgene expression was stable and did not interfere with CD34+ differentiation to committed progenitors. We also show that these buffers can be used in CRISPR-mediated editing of PDCD1 gene locus in 293T and human peripheral blood mononuclear cells. The optimized protocols reported in this study provide a suitable and cost-effective platform for the genetic modification of cells, facilitating the widespread adoption of this technology. PMID:28168187

Vesicle-associated membrane protein 7 (VAMP-7) is essential for target cell killing in a natural killer cell line

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

Marcet-Palacios, Marcelo; Odemuyiwa, Solomon O.; Coughlin, Jason J.

2008-02-15

Natural killer cells recognize and induce apoptosis in foreign, transformed or virus-infected cells through the release of perforin and granzymes from secretory lysosomes. Clinically, NK-cell mediated killing is a major limitation to successful allo- and xenotransplantation. The molecular mechanisms that regulate the fusion of granzyme B-containing secretory lysosomes to the plasma membrane in activated NK cells, prior to target cell killing, are not fully understood. Using the NK cell line YT-Indy as a model, we have investigated the expression of SNAP REceptors (SNAREs), both target (t-) and vesicular (v-) SNAREs, and their function in granzyme B-mediated target cell killing. Ourmore » data showed that YT-Indy cells express VAMP-7 and SNAP-23, but not VAMP-2. VAMP-7 was associated with granzyme B-containing lysosomal granules. Using VAMP-7 small interfering RNA (siRNA), we successfully knocked down the expression of VAMP-7 protein in YT-Indy to less than 10% of untreated cells in 24 h. VAMP7-deficient YT-Indy cells activated via co-culture with Jurkat cells released <1 ng/mL of granzyme B, compared to 1.5-2.5 {mu}g/mL from controls. Using Jurkat cells as targets, we showed a 7-fold reduction in NK cell-mediated killing by VAMP-7 deficient YT-Indy cells. Our results show that VAMP-7 is a crucial component of granzyme B release and target cell killing in the NK cell line YT-Indy. Thus, targeting VAMP-7 expression specifically with siRNA, following transplantation, may be a viable strategy for preventing NK cell-mediated transplant rejection, in vivo.« less

Mitochondrial genome-knockout cells demonstrate a dual mechanism of action for the electron transport complex I inhibitor mycothiazole.

PubMed

Meyer, Kirsten J; Singh, A Jonathan; Cameron, Alanna; Tan, An S; Leahy, Dora C; O'Sullivan, David; Joshi, Praneta; La Flamme, Anne C; Northcote, Peter T; Berridge, Michael V; Miller, John H

2012-04-01

Mycothiazole, a polyketide metabolite isolated from the marine sponge Cacospongia mycofijiensis, is a potent inhibitor of metabolic activity and mitochondrial electron transport chain complex I in sensitive cells, but other cells are relatively insensitive to the drug. Sensitive cell lines (IC(50) 0.36-13.8 nM) include HeLa, P815, RAW 264.7, MDCK, HeLa S3, 143B, 4T1, B16, and CD4/CD8 T cells. Insensitive cell lines (IC(50) 12.2-26.5 μM) include HL-60, LN18, and Jurkat. Thus, there is a 34,000-fold difference in sensitivity between HeLa and HL-60 cells. Some sensitive cell lines show a biphasic response, suggesting more than one mechanism of action. Mitochondrial genome-knockout ρ(0) cell lines are insensitive to mycothiazole, supporting a conditional mitochondrial site of action. Mycothiazole is cytostatic rather than cytotoxic in sensitive cells, has a long lag period of about 12 h, and unlike the complex I inhibitor, rotenone, does not cause G(2)/M cell cycle arrest. Mycothiazole decreases, rather than increases the levels of reactive oxygen species after 24 h. It is concluded that the cytostatic inhibitory effects of mycothiazole on mitochondrial electron transport function in sensitive cell lines may depend on a pre-activation step that is absent in insensitive cell lines with intact mitochondria, and that a second lower-affinity cytotoxic target may also be involved in the metabolic and growth inhibition of cells.

Macroalgae Extracts From Antarctica Have Antimicrobial and Anticancer Potential

PubMed Central

Martins, Rosiane M.; Nedel, Fernanda; Guimarães, Victoria B. S.; da Silva, Adriana F.; Colepicolo, Pio; de Pereira, Claudio M. P.; Lund, Rafael G.

2018-01-01

Background: Macroalgae are sources of bioactive compounds due to the large number of secondary metabolites they synthesize. The Antarctica region is characterized by extreme weather conditions and abundant aggregations of macroalgae. However, current knowledge on their biodiversity and their potential for bio-prospecting is still fledging. This study evaluates the antimicrobial and cytotoxic activity of different extracts of four macroalgae (Cystosphaera jacquinotii, Iridaea cordata, Himantothallus grandifolius, and Pyropia endiviifolia) from the Antarctic region against cancer and non-cancer cell lines. Methods: The antimicrobial activity of macroalgae was evaluated by the broth microdilution method. Extracts were assessed against Staphylococcus aureus ATCC 19095, Enterococcus faecalis ATCC 4083, Escherichia coli ATCC29214, Pseudomonas aeruginosa ATCC 9027, Candida albicans ATCC 62342, and the clinical isolates from the human oral cavity, namely, C. albicans (3), C. parapsilosis, C. glabrata, C. lipolytica, and C. famata. Cytotoxicity against human epidermoid carcinoma (A-431) and mouse embryonic fibroblast (NIH/3T3) cell lines was evaluated with MTT colorimetric assay. Results: An ethyl acetate extract of H. grandifolius showed noticeable antifungal activity against all fungal strains tested, including fluconazole-resistant samples. Cytotoxicity investigation with a cancer cell line revealed that the ethyl acetate extract of I. cordata was highly cytotoxic against A-431 cancer cell line, increasing the inhibitory ratio to 91.1 and 95.6% after 24 and 48 h exposure, respectively, for a concentration of 500 μg mL−1. Most of the algal extracts tested showed little or no cytotoxicity against fibroblasts. Conclusion: Data suggest that macroalgae extracts from Antarctica may represent a source of therapeutic agents. HIGHLIGHTS Different macroalgae samples from Antarctica were collected and the lyophilized biomass of each macroalgae was extracted sequentially with different solventsThe antimicrobial and anticancer potential of macroalgae extracts were evaluatedEthyl acetate extract of H. grandifolius showed noticeable antifungal activity against all the fungal strains tested, including fluconazole-resistant samplesEthyl acetate extract of I. cordata was highly cytotoxic against the A-431 cancer cell lineMost of the algal extracts tested showed little or no cytotoxicity against normal cell lines PMID:29568291

Tuft (caveolated) cells in two human colon carcinoma cell lines.

PubMed

Barkla, D H; Whitehead, R H; Foster, H; Tutton, P J

1988-09-01

The presence of an unusual cell type in two human colon carcinoma cell lines is reported. The cells show the same morphology as "tuft" (caveolated) cells present in normal gastrointestinal epithelium. Tuft cells were seen in cell line LIM 1863 growing in vitro and in human colon carcinoma cell line LIM 2210 growing as subcutaneous solid tumour xenografts in nude mice. Characteristic morphologic features of tuft cells included a wide base, narrow apex and a tuft of long microvilli projecting from the apical surface. The microvilli are attached by a core of long microfilaments passing deep into the apical cytoplasm. Between the microvilli are parallel arrays of vesicles (caveoli) containing flocculent material. Two different but not mutually exclusive explanations for the presence of tuft cells are proposed. The first explanation is that tuft cells came from the resected tumour and have survived by mitotic division during subsequent passages. The second explanation suggests that tuft cells are the progeny of undifferentiated tumour cells. Descriptions of tuft cells in colon carcinomas are uncommon and possible reasons for this are presented. The morphology of tuft cells is consistent with that of a highly differentiated cell specialised for absorption, and these new models provide an opportunity to further investigate the structure and function of tuft cells.

Generation of human cortical neurons from a new immortal fetal neural stem cell line.

PubMed

Cacci, E; Villa, A; Parmar, M; Cavallaro, M; Mandahl, N; Lindvall, O; Martinez-Serrano, A; Kokaia, Z

2007-02-01

Isolation and expansion of neural stem cells (NSCs) of human origin are crucial for successful development of cell therapy approaches in neurodegenerative diseases. Different epigenetic and genetic immortalization strategies have been established for long-term maintenance and expansion of these cells in vitro. Here we report the generation of a new, clonal NSC (hc-NSC) line, derived from human fetal cortical tissue, based on v-myc immortalization. Using immunocytochemistry, we show that these cells retain the characteristics of NSCs after more than 50 passages. Under proliferation conditions, when supplemented with epidermal and basic fibroblast growth factors, the hc-NSCs expressed neural stem/progenitor cell markers like nestin, vimentin and Sox2. When growth factors were withdrawn, proliferation and expression of v-myc and telomerase were dramatically reduced, and the hc-NSCs differentiated into glia and neurons (mostly glutamatergic and GABAergic, as well as tyrosine hydroxylase-positive, presumably dopaminergic neurons). RT-PCR analysis showed that the hc-NSCs retained expression of Pax6, Emx2 and Neurogenin2, which are genes associated with regionalization and cell commitment in cortical precursors during brain development. Our data indicate that this hc-NSC line could be useful for exploring the potential of human NSCs to replace dead or damaged cortical cells in animal models of acute and chronic neurodegenerative diseases. Taking advantage of its clonality and homogeneity, this cell line will also be a valuable experimental tool to study the regulatory role of intrinsic and extrinsic factors in human NSC biology.

