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Sample records for cell lines mitochondria

  1. Warburg and Crabtree effects in premalignant Barrett's esophagus cell lines with active mitochondria.

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    Martin T Suchorolski

    Full Text Available 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.

  2. Bioenergetic and antiapoptotic properties of mitochondria from cultured human prostate cancer cell lines PC-3, DU145 and LNCaP.

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    Alexander Panov

    Full Text Available The purpose of this work was to reveal the metabolic features of mitochondria that might be essential for inhibition of apoptotic potential in prostate cancer cells. We studied mitochondria isolated from normal prostate epithelial cells (PrEC, metastatic prostate cancer cell lines LNCaP, PC-3, DU145; and non-prostate cancer cells - human fibrosarcoma HT1080 cells; and normal human lymphoblastoid cells. PrEC cells contained 2 to 4 times less mitochondria per gram of cells than the three PC cell lines. Respiratory activities of PrEC cell mitochondria were 5-20-fold lower than PC mitochondria, depending on substrates and the metabolic state, due to lower content and lower activity of the respiratory enzyme complexes. Mitochondria from the three metastatic prostate cancer cell lines revealed several features that are distinctive only to these cells: low affinity of Complex I for NADH, 20-30 mV higher electrical membrane potential (ΔΨ. Unprotected with cyclosporine A (CsA the PC-3 mitochondria required 4 times more Ca²⁺ to open the permeability transition pore (mPTP when compared with the PrEC mitochondria, and they did not undergo swelling even in the presence of alamethicin, a large pore forming antibiotic. In the presence of CsA, the PC-3 mitochondria did not open spontaneously the mPTP. We conclude that the low apoptotic potential of the metastatic PC cells may arise from inhibition of the Ca²⁺-dependent permeability transition due to a very high ΔΨ and higher capacity to sequester Ca²⁺. We suggest that due to the high ΔΨ, mitochondrial metabolism of the metastatic prostate cancer cells is predominantly based on utilization of glutamate and glutamine, which may promote development of cachexia.

  3. Manipulating the mitochondria activity in human hepatic cell line Huh7 by low-power laser irradiation

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    Lynnyk, Anna; Lunova, Mariia; Jirsa, Milan; Egorova, Daria; Kulikov, Andrei; Kubinová, Šárka; Lunov, Oleg; Dejneka, Alexandr

    2018-01-01

    Low-power laser irradiation of red light has been recognized as a promising tool across a vast variety of biomedical applications. However, deep understanding of the molecular mechanisms behind laser-induced cellular effects remains a significant challenge. Here, we investigated mechanisms involved in the death process in human hepatic cell line Huh7 at a laser irradiation. We decoupled distinct cell death pathways targeted by laser irradiations of different powers. Our data demonstrate that high dose laser irradiation exhibited the highest levels of total reactive oxygen species production, leading to cyclophilin D-related necrosis via the mitochondrial permeability transition. On the contrary, low dose laser irradiation resulted in the nuclear accumulation of superoxide and apoptosis execution. Our findings offer a novel insight into laser-induced cellular responses, and reveal distinct cell death pathways triggered by laser irradiation. The observed link between mitochondria depolarization and triggering ROS could be a fundamental phenomenon in laser-induced cellular responses. PMID:29541521

  4. Role of the mitochondria in immune-mediated apoptotic death of the human pancreatic β cell line βLox5.

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    Yaíma L Lightfoot

    Full Text Available Mitochondria are indispensable in the life and death of many types of eukaryotic cells. In pancreatic beta cells, mitochondria play an essential role in the secretion of insulin, a hormone that regulates blood glucose levels. Unregulated blood glucose is a hallmark symptom of diabetes. The onset of Type 1 diabetes is preceded by autoimmune-mediated destruction of beta cells. However, the exact role of mitochondria has not been assessed in beta cell death. In this study, we examine the role of mitochondria in both Fas- and proinflammatory cytokine-mediated destruction of the human beta cell line, βLox5. IFNγ primed βLox5 cells for apoptosis by elevating cell surface Fas. Consequently, βLox5 cells were killed by caspase-dependent apoptosis by agonistic activation of Fas, but only after priming with IFNγ. This beta cell line undergoes both apoptotic and necrotic cell death after incubation with the combination of the proinflammatory cytokines IFNγ and TNFα. Additionally, both caspase-dependent and -independent mechanisms that require proper mitochondrial function are involved. Mitochondrial contributions to βLox5 cell death were analyzed using mitochondrial DNA (mtDNA depleted βLox5 cells, or βLox5 ρ(0 cells. βLox5 ρ(0 cells are not sensitive to IFNγ and TNFα killing, indicating a direct role for the mitochondria in cytokine-induced cell death of the parental cell line. However, βLox5 ρ(0 cells are susceptible to Fas killing, implicating caspase-dependent extrinsic apoptotic death is the mechanism by which these human beta cells die after Fas ligation. These data support the hypothesis that immune mediators kill βLox5 cells by both mitochondrial-dependent intrinsic and caspase-dependent extrinsic pathways.

  5. Mitochondria in aging cell differentiation

    Czech Academy of Sciences Publication Activity Database

    Palková, Zdena; Váchová, Libuše

    2016-01-01

    Roč. 8, č. 7 (2016), s. 1287-1288 ISSN 1945-4589 R&D Projects: GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:61388971 Keywords : mitochondria * cell differentiation * retrograde signaling Subject RIV: EE - Microbiology, Virology Impact factor: 4.867, year: 2016

  6. 3-Bromopyruvate and sodium citrate induce apoptosis in human gastric cancer cell line MGC-803 by inhibiting glycolysis and promoting mitochondria-regulated apoptosis pathway.

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    Guo, Xingyu; Zhang, Xiaodong; Wang, Tingan; Xian, Shulin; Lu, Yunfei

    2016-06-17

    Cancer cells are mainly dependent on glycolysis to generate adenosine triphosphate (ATP) and intermediates required for cell growth and proliferation. Thus, inhibition of glycolysis might be of therapeutic value in antitumor treatment. Our previously studies had found that both 3-bromopyruvate (BP) and sodium citrate (SCT) can inhibit tumor growth and proliferation in vitro and in vivo. However, the mechanism involved in the BP and SCT mediated antitumor activity is not entirely clear. In this work, it is demonstrated that BP inhibits the enzyme hexokinase (HK) activity and SCT suppresses the phosphofructokinase (PFK) activity respectively, both the two agents decrease viability, ATP generation and lactate content in the human gastric cancer cell line MGC-803. These effects are directly correlated with blockage of glycolysis. Furthermore, BP and SCT can induce the characteristic manifestations of mitochondria-regulated apoptosis, such as down-regulation of anti-apoptosis proteins Bcl-2 and Survivin, up-regulation of pro-apoptosis protein Bax, activation of caspase-3, as well as leakage of cytochrome c (Cyt-c). In summary, our results provided evidences that BP and SCT inhibit the MGC-803 cells growth and proliferation might be correlated with inhibiting glycolysis and promoting mitochondria-regulated apoptosis. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  7. DK1 Induces Apoptosis via Mitochondria-Dependent Signaling Pathway in Human Colon Carcinoma Cell Lines In Vitro

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    Yazmin Hussin

    2018-04-01

    Full Text Available Extensive research has been done in the search for innovative treatments against colon adenocarcinomas; however, the incidence rate of patients remains a major cause of cancer-related deaths in Malaysia. Natural bioactive compounds such as curcumin have been substantially studied as an alternative to anticancer drug therapies and have been surmised as a potent agent but, nevertheless, remain deficient due to its poor cellular uptake. Therefore, efforts now have shifted toward mimicking curcumin to synthesize novel compounds sharing similar effects. A synthetic analog, (Z-3-hydroxy-1-(2-hydroxyphenyl-3-phenylprop-2-ene-1-one (DK1, was recently synthesized and reported to confer improved bioavailability and selectivity toward human breast cancer cells. This study, therefore, aims to assess the anticancer mechanism of DK1 in relation to the induction of in vitro cell death in selected human colon cancer cell lines. Using the3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide(MTT assay, the cytotoxicity of DK1 towards HT29 and SW620 cell lines were investigated. Acridine orange/propidium iodide (AO/PI dual-staining assay and flow cytometry analyses (cell cycle analysis, Annexin/V-FITC and JC-1 assays were incorporated to determine the mode of cell death. To further determine the mechanism of cell death, quantitative real-time polymerase chain reaction (qRT-PCR and proteome profiling were conducted. Results from this study suggest that DK1 induced changes in cell morphology, leading to a decrease in cell viability and subsequent induction of apoptosis. DK1 treatment inhibited cell viability and proliferation 48 h post treatment with IC50 values of 7.5 ± 1.6 µM for HT29 cells and 14.5 ± 4.3 µM for SW620 cells, causing cell cycle arrest with increased accumulation of cell populations at the sub-G0/G1phaseof 74% and 23%, respectively. Flow cytometry analyses showed that DK1 treatment in cancer cells induced apoptosis, as indicated by DNA

  8. 3-Bromopyruvate and sodium citrate induce apoptosis in human gastric cancer cell line MGC-803 by inhibiting glycolysis and promoting mitochondria-regulated apoptosis pathway

    International Nuclear Information System (INIS)

    Guo, Xingyu; Zhang, Xiaodong; Wang, Tingan; Xian, Shulin; Lu, Yunfei

    2016-01-01

    Cancer cells are mainly dependent on glycolysis to generate adenosine triphosphate (ATP) and intermediates required for cell growth and proliferation. Thus, inhibition of glycolysis might be of therapeutic value in antitumor treatment. Our previously studies had found that both 3-bromopyruvate (BP) and sodium citrate (SCT) can inhibit tumor growth and proliferation in vitro and in vivo. However, the mechanism involved in the BP and SCT mediated antitumor activity is not entirely clear. In this work, it is demonstrated that BP inhibits the enzyme hexokinase (HK) activity and SCT suppresses the phosphofructokinase (PFK) activity respectively, both the two agents decrease viability, ATP generation and lactate content in the human gastric cancer cell line MGC-803. These effects are directly correlated with blockage of glycolysis. Furthermore, BP and SCT can induce the characteristic manifestations of mitochondria-regulated apoptosis, such as down-regulation of anti-apoptosis proteins Bcl-2 and Survivin, up-regulation of pro-apoptosis protein Bax, activation of caspase-3, as well as leakage of cytochrome c (Cyt-c). In summary, our results provided evidences that BP and SCT inhibit the MGC-803 cells growth and proliferation might be correlated with inhibiting glycolysis and promoting mitochondria-regulated apoptosis. -- Highlights: •Blockage of glycolysis might be a novel way to anticancer. •Both 3-bromopyruvate and sodium citrate could inhibit glycolysis and regulate mitochondrial pathway in cancer cells. •Both 3-bromopyruvate and sodium citrate would be the novel agents on treatment of gastric cancer.

  9. 3-Bromopyruvate and sodium citrate induce apoptosis in human gastric cancer cell line MGC-803 by inhibiting glycolysis and promoting mitochondria-regulated apoptosis pathway

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    Guo, Xingyu; Zhang, Xiaodong; Wang, Tingan, E-mail: moonsonlife@yahoo.com; Xian, Shulin; Lu, Yunfei, E-mail: doctorlife@126.com

    2016-06-17

    Cancer cells are mainly dependent on glycolysis to generate adenosine triphosphate (ATP) and intermediates required for cell growth and proliferation. Thus, inhibition of glycolysis might be of therapeutic value in antitumor treatment. Our previously studies had found that both 3-bromopyruvate (BP) and sodium citrate (SCT) can inhibit tumor growth and proliferation in vitro and in vivo. However, the mechanism involved in the BP and SCT mediated antitumor activity is not entirely clear. In this work, it is demonstrated that BP inhibits the enzyme hexokinase (HK) activity and SCT suppresses the phosphofructokinase (PFK) activity respectively, both the two agents decrease viability, ATP generation and lactate content in the human gastric cancer cell line MGC-803. These effects are directly correlated with blockage of glycolysis. Furthermore, BP and SCT can induce the characteristic manifestations of mitochondria-regulated apoptosis, such as down-regulation of anti-apoptosis proteins Bcl-2 and Survivin, up-regulation of pro-apoptosis protein Bax, activation of caspase-3, as well as leakage of cytochrome c (Cyt-c). In summary, our results provided evidences that BP and SCT inhibit the MGC-803 cells growth and proliferation might be correlated with inhibiting glycolysis and promoting mitochondria-regulated apoptosis. -- Highlights: •Blockage of glycolysis might be a novel way to anticancer. •Both 3-bromopyruvate and sodium citrate could inhibit glycolysis and regulate mitochondrial pathway in cancer cells. •Both 3-bromopyruvate and sodium citrate would be the novel agents on treatment of gastric cancer.

  10. Doubly uniparental inheritance of mitochondria as a model system for studying germ line formation.

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    Liliana Milani

    Full Text Available BACKGROUND: Doubly Uniparental Inheritance (DUI of mitochondria occurs when both mothers and fathers are capable of transmitting mitochondria to their offspring, in contrast to the typical Strictly Maternal Inheritance (SMI. DUI was found in some bivalve molluscs, in which two mitochondrial genomes are inherited, one through eggs, the other through sperm. During male embryo development, spermatozoon mitochondria aggregate in proximity of the first cleavage furrow and end up in the primordial germ cells, while they are dispersed in female embryos. METHODOLOGY/PRINCIPAL FINDINGS: We used MitoTracker, microtubule staining and transmission electron microscopy to examine the mechanisms of this unusual distribution of sperm mitochondria in the DUI species Ruditapes philippinarum. Our results suggest that in male embryos the midbody deriving from the mitotic spindle of the first division concurs in positioning the aggregate of sperm mitochondria. Furthermore, an immunocytochemical analysis showed that the germ line determinant Vasa segregates close to the first cleavage furrow. CONCLUSIONS/SIGNIFICANCE: In DUI male embryos, spermatozoon mitochondria aggregate in a stable area on the animal-vegetal axis: in organisms with spiral segmentation this zone is not involved in cleavage, so the aggregation is maintained. Moreover, sperm mitochondria reach the same embryonic area in which also germ plasm is transferred. In 2-blastomere embryos, the segregation of sperm mitochondria in the same region with Vasa suggests their contribution in male germ line formation. In DUI male embryos, M-type mitochondria must be recognized by egg factors to be actively transferred in the germ line, where they become dominant replacing the Balbiani body mitochondria. The typical features of germ line assembly point to a common biological mechanism shared by DUI and SMI organisms. Although the molecular dynamics of the segregation of sperm mitochondria in DUI species are unknown

  11. Human cultured cells are capable to incorporate isolated plant mitochondria loaded with exogenous DNA

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    Laktionov P. P.

    2012-07-01

    Full Text Available Aim. To investigate the possibility of human cultured cells to incorporate isolated mitochondria together with exogenous DNA introduced into organelles. Methods. Two approaches were used for this purpose, fluorescent labelling of mitochondria and/or DNA with subsequent analysis of the cells subjected to incubation by microscopy or by quantitative PCR. Results. We have shown that human cultured cells lines, HeLa and HUVEC, are capable to uptake isolated plant mitochondria and that this process depends on the incubation time and concentration of organelles present in medium. The incorporated mitochondria can serve as vehicles to deliver exogenous DNA into human cells, this DNA is then distributed in different cell compartments. Conclusions. These results are preliminary and need further investigations, including testing the possibility of human cells to incorporate the mitochondria of human or animal origin and creating genetic construction which could provide certain selectivity or stability of the transferred exogenous DNA upon cell uptake of the mitochondria as vectors.

  12. Connecting Mitochondria, Metabolism, and Stem Cell Fate

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    Wanet, Anaïs; Arnould, Thierry; Najimi, Mustapha

    2015-01-01

    As sites of cellular respiration and energy production, mitochondria play a central role in cell metabolism. Cell differentiation is associated with an increase in mitochondrial content and activity and with a metabolic shift toward increased oxidative phosphorylation activity. The opposite occurs during reprogramming of somatic cells into induced pluripotent stem cells. Studies have provided evidence of mitochondrial and metabolic changes during the differentiation of both embryonic and somatic (or adult) stem cells (SSCs), such as hematopoietic stem cells, mesenchymal stem cells, and tissue-specific progenitor cells. We thus propose to consider those mitochondrial and metabolic changes as hallmarks of differentiation processes. We review how mitochondrial biogenesis, dynamics, and function are directly involved in embryonic and SSC differentiation and how metabolic and sensing pathways connect mitochondria and metabolism with cell fate and pluripotency. Understanding the basis of the crosstalk between mitochondria and cell fate is of critical importance, given the promising application of stem cells in regenerative medicine. In addition to the development of novel strategies to improve the in vitro lineage-directed differentiation of stem cells, understanding the molecular basis of this interplay could lead to the identification of novel targets to improve the treatment of degenerative diseases. PMID:26134242

  13. Mitochondria and Energetic Depression in Cell Pathophysiology

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    Stephan Zierz

    2009-05-01

    Full Text Available Mitochondrial dysfunction is a hallmark of almost all diseases. Acquired or inherited mutations of the mitochondrial genome DNA may give rise to mitochondrial diseases. Another class of disorders, in which mitochondrial impairments are initiated by extramitochondrial factors, includes neurodegenerative diseases and syndromes resulting from typical pathological processes, such as hypoxia/ischemia, inflammation, intoxications, and carcinogenesis. Both classes of diseases lead to cellular energetic depression (CED, which is characterized by decreased cytosolic phosphorylation potential that suppresses the cell’s ability to do work and control the intracellular Ca2+ homeostasis and its redox state. If progressing, CED leads to cell death, whose type is linked to the functional status of the mitochondria. In the case of limited deterioration, when some amounts of ATP can still be generated due to oxidative phosphorylation (OXPHOS, mitochondria launch the apoptotic cell death program by release of cytochrome c. Following pronounced CED, cytoplasmic ATP levels fall below the thresholds required for processing the ATP-dependent apoptotic cascade and the cell dies from necrosis. Both types of death can be grouped together as a mitochondrial cell death (MCD. However, there exist multiple adaptive reactions aimed at protecting cells against CED. In this context, a metabolic shift characterized by suppression of OXPHOS combined with activation of aerobic glycolysis as the main pathway for ATP synthesis (Warburg effect is of central importance. Whereas this type of adaptation is sufficiently effective to avoid CED and to control the cellular redox state, thereby ensuring the cell survival, it also favors the avoidance of apoptotic cell death. This scenario may underlie uncontrolled cellular proliferation and growth, eventually resulting in carcinogenesis.