Epstein-Barr virus (EBV) provides survival factors to EBV+ diffuse large B-cell lymphoma (DLBCL) lines and modulates cytokine induced specific chemotaxis in EBV+  DLBCL.

PubMed

Wu, Liang; Ehlin-Henriksson, Barbro; Zhou, Xiaoying; Zhu, Hong; Ernberg, Ingemar; Kis, Lorand L; Klein, George

2017-12-01

Diffuse large B-cell lymphoma (DLBCL), the most common type of malignant lymphoma, accounts for 30% of adult non-Hodgkin lymphomas. Epstein-Barr virus (EBV) -positive DLBCL of the elderly is a newly recognized subtype that accounts for 8-10% of DLBCLs in Asian countries, but is less common in Western populations. Five DLBCL-derived cell lines were employed to characterize patterns of EBV latent gene expression, as well as response to cytokines and chemotaxis. Interleukin-4 and interleukin-21 modified LMP1, EBNA1 and EBNA2 expression depending on cell phenotype and type of EBV latent programme (type I, II or III). These cytokines also affected CXCR4- or CCR7-mediated chemotaxis in two of the cell lines, Farage (type III) and Val (type II). Further, we investigated the effect of EBV by using dominant-negative EBV nuclear antigen 1(dnEBNA1) to eliminate EBV genomes. This resulted in decreased chemotaxis. By employing an alternative way to eliminate EBV genomes, Roscovitine, we show an increase of apoptosis in the EBV-positive lines. These results show that EBV plays an important role in EBV-positive DLBCL lines with regard to survival and chemotactic response. Our findings provide evidence for the impact of microenvironment on EBV-carrying DLBCL cells and might have therapeutic implications. © 2017 John Wiley & Sons Ltd.

Early exposure to interleukin-21 limits rapidly generated anti-Epstein-Barr virus T-cell line differentiation.

PubMed

Orio, Julie; Carli, Cédric; Janelle, Valérie; Giroux, Martin; Taillefer, Julie; Goupil, Mathieu; Richaud, Manon; Roy, Denis-Claude; Delisle, Jean-Sébastien

2015-04-01

The adoptive transfer of ex vivo-expanded Epstein-Barr virus (EBV)-specific T-cell lines is an attractive strategy to treat EBV-related neoplasms. Current evidence suggests that for adoptive immunotherapy in general, clinical responses are superior if the transferred cells have not reached a late or terminal effector differentiation phenotype before infusion. The cytokine interleukin (IL)-21 has shown great promise at limiting late T-cell differentiation in vitro, but this remains to be demonstrated in anti-viral T-cell lines. We adapted a clinically validated protocol to rapidly generate EBV-specific T-cell lines in 12 to 14 days and tested whether the addition of IL-21 at the initiation of the culture would affect T-cell expansion and differentiation. We generated clinical-scale EBV-restricted T-cell line expansion with balanced T-cell subset ratios. The addition of IL-21 at the beginning of the culture decreased both T-cell expansion and effector memory T-cell accumulation, with a relative increase in less-differentiated T cells. Within CD4 T-cell subsets, exogenous IL-21 was notably associated with the cell surface expression of CD27 and high KLF2 transcript levels, further arguing for a role of IL-21 in the control of late T-cell differentiation. Our results show that IL-21 has profound effects on T-cell differentiation in a rapid T-cell line generation protocol and as such should be further explored as a novel approach to program anti-viral T cells with features associated with early differentiation and optimal therapeutic efficacy. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Combined effect of microRNA, nutraceuticals and drug on pancreatic cancer cell lines.

PubMed

Pandita, Archana; Manvati, Siddharth; Singh, Shashank K; Vaishnavi, Samantha; Bamezai, Rameshwar N K

2015-05-25

We proposed to investigate the combination effect of microRNA, nutraceuticals and drug (MND), in two pancreatic cancer cell lines to assess the therapeutic potential. MIA PaCa-2 and PANC-1 cells transfected with miR-101 or miR-24-2 were treated with Betulinic acid or Thymoquinone and gemcitabine independently and in combination and assessed for the extent of synergism in both experimental and control conditions, considering significance at the p value of <0.05. miR-101 or miR-24-2 over-expressing cells when treated with lower than IC50 doses of the dietary compounds and drug showed a reduced (37-50%) viability in two cell lines with differential synergistic effect and the outcome for Pro-caspase3, Poly (ADP-ribose) polymerase (PARP) cleavage and PKM2 expression. Two independent microRNA backgrounds showed promise in therapeutic intervention of gemcitabine sensitive, MIA PaCa-2 and resistant, PANC-1 pancreatic cancer cells, in combination with dietary agents and drug. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Novel menadione hybrids: Synthesis, anticancer activity, and cell-based studies.

PubMed

Prasad, Chakka Vara; Nayak, Vadithe Lakshma; Ramakrishna, Sistla; Mallavadhani, Uppuluri Venkata

2018-01-01

A series of novel menadione-based triazole hybrids were designed and synthesized by employing copper-catalyzed azide-alkyne cycloaddition (CuAAC). All the synthesized hybrids were characterized by their spectral data ( 1 H NMR, 13 C NMR, IR, and HRMS). The synthesized compounds were evaluated for their anticancer activity against five selected cancer cell lines including lung (A549), prostate (DU-145), cervical (Hela), breast (MCF-7), and mouse melanoma (B-16) using MTT assay. The screening results showed that majority of the synthesized compounds displayed significant anticancer activity. Among the tested compounds, the triazoles 5 and 6 exhibited potent activity against all cell lines. In particular, compound 6 showed higher potency than the standard tamoxifen and parent menadione against MCF-7 cell line. Flow cytometric analysis revealed that compound 6 arrested cell cycle at G0/G1 phase and induced apoptotic cell death which was further confirmed by Hoechst staining, measurement of mitochondrial membrane potential (ΔΨm) and Annexin-V-FITC assay. Thus, compound 6 can be considered as lead molecule for further development as potent anticancer therapeutic agent. © 2017 John Wiley & Sons A/S.

Effects of phytoestrogens on the trophoblast tumour cell lines BeWo and Jeg3.

PubMed

Plessow, D; Waldschläger, J; Richter, D U; Jeschke, U; Bruer, G; Briese, V; Friese, K

2003-01-01

Phytoestrogens are a diverse group of nonsteroidal plant compounds that occur naturally in many plants. Because they possess a ring system similar to estrogens they are able to bind to estrogen receptors in humans. With this study we tested the effects of the phytoestrogens genistein and daidzein in cell proliferation and the production of progesterone and hCG in trophoblast tumour cells of the cell lines BeWo and Jeg3. The phytoestrogens genistein and daidzein were incubated in different concentrations with trophoblast tumour cells. Untreated cells were used as controls. At designated times, aliquots were removed and tested for progesterone and hCG. In addition we tested the effects of phytoestrogens on cell proliferation. Different concentrations of genistein and daidzein were cultivated with trophoblast tumour cells. After designated times, 1 microCi thymidin-(methyl-3H) was added. Methyl-3H thymidin incorporation was tested and compared to incorporation results of untreated cells. With this study we could show that the production of the steroid hormone progesterone and the protein hormone hCG is influenced by the phytoestrogens genistein and daidzein in trophoblast tumour cells of the cell lines BeWo and Jeg3. We found a correlation between the effects on the proliferation and the production of progesterone and hCG at high concentrations of genistein and daidzein in the cell lines tested. With low concentrations of genistein and daidzein we observed a stimulation of the production of hCG and a weak inhibition of proliferation in both cell lines BeWo and Jeg3. The results obtained with this study suggest that only high doses of phytoestrogens (> 1 mumol/ml) can reduce the proliferation of trophoblast tumour cells significantly. Low doses of phytoestrogens induced a higher hCG production in both cell lines tested. Although high hCG production did not lead to a higher proliferation rate of the tumour cells tested, hCG is able to induce neovascularisation in tumour cells. In summary, with this in vitro study we showed that high doses of phytoestrogens inhibit proliferation and progesterone production in trophoblast tumour cells. High doses of phytoestrogens could be useful candidates for special diet programs for prevention and surgery for patients with this type of disease. In addition we found a useful cell culture model for the testing of new types of phytoestrogens.