  14. Calcium regulates cell death in cancer: Roles of the mitochondria and mitochondria-associated membranes (MAMs).

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    Danese, Alberto; Patergnani, Simone; Bonora, Massimo; Wieckowski, Mariusz R; Previati, Maurizio; Giorgi, Carlotta; Pinton, Paolo

    2017-08-01

    Until 1972, the term 'apoptosis' was used to differentiate the programmed cell death that naturally occurs in organismal development from the acute tissue death referred to as necrosis. Many studies on cell death and programmed cell death have been published and most are, at least to some degree, related to cancer. Some key proteins and molecular pathways implicated in cell death have been analyzed, whereas others are still being actively researched; therefore, an increasing number of cellular compartments and organelles are being implicated in cell death and cancer. Here, we discuss the mitochondria and subdomains of the endoplasmic reticulum (ER) that interact with mitochondria, the mitochondria-associated membranes (MAMs), which have been identified as critical hubs in the regulation of cell death and tumor growth. MAMs-dependent calcium (Ca 2+ ) release from the ER allows selective Ca 2+ uptake by the mitochondria. The perturbation of Ca 2+ homeostasis in cancer cells is correlated with sustained cell proliferation and the inhibition of cell death through the modulation of Ca 2+ signaling. This article is part of a Special Issue entitled Mitochondria in Cancer, edited by Giuseppe Gasparre, Rodrigue Rossignol and Pierre Sonveaux. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Localization of mitochondria in living cells with rhodamine 123.

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    Johnson, L V; Walsh, M L; Chen, L B

    1980-01-01

    The laser dye rhodamine 123 is shown to be a specific probe for the localization of mitochondria in living cells. By virtue of its selectivity for mitochondria and its fluorescent properties, the detectability of mitochondria stained with rhodamine 123 is significantly improved over that provided by conventional light microscopic techniques. With the use of rhodamine 123, it is possible to detect alterations in mitochondrial distribution following transformation by Rous sarcoma virus and changes in the shape and organization of mitochondria induced by colchicine treatment. Images PMID:6965798

  16. Detection of PIWI and piRNAs in the mitochondria of mammalian cancer cells

    International Nuclear Information System (INIS)

    Kwon, ChangHyuk; Tak, Hyosun; Rho, Mina; Chang, Hae Ryung; Kim, Yon Hui; Kim, Kyung Tae; Balch, Curt; Lee, Eun Kyung; Nam, Seungyoon

    2014-01-01

    Highlights: • piRNA sequences were mapped to human mitochondrial (mt) genome. • We inspected small RNA-Seq datasets from somatic cell mt subcellular fractions. • Piwi and piRNA transcripts are present in mammalian somatic cancer cell mt fractions. - Abstract: Piwi-interacting RNAs (piRNAs) are 26–31 nt small noncoding RNAs that are processed from their longer precursor transcripts by Piwi proteins. Localization of Piwi and piRNA has been reported mostly in nucleus and cytoplasm of higher eukaryotes germ-line cells, where it is believed that known piRNA sequences are located in repeat regions of nuclear genome in germ-line cells. However, localization of PIWI and piRNA in mammalian somatic cell mitochondria yet remains largely unknown. We identified 29 piRNA sequence alignments from various regions of the human mitochondrial genome. Twelve out 29 piRNA sequences matched stem-loop fragment sequences of seven distinct tRNAs. We observed their actual expression in mitochondria subcellular fractions by inspecting mitochondrial-specific small RNA-Seq datasets. Of interest, the majority of the 29 piRNAs overlapped with multiple longer transcripts (expressed sequence tags) that are unique to the human mitochondrial genome. The presence of mature piRNAs in mitochondria was detected by qRT-PCR of mitochondrial subcellular RNAs. Further validation showed detection of Piwi by colocalization using anti-Piwil1 and mitochondria organelle-specific protein antibodies

  17. Detection of PIWI and piRNAs in the mitochondria of mammalian cancer cells

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    Kwon, ChangHyuk, E-mail: netbuyer@hanmail.net [Cancer Genomics Branch, National Cancer Center, Goyang 410-769 (Korea, Republic of); Tak, Hyosun, E-mail: chuberry@naver.com [Department of Biochemistry, College of Medicine, Catholic University of Korea, Seoul 137-701 (Korea, Republic of); Rho, Mina, E-mail: minarho@hanyang.ac.kr [Department of Computer Science, Hanyang University, Seoul 133-791 (Korea, Republic of); Chang, Hae Ryung, E-mail: heyhae@ncc.re.kr [New Experimental Therapeutics Branch, National Cancer Center, Goyang 410-769 (Korea, Republic of); Kim, Yon Hui, E-mail: yhkim@ncc.re.kr [New Experimental Therapeutics Branch, National Cancer Center, Goyang 410-769 (Korea, Republic of); Kim, Kyung Tae, E-mail: bioktkim@ncc.re.kr [Molecular Epidemiology Branch, National Cancer Center, Goyang 410-769 (Korea, Republic of); Balch, Curt, E-mail: curt.balch@gmail.com [Medical Sciences Program, Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Bloomington, IN 47405 (United States); Lee, Eun Kyung, E-mail: leeek@catholic.ac.kr [Department of Biochemistry, College of Medicine, Catholic University of Korea, Seoul 137-701 (Korea, Republic of); Nam, Seungyoon, E-mail: seungyoon.nam@ncc.re.kr [Cancer Genomics Branch, National Cancer Center, Goyang 410-769 (Korea, Republic of)

    2014-03-28

    Highlights: • piRNA sequences were mapped to human mitochondrial (mt) genome. • We inspected small RNA-Seq datasets from somatic cell mt subcellular fractions. • Piwi and piRNA transcripts are present in mammalian somatic cancer cell mt fractions. - Abstract: Piwi-interacting RNAs (piRNAs) are 26–31 nt small noncoding RNAs that are processed from their longer precursor transcripts by Piwi proteins. Localization of Piwi and piRNA has been reported mostly in nucleus and cytoplasm of higher eukaryotes germ-line cells, where it is believed that known piRNA sequences are located in repeat regions of nuclear genome in germ-line cells. However, localization of PIWI and piRNA in mammalian somatic cell mitochondria yet remains largely unknown. We identified 29 piRNA sequence alignments from various regions of the human mitochondrial genome. Twelve out 29 piRNA sequences matched stem-loop fragment sequences of seven distinct tRNAs. We observed their actual expression in mitochondria subcellular fractions by inspecting mitochondrial-specific small RNA-Seq datasets. Of interest, the majority of the 29 piRNAs overlapped with multiple longer transcripts (expressed sequence tags) that are unique to the human mitochondrial genome. The presence of mature piRNAs in mitochondria was detected by qRT-PCR of mitochondrial subcellular RNAs. Further validation showed detection of Piwi by colocalization using anti-Piwil1 and mitochondria organelle-specific protein antibodies.

  18. New nanocomposites for SERS studies of living cells and mitochondria

    DEFF Research Database (Denmark)

    Sarycheva, A. S.; Brazhe, N. A.; Baizhumanov, A. A.

    2016-01-01

    A great enhancement in Raman scattering (SERS) from heme-containing submembrane biomolecules inside intact erythrocytes and functional mitochondria is demonstrated for the first time using silver–silica beads prepared using a new method involving aerosol pyrolysis with aqueous diamminesilver...... molecules. The SERS spectra of functional mitochondria are sensitive to the activity of the mitochondrial electron transport chain, thus making the method a novel label-free approach to monitor the redox state and conformation of cytochromes in their natural cell environment. The developed nanocomposites...

  19. Mitochondria can Power Cells to Life and Death

    Indian Academy of Sciences (India)

    molecular basis of pathogenicity and ... Mitochondria were discovered by R Altman in 1890 and the word was coined ... Diabetes mellitus ... 2 x 103 per cell) depending upon where and in which tissue they exist. They can .... is released, which then gears up other types of caspases to orchestrate .... Cellular Biology, CDFD.

  20. Mitochondria: An intriguing target for killing tumour-initiating cells

    Czech Academy of Sciences Publication Activity Database

    Yan, B.; Dong, L.; Neužil, Jiří

    2016-01-01

    Roč. 26, JAN 2016 (2016), s. 86-93 ISSN 1567-7249 R&D Projects: GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:86652036 Keywords : Tumour-initiating cells * ALPHA-TOCOPHERYL SUCCINATE * Therapeutic resistance * Mitochondria Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.704, year: 2016

  1. Modeling population dynamics of mitochondria in mammalian cells

    Science.gov (United States)

    Kornick, Kellianne; Das, Moumita

    Mitochondria are organelles located inside eukaryotic cells and are essential for several key cellular processes such as energy (ATP) production, cell signaling, differentiation, and apoptosis. All organisms are believed to have low levels of variation in mitochondrial DNA (mtDNA), and alterations in mtDNA are connected to a range of human health conditions, including epilepsy, heart failure, Parkinsons disease, diabetes, and multiple sclerosis. Therefore, understanding how changes in mtDNA accumulate over time and are correlated to changes in mitochondrial function and cell properties can have a profound impact on our understanding of cell physiology and the origins of some diseases. Motivated by this, we develop and study a mathematical model to determine which cellular parameters have the largest impact on mtDNA population dynamics. The model consists of coupled ODEs to describe subpopulations of healthy and dysfunctional mitochondria subject to mitochondrial fission, fusion, autophagy, and mutation. We study the time evolution and stability of each sub-population under specific selection biases and pressures by tuning specific terms in our model. Our results may provide insights into how sub-populations of mitochondria survive and evolve under different selection pressures. This work was supported by a Grant from the Moore Foundation.

  2. The role of mitochondria in yeast programmed cell death

    International Nuclear Information System (INIS)

    Guaragnella, Nicoletta; Ždralević, Maša; Antonacci, Lucia; Passarella, Salvatore; Marra, Ersilia; Giannattasio, Sergio

    2012-01-01

    Mammalian apoptosis and yeast programmed cell death (PCD) share a variety of features including reactive oxygen species production, protease activity and a major role played by mitochondria. In view of this, and of the distinctive characteristics differentiating yeast and multicellular organism PCD, the mitochondrial contribution to cell death in the genetically tractable yeast Saccharomyces cerevisiae has been intensively investigated. In this mini-review we report whether and how yeast mitochondrial function and proteins belonging to oxidative phosphorylation, protein trafficking into and out of mitochondria, and mitochondrial dynamics, play a role in PCD. Since in PCD many processes take place over time, emphasis will be placed on an experimental model based on acetic acid-induced PCD (AA-PCD) which has the unique feature of having been investigated as a function of time. As will be described there are at least two AA-PCD pathways each with a multifaceted role played by mitochondrial components, in particular by cytochrome c.

  3. The mitochondria-mediate apoptosis of Lepidopteran cells induced by azadirachtin.

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    Jingfei Huang

    Full Text Available Mitochondria have been shown to play an important role in apoptosis using mammalian cell lines. However, this seems not to be the case in Drosophila, an insect model organism; thus more in-depth studies of insect cell apoptosis are necessary. In the present study, mitochondrial involvement during azadirachtin- and camptothecin-induced apoptosis in Spodoptera frugiperda Sf9 cells (isolated from Spodoptera frugiperda pupal ovarian tissue was investigated. The results showed that both azadirachtin and camptothecin could induce apoptosis in Sf9 cells. Reactive oxygen species (ROS generation, activation of mitochondrial permeability transition pores (MPTPs and loss of mitochondrial membrane potential (MMP were observed very early during apoptosis and were followed subsequently by the release of cytochrome-c from the mitochondria. Furthermore, the results also revealed that the opening of MPTPs and the loss of MMP induced by azadirachtin could be significantly inhibited by the permeability transition pore (PTP inhibitor cyclosporin A (CsA, which was used to identify the key role of mitochondria in the apoptosis of Sf9 cells. However, in camptothecin-treated Sf9 cells, CsA could not suppress the opening of MPTPs and the loss of MMP when apoptosis was induced. The data from caspase-3 and caspase-9 activity assays and detection of apoptosis by morphological observation and flow cytometry also uncovered the different effect of CsA on the two botanical apoptosis inducers. Although different mechanisms of apoptosis induction exist, our study revealed that mitochondria play a crucial role in insect cell line apoptosis.

  4. The mitochondria-mediate apoptosis of Lepidopteran cells induced by azadirachtin.

    Science.gov (United States)

    Huang, Jingfei; Lv, Chaojun; Hu, Meiying; Zhong, Guohua

    2013-01-01

    Mitochondria have been shown to play an important role in apoptosis using mammalian cell lines. However, this seems not to be the case in Drosophila, an insect model organism; thus more in-depth studies of insect cell apoptosis are necessary. In the present study, mitochondrial involvement during azadirachtin- and camptothecin-induced apoptosis in Spodoptera frugiperda Sf9 cells (isolated from Spodoptera frugiperda pupal ovarian tissue) was investigated. The results showed that both azadirachtin and camptothecin could induce apoptosis in Sf9 cells. Reactive oxygen species (ROS) generation, activation of mitochondrial permeability transition pores (MPTPs) and loss of mitochondrial membrane potential (MMP) were observed very early during apoptosis and were followed subsequently by the release of cytochrome-c from the mitochondria. Furthermore, the results also revealed that the opening of MPTPs and the loss of MMP induced by azadirachtin could be significantly inhibited by the permeability transition pore (PTP) inhibitor cyclosporin A (CsA), which was used to identify the key role of mitochondria in the apoptosis of Sf9 cells. However, in camptothecin-treated Sf9 cells, CsA could not suppress the opening of MPTPs and the loss of MMP when apoptosis was induced. The data from caspase-3 and caspase-9 activity assays and detection of apoptosis by morphological observation and flow cytometry also uncovered the different effect of CsA on the two botanical apoptosis inducers. Although different mechanisms of apoptosis induction exist, our study revealed that mitochondria play a crucial role in insect cell line apoptosis.

  5. Computational properties of mitochondria in T cell activation and fate.

    Science.gov (United States)

    Uzhachenko, Roman; Shanker, Anil; Dupont, Geneviève

    2016-11-01

    In this article, we review how mitochondrial Ca 2+ transport (mitochondrial Ca 2+ uptake and Na + /Ca 2+ exchange) is involved in T cell biology, including activation and differentiation through shaping cellular Ca 2+ signals. Based on recent observations, we propose that the Ca 2+ crosstalk between mitochondria, endoplasmic reticulum and cytoplasm may form a proportional-integral-derivative (PID) controller. This PID mechanism (which is well known in engineering) could be responsible for computing cellular decisions. In addition, we point out the importance of analogue and digital signal processing in T cell life and implication of mitochondrial Ca 2+ transport in this process. © 2016 The Authors.

  6. Pluripotent Stem Cell Metabolism and Mitochondria: Beyond ATP

    Directory of Open Access Journals (Sweden)

    Jarmon G. Lees

    2017-01-01

    Full Text Available Metabolism is central to embryonic stem cell (ESC pluripotency and differentiation, with distinct profiles apparent under different nutrient milieu, and conditions that maintain alternate cell states. The significance of altered nutrient availability, particularly oxygen, and metabolic pathway activity has been highlighted by extensive studies of their impact on preimplantation embryo development, physiology, and viability. ESC similarly modulate their metabolism in response to altered metabolite levels, with changes in nutrient availability shown to have a lasting impact on derived cell identity through the regulation of the epigenetic landscape. Further, the preferential use of glucose and anaplerotic glutamine metabolism serves to not only support cell growth and proliferation but also minimise reactive oxygen species production. However, the perinuclear localisation of spherical, electron-poor mitochondria in ESC is proposed to sustain ESC nuclear-mitochondrial crosstalk and a mitochondrial-H2O2 presence, to facilitate signalling to support self-renewal through the stabilisation of HIFα, a process that may be favoured under physiological oxygen. The environment in which a cell is grown is therefore a critical regulator and determinant of cell fate, with metabolism, and particularly mitochondria, acting as an interface between the environment and the epigenome.

  7. Identification of nitric oxide in mitochondria of myometrium cell

    Directory of Open Access Journals (Sweden)

    Danylovych Yu. V.

    2015-06-01

    Full Text Available Aim. To demonstrate the possibility of NO synthesis in intact myocytes of uterus. Methods. Confocal scanning microscopy method, NO-sensitive fluorescent probe DAF-FM, MitoTracker Orange CM-H2TMRos. Results. The basal production of NO in intact myocytes was shown using DAF-FM. Incubation of myocytes with NO donor – sodium nitroprusside (SNP – led to an increase of the DAF-FM-T fluorescent signal. On the contrary, the addition of NO-synthase inhibitor – N-nitro-L-arginine (NA – results in the reduction of fluorescent intensity. It was demonstrated colocalizition of specific probe for mitochondria MitoTracker Orange CM-H2TMRos and NO-sensitive dye DAF-FM. Conclusions. For the first time it has been demonstrated the presence of NO in smooth muscle cell mitochondria using laser confocal microscopy, NO-sensitive probe DAF-FM and specific marker of the functionally active mitochondria MitoTracker Orange CM-H2TMRos.