A review of the emerging potential therapy for neurological disorders: human embryonic stem cell therapy

PubMed Central

Shroff, Geeta; Dhanda Titus, Jyoti; Shroff, Rhea

2017-01-01

The first human embryonic stem cell (hESC) line was developed in the late nineties. hESCs are capable of proliferating indefinitely and differentiate into all the three embryonic germ layers. Further, the differentiation of hESC lines into neural precursor cells and neurons, astrocytes and oligodendrocytes showed their potential in treating several incurable neurological disorders such as spinal cord injury (SCI), cerebral palsy (CP), Parkinson’s disease (PD). In this review, we will discuss the global scenario of research and therapeutic use of hESCs in the treatment of neurological disorders. Following this, we will discuss the development of a unique hESC line, how it differs from the other available hESC lines and its use in the treatment of neurological disorders. hESCs were isolated from mixture of neuronal and non-neuronal progenitor cells in their pre progenitor state in a Good Laboratory Practices, Good Tissue Practices and Good Manufacturing Practices compliant laboratory. Blastomere cells have served as a source to derive the hESCs and the xeno-free culture was demonstrated to be more safe and effective in clinical therapeutic application of hESCs. All the patients showed a remarkable improvement in their conditions and no serious adverse events were reported. This study concluded that hESC lines could be scalable and used in the treatment of various neurological disorders such as SCI, CP, and PD. PMID:28533935

Changes in Cell Wall Properties Coincide with Overexpression of Extensin Fusion Proteins in Suspension Cultured Tobacco Cells

DOE PAGES

Tan, Li; Pu, Yunqiao; Pattathil, Sivakumar; ...

2014-12-23

Extensins are one subfamily of the cell wall hydroxyproline-rich glycoproteins, containing characteristic SerHyp4 glycosylation motifs and intermolecular cross-linking motifs such as the TyrXaaTyr sequence. Extensins are believed to form a cross-linked network in the plant cell wall through the tyrosine-derivatives isodityrosine, pulcherosine, and di-isodityrosine. Overexpression of three synthetic genes encoding different elastin-arabinogalactan protein-extensin hybrids in tobacco suspension cultured cells yielded novel cross-linking glycoproteins that shared features of the extensins, arabinogalactan proteins and elastin. The cell wall properties of the three transgenic cell lines were all changed, but in different ways. One transgenic cell line showed decreased cellulose crystallinity and increasedmore » wall xyloglucan content; the second transgenic cell line contained dramatically increased hydration capacity and notably increased cell wall biomass, increased di-isodityrosine, and increased protein content; the third transgenic cell line displayed wall phenotypes similar to wild type cells, except changed xyloglucan epitope extractability. In conclusion, these data indicate that overexpression of modified extensins may be a route to engineer plants for bioenergy and biomaterial production.« less

Cytotoxic Effect on Human Myeloma Cells and Leukemic Cells by the Agaricus blazei Murill Based Mushroom Extract, Andosan™

PubMed Central

Holien, Toril; Mirlashari, Mohammad Reza; Misund, Kristine

2017-01-01

Agaricus blazei Murill is an edible mushroom of the Basidiomycetes family, which has been found to contain a number of compounds with antitumor properties, such as proteoglycans and ergosterol. In the present investigation, we show that the commercial mushroom product Andosan, which contains 82.4% Agaricus blazei Murill, together with medicinal mushrooms Hericium erinaceus (14.7%) and Grifola frondosa (2.9%), has a cytotoxic effect on primary myeloma cells, other myeloma cell lines, and leukemia cell lines in vitro. Although the exact content and hence the mechanisms of action of the Andosan extract are unknown, we have found in this investigation indications of cell cycle arrest when myeloma cell lines are cultivated with Andosan. This may be one of the possible explanations for the cytotoxic effects of Andosan. PMID:29238712

Cytotoxic Effect on Human Myeloma Cells and Leukemic Cells by the Agaricus blazei Murill Based Mushroom Extract, Andosan™.

PubMed

Tangen, Jon-Magnus; Holien, Toril; Mirlashari, Mohammad Reza; Misund, Kristine; Hetland, Geir

2017-01-01

Agaricus blazei Murill is an edible mushroom of the Basidiomycetes family, which has been found to contain a number of compounds with antitumor properties, such as proteoglycans and ergosterol. In the present investigation, we show that the commercial mushroom product Andosan, which contains 82.4% Agaricus blazei Murill, together with medicinal mushrooms Hericium erinaceus (14.7%) and Grifola frondosa (2.9%), has a cytotoxic effect on primary myeloma cells, other myeloma cell lines, and leukemia cell lines in vitro. Although the exact content and hence the mechanisms of action of the Andosan extract are unknown, we have found in this investigation indications of cell cycle arrest when myeloma cell lines are cultivated with Andosan. This may be one of the possible explanations for the cytotoxic effects of Andosan.

Systemic Analysis of Heat Shock Response Induced by Heat Shock and a Proteasome Inhibitor MG132

PubMed Central

Kim, Hee-Jung; Joo, Hye Joon; Kim, Yung Hee; Ahn, Soyeon; Chang, Jun; Hwang, Kyu-Baek; Lee, Dong-Hee; Lee, Kong-Joo

2011-01-01

The molecular basis of heat shock response (HSR), a cellular defense mechanism against various stresses, is not well understood. In this, the first comprehensive analysis of gene expression changes in response to heat shock and MG132 (a proteasome inhibitor), both of which are known to induce heat shock proteins (Hsps), we compared the responses of normal mouse fibrosarcoma cell line, RIF- 1, and its thermotolerant variant cell line, TR-RIF-1 (TR), to the two stresses. The cellular responses we examined included Hsp expressions, cell viability, total protein synthesis patterns, and accumulation of poly-ubiquitinated proteins. We also compared the mRNA expression profiles and kinetics, in the two cell lines exposed to the two stresses, using microarray analysis. In contrast to RIF-1 cells, TR cells resist heat shock caused changes in cell viability and whole-cell protein synthesis. The patterns of total cellular protein synthesis and accumulation of poly-ubiquitinated proteins in the two cell lines were distinct, depending on the stress and the cell line. Microarray analysis revealed that the gene expression pattern of TR cells was faster and more transient than that of RIF-1 cells, in response to heat shock, while both RIF-1 and TR cells showed similar kinetics of mRNA expression in response to MG132. We also found that 2,208 genes were up-regulated more than 2 fold and could sort them into three groups: 1) genes regulated by both heat shock and MG132, (e.g. chaperones); 2) those regulated only by heat shock (e.g. DNA binding proteins including histones); and 3) those regulated only by MG132 (e.g. innate immunity and defense related molecules). This study shows that heat shock and MG132 share some aspects of HSR signaling pathway, at the same time, inducing distinct stress response signaling pathways, triggered by distinct abnormal proteins. PMID:21738571

Synthesis and biological evaluation of pyrimidine bridged combretastatin derivatives as potential anticancer agents and mechanistic studies.

PubMed

Kumar, Bhupinder; Sharma, Praveen; Gupta, Vivek Prakash; Khullar, Madhu; Singh, Sandeep; Dogra, Nilambra; Kumar, Vinod

2018-08-01

A number of pyrimidine bridged combretastatin derivatives were designed, synthesized and evaluated for anticancer activities against breast cancer (MCF-7) and lung cancer (A549) cell lines using MTT assays. Most of the synthesized compounds displayed good anticancer activity with IC 50 values in low micro-molar range. Compounds 4a and 4p were found most potent in the series with IC 50 values of 4.67 µM & 3.38 µM and 4.63 µM & 3.71 µM against MCF7 and A549 cancer cell lines, respectively. Biological evaluation of these compounds showed that selective cancer cell toxicity (in vitro using human lung and breast cancer cell lines) might be due to the inhibition of antioxidant enzymes instigating elevated ROS levels which triggers intrinsic apoptotic pathways. These compounds were found nontoxic to the normal human primary cells. Compound 4a, was found to be competitive inhibitor of colchicine and in the tubulin binding assay it showed tubulin polymerization inhibition potential comparable to colchicine. The molecular modeling studies also showed that the synthesized compounds fit well in the colchicine-binding pocket. Copyright © 2018 Elsevier Inc. All rights reserved.

Cytotoxic activity of vitamins K1, K2 and K3 against human oral tumor cell lines.

PubMed

Okayasu, H; Ishihara, M; Satoh, K; Sakagami, H

2001-01-01

Vitamin K1, K2 and K3 were compared for their cytotoxic activity, radical generation and O2- scavenging activity. Among these compounds, vitamin K3 showed the highest cytotoxic activity against human oral tumor cell lines (HSC-2, HSG), human promyelocytic leukemic cell line (HL-60) and human gingival fibroblast (HGF). Vitamin K3 induced internucleosomal DNA fragmentation in HL-60 cells, but not in HSC-2 or HSG cells. The cytotoxic activity of vitamins K2 and K1 was one and two orders lower, respectively, than K3. Vitamin K2, but not vitamin K3, showed tumor-specific cytotoxic action. ESR spectroscopy showed that only vitamin K3 produced radical(s) under alkaline condition and most potently enhanced the radical intensity of sodium ascorbate and scavenged O2- (generated by hypoxanthine-xanthine oxidase reaction system); vitamin K2 was much less active whereas vitamin K1 was inactive. These data suggest that the cytotoxic activity of vitamin K3 is generated by radical-mediated oxidation mechanism and that this vitamin has two opposing actions (that is, antioxidant and prooxidant), depending on the experimental conditions.

Plectin deficiency in liver cancer cells promotes cell migration and sensitivity to sorafenib treatment.

PubMed

Cheng, Chiung-Chi; Chao, Wei-Ting; Liao, Chen-Chun; Tseng, Yu-Hui; Lai, Yen-Chang Clark; Lai, Yih-Shyong; Hsu, Yung-Hsiang; Liu, Yi-Hsiang

2018-01-02

Plectin involved in activation of kinases in cell signaling pathway and plays important role in cell morphology and migration. Plectin knockdown promotes cell migration by activating focal adhesion kinase and Rac1-GTPase activity in liver cells. Sorafenib is a multi-targeting tyrosine kinase inhibitor that improves patient survival on hepatocellular carcinoma. The aim of this study is to investigate the correlation between the expression of plectin and cell migration as well as the sensitivity of hepatoma cell lines exposing to sorafenib. Hepatoma cell lines PLC/PRF/5 and HepG2 were used to examine the level of plectin expression and cell migration in comparison with Chang liver cell line. In addition, sensitivity of the 3 cell lines to sorafenib treatment was also measured. Expression of plectin was lower in PLC/PRF/5 and HepG2 hepatoma cells than that of Chang liver cells whereas HepG2 and PLC/PRF/5 cells exhibit higher rate of cell migration in trans-well migration assay. Immunohistofluorecent staining on E-cadherin revealed the highest rate of collective cell migration in HepG2 cells and the lowest was found in Chang liver cells. Likewise, HepG2 cell line was most sensitive to sorafenib treatment and Chang liver cells exhibited the least sensitivity. The drug sensitivity to sorafenib treatment showed inverse correlation with the expression of plectin. We suggest that plectin deficiency and increased E-cadherin in hepatoma cells were associated with higher rates of cell motility, collective cell migration as well as higher drug sensitivity to sorafenib treatment.

Active microwave negative-index metamaterial transmission line with gain.

PubMed

Jiang, Tao; Chang, Kihun; Si, Li-Ming; Ran, Lixin; Xin, Hao

2011-11-11

We studied the active metamaterial transmission line at microwave frequency. The active composite right-handed or left-handed transmission line was designed to incorporate a germanium tunnel diode with a negative differential resistance property as the gain device at the unit cell level. Measurements of the fabricated planar transmission line structures with one-, two-, and three-unit cells showed that the addition of the dc pumped tunnel diodes not only provided gain but also maintained the left handedness of the transmission line metamaterial. Simulation results agree well with experimental observation. This work demonstrated that negative index material can be obtained with a net gain when an external source is incorporated.

MicroRNA-145 is regulated by DNA methylation and p53 gene mutation in prostate cancer

PubMed Central

Suh, Seong O.; Chen, Yi; Zaman, Mohd Saif; Hirata, Hiroshi; Yamamura, Soichiro; Shahryari, Varahram; Liu, Jan; Tabatabai, Z.Laura; Kakar, Sanjay; Deng, Guoren; Tanaka, Yuichiro; Dahiya, Rajvir

2011-01-01

MiR-145 is downregulated in various cancers including prostate cancer. However, the underlying mechanisms of miR-145 downregulation are not fully understood. Here, we reported that miR-145 was silenced through DNA hypermethylation and p53 mutation status in laser capture microdissected (LCM) prostate cancer and matched adjacent normal tissues. In 22 of 27 (81%) prostate tissues, miR-145 was significantly downregulated in the cancer compared with the normal tissues. Further studies on miR-145 downregulation mechanism showed that miR-145 is methylated at the promoter region in both prostate cancer tissues and 50 different types of cancer cell lines. In seven cancer cell lines with miR-145 hypermethylation, 5-aza-2′-deoxycytidine treatment dramatically induced miR-145 expression. Interestingly, we also found a significant correlation between miR-145 expression and the status of p53 gene in both LCM prostate tissues and 47 cancer cell lines. In 29 cell lines with mutant p53, miR-145 levels were downregulated in 28 lines (97%), whereas in 18 cell lines with wild-type p53 (WT p53), miR-145 levels were downregulated in only 6 lines (33%, P < 0.001). Electrophoretic mobility shift assay showed that p53 binds to the p53 response element upstream of miR-145, but the binding was inhibited by hypermethylation. To further confirm that p53 binding to miR-145 could regulate miR-145 expression, we transfected WT p53 and MUT p53 into PC-3 cells and found that miR-145 is upregulated by WT p53 but not with MUTp53. The apoptotic cells are increased after WT p53 transfection. In summary, this is the first report documenting that downregulation of miR-145 is through DNA methylation and p53 mutation pathways in prostate cancer. PMID:21349819

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

PubMed Central

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

2015-01-01

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

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

Kiradzhiyska, D. D., E-mail: denica.kiradjiiska@gmail.com; Mantcheva, R. D., E-mail: r-manch@abv.bg; Feodorova, Y. N.

Materials for medical implants should have suitable mechanical properties, excellent biocompatibility and high corrosion resistance. They should not stimulate allergic and immunologic reactions and should not cause cancer. The use of aluminum as a construction material in implantology is continuously expanding. There are various methods for surface treatment to improve its biocompatibility. In this study aluminum samples anodized in 15% H{sub 2} SO{sub 4} or treated with positive or negative corona discharge were investigated. PDL-cell line of immortalized cells, precursors of periodontal ligament and RAW 264.7 cell line from mouse macrophages are used for the bioassays. The results show thatmore » 10 and 20 μm thick oxide film provides better development of the PLD cells, compared to untreated aluminum. Metal surfaces with 10 μm thick oxide film show the best properties in terms of cells vitality, proliferation and growth. Polymer treated but uncharged samples show good results.« less

Nitrophenols isolated from diesel exhaust particles promote the growth of MCF-7 breast adenocarcinoma cells

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

Furuta, Chie; Laboratory of Veterinary Physiology, Department of Veterinary Medicine, Faculty of Agriculture, Tokyo University of Agriculture and Technology, Tokyo 183-8509; Suzuki, Akira K.

2008-08-01

Diesel exhaust particles (DEPs) cause many adverse health problems, and reports indicate increased risk of breast cancer in men and women through exposure to gasoline and vehicle exhaust. However, DEPs include vast numbers of compounds, and the specific compound(s) responsible for these actions are not clear. We recently isolated two nitrophenols from DEPs-3-methyl-4-nitrophenol (4-nitro-m-cresol; PNMC) and 4-nitro-3-phenylphenol (PNMPP)-and showed that they had estrogenic and anti-androgenic activities. Here, we tried to clarify the involvement of these two nitrophenols in promoting the growth of the MCF-7 breast cancer cell line. First, comet assay was used to detect the genotoxicity of PNMC andmore » PNMPP in a CHO cell line. At all doses tested, PNMC and PNMPP showed negative genotoxicity, indicating that they had no tumor initiating activity. Next, the estrogen-responsive breast cancer cell line MCF-7 was used to assess cell proliferation. Proliferation of MCF-7 cells was stimulated by PNMC, PNMPP, and estradiol-17{beta} and the anti-estrogens 4-hydroxytamoxifen and ICI 182,780 inhibited the proliferation. To further investigate transcriptional activity through the estrogen receptor, MCF-7 cells were transfected with a receptor gene that allowed expression of luciferase enzyme under the control of the estrogen regulatory element. PNMC and PNMPP induced luciferase activity in a dose-dependent manner at submicromolar concentrations. ICI 182,780 inhibited the luciferase activity induced by PNMC and PNMPP. These results clearly indicate that PNMC and PNMPP do not show genotoxicity but act as tumor promoters in an estrogen receptor {alpha}-predominant breast cancer cell line.« less

Antiproliferative and apoptotic activities of extracts of Asclepias subulata.