  8. Shikonin Directly Targets Mitochondria and Causes Mitochondrial Dysfunction in Cancer Cells

    Directory of Open Access Journals (Sweden)

    Benjamin Wiench

    2012-01-01

    Full Text Available Chemotherapy is a mainstay of cancer treatment. Due to increased drug resistance and the severe side effects of currently used therapeutics, new candidate compounds are required for improvement of therapy success. Shikonin, a natural naphthoquinone, was used in traditional Chinese medicine for the treatment of different inflammatory diseases and recent studies revealed the anticancer activities of shikonin. We found that shikonin has strong cytotoxic effects on 15 cancer cell lines, including multidrug-resistant cell lines. Transcriptome-wide mRNA expression studies showed that shikonin induced genetic pathways regulating cell cycle, mitochondrial function, levels of reactive oxygen species, and cytoskeletal formation. Taking advantage of the inherent fluorescence of shikonin, we analyzed its uptake and distribution in live cells with high spatial and temporal resolution using flow cytometry and confocal microscopy. Shikonin was specifically accumulated in the mitochondria, and this accumulation was associated with a shikonin-dependent deregulation of cellular Ca2+ and ROS levels. This deregulation led to a breakdown of the mitochondrial membrane potential, dysfunction of microtubules, cell-cycle arrest, and ultimately induction of apoptosis. Seeing as both the metabolism and the structure of mitochondria show marked differences between cancer cells and normal cells, shikonin is a promising candidate for the next generation of chemotherapy.

  9. Correlation between chloride flux via the mitochondria-rich cells and transepithelial water movement in isolated frog skin (Rana esculenta)

    DEFF Research Database (Denmark)

    Nielsen, Robert

    1995-01-01

    Antidiuretic hormone; chloride transport; electroosmosis; Frog skin; Intercalated cells; Local osmosis; Mitochondria-rich cells.......Antidiuretic hormone; chloride transport; electroosmosis; Frog skin; Intercalated cells; Local osmosis; Mitochondria-rich cells....

  10. Sensitivity of mitochondria of the mouse liver cells to radiation

    International Nuclear Information System (INIS)

    Shima, Akihiro

    1974-01-01

    In order to study the sensitivity of mitochondria (Mt) of the liver cells to radiation, 0.4 mg of riboflavine (RF) was intraperitoneally injected into mice which had been fed RF deficient food for 13 weeks. Three hours later 400 R of X-ray (190 KVP, 25 mA, 0.5 mmCu, 0.5 mmAl filter, FSD 61.5 cm, and HVL 0.80 mmCu) were irradiated to the whole body, and giant Mt of the liver cells were observed. When the liver cells were observed 24 hours after injection, neither giant Mt nor mitotic findings of Mt were found. All Mt observed were small (1.2 μ), although mice received 400 R of X-ray. (Serizawa, K.)

  11. Sensitivity of mitochondria of the mouse liver cells to radiation

    Energy Technology Data Exchange (ETDEWEB)

    Shima, A [Tokyo Univ. (Japan). Faculty of Science

    1974-06-01

    In order to study the sensitivity of mitochondria (Mt) of the liver cells to radiation, 0.4 mg of riboflavine (RF) was intraperitoneally injected into mice which had been fed RF deficient food for 13 weeks. Three hours later 400 R of X-ray (190 KVP, 25 mA, 0.5 mmCu, 0.5 mmAl filter, FSD 61.5 cm, and HVL 0.80 mmCu) were irradiated to the whole body, and giant Mt of the liver cells were observed. When the liver cells were observed 24 hours after injection, neither giant Mt nor mitotic findings of Mt were found. All Mt observed were small (1.2 ..mu..), although mice received 400 R of X-ray.

  12. Action of diclofenac on kidney mitochondria and cells

    International Nuclear Information System (INIS)

    Ng, Lin Eng; Vincent, Annette S.; Halliwell, Barry; Wong, Kim Ping

    2006-01-01

    The mitochondrial membrane potential measured in isolated rat kidney mitochondria and in digitonin-permeabilized MDCK type II cells pre-energized with succinate, glutamate, and/or malate was reduced by micromolar diclofenac dose-dependently. However, ATP biosynthesis from glutamate/malate was significantly more compromised compared to that from succinate. Inhibition of the malate-aspartate shuttle by diclofenac with a resultant decrease in the ability of mitochondria to generate NAD(P)H was demonstrated. Diclofenac however had no effect on the activities of NADH dehydrogenase, glutamate dehydrogenase, and malate dehydrogenase. In conclusion, decreased NAD(P)H production due to an inhibition of the entry of malate and glutamate via the malate-aspartate shuttle explained the more pronounced decreased rate of ATP biosynthesis from glutamate and malate by diclofenac. This drug, therefore affects the bioavailability of two major respiratory complex I substrates which would normally contribute substantially to supplying the reducing equivalents for mitochondrial electron transport for generation of ATP in the renal cell

  13. Mitochondria and the non-genetic origins of cell-to-cell variability: More is different.

    Science.gov (United States)

    Guantes, Raúl; Díaz-Colunga, Juan; Iborra, Francisco J

    2016-01-01

    Gene expression activity is heterogeneous in a population of isogenic cells. Identifying the molecular basis of this variability will improve our understanding of phenomena like tumor resistance to drugs, virus infection, or cell fate choice. The complexity of the molecular steps and machines involved in transcription and translation could introduce sources of randomness at many levels, but a common constraint to most of these processes is its energy dependence. In eukaryotic cells, most of this energy is provided by mitochondria. A clonal population of cells may show a large variability in the number and functionality of mitochondria. Here, we discuss how differences in the mitochondrial content of each cell contribute to heterogeneity in gene products. Changes in the amount of mitochondria can also entail drastic alterations of a cell's gene expression program, which ultimately leads to phenotypic diversity. Also watch the Video Abstract. © 2015 WILEY Periodicals, Inc.

  14. The oncolytic peptide LTX-315 induces cell death and DAMP release by mitochondria distortion in human melanoma cells

    Science.gov (United States)

    Eike, Liv-Marie; Yang, Nannan; Rekdal, Øystein; Sveinbjørnsson, Baldur

    2015-01-01

    Host defense peptides (HDPs) are naturally occurring molecules found in most species, in which they play a significant role in the first line defense against intruding pathogens, and several HDPs have been shown to possess anticancer activity. Structure-activity relationship studies on the HDP bovine lactoferricin revealed a de novo design of a nonamer peptide LTX-315, with oncolytic properties. In the present study, we investigated the oncolytic activity of LTX-315 in human melanoma cells (A375). LTX-315 induced a rapid plasma membrane disruption and cell death within 2 hours. At a low concentration, fluorescence-labeled LTX-315 was internalized and accumulated in cytoplasmic vacuoles in close proximity to the mitochondria. The mitochondrial membrane potential was shown to depolarize as a consequence of LTX-315 treatment and at ultrastructural level, the mitochondria morphology was significantly altered. Release of danger signals (DAMPs) such as ATP, Cytochrome C and HMGB1 into the cell supernatant of cultured cells was evident minutes after peptide treatment. The oncolytic effect of LTX-315 involving perturbation of both the cell membrane and the mitochondria with subsequent release of DAMPs may highlight the ability of LTX-315 to induce complete regression and long-term protective immune responses as previously reported in experimental animal models. PMID:26472184

  15. Ion transport by mitochondria-rich cells in toad skin

    DEFF Research Database (Denmark)

    Larsen, Erik Hviid; Ussing, H H; Spring, K R

    1987-01-01

    The optical sectioning video imaging technique was used for measurements of the volume of mitochondria-rich (m.r.) cells of the isolated epithelium of toad skin. Under short-circuit conditions, cell volume decreased by about 14% in response to bilateral exposure to Cl-free (gluconate substitution....... Unilateral exposure to a Cl-free solution did not prevent ouabain-induced cell swelling. It is concluded that m.r. cells have an amiloride-blockable Na conductance in the apical membrane, a ouabain-sensitive Na pump in the basolateral membrane, and a passive Cl permeability in both membranes. From...... the initial rate of ouabain-induced cell volume increase the active Na current carried by a single m.r. cell was estimated to be 9.9 +/- 1.3 pA. Voltage clamping of the preparation in the physiological range of potentials (0 to -100 mV, serosa grounded) resulted in a cell volume increase with a time course...

  16. Involvement of S6K1 in mitochondria function and structure in HeLa cells.

    Science.gov (United States)

    Park, Jisoo; Tran, Quangdon; Mun, Kisun; Masuda, Kouhei; Kwon, So Hee; Kim, Seon-Hwan; Kim, Dong-Hoon; Thomas, George; Park, Jongsun

    2016-12-01

    The major biological function of mitochondria is to generate cellular energy through oxidative phosphorylation. Apart from cellular respiration, mitochondria also play a key role in signaling processes, including aging and cancer metabolism. It has been shown that S6K1-knockout mice are resistant to obesity due to enhanced beta-oxidation, with an increased number of large mitochondria. Therefore, in this report, the possible involvement of S6K1 in regulating mitochondria dynamics and function has been investigated in stable lenti-shS6K1-HeLa cells. Interestingly, S6K1-stably depleted HeLa cells showed phenotypical changes in mitochondria morphology. This observation was further confirmed by detailed image analysis of mitochondria shape. Corresponding molecular changes were also observed in these cells, such as the induction of mitochondrial fission proteins (Drp1 and Fis1). Oxygen consumption is elevated in S6K1-depeleted HeLa cells and FL5.12 cells. In addition, S6K1 depletion leads to enhancement of ATP production in cytoplasm and mitochondria. However, the relative ratio of mitochondrial ATP to cytoplasmic ATP is actually decreased in lenti-shS6K1-HeLa cells compared to control cells. Lastly, induction of mitophagy was found in lenti-shS6K1-HeLa cells with corresponding changes of mitochondria shape on electron microscope analysis. Taken together, our results indicate that S6K1 is involved in the regulation of mitochondria morphology and function in HeLa cells. This study will provide novel insights into S6K1 function in mitochondria-mediated cellular signaling. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Acrolein-induced cell death in PC12 cells: role of mitochondria-mediated oxidative stress.

    Science.gov (United States)

    Luo, Jian; Robinson, J Paul; Shi, Riyi

    2005-12-01

    Oxidative stress has been implicated in acrolein cytotoxicity in various cell types, including mammalian spinal cord tissue. In this study we report that acrolein also decreases PC12 cell viability in a reactive oxygen species (ROS)-dependent manner. Specifically, acrolein-induced cell death, mainly necrosis, is accompanied by the accumulation of cellular ROS. Elevating ROS scavengers can alleviate acrolein-induced cell death. Furthermore, we show that exposure to acrolein leads to mitochondrial dysfunction, denoted by the loss of mitochondrial transmembrane potential, reduction of cellular oxygen consumption, and decrease of ATP level. This raises the possibility that the cellular accumulation of ROS could result from the increased production of ROS in the mitochondria of PC12 cells as a result of exposure to acrolein. The acrolein-induced significant decrease of ATP production in mitochondria may also explain why necrosis, not apoptosis, is the dominant type of cell death. In conclusion, our data suggest that one possible mechanism of acrolein-induced cell death could be through mitochondria as its initial target. The subsequent increase of ROS then inflicts cell death and further worsens mitochondria function. Such mechanism may play an important role in CNS trauma and neurodegenerative diseases.

  18. Role of mitochondria-associated hexokinase II in cancer cell death induced by 3-Bromopyruvate

    Science.gov (United States)

    Chen, Zhao; Zhang, Hui; Lu, Weiqin; Huang, Peng

    2009-01-01

    Summary It has long been observed that cancer cells rely more on glycolysis to generate ATP and actively use certain glycolytic metabolic intermediates for biosynthesis. Hexokinase II (HKII) is a key glycolytic enzyme that plays a role in the regulation of the mitochondria-initiated apoptotic cell death. As a potent inhibitor of hexokinase, 3-bromopyruvate (3-BrPA) is known to inhibit cancer cell energy metabolism and trigger cell death, supposedly through depletion of cellular ATP. The current study showed that 3-BrPA caused a covalent modification of HKII protein and directly triggered its dissociation from mitochondria, leading to a specific release of apoptosis-inducing factor (AIF) from the mitochondria to cytosol and eventual cell death. Co-immunoprecipitation revealed a physical interaction between HKII and AIF. Using a competitive peptide of HKII, we showed that the dissociation of hexokinase II from mitochondria alone could cause apoptotic cell death, especially in the mitochondria-deficient ρ0 cells that highly express HKII. Interestingly, the dissociation of HKII itself did no directly affect the mitochondrial membrane potential, ROS generation, and oxidative phosphorylation. Our study suggests that the physical association between HKII and AIF is important for the normal localization of AIF in the mitochondria, and disruption of this protein complex by 3-BrPA leads to their release from the mitochondria and eventual cell death. PMID:19285479

  19. Mitochondria-Associated Membranes As Networking Platforms and Regulators of Cancer Cell Fate

    Directory of Open Access Journals (Sweden)

    Maria Livia Sassano

    2017-08-01

    Full Text Available The tight cross talk between two essential organelles of the cell, the endoplasmic reticulum (ER and mitochondria, is spatially and functionally regulated by specific microdomains known as the mitochondria-associated membranes (MAMs. MAMs are hot spots of Ca2+ transfer between the ER and mitochondria, and emerging data indicate their vital role in the regulation of fundamental physiological processes, chief among them mitochondria bioenergetics, proteostasis, cell death, and autophagy. Moreover, and perhaps not surprisingly, it has become clear that signaling events regulated at the ER–mitochondria intersection regulate key processes in oncogenesis and in the response of cancer cells to therapeutics. ER–mitochondria appositions have been shown to dynamically recruit oncogenes and tumor suppressors, modulating their activity and protein complex formation, adapt the bioenergetic demand of cancer cells and to regulate cell death pathways and redox signaling in cancer cells. In this review, we discuss some emerging players of the ER–mitochondria contact sites in mammalian cells, the key processes they regulate and recent evidence highlighting the role of MAMs in shaping cell-autonomous and non-autonomous signals that regulate cancer growth.

  20. ONC201 kills breast cancer cells in vitro by targeting mitochondria.

    Science.gov (United States)

    Greer, Yoshimi Endo; Porat-Shliom, Natalie; Nagashima, Kunio; Stuelten, Christina; Crooks, Dan; Koparde, Vishal N; Gilbert, Samuel F; Islam, Celia; Ubaldini, Ashley; Ji, Yun; Gattinoni, Luca; Soheilian, Ferri; Wang, Xiantao; Hafner, Markus; Shetty, Jyoti; Tran, Bao; Jailwala, Parthav; Cam, Maggie; Lang, Martin; Voeller, Donna; Reinhold, William C; Rajapakse, Vinodh; Pommier, Yves; Weigert, Roberto; Linehan, W Marston; Lipkowitz, Stanley

    2018-04-06

    We report a novel mechanism of action of ONC201 as a mitochondria-targeting drug in cancer cells. ONC201 was originally identified as a small molecule that induces transcription of TNF-related apoptosis-inducing ligand (TRAIL) and subsequently kills cancer cells by activating TRAIL death receptors. In this study, we examined ONC201 toxicity on multiple human breast and endometrial cancer cell lines. ONC201 attenuated cell viability in all cancer cell lines tested. Unexpectedly, ONC201 toxicity was not dependent on either TRAIL receptors nor caspases. Time-lapse live cell imaging revealed that ONC201 induces cell membrane ballooning followed by rupture, distinct from the morphology of cells undergoing apoptosis. Further investigation found that ONC201 induces phosphorylation of AMP-dependent kinase and ATP loss. Cytotoxicity and ATP depletion were significantly enhanced in the absence of glucose, suggesting that ONC201 targets mitochondrial respiration. Further analysis indicated that ONC201 indirectly inhibits mitochondrial respiration. Confocal and electron microscopic analysis demonstrated that ONC201 triggers mitochondrial structural damage and functional impairment. Moreover, ONC201 decreased mitochondrial DNA (mtDNA). RNAseq analysis revealed that ONC201 suppresses expression of multiple mtDNA-encoded genes and nuclear-encoded mitochondrial genes involved in oxidative phosphorylation and other mitochondrial functions. Importantly, fumarate hydratase deficient cancer cells and multiple cancer cell lines with reduced amounts of mtDNA were resistant to ONC201. These results indicate that cells not dependent on mitochondrial respiration are ONC201-resistant. Our data demonstrate that ONC201 kills cancer cells by disrupting mitochondrial function and further suggests that cancer cells that are dependent on glycolysis will be resistant to ONC201.