PubMed

Rascón Valenzuela, Luisa Alondra; Jiménez Estrada, Manuel; Velázquez Contreras, Carlos Arturo; Garibay Escobar, Adriana; Medina Juárez, Luis Angel; Gámez Meza, Nohemi; Robles Zepeda, Ramón Enrique

2015-01-01

Asclepias subulata Decne. (Apocynaceae) is a shrub used in the Mexican traditional medicine for the treatment of cancer. The objective of this study was to evaluate the antiproliferative activity of methanol extract of aerial parts of A. subulata and its fractions against different cancer cell lines. Additionally, we analyzed the mechanism of action of the active fractions. Methanol extract fractions were prepared by serial extraction with n-hexane, ethyl acetate, and ethanol. The antiproliferative activity of methanol extract and its fractions was evaluated, against several murine (M12.C3.F6, RAW 264.7, and L929) and human (HeLa, A549, PC-3, LS 180, and ARPE-19) cell lines by the MTT assay, using concentrations of 0.4-400 µg/mL for 48 h. Ethanol and residual fractions were separated using silica gel column. Apoptosis induction of cancer cells was evaluated by Annexin and JC-1 staining using flow cytometry. Methanol extract and its fractions showed antiproliferative activity against all human cancer cell lines tested. Methanol extract had the highest antiproliferative activity on A549 and HeLa cells (IC50 values < 0.4 and 8.7 µg/mL, respectively). Ethanol and residual fractions exerted significant antiproliferative effect on A549 (IC50 < 0.4 µg/mL) and PC3 cells (IC50 1.4 and 5.1 µg/mL). Apoptotic assays showed that CEF7, CEF9, CRF6, and CRF5 fractions induced mitochondrial depolarization in A549 cells, 70, 73, 77, and 80%, respectively. Those fractions triggered the apoptosis mitochondrial pathway. Our data show that A. subulata extracts have potent antiproliferative properties on human cancer cell lines. This plant should be considered an important source of potent anticancer compounds.

Targeting malignant B cells with an immunotoxin against ROR1

PubMed Central

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

2012-01-01

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

Glioblastoma entities express subtle differences in molecular composition and response to treatment

PubMed Central

Balça-Silva, Joana; Matias, Diana; Do Carmo, Anália; Dubois, Luiz Gustavo; Gonçalves, Ana Cristina; Girão, Henrique; Silva Canedo, Nathalie Henriques; Correia, Ana Helena; De Souza, Jorge Marcondes; Sarmento-Ribeiro, Ana Bela; Lopes, Maria Celeste; Moura-Neto, Vivaldo

2017-01-01

Glioblastoma (GBM) is a grade IV astrocytoma. GBM patients show resistance to chemotherapy such as temozolomide (TMZ), the gold standard treatment. In order to simulate the molecular mechanisms behind the different chemotherapeutic responses in GBM patients we compared the cellular heterogeneity and chemotherapeutic resistance mechanisms in different GBM cell lines. We isolated and characterized a human GBM cell line obtained from a GBM patient, named GBM11. We studied the GBM11 behaviour when treated with Tamoxifen (TMX) that, among other functions, is a protein kinase C (PKC) inhibitor, alone and in combination with TMZ in comparison with the responses of U87 and U118 human GBM cell lines. We evaluated the cell death, cell cycle arrest and cell proliferation, mainly through PKC expression, by flow cytometry and western blot analysis and, ultimately, cell migration capability and F-actin filament disorganization by fluorescence microscopy. We demonstrated that the constitutive activation of p-PKC seems to be one of the main metabolic implicated on GBM malignancy. Despite of its higher resistance, possibly due to the overexpression of P-glycoprotein and stem-like cell markers, GBM11 cells presented a subtle different chemotherapeutic response compared to U87 and U118 cells. The GBM11, U87, U118 cell lines show subtle molecular differences, which clearly indicate the characterization of GBM heterogeneity, one of the main reasons for tumor resistance. The adding of cellular heterogeneity in molecular behaviour constitutes a step closer in the understanding of resistant molecular mechanisms in GBM, and can circumvents the eventual impaired therapy. PMID:28714013

E-cigarette vapour is not inert and exposure can lead to cell damage.

PubMed

Holliday, Richard; Kist, Ralf; Bauld, Linda

2016-03-01

In vitro experiments were performed on normal epithelial cells as well as head and neck squamous cell carcinoma (HNSCC) cell lines. The widely available cell line HaCat, a spontaneously transformed immortal keratinocyte and the HNSCC cell lines HN30 and UMSCC10B were used. Cells were exposed to nicotine-containing and nicotine-free vapour extract from two popular e-cigarette brands for periods ranging from 48 hours to eight weeks. Cytotoxicity was assessed using Annexin V flow cytometric analysis, trypan blue exclusion and clonogenic assays. Genotoxicity in the form of DNA strand breaks was quantified using the neutral comet assay and γ-H2AX immunostaining. E-cigarette-exposed cells showed significantly reduced cell viability and clonogenic survival, along with increased rates of apoptosis and necrosis, regardless of e-cigarette vapour nicotine content. They also exhibited significantly increased comet tail length and accumulation of γ-H2AX foci, demonstrating increased DNA strand breaks. In conclusion, our study strongly suggests that electronic cigarettes are not as safe as their marketing makes them appear to the public. Our in vitro experiments employing two brands of e-cigs show that at biologically relevant doses, vapourised e-cig liquids induce increased DNA strand breaks and cell death, and decreased clono- genic survival in both normal epithelial and HNSCC cell lines independently of nicotine content. Further research is needed to definitively determine the long-term effects of e-cig usage, as well as whether the DNA damage shown in our study as a result of e-cig exposure will lead to mutations that ultimately result in cancer.

Evaluation of anticancer potential of Bacopa monnieri L. against MCF-7 and MDA-MB 231 cell line

PubMed Central

Mallick, Md. Nasar; Akhtar, Md. Salman; Najm, Mohd. Zeeshan; Tamboli, E. T.; Ahmad, Sayeed; Husain, Syed Akhtar

2015-01-01

Background: The ethanolic extract of Bacopa monnieri contains bacoside A and B, brahmin, cucurbitacins, and betulinic acid. Currently, cucurbitacins have also been reported for their strong anti-tumorigenic and anti-proliferative activity by inducing cell cycle arrest at the G2/M phase and formation of multiplied cells. The present study was carried out to evaluate the in vitro cytotoxic activity of ethanolic extract of dichloromethane (DCM) fraction of B. monnieri on two different cell lines. Materials and Methods: The ethanolic extract of B. monnieri was prepared using soxhlet extraction method and different fractions (hexane, DCM, methanol, acetone, and water) of ethanolic extracts were prepared. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay of ethanolic extract and of all fractions was carried out on MCF-7 and MDA-MB 231 cell lines. The presence of cucurbitacins and betulinic acid in these fractions was confirmed by high-performance thin layer chromatography. Results: The IC50 values of ethanolic extract of B. monnieri in MCF-7 and MDA-MB 231 cell lines were 72.0 μg/mL and 75.0 μg/mL, respectively. The DCM fraction of B. monnieri showed maximum cytotoxic activity among all fraction upto 72 h and was found to be 57.0 μg/mL and 42.0 μg/mL, respectively. Conclusion: The results showed good cytotoxic activity in DCM fraction in both the cell lines may be due to the presence of cucurbitacins and betulinic acid in DCM fraction. PMID:26681894

Evaluation of anticancer potential of Bacopa monnieri L. against MCF-7 and MDA-MB 231 cell line.

PubMed

Mallick, Md Nasar; Akhtar, Md Salman; Najm, Mohd Zeeshan; Tamboli, E T; Ahmad, Sayeed; Husain, Syed Akhtar

2015-01-01

The ethanolic extract of Bacopa monnieri contains bacoside A and B, brahmin, cucurbitacins, and betulinic acid. Currently, cucurbitacins have also been reported for their strong anti-tumorigenic and anti-proliferative activity by inducing cell cycle arrest at the G2/M phase and formation of multiplied cells. The present study was carried out to evaluate the in vitro cytotoxic activity of ethanolic extract of dichloromethane (DCM) fraction of B. monnieri on two different cell lines. The ethanolic extract of B. monnieri was prepared using soxhlet extraction method and different fractions (hexane, DCM, methanol, acetone, and water) of ethanolic extracts were prepared. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay of ethanolic extract and of all fractions was carried out on MCF-7 and MDA-MB 231 cell lines. The presence of cucurbitacins and betulinic acid in these fractions was confirmed by high-performance thin layer chromatography. The IC50 values of ethanolic extract of B. monnieri in MCF-7 and MDA-MB 231 cell lines were 72.0 μg/mL and 75.0 μg/mL, respectively. The DCM fraction of B. monnieri showed maximum cytotoxic activity among all fraction upto 72 h and was found to be 57.0 μg/mL and 42.0 μg/mL, respectively. The results showed good cytotoxic activity in DCM fraction in both the cell lines may be due to the presence of cucurbitacins and betulinic acid in DCM fraction.

System analysis of the regulation of the immune response by CD147 and FOXC1 in cancer cell lines

PubMed Central

Kong, Ling-Min; Wei, Ding; Xu, Jing; Wang, Zi-Ling; Bian, Huijie; Chen, Zhi-Nan

2018-01-01

CD147, encoded by BSG, is a highly glycosylated transmembrane protein that belongs to the immunological superfamily and expressed on the surface of many types of cancer cells. While CD147 is best known as a potent inducer of extracellular matrix metalloproteinases, it can also function as a key mediator of inflammatory and immune responses. To systematically elucidate the function of CD147 in cancer cells, we performed an analysis of genome-wide profiling across the Cancer Cell Line Encyclopedia (CCLE). We showed that CD147 mRNA expression was much higher than that of most other genes in cancer cell lines. CD147 varied widely across these cell lines, with the highest levels in the ovary (COLO704) and stomach (SNU668), intermediate levels in the lung (RERFLCKJ, NCIH596 and NCIH1651) and lowest levels in hematopoietic and lymphoid tissue (UT7, HEL9217, HEL and MHHCALL3) and the kidney (A704 and SLR20). Genome-wide analyses showed that CD147 expression was significantly negatively correlated with immune-related genes. Our findings implicated CD147 as a novel regulator of immune-related genes and suggest its important role as a master regulator of immune-related responses in cancer cell lines. We also found a high correlation between the expression of CD147 and FOXC1, and proved that CD147 was a direct transcriptional target of FOXC1. Our findings demonstrate that FOXC1 is a novel regulator of CD147 and confirms its role as a master regulator of the immune response. PMID:29560120

High hydrostatic pressure affects antigenic pool in tumor cells: Implication for dendritic cell-based cancer immunotherapy.

PubMed

Urbanova, Linda; Hradilova, Nada; Moserova, Irena; Vosahlikova, Sarka; Sadilkova, Lenka; Hensler, Michal; Spisek, Radek; Adkins, Irena

2017-07-01

High hydrostatic pressure (HHP) can be used to generate dendritic cell (DC)-based active immunotherapy for prostate, lung and ovarian cancer. We showed here that HHP treatment of selected human cancer cell lines leads to a degradation of tumor antigens which depends on the magnitude of HHP applied and on the cancer cell line origin. Whereas prostate or ovarian cell lines displayed little protein antigen degradation with HHP treatment up to 300MPa after 2h, tumor antigens are hardly detected in lung cancer cell line after treatment with HHP 250MPa at the same time. On the other hand, quick reduction of tumor antigen-coding mRNA was observed at HHP 200MPa immediately after treatment in all cell lines tested. To optimize the DC-based active cellular therapy protocol for HHP-sensitive cell lines the immunogenicity of HHP-treated lung cancer cells at 150, 200 and 250MPa was compared. Lung cancer cells treated with HHP 150MPa display characteristics of immunogenic cell death, however cells are not efficiently phagocytosed by DC. Despite induction of the highest number of antigen-specific CD8 + T cells, 150 MPa-treated lung cancer cells survive in high numbers. This excludes their use in DC vaccine manufacturing. HHP of 200MPa treatment of lung cancer cells ensures the optimal ratio of efficient immunogenic killing and delivery of protein antigens in DC. These results represent an important pre-clinical data for generation of immunogenic killed lung cancer cells in ongoing NSCLC Phase I/II clinical trial using DC-based active cellular immunotherapy (DCVAC/LuCa). Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

Liposomal nanoparticles as a drug delivery vehicle against osteosarcoma

NASA Astrophysics Data System (ADS)

Dhule, Santosh Subhashrao

The delivery of curcumin, a broad-spectrum anticancer drug, has been explored in the form of liposomal nanoparticles to treat osteosarcoma (OS). Curcumin is water insoluble and an effective delivery route is through encapsulation in cyclodextrins followed by a second encapsulation in liposomes. Liposomal curcumin's potential was evaluated against cancer models of mesenchymal (OS) and epithelial origin (breast cancer). The resulting 2-Hydroxypropyl-gamma-cyclodextrin/curcumin - liposome complex shows promising anticancer potential both in vitro and in vivo against KHOS OS cell line and MCF-7 breast cancer cell line. An interesting aspect is that liposomal curcumin initiates the caspase cascade that leads to apoptotic cell death in vitro in comparison with DMSO-curcumin induced autophagic cell death. In addition, the efficiency of the liposomal curcumin formulation was confirmed in vivo using a xenograft OS model. Curcumin-loaded gamma-cyclodextrin liposomes indicate significant potential as delivery vehicles for the treatment of cancers of different tissue origin. The second part of this study examines the anti-tumor potential of curcumin and C6 ceramide (C6) against osteosarcoma cell lines when both are encapsulated in the bilayer of liposomal nanoparticles. Curcumin in combination with C6 showed 1.5 times enhanced cytotoxic effect in the case of MG-63 and KHOS OS cell lines, in comparison with systems with curcumin alone. Interestingly, C6-curcumin liposomes were found to be less toxic on untransformed human cells in comparison to OS cell lines. In addition, cell cycle assays on a KHOS cell line after treatment revealed that curcumin only liposomes induced G 2/M arrest by upregulation of cyclin B1, while C6 only liposomes induced G1 arrest by downregulation of cyclin D1. C6-curcumin liposomes induced G2/M arrest and showed a combined effect in the expression levels of cyclin D1 and cyclin B1. Using pegylated liposomes to increase the plasma half-life and tagging with folate for targeted delivery in vivo, a significant reduction in tumor size was observed with C6-curcumin-folate liposomes. The encapsulation of two water insoluble drugs, curcumin and C6, in the lipid bilayer of liposomes enhances the cytotoxic effect and validates the potential of combined drug therapy.

Dual function of active constituents from bark of Ficus racemosa L in wound healing.

PubMed

Bopage, Nisansala Swarnamali; Kamal Bandara Gunaherath, G M; Jayawardena, Kithsiri Hector; Wijeyaratne, Sushila Chandrani; Abeysekera, Ajita Mahendra; Somaratne, Seneviratne

2018-01-25

Different parts including the latex of Ficus racemosa L. has been used as a medicine for wound healing in the Ayurveda and in the indigenous system of medicine in Sri Lanka. This plant has been evaluated for its wound healing potential using animal models. The aim of this study was to obtain an insight into the wound healing process and identify the potential wound healing active substance/s present in F. racemosa L. bark using scratch wound assay (SWA) as the in-vitro assay method. Stem bark extracts of F. racemosa were evaluated using scratch wound assay (SWA) on Baby Hamster Kidney (BHK 21) and Madin-Darby Canine Kidney (MDCK) cell lines and Kirby Bauer disc diffusion assay on common bacteria and fungi for cell migration enhancing ability and antimicrobial activity respectively. Dichloromethane and hexanes extracts which showed cell migration enhancement activity on SWA were subjected to bioactivity directed fractionation using column chromatography followed by preparative thin layer chromatography to identify the compounds responsible for the cell migration enhancement activity. Dichloromethane and hexanes extracts showed cell migration enhancement activity on both cell lines, while EtOAc and MeOH extracts showed antibacterial activity against Staphylococcus and Bacillus species and antifungal activity against Saccharomyces spp. and Candida albicans. Lupeol (1) and β-sitosterol (2) were isolated as the potential wound healing active compounds which exhibited significant cell migration enhancement activity on BHK 21 and MDCK cell lines (> 80%) in par with the positive control, asiaticoside at a concentration of 25 μM. The optimum concentration of each compound required for the maximum wound healing has been determined as 30 μM and 35 μM for 1 and 2 respectively on both cell lines. It is also established that lupeol acetate (3) isolated from the hexanes extract act as a pro-drug by undergoing hydrolysis into lupeol in the vicinity of cells. Different chemical constituents present in stem bark of Ficus racemosa L show enhancement of cell migration (which corresponds to the cell proliferation) as well as antimicrobial activity. This dual action of F. racemosa stem bark provides scientific support for its traditional use in wound healing.

Everolimus affects vasculogenic mimicry in renal carcinoma resistant to sunitinib

PubMed Central

Serova, Maria; Tijeras-Raballand, Annemilaï; Santos, Celia Dos; Martinet, Matthieu; Neuzillet, Cindy; Lopez, Alfred; Mitchell, Dianne C.; Bryan, Brad A.; Gapihan, Guillaume; Janin, Anne; Bousquet, Guilhem; Riveiro, Maria Eugenia; Bieche, Ivan; Faivre, Sandrine

2016-01-01

Angiogenesis is hallmark of clear cell renal cell carcinogenesis. Anti-angiogenic therapies have been successful in improving disease outcome; however, most patients treated with anti-angiogenic agents will eventually progress. In this study we report that clear cell renal cell carcinoma was associated with vasculogenic mimicry in both mice and human with tumor cells expressing endothelial markers in the vicinity of tumor vessels. We show that vasculogenic mimicry was efficiently targeted by sunitinib but eventually associated with tumor resistance and a more aggressive phenotype both in vitro and in vivo. Re-challenging these resistant tumors in mice, we showed that second-line treatment with everolimus particularly affected vasculogenic mimicry and tumor cell differentiation compared to sorafenib and axitinib. Finally, our results highlighted the phenotypic and genotypic changes at the tumor cell and microenvironment levels during sunitinib response and progression and the subsequent improvement second-line therapies bring to the current renal cell carcinoma treatment paradigm. PMID:27509260

CD40 expression in Wehi-164 cell line

PubMed Central

Ebadi, Padideh; Pourfathollah, Ali Akbar; Soheili, Zahra Soheila; Moazzeni, Seyed Mohammad

2010-01-01

CD40-CD154 interaction is an important process for cellular and humoral immunity regulation and can be effective in the body’s defense against tumors. In the present study, we evaluated the expression of CD40 in Wehi-164 cell line. CD40 expressions on the cell surface and in the cytoplasm were assessed by flow cytometry and intracellular staining assay, respectively. Also, the mRNA expression was identified by real time-PCR. The obtained results showed the high mRNA and cytoplasmic protein expression of CD40 but no surface expression. These results suggest that the Wehi-164 cell line down regulates expression of CD40 on the surface for evasion of immune system. PMID:20496113

CD40 expression in Wehi-164 cell line.