  1. Branchial mitochondria-rich cells in the dogfish Squalus acanthias.

    Science.gov (United States)

    Wilson, Jonathan M; Morgan, John D; Vogl, A Wayne; Randall, David J

    2002-06-01

    In marine teleost fishes, the gill mitochondria-rich cells (MRCs) are responsible for NaCl elimination; however, in elasmobranch fishes, the specialized rectal gland is considered to be the most important site for salt secretion. The role of the gills in elasmobranch ion regulation, although clearly shown to be secondary, is not well characterized. In the present study, we investigated some morphological properties of the branchial MRCs and the localization, and activity of the important ionoregulatory enzyme Na(+)/K(+)-ATPase, under control conditions and following rectal gland removal (1 month) in the spiny dogfish, Squalus acanthias. A clear correlation can be made between MRC numbers and the levels of Na(+)/K(+)-ATPase activity in crude gill homogenates (r(2)=-0.69). Strong Na(+)/K(+)-ATPase immunoreactivity is also clearly associated with the basolateral membrane of these MRCs. In addition, the dogfish were able to maintain ionic balance after rectal gland removal. These results all suggest a possible role of the dogfish gill in salt secretion. MRCs were, however, unresponsive to rectal gland removal in terms of changes in number, fine structure and Na(+)/K(+)-ATPase activity, as might be expected if they were compensating for the loss of salt secretion by the rectal gland. Thus, the specific role that these MRCs play in ion regulation in the dogfish remains to be determined

  2. PEGylated anticancer-carbon nanotubes complex targeting mitochondria of lung cancer cells

    Science.gov (United States)

    Kim, Sang-Woo; Lee, Yeon Kyung; Lee, Jong Yeon; Hong, Jeong Hee; Khang, Dongwoo

    2017-11-01

    Although activating apoptosis in cancer cells by targeting the mitochondria is an effective strategy for cancer therapy, insufficient targeting of the mitochondria in cancer cells restricts the availability in clinical treatment. Here, we report on a polyethylene glycol-coated carbon nanotube (CNT)-ABT737 nanodrug that improves the mitochondrial targeting of lung cancer cells. The polyethylene glycol-coated CNT-ABT737 nanodrug internalized into the early endosomes via macropinocytosis and clathrin-mediated endocytosis in advance of early endosomal escape and delivered into the mitochondria. Cytosol release of the nanodrug led to apoptosis of lung cancer cells by abruption of the mitochondrial membrane potential, inducing Bcl-2-mediated apoptosis and generating intracellular reactive oxygen species. As such, this study provides an effective strategy for increasing the anti-lung cancer efficacy by increasing mitochondria accumulation rate of cytosol released anticancer nanodrugs.

  3. Mitochondria-targeted superoxide dismutase (SOD2) regulates radiation resistance and radiation stress response in HeLa cells

    International Nuclear Information System (INIS)

    Hosoki, Ayaka; Yonekura, Shin-Ichiro; Zhao, Qing-Li

    2012-01-01

    Reactive oxygen species (ROS) act as a mediator of ionizing radiation-induced cellular damage. Previous studies have indicated that MnSOD (SOD2) plays a critical role in protection against ionizing radiation in mammalian cells. In this study, we constructed two types of stable HeLa cell lines overexpressing SOD2, HeLa S3/SOD2 and T-REx HeLa/SOD2, to elucidate the mechanisms underlying the protection against radiation by SOD2. SOD2 overexpression in mitochondria enhanced the survival of HeLa S3 and T-REx HeLa cells following γ-irradiation. The levels of γH2AX significantly decreased in HeLa S3/SOD2 and T-REx HeLa/SOD2 cells compared with those in the control cells. MitoSox TM Red assays showed that both lines of SOD2-expressing cells showed suppression of the superoxide generation in mitochondria. Furthermore, flow cytometry with a fluorescent probe (2',7'-dichlorofluorescein) revealed that the cellular levels of ROS increased in HeLa S3 cells during post-irradiation incubation, but the increase was markedly attenuated in HeLa S3/SOD2 cells. DNA microarray analysis revealed that, of 47,000 probe sets analyzed, 117 and 166 probes showed more than 2-fold changes after 5.5 Gy of γ-irradiation in control and HeLa S3/SOD2 cells, respectively. Pathway analysis revealed different expression profiles in irradiated control cells and irradiated SOD2-overexpressing cells. These results indicate that SOD2 protects HeLa cells against cellular effects of γ-rays through suppressing oxidative stress in irradiated cells caused by ROS generated in the mitochondria and through regulating the expression of genes which play a critical role in protection against ionizing radiation. (author)

  4. Edaravone Decreases Paraquat Toxicity in A549 Cells and Lung Isolated Mitochondria

    OpenAIRE

    Shokrzadeh, Mohammad; Shaki, Fatemeh; Mohammadi, Ebrahim; Rezagholizadeh, Neda; Ebrahimi, Fatemeh

    2014-01-01

    Edaravone, an antioxidant and radical scavenger, showed protective effects against oxidative stress-like condition. Paraquat (PQ) is toxic herbicide considerable evidence suggests that oxidative stress and mitochondrial dysfunction contribute to PQ toxicity. In this study, protective effect of edaravone against PQ induced toxicity and reactive oxygen species (ROS) generation in A549 cells and lung isolated mitochondria were evaluated. A549 cells and lung isolated mitochondria were divided int...

  5. Structure of cells chloroplasts and mitochondria of cotton leaves following gamma irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Arslanova, S V [AN Uzbekskoj SSR, Tashkent. Inst. Ehksperimental' noj Biologii Rastenij

    1975-01-01

    The article investigates the structural changes in the plastides and mitochondria of cotton leaf cells after irradiation. Cotton seeds that had been moistened for 24 hours were irradiated by a gamma source with a dose of 10 kR (intensity: 19 R/s.). For the study of the plastides and mitochondria of the leaf cells samples were taken in the cotyledonous leaf and flowering phases of the cotton. The cells of the cotton leaf mesophillum in the standard consists of chloroplast with developed interior structures. Study of the ultrastructure of the cells of the mesophilic tissue of the cotyledonous leaf in irradiated cotton plants showed that the chloroplastide membranes are not damaged. A change in the form of the chloroplasts, an accumulation of starch and plastic substances in the chloroplasts, and a reduction in the number of inter-grain bonds were noted. It was discovered that gamma irradiation produces an excessive build-up of starch in the chloroplasts. The mitochondria are often located close to the plastides. The optical density is typical of the matrix of the mitochondria in non-irradiated plants. After cotton seeds that have sprouted are irradiated with a dose of 10 kR in the cotyledonous leaf phase, part of the mitochondria swells. The matrix becomes more transparent, and the number of chrysts decreases. Part of the mitochondria remains intact. The optical density and internal membranes of the mitochondria remain the same as in the control group. The disturbances of the chloroplast and the mitochondria are also observed in the budding and flowering phases (under conditions of a natural day). It was noted that a shortened day facilitated to some extent a normalization of metabolism, and this produced in turn a normal development of the chloroplasts, leaf mitochondria and ATF generation, which reduces the final biological effect of the radiation.

  6. The antineoplastic agent α-bisabolol promotes cell death by inducing pores in mitochondria and lysosomes.

    Science.gov (United States)

    Rigo, Antonella; Vinante, Fabrizio

    2016-08-01

    The sesquiterpene α-bisabolol (α-BSB) has been shown to be an effective cytotoxic agent for a variety of human cancer cells in culture and animal models. However, much of its intracellular action remains elusive. We evaluated the cytotoxic action of α-BSB against CML-T1, Jurkat and HeLa cell lines, as preclinical models for myeloid, lymphoid and epithelial neoplasias. The approach included single cell analysis (flow cytometry, immunocytology) combined with cytotoxicity and proliferation assays to characterize organelle damage, autophagy, cytostatic effect, and apoptosis. The study focuses on the relevant steps in the cytotoxic cascade triggered by α-BSB: (1) the lipid rafts through which α-BSB enters the cells, (2) the opening of pores in the mitochondria and lysosomes, (3) the activation of both caspase-dependent and caspase-independent cell death pathways, (4) the induction of autophagy and (5) apoptosis. The effectiveness of α-BSB as an agent against tumor cells is grounded on its capability to act on different layers of cell regulation to elicit different concurrent death signals, thereby neutralizing a variety of aberrant survival mechanisms leading to treatment resistance in neoplastic cell.

  7. Diabetes-Induced Dysfunction of Mitochondria and Stem Cells in Skeletal Muscle and the Nervous System

    Science.gov (United States)

    Fujimaki, Shin; Kuwabara, Tomoko

    2017-01-01

    Diabetes mellitus is one of the most common metabolic diseases spread all over the world, which results in hyperglycemia caused by the breakdown of insulin secretion or insulin action or both. Diabetes has been reported to disrupt the functions and dynamics of mitochondria, which play a fundamental role in regulating metabolic pathways and are crucial to maintain appropriate energy balance. Similar to mitochondria, the functions and the abilities of stem cells are attenuated under diabetic condition in several tissues. In recent years, several studies have suggested that the regulation of mitochondria functions and dynamics is critical for the precise differentiation of stem cells. Importantly, physical exercise is very useful for preventing the diabetic alteration by improving the functions of both mitochondria and stem cells. In the present review, we provide an overview of the diabetic alterations of mitochondria and stem cells and the preventive effects of physical exercise on diabetes, focused on skeletal muscle and the nervous system. We propose physical exercise as a countermeasure for the dysfunction of mitochondria and stem cells in several target tissues under diabetes complication and to improve the physiological function of patients with diabetes, resulting in their quality of life being maintained. PMID:29036909

  8. Porcine parvovirus infection induces apoptosis in PK-15 cells through activation of p53 and mitochondria-mediated pathway

    International Nuclear Information System (INIS)

    Zhang, Hongling; Huang, Yong; Du, Qian; Luo, Xiaomao; Zhang, Liang; Zhao, Xiaomin; Tong, Dewen

    2015-01-01

    Highlights: • PPV reduces PK-15 cells viability by inducing apoptosis. • PPV infection induces apoptosis through mitochondria-mediated pathway. • PPV infection activates p53 to regulate the mitochondria apoptotic signaling. - Abstract: Porcine parvovirus (PPV) infection has been reported to induce the cytopathic effects (CPE) in some special host cells and contribute the occurrence of porcine parvovirus disease, but the molecular mechanisms underlying PPV-induced CPE are not clear. In this study, we investigated the morphological and molecular changes of porcine kidney cell line (PK-15 cells) infected with PPV. The results showed that PPV infection inhibited the viability of PK-15 cells in a time and concentration dependent manner. PPV infection induced typical apoptotic features including chromatin condensation, apoptotic body formation, nuclear fragmentation, and Annexin V-binding activity. Further studies showed that Bax was increased and translocated to mitochondria, whereas Bcl-2 was decreased in PPV-infected cells, which caused mitochondrial outer-membrane permeabilization, resulting in the release of mitochondrial cytochrome c, followed by caspase-9 and caspase-3 activation. However, the expression of Fas and Fas ligand (FasL) did not appear significant changes in the process of PPV-induced apoptosis. Moreover, PPV infection activated p53 signaling, which was involved in the activation of apoptotic signaling induced by PPV infection via regulation of Bax and Bcl-2. Taken together, our results demonstrated that PPV infection induced apoptosis in PK-15 cells through activation of p53 and mitochondria-mediated apoptosis pathway. This study may contribute to shed light on the molecular pathogenesis of PPV infection

  9. Porcine parvovirus infection induces apoptosis in PK-15 cells through activation of p53 and mitochondria-mediated pathway

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Hongling; Huang, Yong; Du, Qian; Luo, Xiaomao; Zhang, Liang; Zhao, Xiaomin; Tong, Dewen, E-mail: dwtong@nwsuaf.edu.cn

    2015-01-09

    Highlights: • PPV reduces PK-15 cells viability by inducing apoptosis. • PPV infection induces apoptosis through mitochondria-mediated pathway. • PPV infection activates p53 to regulate the mitochondria apoptotic signaling. - Abstract: Porcine parvovirus (PPV) infection has been reported to induce the cytopathic effects (CPE) in some special host cells and contribute the occurrence of porcine parvovirus disease, but the molecular mechanisms underlying PPV-induced CPE are not clear. In this study, we investigated the morphological and molecular changes of porcine kidney cell line (PK-15 cells) infected with PPV. The results showed that PPV infection inhibited the viability of PK-15 cells in a time and concentration dependent manner. PPV infection induced typical apoptotic features including chromatin condensation, apoptotic body formation, nuclear fragmentation, and Annexin V-binding activity. Further studies showed that Bax was increased and translocated to mitochondria, whereas Bcl-2 was decreased in PPV-infected cells, which caused mitochondrial outer-membrane permeabilization, resulting in the release of mitochondrial cytochrome c, followed by caspase-9 and caspase-3 activation. However, the expression of Fas and Fas ligand (FasL) did not appear significant changes in the process of PPV-induced apoptosis. Moreover, PPV infection activated p53 signaling, which was involved in the activation of apoptotic signaling induced by PPV infection via regulation of Bax and Bcl-2. Taken together, our results demonstrated that PPV infection induced apoptosis in PK-15 cells through activation of p53 and mitochondria-mediated apoptosis pathway. This study may contribute to shed light on the molecular pathogenesis of PPV infection.

  10. Feedback regulation of mitochondria by caspase-9 in the B cell receptor-mediated apoptosis.

    Science.gov (United States)

    Eeva, J; Nuutinen, U; Ropponen, A; Mättö, M; Eray, M; Pellinen, R; Wahlfors, J; Pelkonen, J

    2009-12-01

    During the germinal centre reaction (GC), B cells with non-functional or self-reactive antigen receptors are negatively selected by apoptosis to generate B cell repertoire with appropriate antigen specificities. We studied the molecular mechanism of Fas/CD95- and B cell receptor (BCR)-induced apoptosis to shed light on the signalling events involved in the negative selection of GC B cells. As an experimental model, we used human follicular lymphoma (FL) cell line HF1A3, which originates from a GC B cell, and transfected HF1A3 cell lines overexpressing Bcl-x(L), c-FLIP(long) or dominant negative (DN) caspase-9. Fas-induced apoptosis was dependent on the caspase-8 activation, since the overexpression of c-FLIP(long), a natural inhibitor of caspase-8 activation, blocked apoptosis induced by Fas. In contrast, caspase-9 activation was not involved in Fas-induced apoptosis. BCR-induced apoptosis showed the typical characteristics of mitochondria-dependent (intrinsic) apoptosis. Firstly, the activation of caspase-9 was involved in BCR-induced DNA fragmentation, while caspase-8 showed only marginal role. Secondly, overexpression of Bcl-x(L) could block all apoptotic changes induced by BCR. As a novel finding, we demonstrate that caspase-9 can enhance the cytochrome-c release and collapse of mitochondrial membrane potential (DeltaPsi(m)) during BCR-induced apoptosis. The requirement of different signalling pathways in apoptosis induced by BCR and Fas may be relevant, since Fas- and BCR-induced apoptosis can thus be regulated independently, and targeted to different subsets of GC B cells.

  11. Mitochondria are the primary target in the induction of apoptosis by chiral ruthenium(II) polypyridyl complexes in cancer cells.

    Science.gov (United States)

    Wang, Jin-Quan; Zhang, Ping-Yu; Qian, Chen; Hou, Xiao-Juan; Ji, Liang-Nian; Chao, Hui

    2014-03-01

    A series of novel chiral ruthenium(II) polypyridyl complexes (Δ-Ru1, Λ-Ru1, Δ-Ru2, Λ-Ru2, Δ-Ru3, Λ-Ru3) were synthesized and evaluated to determine their antiproliferative activities. Colocalization, inductively coupled plasma mass spectrometry, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay studies showed that these ruthenium(II) complexes accumulated preferentially in the mitochondria and exhibited cytotoxicity against various cancer cells in vitro. The complex Δ-Ru1 is of particular interest because it was found to have half-maximal inhibitory concentrations comparable to those of cisplatin and better activity than cisplatin against a cisplatin-resistant cell line, A549-CP/R. Δ-Ru1 induced alterations in the mitochondrial membrane potential and triggered intrinsic mitochondria-mediated apoptosis in HeLa cells, which involved the regulation of Bcl-2 family members and the activation of caspases. Taken together, these data suggest that Δ-Ru1 may be a novel mitochondria-targeting anticancer agent.

  12. Biochemistry, proteomics, and phosphoproteomics of plant mitochondria from non-photosynthetic cells

    DEFF Research Database (Denmark)

    Havelund, Jesper; Thelen, Jay J.; Møller, Ian Max

    2013-01-01

    of mitochondria and general biochemical properties such as oxidative phosphorylation. We will then mention a few adaptive properties in response to water stress, seed maturation and germination and the ability to function under hypoxic conditions. The discussion will mainly focus on Arabidopsis cell cultures......Mitochondria fulfill some basic roles in all plant cells. They supply the cell with energy in the form of ATP and reducing equivalents (NAD(P)H) and they provide the cell with intermediates for a range of biosynthetic pathways. In addition to this, mitochondria contribute to a number of specialized......, etiolated germinating rice seedlings and potato tubers as model plants. It will cover the general proteome as well as the posttranslational modification protein phosphorylation. To date 64 phosphorylated mitochondrial proteins with a total of 103 phosphorylation sites have been identified....