PubMed

Karimi, Mohammad Hossein; Ebadi, Padideh; Pourfathollah, Ali Akbar; Soheili, Zahra Soheila; Moazzeni, Seyed Mohammad

2010-07-01

CD40-CD154 interaction is an important process for cellular and humoral immunity regulation and can be effective in the body's defense against tumors. In the present study, we evaluated the expression of CD40 in Wehi-164 cell line. CD40 expressions on the cell surface and in the cytoplasm were assessed by flow cytometry and intracellular staining assay, respectively. Also, the mRNA expression was identified by real time-PCR. The obtained results showed the high mRNA and cytoplasmic protein expression of CD40 but no surface expression. These results suggest that the Wehi-164 cell line down regulates expression of CD40 on the surface for evasion of immune system.

[Experimental study on aging effect of Angelica sinensis polysaccharides combined with cytarabine on human leukemia KG1alpha cell lines].

PubMed

Xu, Chun-Yan; Geng, Shan; Liu, Jun; Zhu, Jia-Hong; Zhang, Xian-Ping; Jiang, Rong; Wang, Ya-Ping

2014-04-01

The latest findings of our laboratory showed that Angelica sinensis polysaccharide (ASP) showed a definite effect in regulating the aging of hematopoietic stem cells. Leukemia is a type of malignant hematopoietic tumor in hematopoietic stem cells. There have been no relevant reports about ASP's effect in regulating the aging of leukemia cells. In this study, human acute myeloid leukemia (AML) KG1alpha cell lines in logarithmic growth phase were taken as the study object, and were divided into the ASP group, the cytarabine (Ara-C) group, the ASP + Ara-C group and the control group. The groups were respectively treated with different concentration of ASP, Ara-C and ASP + Ara-C for different periods, with the aim to study the effect of ASP combined with Ara-C in regulating the aging of human acute myeloid leukemia KG1alpha cell lines and its relevant mechanism. The results showed that ASP, Ara-C and ASP + Ara-C could obviously inhibit KG1alpha cell proliferation in vitro, block the cells in G0/G1 phase. The cells showed the aging morphological feature. The percentage of positive stained aging cells was dramatically increased, and could significantly up-regulate the expression of aging-related proteins P16 and RB, which were more obvious in the ASP + Ara-C group. In conclusion, the aging mechanism of KG1alpha cell induced by ASP and Ara-C may be related to the regulation of the expression of aging-related proteins, suggesting that the combined administration of ASP and anticancer drugs plays a better role in the treatment of leukemia .

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

Fontenot, Krystal; Naragoni, Srivatcha; Claville, Michelle

Kola acuminate, also known as Bizzy Nut or Kola Nut, is a natural product that contains bioactive chemicals that possess hormonal properties. The purpose of this study was to characterize the putative phytoestrogenic compounds present in Bizzy Nut for estrogenic-like activity. As an initial step, five extracts (E1 - hexane, E2 - ether, E3 - acetone, E4 - methanol and E5 - water) were sequentially generated using solid-liquid phase extraction and their bioactivity was examined in MCF-7, MDA-MB-468 and LNCaP cancer cell models. MTT cell viability, dye exclusion, caspase activity and microscopic assessment of apoptotic cells demonstrated that extracts ofmore » Bizzy were cytotoxic to MCF-7, MDA-MB 468 and LNCaP cells. In MCF-7 cells, the acetone extract (E3) at 100 ppm elicited a potent cytotoxic response with a growth-inhibitory concentration (GI{sub 50}) of 67 ppm. In contrast, E3 stimulated growth in LNCaP cells. The ether extract (E2) showed a dose-dependent cytotoxic response with a GI{sub 50} of 13 ppm in the LNCaP cell line. Examination of the apoptotic response induced by E2 and E3 paralleled the level of cell cytotoxicity observed in both cell lines. The methanol extract (E4) was the only extract that showed a time-, dose-, and estrogen-receptor-dependent stimulation of pS2 gene expression. On the other hand, the acetone extract (E3), which showed the highest degree of cytotoxicity, showed no transcription stimulation of pS2 in MCF-7 cells. Altogether, these data indicate that Bizzy contains unique active hormonal compounds that have specific biological properties that are cell line-dependent.« less

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

PubMed

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

2018-07-01

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

New oxirane derivatives of 1,4-naphthoquinones and their evaluation against T. cruzi epimastigote forms.

PubMed

Carneiro, Paula F; do Nascimento, Samara B; Pinto, Antonio V; Pinto, Maria do Carmo F R; Lechuga, Guilherme C; Santos, Dilvani O; dos Santos Júnior, Helvécio M; Resende, Jackson A L C; Bourguignon, Saulo C; Ferreira, Vitor F

2012-08-15

New oxirane derivatives were synthesized using six naphthoquinones as the starting materials. Our biological results showed that these oxiranes acted as trypanocidal agents against Trypanosoma cruzi with minimal cytotoxicity in the VERO cell line compared to naphthoquinones. In particular, oxirane derivative 14 showed low cytotoxicity in a mammalian cell line and exhibited better activity against epimastigote forms of T.cruzi than the current drug used to treat Chagas disease, benznidazole. Copyright © 2012 Elsevier Ltd. All rights reserved.

Synthesis and cytotoxic activity of two steroids: icogenin aglycone analogs.

PubMed

Guan, Yu-Yao; Li, Shu-Zhen; Lei, Ping-Sheng

2017-05-01

During the process of icogenin analog research, we obtained two cytotoxic steroids: compound 4 and compound 6 casually. Their in vitro antitumor activities were tested by the standard MTT assay. The results disclosed that compound 4 (IC 50  = 3.65-6.90 μM) showed potential antitumor activities against HELA, KB cell lines and compound 6 (IC 50  = 2.40-9.05 μM) showed potential antitumor activities against HELA, BGC-823, KB, A549, HCT-8 cell lines.

5-Fluorouracil-induced apoptosis in cultured oral cancer cells.

PubMed

Tong, D; Poot, M; Hu, D; Oda, D

2000-03-01

Chemotherapy is commonly used to treat advanced oral squamous cell carcinoma (SCC) and is known to kill cancer cells through apoptosis. Our hypothesis states that 5-fluorouracil (5FU) also kills cultured oral epithelial cells through programmed cell death or apoptosis. Cultured oral cancer cells were exposed to an optimum dose of 20 mg/ml of 5FU. Cells were analyzed for changes in cell cycle distribution and induction of cell death including apoptosis. Normal control, human papilloma virus-immortalized (PP), ATCC SCC cell line (CA1) and two primary oral SCC cell lines (CA3 and -4) were studied. Inhibition of apoptosis by a pan-caspase inhibitor was used. SYTO 11 flow cytometry showed increased apoptosis in all 5FU-treated cell cultures compared to untreated controls. The results show biological variation in apoptotic response. CA1 had the lowest apoptotic rate of the cancer cell lines at 1.5%. Next lowest was CA3, followed by CA4 and PP. In addition, alteration in the G1 and S phase fractions were found. Untreated CA1 showed 28% G1, 53% S compared to 43% G1, and 40% S of treated. We investigated the pathway of apoptosis using the pan-caspase inhibitor IDN-1529 by methylthiazolyl diphenyl tetrazolium bromide (MTT) colorimetric analysis. Results showed mild inhibition of cell death when cells were incubated with 50 microM IDN-1529 for 24 h. This suggests a probable caspase-dependent apoptotic pathway. In conclusion, our data suggest that 5FU induces oral cancer cell death through apoptosis and that biological variation exists between normal and cancer cells and between different types of cancer cells themselves. Our data indicate that cultures of a useful in vitro model for chemosensitivity assays are possible. Our results also suggest a caspase-dependent pathway for chemocytotoxicity in oral SCC.

Modulation of growth, differentiation, and mucous glycoprotein synthesis by retinyl acetate in cloned carcinoma cell lines. [Rats

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

Marchok, A.C.; Clark, J.N.; Klein-Szanto, A.