  13. Mitochondria-targeted vitamin E analogs inhibit breast cancer cell energy metabolism and promote cell death

    International Nuclear Information System (INIS)

    Cheng, Gang; Zielonka, Jacek; McAllister, Donna M; Mackinnon, A Craig Jr; Joseph, Joy; Dwinell, Michael B; Kalyanaraman, Balaraman

    2013-01-01

    Recent research has revealed that targeting mitochondrial bioenergetic metabolism is a promising chemotherapeutic strategy. Key to successful implementation of this chemotherapeutic strategy is the use of new and improved mitochondria-targeted cationic agents that selectively inhibit energy metabolism in breast cancer cells, while exerting little or no long-term cytotoxic effect in normal cells. In this study, we investigated the cytotoxicity and alterations in bioenergetic metabolism induced by mitochondria-targeted vitamin E analog (Mito-chromanol, Mito-ChM) and its acetylated ester analog (Mito-ChMAc). Assays of cell death, colony formation, mitochondrial bioenergetic function, intracellular ATP levels, intracellular and tissue concentrations of tested compounds, and in vivo tumor growth were performed. Both Mito-ChM and Mito-ChMAc selectively depleted intracellular ATP and caused prolonged inhibition of ATP-linked oxygen consumption rate in breast cancer cells, but not in non-cancerous cells. These effects were significantly augmented by inhibition of glycolysis. Mito-ChM and Mito-ChMAc exhibited anti-proliferative effects and cytotoxicity in several breast cancer cells with different genetic background. Furthermore, Mito-ChM selectively accumulated in tumor tissue and inhibited tumor growth in a xenograft model of human breast cancer. We conclude that mitochondria-targeted small molecular weight chromanols exhibit selective anti-proliferative effects and cytotoxicity in multiple breast cancer cells, and that esterification of the hydroxyl group in mito-chromanols is not a critical requirement for its anti-proliferative and cytotoxic effect

  14. Electron microscopic radioautographic studies on macromolecular synthesis in mitochondria of animal cells in aging

    International Nuclear Information System (INIS)

    Nagata, Tetsuji

    2010-01-01

    Study aging changes of intramitochondrial DNA, RNA, protein synthesis of mouse organs during the development and aging, 30 groups of developing and aging mice (3 individuals each), from fetal day 19 to postnatal newborn at day 1, 3, 9, 14 and adult at month 1, 2, 6, 12 to 24, were injected with either 3 H-thymidine, 3 H-uriidine, or 3 H-leucine, sacrificed 1 h later and liver, adrenal, lung and testis tissues observed by electron microscopic radioautography. Accordingly, numbers of mitochondria per cell profile area, numbers of labeled mitochondria and the mitochondrial labeling index labeled with 3 H-labeled precursors showing DNA, RNA, protein synthesis in these cells (hepatocytes, 3 zones of the adrenal cortices - zona glomerulosa, fasciculata and reticularis -, adrenal medullary cells, pulmonary cells and testis cells) were counted per cells and compared among the respective developing and aging groups. The numbers of mitochondria in these cells increased from fetal day 19 to postnatal month 1 and 2. However, the numbers of labeled mitochondria and the labeling indices of intramitochondrial DNA, RNA, protein syntheses incorporating the 3 H-labeled precursors in the described tissue cells increased from fetal day 19 to postnatal month 1 and decreased to month 24. These data support that the activity of intramitochnodrial DNA, RNA, protein syntheses in cells of these tissues increased and decreased by development and aging in mice. The intramitochondrial DNA, RNA and protein syntheses in some other organs were also reviewed and discussed. (author)

  15. Electron microscopic radioautographic studies on macromolecular synthesis in mitochondria of animal cells in aging

    Energy Technology Data Exchange (ETDEWEB)

    Nagata, Tetsuji, E-mail: nagata@kowagakuen.ac.j [Shinshu Univ. School of Medicine, Matsumoto (Japan). Dept. of Anatomy and Cell Biology

    2010-07-01

    Study aging changes of intramitochondrial DNA, RNA, protein synthesis of mouse organs during the development and aging, 30 groups of developing and aging mice (3 individuals each), from fetal day 19 to postnatal newborn at day 1, 3, 9, 14 and adult at month 1, 2, 6, 12 to 24, were injected with either {sup 3}H-thymidine, {sup 3}H-uriidine, or {sup 3}H-leucine, sacrificed 1 h later and liver, adrenal, lung and testis tissues observed by electron microscopic radioautography. Accordingly, numbers of mitochondria per cell profile area, numbers of labeled mitochondria and the mitochondrial labeling index labeled with {sup 3}H-labeled precursors showing DNA, RNA, protein synthesis in these cells (hepatocytes, 3 zones of the adrenal cortices - zona glomerulosa, fasciculata and reticularis -, adrenal medullary cells, pulmonary cells and testis cells) were counted per cells and compared among the respective developing and aging groups. The numbers of mitochondria in these cells increased from fetal day 19 to postnatal month 1 and 2. However, the numbers of labeled mitochondria and the labeling indices of intramitochondrial DNA, RNA, protein syntheses incorporating the {sup 3}H-labeled precursors in the described tissue cells increased from fetal day 19 to postnatal month 1 and decreased to month 24. These data support that the activity of intramitochnodrial DNA, RNA, protein syntheses in cells of these tissues increased and decreased by development and aging in mice. The intramitochondrial DNA, RNA and protein syntheses in some other organs were also reviewed and discussed. (author)

  16. Extracts of Artocarpus communis Induce Mitochondria-Associated Apoptosis via Pro-oxidative Activity in Human Glioblastoma Cells

    Directory of Open Access Journals (Sweden)

    Chiang-Wen Lee

    2018-05-01

    Full Text Available Glioblastoma multiforme (GBM is an extremely aggressive and devastating malignant tumor in the central nervous system. Its incidence is increasing and the prognosis is poor. Artocarpin is a natural prenylated flavonoid with various anti-inflammatory and anti-tumor properties. Studies have shown that artocarpin is associated with cell death of primary glioblastoma cells. However, the in vivo effects and the cellular and molecular mechanisms modulating the anticancer activities of artocarpin remain unknown. In this study, we demonstrated that treating the glioblastoma cell lines U87 and U118 cells with artocarpin induced apoptosis. Artocarpin-induced apoptosis is associated with caspase activation and poly (ADP-ribose polymerase (PARP cleavage and is mediated by the mitochondrial pathway. This is associated with mitochondrial depolarization, mitochondrial-derived reactive oxidative species (ROS production, cytochrome c release, Bad and Bax upregulations, and Bcl-2 downregulation. Artocarpin induced NADPH oxidase/ROS generation plays an important role in the mitochondrial pathway activation. Furthermore, we found artocarpin-induced ROS production in mitochondria is associated with Akt- and ERK1/2 activation. After treatment with artocarpin, ROS causes PI3K/Akt/ERK1/2-induced cell death of these tumor cells. These observations were further verified by the results from the implantation of both U87 and U118 cells into in vivo mouse. In conclusion, our findings suggest that artocarpin induces mitochondria-associated apoptosis of glioma cells, suggesting that artocarpine can be a potential chemotherapeutic agent for future GBM treatment.

  17. Quantitative control of mitochondria transfer between live single cells using a microfluidic device

    Directory of Open Access Journals (Sweden)

    Ken-Ichi Wada

    2017-12-01

    Full Text Available Quantitative control of mitochondria transfer between live cells is a promising approach for genetic manipulation of mitochondrial DNA (mtDNA because single mitochondrion transfer to a mtDNA-less (ρ0 cell potentially leads to homoplasmy of mtDNA. In this paper, we describe a method for quantitative control of mitochondria transfer between live single cells. For this purpose, we fabricated novel microfluidic devices having cell paring structures with a 4.1, 5.6 or 10.0 μm-length microtunnel. When cells were fused through a microtunnel using the Sendai virus envelope-based method, a strictured cytoplasmic connection was achieved with a length corresponding to that of the microtunnel. Elongation of the cytoplasmic connection led to a decrease in mitochondria transfer to the fusion partner. Moreover, some cell pairs that fused through a 10.0 μm-length microtunnel showed single mitochondrion transfer. Fused cells were spontaneously disconnected from each other when they were recovered in a normal culture medium. These results suggest that our cell fusion method can perform quantitative control of mitochondria transfer that includes a single mitochondrion transfer.

  18. Radiosensitivity of mesothelioma cell lines

    International Nuclear Information System (INIS)

    Haekkinen, A.M.; Laasonen, A.; Linnainmaa, K.; Mattson, K.; Pyrhoenen, S.

    1996-01-01

    The present study was carried out in order to examine the radiosensitivity of malignant pleural mesothelioma cell lines. Cell kinetics, radiation-induced delay of the cell cycle and DNA ploidy of the cell lines were also determined. For comparison an HeLa and a human foetal fibroblast cell line were simultaneously explored. Six previously cytogenetically and histologically characterized mesothelioma tumor cell lines were applied. A rapid tiazolyl blue microtiter (MTT) assay was used to analyze radiosensitivity and cell kinetics and DNA ploidy of the cultured cells were determined by flow cytometry. The survival fraction after a dose of 2 Gy (SF2), parameters α and β of the linear quadratic model (LQ-model) and mean inactivation dose (D MID ) were also estimated. The DNA index of four cell lines equaled 1.0 and two cell lines equaled 1.5 and 1.6. Different mesothelioma cell lines showed a great variation in radiosensitivity. Mean survival fraction after a radiation dose of 2 Gy (SF2) was 0.60 and ranged from 0.36 to 0.81 and mean α value was 0.26 (range 0.48-0.083). The SF2 of the most sensitive diploid mesothelioma cell line was 0.36: Less than that of the foetal fibroblast cell line (0.49). The survival fractions (0.81 and 0.74) of the two most resistant cell lines, which also were aneuploid, were equal to that of the HeLa cell line (0.78). The α/β ratios of the most sensitive cell lines were almost an order of magnitude greater than those of the two most resistant cell lines. Radiation-induced delay of the most resistant aneuploid cell line was similar to that of HeLa cells but in the most sensitive (diploid cells) there was practically no entry into the G1 phase following the 2 Gy radiation dose during 36 h. (orig.)

  19. Ca2+ Transport by Mitochondria from L1210 Mouse Ascites Tumor Cells

    Science.gov (United States)

    Reynafarje, Baltazar; Lehninger, Albert L.

    1973-01-01

    Mitochondria isolated from the ascites form of L1210 mouse leukemia cells readily accumulate Ca2+ from the suspending medium and eject H+ during oxidation of succinate in the presence of phosphate and Mg2+, with normal stoichiometry between Ca2+ uptake and electron transport. Ca2+ loads up to 1600 ng-atoms per mg of protein are attained. As is the case in mitochondria from normal tissues, Ca2+ uptake takes precedence over oxidative phosphorylation. However, Ca2+ transport by the L-1210 mitochondria is unusual in other respects, which may possibly have general significance in tumor cells. The apparent affinity of the L1210 mitochondria for Ca2+ in stimulation of oxygen uptake is about 3-fold greater than in normal liver mitochondria; moreover, the maximal rate of Ca2+ transport is also considerably higher. Furthermore, when Ca2+ pulses are added to L1210 mitochondria in the absence of phosphate or other permeant anions, much larger amounts of Ca2+ are bound and H+ ejected per atom of oxygen consumed than in the presence of phosphate; up to 7 Ca2+ ions are bound per pair of electrons passing each energy-conserving site of the electron-transport chain. Such “superstoichiometry” of Ca2+ uptake can be accounted for by two distinct types of respiration-dependent interaction of Ca2+ with the L1210 mitochondria. One is the stimulation of oxygen consumption, which is achieved by relatively low concentrations of Ca2+ (Km ≅ 8 μM) and is accompanied by binding of Ca2+ up to 40 ng-atoms per mg of protein. The second process, also dependent on electron transport, is the binding of further Ca2+ from the medium in exchange with previously stored membrane-bound protons, in which the affinity for Ca2+ is much lower (Km ≅ 120 μM). PMID:4515933

  20. CLO : The cell line ontology

    NARCIS (Netherlands)

    Sarntivijai, Sirarat; Lin, Yu; Xiang, Zuoshuang; Meehan, Terrence F.; Diehl, Alexander D.; Vempati, Uma D.; Schuerer, Stephan C.; Pang, Chao; Malone, James; Parkinson, Helen; Liu, Yue; Takatsuki, Terue; Saijo, Kaoru; Masuya, Hiroshi; Nakamura, Yukio; Brush, Matthew H.; Haendel, Melissa A.; Zheng, Jie; Stoeckert, Christian J.; Peters, Bjoern; Mungall, Christopher J.; Carey, Thomas E.; States, David J.; Athey, Brian D.; He, Yongqun

    2014-01-01

    Background: Cell lines have been widely used in biomedical research. The community-based Cell Line Ontology (CLO) is a member of the OBO Foundry library that covers the domain of cell lines. Since its publication two years ago, significant updates have been made, including new groups joining the CLO

  1. Biochemistry, proteomics and phosphoproteomics of plant mitochondria from non-photosynthetic cells

    Directory of Open Access Journals (Sweden)

    Jesper Foged Havelund

    2013-03-01

    Full Text Available Mitochondria fulfill some basic roles in all plant cells. They supply the cell with energy in the form of ATP and reducing equivalents (NAD(PH and they provide the cell with intermediates for a range of biosynthetic pathways. In addition to this, mitochondria contribute to a number of specialized functions depending on the tissue and cell type, as well as environmental conditions. We will here review the biochemistry and proteomics of mitochondria from non-green cells and organs, which differ from those of photosynthetic organs in a number of respects. We will briefly cover purification of mitochondria and general biochemical properties such as oxidative phosphorylation. We will then mention a few adaptive properties in response to water stress, seed maturation and germination and the ability to function under hypoxic conditions. The discussion will mainly focus on Arabidopsis cell cultures, etiolated germinating rice seedlings and potato tubers as model plants. It will cover the general proteome as well as the posttranslational modification protein phosphorylation. To date 64 phosphorylated mitochondrial proteins with a total of 103 phosphorylation sites have been identified.

  2. HIV-1 infection and first line ART induced differential responses in mitochondria from blood lymphocytes and monocytes: the ANRS EP45 "Aging" study.

    Directory of Open Access Journals (Sweden)

    Sophie Perrin

    Full Text Available The ANRS EP45 "Aging" study investigates the cellular mechanisms involved in the accelerated aging of HIV-1 infected and treated patients. The data reported focus on mitochondria, organelles known to be involved in cell senescence.49 HIV-1 infected patients untreated with antiretroviral therapy, together with 49 seronegative age- and sex-matched control subjects and 81 HIV-1 infected and treated patients, were recruited by 3 AIDS centres (Marseille, Montpellier, Nice; France; http://clinicaltrials.gov/, NCT01038999. In more than 88% of treated patients, the viral load was 500/mm(3. ROS (reactive oxygen species production and ΔΨm (inner membrane potential were measured by flow cytometry in blood lymphocytes and monocytes (functional parameters. Three mitochondrial network quantitative morphological parameters were computed using confocal microscopy and image analysis. Three PBMC mitochondrial proteins (porin and subunits 2 and 4 of cytochrome C oxidase encoded by mtDNA or nuclear DNA, respectively were analysed by western blotting.Quantitative changes in PBMC mitochondrial proteins were not induced by either HIV-1 infection or ART. Discriminant analysis integrating functional (ROS production and ΔΨm or morphological (network volume density, fragmentation and branching parameters revealed HIV-1 infection and ART differential effects according to cell type. First line ART tended to rescue lymphocyte mitochondrial parameters altered by viral infection, but induced slight changes in monocytes. No statistical difference was found between the effects of three ART regimens on mitochondrial parameters. Correlations between functional parameters and viral load confirmed the damaging effects of HIV-1 in lymphocyte mitochondria.In patients considered to be clinically stable, mitochondria exhibited functional and morphological modifications in PBMCs resulting from either direct or indirect effects of HIV-1 infection (lymphocytes, or from first line ART

  3. [Oxidative power and intracellular distribution of mitochondria control cell oxygen regime when arterial hypoxemia occurs].

    Science.gov (United States)

    Liabakh, E G; Lissov, P N

    2012-01-01

    The regulatory impact of the mitochondria spatial distribution and enlargement in their oxidative power qO2 on the tissue oxygenation of skeletal muscle during hypoxia were studied. Investigations were performed by the mathematical modeling of 3D O2 diffusion-reaction in muscle fiber. The oxygen consumption rate VO2 and tissue pO2 were analyzed in response to a decrease in arterial blood oxygen concentration from 19.5 to 10 vol. % at a moderate load (3.5 ml/min per 100 g). The cells with evenly (case 1) and unevenly (case 2) distributed mitochondria were considered. According to calculations due to a rise in mitochondria oxidative power from 3.5 to 6.5 ml/min. per 100 g of tissue it is possible to maintain muscle oxygen V(O2) at constant level of 3.5 ml/min per 100 g despite a decrease in O2 delivery. Minimum value of tissue pO2 was about 0 and an area of hypoxia appeared inside the cell in case 1. But hypoxia disappeared and minimum value of pO2 increased from 0 to 4 mm Hg if mitochondria were distributed unevenly (case 2). It is shown that the possibilities of such regulation were limited and depended on the ratio of "the degree of hypoxemia--the level of oxygen delivery." It was assumed that an increase in mitochondria enzyme activity and mitochondria migration to the places of the greatest oxygen consumption rate can improve oxygen regime in the cells in terms of their adaptation to hypoxia. It is possible that changes in mitochondrial oxidative power and their intracellular redistribution may be considered as a new dimension in regulation of cell oxygen regime.

  4. Nickel (II)-induced cytotoxicity and apoptosis in human proximal tubule cells through a ROS- and mitochondria-mediated pathway

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Yi-Fen; Shyu, Huey-Wen [Department of Medical Laboratory Sciences and Biotechnology, Fooyin University, Kaohsiung, Taiwan (China); Chang, Yi-Chuang [Department of Nursing, Fooyin University, Kaohsiung, Taiwan (China); Tseng, Wei-Chang [Department of Medical Laboratory Sciences and Biotechnology, Fooyin University, Kaohsiung, Taiwan (China); Huang, Yeou-Lih [Department of Medical Laboratory Science and Biotechnology, Kaohsiung Medical University, Kaohsiung, Taiwan (China); Lin, Kuan-Hua; Chou, Miao-Chen; Liu, Heng-Ling [Department of Medical Laboratory Sciences and Biotechnology, Fooyin University, Kaohsiung, Taiwan (China); Chen, Chang-Yu, E-mail: mt037@mail.fy.edu.tw [Department of Medical Laboratory Sciences and Biotechnology, Fooyin University, Kaohsiung, Taiwan (China)

    2012-03-01

    Nickel compounds are known to be toxic and carcinogenic in kidney and lung. In this present study, we investigated the roles of reactive oxygen species (ROS) and mitochondria in nickel (II) acetate-induced cytotoxicity and apoptosis in the HK-2 human renal cell line. The results showed that the cytotoxic effects of nickel (II) involved significant cell death and DNA damage. Nickel (II) increased the generation of ROS and induced a noticeable reduction of mitochondrial membrane potential (MMP). Analysis of the sub-G1 phase showed a significant increase in apoptosis in HK-2 cells after nickel (II) treatment. Pretreatment with N-acetylcysteine (NAC) not only inhibited nickel (II)-induced cell death and DNA damage, but also significantly prevented nickel (II)-induced loss of MMP and apoptosis. Cell apoptosis triggered by nickel (II) was characterized by the reduced protein expression of Bcl-2 and Bcl-xL and the induced the protein expression of Bad, Bcl-Xs, Bax, cytochrome c and caspases 9, 3 and 6. The regulation of the expression of Bcl-2-family proteins, the release of cytochrome c and the activation of caspases 9, 3 and 6 were inhibited in the presence of NAC. These results suggest that nickel (II) induces cytotoxicity and apoptosis in HK-2 cells via ROS generation and that the mitochondria-mediated apoptotic signaling pathway may be involved in the positive regulation of nickel (II)-induced renal cytotoxicity.

  5. Nickel (II)-induced cytotoxicity and apoptosis in human proximal tubule cells through a ROS- and mitochondria-mediated pathway

    International Nuclear Information System (INIS)

    Wang, Yi-Fen; Shyu, Huey-Wen; Chang, Yi-Chuang; Tseng, Wei-Chang; Huang, Yeou-Lih; Lin, Kuan-Hua; Chou, Miao-Chen; Liu, Heng-Ling; Chen, Chang-Yu

    2012-01-01

    Nickel compounds are known to be toxic and carcinogenic in kidney and lung. In this present study, we investigated the roles of reactive oxygen species (ROS) and mitochondria in nickel (II) acetate-induced cytotoxicity and apoptosis in the HK-2 human renal cell line. The results showed that the cytotoxic effects of nickel (II) involved significant cell death and DNA damage. Nickel (II) increased the generation of ROS and induced a noticeable reduction of mitochondrial membrane potential (MMP). Analysis of the sub-G1 phase showed a significant increase in apoptosis in HK-2 cells after nickel (II) treatment. Pretreatment with N-acetylcysteine (NAC) not only inhibited nickel (II)-induced cell death and DNA damage, but also significantly prevented nickel (II)-induced loss of MMP and apoptosis. Cell apoptosis triggered by nickel (II) was characterized by the reduced protein expression of Bcl-2 and Bcl-xL and the induced the protein expression of Bad, Bcl-Xs, Bax, cytochrome c and caspases 9, 3 and 6. The regulation of the expression of Bcl-2-family proteins, the release of cytochrome c and the activation of caspases 9, 3 and 6 were inhibited in the presence of NAC. These results suggest that nickel (II) induces cytotoxicity and apoptosis in HK-2 cells via ROS generation and that the mitochondria-mediated apoptotic signaling pathway may be involved in the positive regulation of nickel (II)-induced renal cytotoxicity.

  6. A receptor tyrosine kinase inhibitor, Tyrphostin A9 induces cancer cell death through Drp1 dependent mitochondria fragmentation

    International Nuclear Information System (INIS)

    Park, So Jung; Park, Young Jun; Shin, Ji Hyun; Kim, Eun Sung; Hwang, Jung Jin; Jin, Dong-Hoon; Kim, Jin Cheon; Cho, Dong-Hyung

    2011-01-01

    Highlights: → We screened and identified Tyrphostin A9, a receptor tyrosine kinase inhibitor as a strong mitochondria fission inducer. → Tyrphostin A9 treatment promotes mitochondria dysfunction and contributes to cytotoxicity in cancer cells. → Tyrphostin A9 induces apoptotic cell death through a Drp1-mediated pathway. → Our studies suggest that Tyrphostin A9 induces mitochondria fragmentation and apoptotic cell death via Drp1 dependently. -- Abstract: Mitochondria dynamics controls not only their morphology but also functions of mitochondria. Therefore, an imbalance of the dynamics eventually leads to mitochondria disruption and cell death. To identify specific regulators of mitochondria dynamics, we screened a bioactive chemical compound library and selected Tyrphostin A9, a tyrosine kinase inhibitor, as a potent inducer of mitochondrial fission. Tyrphostin A9 treatment resulted in the formation of fragmented mitochondria filament. In addition, cellular ATP level was decreased and the mitochondrial membrane potential was collapsed in Tyr A9-treated cells. Suppression of Drp1 activity by siRNA or over-expression of a dominant negative mutant of Drp1 inhibited both mitochondrial fragmentation and cell death induced by Tyrpohotin A9. Moreover, treatment of Tyrphostin A9 also evoked mitochondrial fragmentation in other cells including the neuroblastomas. Taken together, these results suggest that Tyrphostin A9 induces Drp1-mediated mitochondrial fission and apoptotic cell death.

  7. Subcellular Distribution of NAD+ between Cytosol and Mitochondria Determines the Metabolic Profile of Human Cells*

    Science.gov (United States)

    VanLinden, Magali R.; Dölle, Christian; Pettersen, Ina K. N.; Kulikova, Veronika A.; Niere, Marc; Agrimi, Gennaro; Dyrstad, Sissel E.; Palmieri, Ferdinando; Nikiforov, Andrey A.; Tronstad, Karl Johan; Ziegler, Mathias

    2015-01-01

    The mitochondrial NAD pool is particularly important for the maintenance of vital cellular functions. Although at least in some fungi and plants, mitochondrial NAD is imported from the cytosol by carrier proteins, in mammals, the mechanism of how this organellar pool is generated has remained obscure. A transporter mediating NAD import into mammalian mitochondria has not been identified. In contrast, human recombinant NMNAT3 localizes to the mitochondrial matrix and is able to catalyze NAD+ biosynthesis in vitro. However, whether the endogenous NMNAT3 protein is functionally effective at generating NAD+ in mitochondria of intact human cells still remains to be demonstrated. To modulate mitochondrial NAD+ content, we have expressed plant and yeast mitochondrial NAD+ carriers in human cells and observed a profound increase in mitochondrial NAD+. None of the closest human homologs of these carriers had any detectable effect on mitochondrial NAD+ content. Surprisingly, constitutive redistribution of NAD+ from the cytosol to the mitochondria by stable expression of the Arabidopsis thaliana mitochondrial NAD+ transporter NDT2 in HEK293 cells resulted in dramatic growth retardation and a metabolic shift from oxidative phosphorylation to glycolysis, despite the elevated mitochondrial NAD+ levels. These results suggest that a mitochondrial NAD+ transporter, similar to the known one from A. thaliana, is likely absent and could even be harmful in human cells. We provide further support for the alternative possibility, namely intramitochondrial NAD+ synthesis, by demonstrating the presence of endogenous NMNAT3 in the mitochondria of human cells. PMID:26432643

  8. Subcellular Distribution of NAD+ between Cytosol and Mitochondria Determines the Metabolic Profile of Human Cells.

    Science.gov (United States)

    VanLinden, Magali R; Dölle, Christian; Pettersen, Ina K N; Kulikova, Veronika A; Niere, Marc; Agrimi, Gennaro; Dyrstad, Sissel E; Palmieri, Ferdinando; Nikiforov, Andrey A; Tronstad, Karl Johan; Ziegler, Mathias

    2015-11-13

    The mitochondrial NAD pool is particularly important for the maintenance of vital cellular functions. Although at least in some fungi and plants, mitochondrial NAD is imported from the cytosol by carrier proteins, in mammals, the mechanism of how this organellar pool is generated has remained obscure. A transporter mediating NAD import into mammalian mitochondria has not been identified. In contrast, human recombinant NMNAT3 localizes to the mitochondrial matrix and is able to catalyze NAD(+) biosynthesis in vitro. However, whether the endogenous NMNAT3 protein is functionally effective at generating NAD(+) in mitochondria of intact human cells still remains to be demonstrated. To modulate mitochondrial NAD(+) content, we have expressed plant and yeast mitochondrial NAD(+) carriers in human cells and observed a profound increase in mitochondrial NAD(+). None of the closest human homologs of these carriers had any detectable effect on mitochondrial NAD(+) content. Surprisingly, constitutive redistribution of NAD(+) from the cytosol to the mitochondria by stable expression of the Arabidopsis thaliana mitochondrial NAD(+) transporter NDT2 in HEK293 cells resulted in dramatic growth retardation and a metabolic shift from oxidative phosphorylation to glycolysis, despite the elevated mitochondrial NAD(+) levels. These results suggest that a mitochondrial NAD(+) transporter, similar to the known one from A. thaliana, is likely absent and could even be harmful in human cells. We provide further support for the alternative possibility, namely intramitochondrial NAD(+) synthesis, by demonstrating the presence of endogenous NMNAT3 in the mitochondria of human cells. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Heptachlor induced mitochondria-mediated cell death via impairing electron transport chain complex III

    International Nuclear Information System (INIS)

    Hong, Seokheon; Kim, Joo Yeon; Hwang, Joohyun; Shin, Ki Soon; Kang, Shin Jung

    2013-01-01

    Highlights: •Heptachlor inhibited mitochondrial electron transport chain complex III activity. •Heptachlor promoted generation of reactive oxygen species. •Heptachlor induced Bax activation. •Heptachlor induced mitochondria-mediated and caspase-dependent apoptosis. -- Abstract: Environmental toxins like pesticides have been implicated in the pathogenesis of Parkinson’s disease (PD). Epidemiological studies suggested that exposures to organochlorine pesticides have an association with an increased PD risk. In the present study, we examined the mechanism of toxicity induced by an organochlorine pesticide heptachlor. In a human dopaminergic neuroblastoma SH-SY5Y cells, heptachlor induced both morphological and functional damages in mitochondria. Interestingly, the compound inhibited mitochondrial electron transport chain complex III activity. Rapid generation of reactive oxygen species and the activation of Bax were then detected. Subsequently, mitochondria-mediated, caspase-dependent apoptosis followed. Our results raise a possibility that an organochlorine pesticide heptachlor can act as a neurotoxicant associated with PD

  10. Heptachlor induced mitochondria-mediated cell death via impairing electron transport chain complex III

    Energy Technology Data Exchange (ETDEWEB)

    Hong, Seokheon; Kim, Joo Yeon; Hwang, Joohyun [Department of Molecular Biology, Sejong University, Seoul 143-747 (Korea, Republic of); Shin, Ki Soon [Department of Biology, Department of Life and Nanopharmaceutical Sciences, Kyung Hee University, Seoul 130-701 (Korea, Republic of); Kang, Shin Jung, E-mail: sjkang@sejong.ac.kr [Department of Molecular Biology, Sejong University, Seoul 143-747 (Korea, Republic of)

    2013-08-09

    Highlights: •Heptachlor inhibited mitochondrial electron transport chain complex III activity. •Heptachlor promoted generation of reactive oxygen species. •Heptachlor induced Bax activation. •Heptachlor induced mitochondria-mediated and caspase-dependent apoptosis. -- Abstract: Environmental toxins like pesticides have been implicated in the pathogenesis of Parkinson’s disease (PD). Epidemiological studies suggested that exposures to organochlorine pesticides have an association with an increased PD risk. In the present study, we examined the mechanism of toxicity induced by an organochlorine pesticide heptachlor. In a human dopaminergic neuroblastoma SH-SY5Y cells, heptachlor induced both morphological and functional damages in mitochondria. Interestingly, the compound inhibited mitochondrial electron transport chain complex III activity. Rapid generation of reactive oxygen species and the activation of Bax were then detected. Subsequently, mitochondria-mediated, caspase-dependent apoptosis followed. Our results raise a possibility that an organochlorine pesticide heptachlor can act as a neurotoxicant associated with PD.

  11. Caspase-8 acts as a key upstream executor of mitochondria during justicidin A-induced apoptosis in human hepatoma cells.

    Science.gov (United States)

    Su, Chun-Li; Huang, Lynn L H; Huang, Li-Min; Lee, Jenq-Chang; Lin, Chun-Nan; Won, Shen-Jeu

    2006-05-29

    Justicia procumbens is a traditional Taiwanese herbal remedy used to treat fever, pain, and cancer. Justicidin A, isolated from Justicia procumbens, has been reported to suppress in vitro growth of several tumor cell lines as well as hepatoma cells. In this study, justicidin A activated caspase-8 to increase tBid, disrupted mitochondrial membrane potential (Delta psi(m)), and caused the release of cytochrome c and Smac/DIABLO in Hep 3B and Hep G2 cells. Justicidin A also reduced Bcl-x(L) and increased Bax and Bak in mitochondria. Caspase-8 inhibitor (Z-IETD) attenuated the justicidin A-induced disruption of Delta psi(m). Growth of Hep 3B implanted in NOD-SCID mice was suppressed significantly by oral justicidin A (20 mg/kg/day). These results indicate that justicidin A-induced apoptosis in these cells proceeds via caspase-8 and is followed by mitochondrial disruption.

  12. Erythroid cell mitochondria receive endosomal iron by a "kiss-and-run" mechanism.

    Science.gov (United States)

    Hamdi, Amel; Roshan, Tariq M; Kahawita, Tanya M; Mason, Anne B; Sheftel, Alex D; Ponka, Prem

    2016-12-01

    In erythroid cells, more than 90% of transferrin-derived iron enters mitochondria where ferrochelatase inserts Fe 2+ into protoporphyrin IX. However, the path of iron from endosomes to mitochondrial ferrochelatase remains elusive. The prevailing opinion is that, after its export from endosomes, the redox-active metal spreads into the cytosol and mysteriously finds its way into mitochondria through passive diffusion. In contrast, this study supports the hypothesis that the highly efficient transport of iron toward ferrochelatase in erythroid cells requires a direct interaction between transferrin-endosomes and mitochondria (the "kiss-and-run" hypothesis). Using a novel method (flow sub-cytometry), we analyze lysates of reticulocytes after labeling these organelles with different fluorophores. We have identified a double-labeled population definitively representing endosomes interacting with mitochondria, as demonstrated by confocal microscopy. Moreover, we conclude that this endosome-mitochondrion association is reversible, since a "chase" with unlabeled holotransferrin causes a time-dependent decrease in the size of the double-labeled population. Importantly, the dissociation of endosomes from mitochondria does not occur in the absence of holotransferrin. Additionally, mutated recombinant holotransferrin, that cannot release iron, significantly decreases the uptake of 59 Fe by reticulocytes and diminishes 59 Fe incorporation into heme. This suggests that endosomes, which are unable to provide iron to mitochondria, cause a "traffic jam" leading to decreased endocytosis of holotransferrin. Altogether, our results suggest that a molecular mechanism exists to coordinate the iron status of endosomal transferrin with its trafficking. Besides its contribution to the field of iron metabolism, this study provides evidence for a new intracellular trafficking pathway of organelles. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Are maternal mitochondria the selfish entities that are masters of the cells of eukaryotic multicellular organisms?

    Science.gov (United States)

    Barlow, Peter W; Baldelli, E; Baluška, Frantisek

    2009-01-01

    The Energide concept, as well as the endosymbiotic theory of eukaryotic cell organization and evolution, proposes that present-day cells of eukaryotic organisms are mosaics of specialized and cooperating units, or organelles. Some of these units were originally free-living prokaryotes, which were engulfed during evolutionary time. Mitochondria represent one of these types of previously independent organisms, the Energide, is another type. This new perspective on the organization of the cell has been further expanded to reveal the concept of a public milieu, the cytosol, in which Energides and mitochondria live, each with their own private internal milieu. The present paper discusses how the endosymbiotic theory implicates a new hypothesis about the hierarchical and communicational organization of the integrated prokaryotic components of the eukaryotic cell and provides a new angle from which to consider the theory of evolution and its bearing upon cellular complexity. Thus, it is proposed that the “selfish gene” hypothesis of Dawkins1 is not the only possible perspective for comprehending genomic and cellular evolution. Our proposal is that maternal mitochondria are the selfish “master” entities of the eukaryotic cell with respect not only to their propagation from cell-to-cell and from generation-to-generation but also to their regulation of all other cellular functions. However, it should be recognized that the concept of “master” and “servant” cell components is a metaphor; in present-day living organisms their organellar components are considered to be interdependent and inseparable. PMID:19513277

  14. Repercussion of mitochondria deformity induced by anti-Hsp90 drug 17AAG in human tumor cells

    KAUST Repository

    Vishal, Chaturvedi; Kumar, Jonnala Ujwal; Veera Brahmendra Swamy, Cherukuvada; Nandini, Rangaraj; Srinivas, Gunda; Kumaresan, Rathinam; Shashi, Singh; Sreedhar, Amere Subbarao

    2011-01-01

    Inhibiting Hsp90 chaperone roles using 17AAG induces cytostasis or apoptosis in tumor cells through destabilization of several mutated cancer promoting proteins. Although mitochondria are central in deciding the fate of cells, 17AAG induced effects

  15. Corn silk maysin induces apoptotic cell death in PC-3 prostate cancer cells via mitochondria-dependent pathway.

    Science.gov (United States)

    Lee, Jisun; Lee, Seul; Kim, Sun-Lim; Choi, Ji Won; Seo, Jeong Yeon; Choi, Doo Jin; Park, Yong Il

    2014-12-05

    Despite recent advances in prostate cancer diagnostics and therapeutics, the overall survival rate still remains low. This study was aimed to assess potential anti-cancer activity of maysin, a major flavonoid of corn silk (CS, Zea mays L.), in androgen-independent human prostate cancer cells (PC-3). Maysin was isolated from CS of Kwangpyeongok, a Korean hybrid corn, via methanol extraction and preparative C18 reverse phase column chromatography. Maysin cytotoxicity was determined by either monitoring cell viability in various cancer cell lines by MTT assay or morphological changes. Apoptotic cell death was assessed by annexin V-FITC/PI double staining, depolarization of mitochondrial membrane potential (MMP), expression levels of Bcl-2 and pro-caspase-3 and by terminal transferase mediated dUTP-fluorescein nick end labeling (TUNEL) staining. Underlying mechanism in maysin-induced apoptosis of PC-3 cells was explored by evaluating its effects on Akt and ERK pathway. Maysin dose-dependently reduced the PC-3 cell viability, with an 87% reduction at 200 μg/ml. Maysin treatment significantly induced apoptotic cell death, DNA fragmentation, depolarization of MMP, and reduction in Bcl-2 and pro-caspase-3 expression levels. Maysin also significantly attenuated phosphorylation of Akt and ERK. A combined treatment with maysin and other known anti-cancer agents, including 5-FU, etoposide, cisplatin, or camptothecin, synergistically enhanced PC-3 cell death. These results suggested for the first time that maysin inhibits the PC-3 cancer cell growth via stimulation of mitochondria-dependent apoptotic cell death and may have a strong therapeutic potential for the treatment of either chemo-resistant or androgen-independent human prostate cancer. Copyright © 2014 Elsevier Inc. All rights reserved.