1981-06-01

The ability of retinyl acetate to alter growth, differentiation, and synthesis of mucous glycoproteins in cell lines cloned from an adenocarcinoma (T-8) and a squamous cell carcinoma (1000 WT) was investigated with the use of F344 rats. Growth rate was inhibited approximately 25 and 50% in 6.6 x 10/sup -6/ and 3.3 x 10/sup -5/ M retinyl acetate, respectively, in both cell lines. Retinyl acetate induced the formation of numerous vacuoles and periodic acid-silver methenamine-positive granules in both T-8 and 1000 WT cells. After T-8 cells were cultured for 7 days in retinyl acetate, (/sup 3/H)glucosamine incorporation increased 133- tomore » 147-fold and (/sup 14/C)serine incorporation increased twelvefold to twentyfold in the high-molecular-weight mucous glycoprotein fraction (peak A) from the cell cytosol. In 1000 WT cells, (/sup 3/H)glucosamine incorporation increased only 4.2- to 7.5-fold, and (/sup 14/C)serine incorporation increased only 2.6- to 4.6-fold under the same culture conditions. Thus T-8 cells showed a marked increase in the synthesis and secretion of mucins, whereas 1000 WT cells showed a comparatively small but significant increase.« less

An Atypical Acidophil Cell Line Tumor Showing Focal Differentiation Toward Both Growth Hormone and Prolactin Cells.

PubMed

Naritaka, Heiji; Kameya, Toru; Sato, Yuichi; Furuhata, Shigeru; Okui, Junichi; Kamiguchi, Yuji; Otani, Mitsuhiro; Toya, Shigeo

1995-01-01

We report a case of giant pituitary adenoma in a child. Computerized tomography (CT) scan revealed a suprasellar extension tumor mass with hydrocephalus. There was no clinical evidence of acromegaly, gigantism, and other hormonal symptoms. Endocrinologic studies showed within normal value of serum growth hormone (GH: 4.2 ng/mL) and slightly increased levels of prolactin (PRL: 78 ng/mL) and other pituitary hormone values were within normal range. On suppression test by bromocryptin, both GH and PRL levels were reduced. Histopathological findings revealed that the tumor consisted of predominantly chromophobic and partly eosinophilic adenoma cells. Immunohistochemical staining detected GH and PRL in a small number of distinctly different adenoma cells, respectively. Nonradioactive in situ hybridization (ISH) also showed GH and PRL mRNA expression in identical immunopositive cells. Electron microscopy (EM) demonstrated adenoma cells with moderate or small numbers of two types of dense granules and without fibrous body which are characteristic of sparsely granulated GH-cell adenomas. The adenoma does not fit into any classification but may be an atypical acidophil cell line tumor showing focal differentiation toward both GH and PRL cells.

The Natural Flavonoid Fisetin Inhibits Cellular Proliferation of Hepatic, Colorectal, and Pancreatic Cancer Cells through Modulation of Multiple Signaling Pathways.

PubMed

Youns, Mаhmoud; Abdel Halim Hegazy, Wael

2017-01-01

Digestive cancers are major causes of mortality and morbidity worldwide. Fisetin, a naturally occurring flavonoid, has been previously shown anti-proliferative, anti-cancer, neuroprotective, and antioxidant activities. In our study, the anti-tumor activities in addition to regulatory effects of fisetin on some cancer cell lines were investigated. Data presented here showed that fisetin induces growth inhibition, and apoptosis in hepatic (HepG-2), colorectal (Caco-2) and pancreatic (Suit-2) cancer cell lines. Gene expression results showed that 1307 genes were significantly regulated in their expression in hepatic and pancreatic cell lines. 350 genes were commonly up-regulated and 353 genes were commonly down-regulated. Additionally, 604 genes were oppositely expressed in both tumor cells. CDK5 signaling, NRF2-mediated oxidative stress response, glucocorticoid signaling, and ERK/MAPK signaling were among most prominent signaling pathways modulating the growth inhibitory effects of fisetin on hepatic and pancreatic cancer cells. The present analysis showed, for the first time, that the anti-tumor effect of fisetin was mediated mainly through modulation of multiple signaling pathways and via activation of CDKN1A, SEMA3E, GADD45B and GADD45A and down-regulation of TOP2A, KIF20A, CCNB2 and CCNB1 genes.

The Natural Flavonoid Fisetin Inhibits Cellular Proliferation of Hepatic, Colorectal, and Pancreatic Cancer Cells through Modulation of Multiple Signaling Pathways

PubMed Central

Youns, Mаhmoud; Abdel Halim Hegazy, Wael

2017-01-01

Digestive cancers are major causes of mortality and morbidity worldwide. Fisetin, a naturally occurring flavonoid, has been previously shown anti-proliferative, anti-cancer, neuroprotective, and antioxidant activities. In our study, the anti-tumor activities in addition to regulatory effects of fisetin on some cancer cell lines were investigated. Data presented here showed that fisetin induces growth inhibition, and apoptosis in hepatic (HepG-2), colorectal (Caco-2) and pancreatic (Suit-2) cancer cell lines. Gene expression results showed that 1307 genes were significantly regulated in their expression in hepatic and pancreatic cell lines. 350 genes were commonly up-regulated and 353 genes were commonly down-regulated. Additionally, 604 genes were oppositely expressed in both tumor cells. CDK5 signaling, NRF2-mediated oxidative stress response, glucocorticoid signaling, and ERK/MAPK signaling were among most prominent signaling pathways modulating the growth inhibitory effects of fisetin on hepatic and pancreatic cancer cells. The present analysis showed, for the first time, that the anti-tumor effect of fisetin was mediated mainly through modulation of multiple signaling pathways and via activation of CDKN1A, SEMA3E, GADD45B and GADD45A and down-regulation of TOP2A, KIF20A, CCNB2 and CCNB1 genes. PMID:28052097

Initial cytotoxicity assays of media for sulfate-reducing bacteria: An endodontic biopharmaceutical product under development.

PubMed

Heggendorn, Fabiano Luiz; Silva, Gabriela Cristina de Carvalho; Cardoso, Elisama Azevedo; Castro, Helena Carla; Gonçalves, Lúcio Souza; Dias, Eliane Pedra; Lione, Viviane de Oliveira Freitas; Lutterbach, Márcia Teresa Soares

2016-01-01

This study assessed the cell viability of the inoculation vehicle of BACCOR (a combination of sulfate-reducing bacteria plus a culture media for bacteria), a biopharmaceutical product under development for dental use as aid in fractured endodontic file removal from the root canal. Different culture media for bacteria were evaluated: modified Postgate E (MCP-E mod), Modified Postgate E without Agar-agar (MCP-E w/Ag), Postgate C with Agar-agar (MCP-C Ag) and Postgate C without Agar-agar (MCP-C w/Ag). Cytotoxicity was quantified by the MTT test, exposing L929 and Vero cell lines to the vehicles over 24 h. The exposure of L929 cell line to MCP-E w/Ag resulted in biocompatibility (52% cell viability), while the exposure of the Vero kidney line revealed only MCP-E mod as cytotoxic. When diluted, all the vehicles showed biocompatibility with both cell lines. MCP-E w/Ag was the vehicle chosen for BACCOR, because of its biocompatibility with the cells used.

Cell type-specific roles of Jak3 in IL-2-induced proliferative signal transduction

PubMed Central

Fujii, Hodaka

2007-01-01

Binding of IL-2 to its specific receptor induces activation of two members of Jak family protein tyrosine kinases, Jak1 and Jak3. An IL-2R-reconstituted NIH 3T3 fibroblast cell line proliferates in response to IL-2 only when hematopoietic lineage-specific Jak3 is ectopically expressed. However, the mechanism of Jak3-dependent proliferation in the fibroblast cell line is not known. Here, I showed that Jak3 expression is dispensable for IL-2-induced activation of Jak1 and Stat proteins and expression of nuclear proto-oncogenes in the IL-2R-reconstituted fibroblast cell line. However, Jak3 expression markedly enhanced these IL-2-induced signaling events. In contrast, Jak3 expression was essential for induction of cyclin genes involved in the G1-S transition. These data suggest a critical role of Jak3 in IL-2 signaling in the fibroblast cell line and may provide further insight into the cell type-specific mechanism of cytokine signaling. PMID:17266928

Rationally optimized cryopreservation of multiple mouse embryonic stem cell lines: I--Comparative fundamental cryobiology of multiple mouse embryonic stem cell lines and the implications for embryonic stem cell cryopreservation protocols.

PubMed

Kashuba, Corinna M; Benson, James D; Critser, John K

2014-04-01

The post-thaw recovery of mouse embryonic stem cells (mESCs) is often assumed to be adequate with current methods. However as this publication will show, this recovery of viable cells actually varies significantly by genetic background. Therefore there is a need to improve the efficiency and reduce the variability of current mESC cryopreservation methods. To address this need, we employed the principles of fundamental cryobiology to improve the cryopreservation protocol of four mESC lines from different genetic backgrounds (BALB/c, CBA, FVB, and 129R1 mESCs) through a comparative study characterizing the membrane permeability characteristics and membrane integrity osmotic tolerance limits of each cell line. In the companion paper, these values were used to predict optimal cryoprotectants, cooling rates, warming rates, and plunge temperatures, and then these predicted optimal protocols were validated against standard freezing protocols. Copyright © 2014 Elsevier Inc. All rights reserved.