  16. Radiosensitivity of mesothelioma cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Haekkinen, A.M. [Dept. of Oncology, Univ. Central Hospital, Helsinki (Finland); Laasonen, A. [Dept. of Pathology, Central Hospital of Etelae-Pohjanmaa, Seinaejoki (Finland); Linnainmaa, K. [Dept. of Industrial Hygiene and Toxicology, Inst. of Occupational Health, Helsinki (Finland); Mattson, K. [Dept. Pulmonary Medicine, Univ. Central Hospital, Helsinki (Finland); Pyrhoenen, S. [Dept. of Oncology, Univ. Central Hospital, Helsinki (Finland)

    1996-10-01

    The present study was carried out in order to examine the radiosensitivity of malignant pleural mesothelioma cell lines. Cell kinetics, radiation-induced delay of the cell cycle and DNA ploidy of the cell lines were also determined. For comparison an HeLa and a human foetal fibroblast cell line were simultaneously explored. Six previously cytogenetically and histologically characterized mesothelioma tumor cell lines were applied. A rapid tiazolyl blue microtiter (MTT) assay was used to analyze radiosensitivity and cell kinetics and DNA ploidy of the cultured cells were determined by flow cytometry. The survival fraction after a dose of 2 Gy (SF2), parameters {alpha} and {beta} of the linear quadratic model (LQ-model) and mean inactivation dose (D{sub MID}) were also estimated. The DNA index of four cell lines equaled 1.0 and two cell lines equaled 1.5 and 1.6. Different mesothelioma cell lines showed a great variation in radiosensitivity. Mean survival fraction after a radiation dose of 2 Gy (SF2) was 0.60 and ranged from 0.36 to 0.81 and mean {alpha} value was 0.26 (range 0.48-0.083). The SF2 of the most sensitive diploid mesothelioma cell line was 0.36: Less than that of the foetal fibroblast cell line (0.49). The survival fractions (0.81 and 0.74) of the two most resistant cell lines, which also were aneuploid, were equal to that of the HeLa cell line (0.78). The {alpha}/{beta} ratios of the most sensitive cell lines were almost an order of magnitude greater than those of the two most resistant cell lines. Radiation-induced delay of the most resistant aneuploid cell line was similar to that of HeLa cells but in the most sensitive (diploid cells) there was practically no entry into the G1 phase following the 2 Gy radiation dose during 36 h. (orig.).

  17. Tumor cell phenotype is sustained by selective MAPK oxidation in mitochondria.

    Directory of Open Access Journals (Sweden)

    Soledad Galli

    2008-06-01

    Full Text Available Mitochondria are major cellular sources of hydrogen peroxide (H(2O(2, the production of which is modulated by oxygen availability and the mitochondrial energy state. An increase of steady-state cell H(2O(2 concentration is able to control the transition from proliferating to quiescent phenotypes and to signal the end of proliferation; in tumor cells thereby, low H(2O(2 due to defective mitochondrial metabolism can contribute to sustain proliferation. Mitogen-activated protein kinases (MAPKs orchestrate signal transduction and recent data indicate that are present in mitochondria and regulated by the redox state. On these bases, we investigated the mechanistic connection of tumor mitochondrial dysfunction, H(2O(2 yield, and activation of MAPKs in LP07 murine tumor cells with confocal microscopy, in vivo imaging and directed mutagenesis. Two redox conditions were examined: low 1 microM H(2O(2 increased cell proliferation in ERK1/2-dependent manner whereas high 50 microM H(2O(2 arrested cell cycle by p38 and JNK1/2 activation. Regarding the experimental conditions as a three-compartment model (mitochondria, cytosol, and nuclei, the different responses depended on MAPKs preferential traffic to mitochondria, where a selective activation of either ERK1/2 or p38-JNK1/2 by co-localized upstream kinases (MAPKKs facilitated their further passage to nuclei. As assessed by mass spectra, MAPKs activation and efficient binding to cognate MAPKKs resulted from oxidation of conserved ERK1/2 or p38-JNK1/2 cysteine domains to sulfinic and sulfonic acids at a definite H(2O(2 level. Like this, high H(2O(2 or directed mutation of redox-sensitive ERK2 Cys(214 impeded binding to MEK1/2, caused ERK2 retention in mitochondria and restricted shuttle to nuclei. It is surmised that selective cysteine oxidations adjust the electrostatic forces that participate in a particular MAPK-MAPKK interaction. Considering that tumor mitochondria are dysfunctional, their inability to

  18. Mitochondria-Targeted Nitroxide, Mito-CP, Suppresses Medullary Thyroid Carcinoma Cell Survival In Vitro and In Vivo

    Science.gov (United States)

    Starenki, Dmytro

    2013-01-01

    Context: Medullary thyroid carcinoma (MTC) is a neuroendocrine tumor mainly caused by mutations in the RET proto-oncogene. For MTC therapy, the U.S. Food and Drug Administration recently approved vandetanib and cabozantinib, multikinase inhibitors targeting RET and other tyrosine kinase receptors of vascular endothelial growth factor, epidermal growth factor, or hepatocyte growth factor. Nevertheless, not all patients with the progressive MTC respond to these drugs, requiring the development of additional therapeutic modalities that have distinct activity. Objective: We aimed to evaluate mitochondria-targeted carboxy-proxyl (Mito-CP), a mitochondria-targeted redox-sensitive agent, for its tumor-suppressive efficacy against MTC. Design: In vitro cultures of 2 human MTC cell lines, TT and MZ-CRC-1, and TT xenografts in mice were treated with Mito-CP in comparison with vandetanib. The effects on cell survival/death, RET expression, mitochondrial integrity, and oxidative stress were determined. Results: Contrary to vandetanib, Mito-CP induced RET downregulation and strong cytotoxic effects in both cell lines in vitro, including caspase-dependent apoptosis. These effects were accompanied by mitochondrial membrane depolarization, decreased oxygen consumption, and increased oxidative stress in cells. Intriguingly, Mito-CP–induced cell death, but not RET downregulation, was partially inhibited by the reactive oxygen species scavenger, N-acetyl-cysteine, indicating that Mito-CP mediates tumor-suppressive effects via redox-dependent as well as redox-independent mechanisms. Orally administered Mito-CP effectively suppressed TT xenografts in mice, with an efficacy comparable to vandetanib and relatively low toxicity to animals. Conclusion: Our results suggest that Mito-CP can effectively suppress MTC cell growth/survival via a mechanism distinct from vandetanib effects. Mitochondrial targeting may be a potential strategy for MTC therapy. PMID:23509102

  19. Metabolic shift in lung alveolar cell mitochondria following acrolein exposure.

    Science.gov (United States)

    Agarwal, Amit R; Yin, Fei; Cadenas, Enrique

    2013-11-15

    Acrolein, an α,β unsaturated electrophile, is an environmental pollutant released in ambient air from diesel exhausts and cooking oils. This study examines the role of acrolein in altering mitochondrial function and metabolism in lung-specific cells. RLE-6TN, H441, and primary alveolar type II (pAT2) cells were exposed to acrolein for 4 h, and its effect on mitochondrial oxygen consumption rates was studied by XF Extracellular Flux analysis. Low-dose acrolein exposure decreased mitochondrial respiration in a dose-dependent manner because of alteration in the metabolism of glucose in all the three cell types. Acrolein inhibited glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity, leading to decreased substrate availability for mitochondrial respiration in RLE-6TN, H441, and pAT2 cells; the reduced GAPDH activity was compensated in pAT2 cells by an increase in the activity of glucose-6-phosphate dehydrogenase, the regulatory control of the pentose phosphate pathway. The decrease in pyruvate from glucose metabolism resulted in utilization of alternative sources to support mitochondrial energy production: palmitate-BSA complex increased mitochondrial respiration in RLE-6TN and pAT2 cells. The presence of palmitate in alveolar cells for surfactant biosynthesis may prove to be the alternative fuel source for mitochondrial respiration. Accordingly, a decrease in phosphatidylcholine levels and an increase in phospholipase A2 activity were found in the alveolar cells after acrolein exposure. These findings have implications for understanding the decrease in surfactant levels frequently observed in pathophysiological situations with altered lung function following exposure to environmental toxicants.

  20. Phenethyl Isothiocyanate Induces Apoptotic Cell Death Through the Mitochondria-dependent Pathway in Gefitinib-resistant NCI-H460 Human Lung Cancer Cells In Vitro.

    Science.gov (United States)

    Hsia, Te-Chun; Huang, Yi-Ping; Jiang, Yi-Wen; Chen, Hsin-Yu; Cheng, Zheng-Yu; Hsiao, Yung-Ting; Chen, Cheng-Yen; Peng, Shu-Fen; Chueh, Fu-Shin; Chou, Yu-Cheng; Chung, Jing-Gung

    2018-04-01

    Some lung cancer patients treated with gefitinib develop resistance to this drug resulting in unsatisfactory treatment outcomes. Phenethyl isothiocyanate (PEITC), present in our common cruciferous vegetables, exhibits anticancer activities in many human cancer cell lines. Currently, there is no available information on the possible modification of gefitinib resistance of lung cancer in vitro by PEITC. Thus, the effects of PEITC on gefitinib resistant lung cancer NCI-H460 cells were investigated in vitro. The total cell viability, apoptotic cell death, production of reactive oxygen species (ROS) and Ca 2+ , levels of mitochondria membrane potential (ΔΨ m ) and caspase-3, -8 and -9 activities were measured by flow cytometry assay. PEITC induced chromatin condensation was examined by DAPI staining. PEITC-induced cell morphological changes, decreased total viable cell number and induced apoptotic cell death in NCI-H460 and NCI-H460/G cells. PEITC decreased ROS production in NCI-H460 cells, but increased production in NCI-H460/G cells. PEITC increased Ca 2+ production, decreased the levels of ΔΨ m and increased caspase-3, -8 and -9 activities in both NCI-H460 and NCI-H460/G cells. Western blotting was used to examine the effect of apoptotic cell death associated protein expression in NCI-H460 NCI-H460/G cells after exposure to PEITC. Results showed that PEITC increased expression of cleaved caspase-3, PARP, GADD153, Endo G and pro-apoptotic protein Bax in NCI-H460/G cells. Based on these results, we suggest that PEITC induces apoptotic cell death via the caspase- and mitochondria-dependent pathway in NCI-H460/G cells. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  1. Beet yellow stunt virus in cells of Sonchus oleraceus L. and its relation to host mitochondria.

    Science.gov (United States)

    Esau, K

    1979-10-15

    In Sonchus oleraceus L. (Asteraceae) infected with the beet yellow stunt virus (BYSV) the virions are found in phloem cells, including the sieve elements. In parenchymatous phloem cells, the virus is present mainly in the cytoplasm. In some parenchymatous cells, containing massive accumulations of virus, the flexuous rodlike virus particles are found partly inserted into mitochondrial cristae. The mitochondrial envelope is absent where virus is present in the cristae. A similar relation between virus and host mitochondria apparently has not been recorded for any other plant virus.

  2. Bcl-xL stimulates Bax relocation to mitochondria and primes cells to ABT-737.

    Science.gov (United States)

    Renault, Thibaud T; Teijido, Oscar; Missire, Florent; Ganesan, Yogesh Tengarai; Velours, Gisèle; Arokium, Hubert; Beaumatin, Florian; Llanos, Raul; Athané, Axel; Camougrand, Nadine; Priault, Muriel; Antonsson, Bruno; Dejean, Laurent M; Manon, Stéphen

    2015-07-01

    Bax cytosol-to-mitochondria translocation is a central event of the intrinsic pathway of apoptosis. Bcl-xL is an important regulator of this event and was recently shown to promote the retrotranslocation of mitochondrial Bax to the cytosol. The present study identifies a new aspect of the regulation of Bax localization by Bcl-xL: in addition to its role in Bax inhibition and retrotranslocation, we found that, like with Bcl-2, an increase of Bcl-xL expression levels led to an increase of Bax mitochondrial content. This finding was substantiated both in pro-lymphocytic FL5.12 cells and a yeast reporting system. Bcl-xL-dependent increase of mitochondrial Bax is counterbalanced by retrotranslocation, as we observed that Bcl-xLΔC, which is unable to promote Bax retrotranslocation, was more efficient than the full-length protein in stimulating Bax relocation to mitochondria. Interestingly, cells overexpressing Bcl-xL were more sensitive to apoptosis upon treatment with the BH3-mimetic ABT-737, suggesting that despite its role in Bax inhibition, Bcl-xL also primes mitochondria to permeabilization and cytochrome c release. Copyright © 2015 Elsevier Ltd. All rights reserved.

  3. Inhibition of oxidative phosphorylation in ascites tumor mitochondria and cells by intramitochondrial Ca2+.

    Science.gov (United States)

    Villalobo, A; Lehninger, A L

    1980-03-25

    Accumulation of Ca2+ (+ phosphate) by respiring mitochondria from Ehrlich ascites or AS30-D hepatoma tumor cells inhibits subsequent phosphorylating respiration in response to ADP. The respiratory chain is still functional since a proton-conducting uncoupler produces a normal stimulation of electron transport. The inhibition of phosphorylating respiration is caused by intramitochondrial Ca2+ (+ phosphate). ATP + Mg2+ together, but not singly, prevents the inhibitory action of Ca2+. Neither AMP, GTP, GDP, nor any other nucleoside 5'-triphosphate or 5'-diphosphate could replace ATP in this effect. Phosphorylating respiration on NAD(NADP)-linked substrates was much more susceptible to the inhibitory effect of intramitochondrial Ca2+ than succinate-linked respiration. Significant inhibition of oxidative phosphorylation is given by the endogenous Ca2+ present in freshly isolated tumor mitochondria. The phosphorylating respiration of permeabilized Ehrlich ascites tumor cells is also inhibited by Ca2+ accumulated by the mitochondria in situ. Possible causes of the Ca2+-induced inhibition of oxidative phosphorylation are considered.

  4. ATAD3 proteins: brokers of a mitochondria-endoplasmic reticulum connection in mammalian cells.

    Science.gov (United States)

    Baudier, Jacques

    2018-05-01

    In yeast, a sequence of physical and genetic interactions termed the endoplasmic reticulum (ER)-mitochondria organizing network (ERMIONE) controls mitochondria-ER interactions and mitochondrial biogenesis. Several functions that characterize ERMIONE complexes are conserved in mammalian cells, suggesting that a similar tethering complex must exist in metazoans. Recent studies have identified a new family of nuclear-encoded ATPases associated with diverse cellular activities (AAA+-ATPase) mitochondrial membrane proteins specific to multicellular eukaryotes, called the ATPase family AAA domain-containing protein 3 (ATAD3) proteins (ATAD3A and ATAD3B). These proteins are crucial for normal mitochondrial-ER interactions and lie at the heart of processes underlying mitochondrial biogenesis. ATAD3A orthologues have been studied in flies, worms, and mammals, highlighting the widespread importance of this gene during embryonic development and in adulthood. ATAD3A is a downstream effector of target of rapamycin (TOR) signalling in Drosophila and exhibits typical features of proteins from the ERMIONE-like complex in metazoans. In humans, mutations in the ATAD3A gene represent a new link between altered mitochondrial-ER interaction and recognizable neurological syndromes. The primate-specific ATAD3B protein is a biomarker of pluripotent embryonic stem cells. Through negative regulation of ATAD3A function, ATAD3B supports mitochondrial stemness properties. © 2017 Cambridge Philosophical Society.

  5. Fisetin-induced apoptosis of human oral cancer SCC-4 cells through reactive oxygen species production, endoplasmic reticulum stress, caspase-, and mitochondria-dependent signaling pathways.

    Science.gov (United States)

    Su, Chen-Hsuan; Kuo, Chao-Lin; Lu, Kung-Wen; Yu, Fu-Shun; Ma, Yi-Shih; Yang, Jiun-Long; Chu, Yung-Lin; Chueh, Fu-Shin; Liu, Kuo-Ching; Chung, Jing-Gung

    2017-06-01

    Oral cancer is one of the cancer-related diseases in human populations and its incidence rates are rising worldwide. Fisetin, a flavonoid from natural products, has been shown to exhibit anticancer activities in many human cancer cell lines but the molecular mechanism of fisetin-induced apoptosis in human oral cancer cells is still unclear; thus, in this study, we investigated fisetin-induced cell death and associated signal pathways on human oral cancer SCC-4 cells in vitro. We examined cell morphological changes, total viable cells, and cell cycle distribution by phase contrast microscopy and flow cytometry assays. Reactive oxygen species (ROS), Ca 2+ , mitochondria membrane potential (ΔΨ m ), and caspase-8, -9, and -3 activities were also measured by flow cytometer. Results indicate that fisetin induced cell death through the cell morphological changes, caused G2/M phase arrest, induction of apoptosis, promoted ROS and Ca 2+ production, and decreased the level of ΔΨ m and increased caspase-3, -8, and -9 activities in SCC-4 cells. DAPI staining and DNA gel electrophoresis were also used to confirm fisetin-induced cell apoptosis in SCC-4 cells. Western blotting also found out that Fisetin increased the proapoptotic proteins such as Bax and Bid and decreased the antiapoptotic proteins such as Bcl-2. Furthermore, results also showed that Fisetin increased the cytochrome c, AIF, and Endo G release from mitochondria in SCC-4 cells. We also used ATF-6α, ATF-6β, GADD153, and GRP78 which indicated that fisetin induced cell death through ER stress. Based on those observations, we suggest that fisetin induced cell apoptosis through ER stress, mitochondria-, and caspase-dependent pathways. © 2017 Wiley Periodicals, Inc.

  6. Biogenesis and dynamics of mitochondria during the cell cycle: significance of 3'UTRs.

    Directory of Open Access Journals (Sweden)

    Marta Martínez-Diez

    Full Text Available Nowadays, we are facing a renaissance of mitochondria in cancer biology. However, our knowledge of the basic cell biology and on the timing and mechanisms that control the biosynthesis of mitochondrial constituents during progression through the cell cycle of mammalian cells remain largely unknown. Herein, we document the in vivo changes on mitochondrial morphology and dynamics that accompany cellular mitosis, and illustrate the following key points of the biogenesis of mitochondria during progression of liver cells through the cycle: (i the replication of nuclear and mitochondrial genomes is synchronized during cellular proliferation, (ii the accretion of OXPHOS proteins is asynchronously regulated during proliferation being the synthesis of beta-F1-ATPase and Hsp60 carried out also at G2/M and, (iii the biosynthesis of cardiolipin is achieved during the S phase, although full development of the mitochondrial membrane potential (DeltaPsim is attained at G2/M. Furthermore, we demonstrate using reporter constructs that the mechanism regulating the accretion of beta-F1-ATPase during cellular proliferation is controlled at the level of mRNA translation by the 3'UTR of the transcript. The 3'UTR-driven synthesis of the protein at G2/M is essential for conferring to the daughter cells the original phenotype of the parental cell. Our findings suggest that alterations on this process may promote deregulated beta-F1-ATPase expression in human cancer.

  7. PDGF-AA-induced filamentous mitochondria benefit dermal papilla cells in cellular migration.

    Science.gov (United States)

    Mifude, C; Kaseda, K

    2015-06-01

    Human dermal papilla cells (HDPCs) play essential roles in hair follicular morphogenesis and postnatal hair growth cycles. Previous reports demonstrated that platelet-derived growth factor-AA (PDGF-AA) enhanced the formation of dermal condensates in hair follicular development. Additionally, PDGF-AA induces/maintains the anagen phase of the hair cycle. It is likely that mitochondrial morphology and functions are tightly coupled with maintenance of these energy-demanding activities. However, little is known about the mitochondrial regulation in HDPCs. Thus, we investigated the PDGF-involved mitochondrial regulation in HDPCs. The mitochondrial morphologies of HDPCs were examined in the presence or absence of PDGF-AA under a fluorescent microscope. ATP production and cellular motility were investigated. The relationship between mitochondrial morphology and the cellular functions was discussed. We observed that primary HDPCs contained mitochondria with filamentous and/or rounded morphologies. Both types of mitochondria showed similar membrane potentials. Interestingly, in the presence of PDGF-AA, but not PDGF-BB, the balance between the two morphologies shifted towards the filamentous form. Concomitantly, both mitochondrial enzymatic activity and total cellular ATP level were augmented by PDGF-AA. These two parameters were closely correlated, suggesting the mitochondrial involvement in the PDGF-augmented ATP production. Moreover, PDGF-AA accelerated the migration of HDPCs in a gap-filling assay, but did not change the rate of cellular proliferation. Notably, filamentous mitochondria dominated migrating HDPCs. PDGF-AA benefits HDPCs in the process of migration, by increasing the number of filamentous mitochondria. © 2014 Society of Cosmetic Scientists and the Société Française de Cosmétologie.

  8. Influence of Pulsed Electric Fields and Mitochondria-Cytoskeleton Interactions on Cell Respiration.

    Science.gov (United States)

    Goswami, Ishan; Perry, Justin B; Allen, Mitchell E; Brown, David A; von Spakovsky, Michael R; Verbridge, Scott S

    2018-06-19

    Pulsed electric fields with microsecond pulse width (μsPEFs) are used clinically; namely, irreversible electroporation/Nanoknife is used for soft tissue tumor ablation. The μsPEF pulse parameters used in irreversible electroporation (0.5-1 kV/cm, 80-100 pulses, ∼100 μs each, 1 Hz frequency) may cause an internal field to develop within the cell because of the disruption of the outer cell membrane and subsequent penetration of the electric field. An internal field may disrupt voltage-sensitive mitochondria, although the research literature has been relatively unclear regarding whether such disruptions occur with μsPEFs. This investigation reports the influence of clinically used μsPEF parameters on mitochondrial respiration in live cells. Using a high-throughput Agilent Seahorse machine, it was observed that μsPEF exposure comprising 80 pulses with amplitudes of 600 or 700 V/cm did not alter mitochondrial respiration in 4T1 cells measured after overnight postexposure recovery. To record alterations in mitochondrial function immediately after μsPEF exposure, high-resolution respirometry was used to measure the electron transport chain state via responses to glutamate-malate and ADP and mitochondrial membrane potential via response to carbonyl cyanide-p-trifluoromethoxyphenylhydrazone. In addition to measuring immediate mitochondrial responses to μsPEF exposure, measurements were also made on cells permeabilized using digitonin and those with compromised cytoskeleton due to actin depolymerization via treatment with the drug latrunculin B. The former treatment was used as a control to tease out the effects of plasma membrane permeabilization, whereas the latter was used to investigate indirect effects on the mitochondria that may occur if μsPEFs impact the cytoskeleton on which the mitochondria are anchored. Based on the results, it was concluded that within the pulse parameters tested, μsPEFs alone do not hinder mitochondrial physiology but can be used

  9. Curcumin Analog DK1 Induces Apoptosis in Human Osteosarcoma Cells In Vitro through Mitochondria-Dependent Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Muhammad Nazirul Mubin Aziz

    2018-01-01

    Full Text Available Osteosarcoma is one of the primary malignant bone tumors that confer low survival rates for patients even with intensive regime treatments. Therefore, discovery of novel anti-osteosarcoma drugs derived from natural products that are not harmful to the normal cells remains crucial. Curcumin is one of the natural substances that have been extensively studied due to its anti-cancer properties and is pharmacologically safe considering its ubiquitous consumption for centuries. However, curcumin suffers from a poor circulating bioavailability, which has led to the development of a chemically synthesized curcuminoid analog, namely (Z-3-hydroxy-1-(2-hydroxyphenyl-3-phenylprop-2-en-1-one (DK1. In this study, the cytotoxic effects of the curcumin analog DK1 was investigated in both U-2OS and MG-63 osteosarcoma cell lines using 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT assay and cell death was microscopically examined via acridine orange/propidium iodide (AO/PI double staining. Flow cytometer analysis including Annexin V/Fluorescein isothiocyanate (FITC, cell cycle analysis and JC-1 were adapted to determine the mode of cell death. Subsequently in order to determine the mechanism of cell death, quantitative polymerase chain reaction (qPCR and proteome profiling was carried out to measure the expression of several apoptotic-related genes and proteins. Results indicated that DK1 induced U-2 OS and MG-63 morphological changes and substantially reduced cell numbers through induction of apoptosis. Several apoptotic genes and proteins were steadily expressed after treatment with DK1; including caspase 3, caspase 9, and BAX, which indicated that apoptosis occurred through a mitochondria-dependent signaling pathway. In conclusion, DK1 could be considered as a potential candidate for an anti-osteosarcoma drug in the near future, contingent upon its ability to induce apoptosis in osteosarcoma cell lines.

  10. Superoxide produced in the matrix of mitochondria enhances methylmercury toxicity in human neuroblastoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Mailloux, Ryan J.; Yumvihoze, Emmanuel; Chan, Hing Man, E-mail: laurie.chan@uottawa.ca

    2015-12-15

    The mechanism of intracellular metabolism of methylmercury (MeHg) is not fully known. It has been shown that superoxide (O{sub 2}·{sup −}), the proximal reactive oxygen species (ROS) generated by mitochondria, is responsible for MeHg demethylation. Here, we investigated the impact of different mitochondrial respiratory inhibitors, namely rotenone and antimycin A, on the O{sub 2}·{sup −} mediated degradation of MeHg in human neuroblastoma cells SH-K-SN. We also utilized paraquat (PQ) which generates O{sub 2}·{sup −} in the mitochondrial matrix. We found that the cleavage of the carbon-metal bond in MeHg was highly dependent on the topology of O{sub 2}·{sup −} production by mitochondria. Both rotenone and PQ, which increase O{sub 2}·{sup −} in the mitochondrial matrix at a dose-dependent manner, enhanced the conversion of MeHg to inorganic mercury (iHg). Surprisingly, antimycin A, which prompts emission of O{sub 2}·{sup −} into the intermembrane space, did not have the same effect even though antimycin A induced a dose dependent increase in O{sub 2}·{sup −} emission. Rotenone and PQ also enhanced the toxicity of sub-toxic doses (0.1 μM) MeHg which correlated with the accumulation of iHg in mitochondria and depletion of mitochondrial protein thiols. Taken together, our results demonstrate that MeHg degradation is mediated by mitochondrial O{sub 2}·{sup −}, specifically within the matrix of mitochondria when O{sub 2}·{sup −} is in adequate supply. Our results also show that O{sub 2}·{sup −} amplifies MeHg toxicity specifically through its conversion to iHg and subsequent interaction with protein cysteine thiols (R-SH). The implications of our findings in mercury neurotoxicity are discussed herein. - Highlights: • Superoxide produced in the matrix of mitochondria degrades MeHg. • Superoxide produced in intermembrane space does not degrade MeHg. • Matrix-generated superoxide enhances Hg toxicity by converting MeHg to iHg.

  11. Single-cell-based evaluation of sperm progressive motility via fluorescent assessment of mitochondria membrane potential.

    Science.gov (United States)

    Moscatelli, Natalina; Spagnolo, Barbara; Pisanello, Marco; Lemma, Enrico Domenico; De Vittorio, Massimo; Zara, Vincenzo; Pisanello, Ferruccio; Ferramosca, Alessandra

    2017-12-20

    Sperm cells progressive motility is the most important parameter involved in the fertilization process. Sperm middle piece contains mitochondria, which play a critical role in energy production and whose proper operation ensures the reproductive success. Notably, sperm progressive motility is strictly related to mitochondrial membrane potential (MMP) and consequently to mitochondrial functionality. Although previous studies presented an evaluation of mitochondrial function through MMP assessment in entire sperm cells samples, a quantitative approach at single-cell level could provide more insights in the analysis of semen quality. Here we combine laser scanning confocal microscopy and functional fluorescent staining of mitochondrial membrane to assess MMP distribution among isolated spermatozoa. We found that the sperm fluorescence value increases as a function of growing progressive motility and that such fluorescence is influenced by MMP disruptors, potentially allowing for the discrimination of different quality classes of sperm cells in heterogeneous populations.

  12. Impaired succinic dehydrogenase activity of rat Purkinje cell mitochondria during aging.

    Science.gov (United States)

    Fattoretti, P; Bertoni-Freddari, C; Caselli, U; Paoloni, R; Meier-Ruge, W

    1998-03-16

    The perikaryal Purkinje cell mitochondria positive to the copper ferrocyanide histochemical reaction for succinic dehydrogenase (SDH) have been investigated by means of semiautomatic morphometric methods in rats of 3, 12 and 24 months of age. The number of organelles/microm3 of Purkinje cell cytoplasm (Numeric density: Nv), the average mitochondrial volume (V) and the mitochondrial volume fraction (Volume density: Vv) were the ultrastructural parameters taken into account. Nv was significantly higher at 12 than at 3 and 24 months of age. V was significantly decreased at 12 and 24 months of age, but no difference was envisaged between adult and old rats. Vv was significantly decreased in old animals vs. the other age groups. In young and old rats, the percentage of organelles larger than 0.32 microm3 was 13.5 and 11%, respectively, while these enlarged mitochondria accounted for less than 1% in the adult group. Since SDH activity is of critical importance when energy demand is high, the marked decrease of Vv supports an impaired capacity of the old Purkinje cells to match actual energy supply at sustained transmission of the nervous impulse. However, the high percentage of enlarged organelles found in old rats may witness a morphofunctional compensatory response.

  13. Analysis of the behavior of mitochondria in the ovaries of the earthworm Dendrobaena veneta Rosa 1839.

    Directory of Open Access Journals (Sweden)

    Justyna Faron

    Full Text Available We examined six types of cells that form the ovary of the earthworm Dendrobena veneta ogonia, prooocytes, vitellogenic oocytes, trophocytes, fully grown postvitellogenic oocytes and somatic cells of the gonad. The quantitative stereological method revealed a much higher "volume density" of mitochondria in all of the types of germ-line cells except for the somatic cells. Fluorescent vital stain JC-1, however, showed a much higher oxidative activity of mitochondria in the somatic cells than in the germ-line cells. The distribution of active and inactive mitochondria within the studied cells was assessed using the computer program ImageJ. The analysis showed a higher luminosity of inactive mitochondria in all of the types of germ-line cells and a higher luminosity of active mitochondria in somatic cells. The OXPHOS activity was found in somatic cells mitochondria and in the peripheral mitochondria of the vitellogenic oocytes. The detection of reactive oxygen species (ROS revealed a differentiated distribution of ROS in the different cell types. The amount of ROS substances was lower in somatic cells than in younger germ-line cells. The ROS level was also low in the cytoplasm of fully grown postwitellogenic oocytes. The distribution of the MnSOD enzyme that protects mitochondria against destructive role of ROS substances was high in the oogonia and in prooocytes and it was very high in vitellogenic and postvitellogenic oocytes. However, a much lower level of this protective enzyme was observed in the trophocytes and the lowest level was found in the cytoplasm of somatic cells. The lower mitochondrial activity and higher level of MnSOD activity in germ-line cells when compared to somatic cells testifies to the necessity of the organisms to protect the mitochondria of oocytes against the destructive role of the ROS that are produced during oxidative phosphorylation. The protection of the mitochondria in oocytes is essential for the transfer of healthy

  14. Mitochondria Targeted Nanoscale Zeolitic Imidazole Framework-90 for ATP Imaging in Live Cells.

    Science.gov (United States)

    Deng, Jingjing; Wang, Kai; Wang, Ming; Yu, Ping; Mao, Lanqun

    2017-04-26

    Zeolitic imidazole frameworks (ZIFs) are an emerging class of functional porous materials with promising biomedical applications such as molecular sensing and intracellular drug delivery. We report herein the first example of using nanoscale ZIFs (i.e., ZIF-90), self-assembled from Zn 2+ and imidazole-2-carboxyaldehyde, to target subcellular mitochondria and image dynamics of mitochondrial ATP in live cells. Encapsulation of fluorescent Rhodamine B (RhB) into ZIF-90 suppresses the emission of RhB, while the competitive coordination between ATP and the metal node of ZIF-90 dissembles ZIFs, resulting in the release of RhB for ATP sensing. With this method, we are able to image mitochondrial ATP in live cells and study the ATP level fluctuation in cellular glycolysis and apoptosis processes. The strategy reported here could be further extended to tune nanoscale ZIFs inside live cells for targeted delivery of therapeutics to subcellular organelles for advanced biomedical applications.

  15. Danazol alters mitochondria metabolism of fibrocystic breast Mcf10A cells.

    Science.gov (United States)

    Irgebay, Zhazira; Yeszhan, Banu; Sen, Bhaswati; Tuleukhanov, Sultan; Brooks, Ari D; Sensenig, Richard; Orynbayeva, Zulfiya

    2017-10-01

    Fibrocystic Breast Disease (FBD) or Fibrocystic change (FC) affects about 60% of women at some time during their life. Although usually benign, it is often associated with pain and tenderness (mastalgia). The synthetic steroid danazol has been shown to be effective in reducing the pain associated with FBD, but the cellular and molecular mechanisms for its action have not been elucidated. We investigated the hypothesis that danazol acts by affecting energy metabolism. Effects of danazol on Mcf10A cells homeostasis, including mechanisms of oxidative phosphorylation, cytosolic calcium signaling and oxidative stress, were assessed by high-resolution respirometry and flow cytometry. In addition to fast physiological responses the associated genomic modulations were evaluated by Affimetrix microarray analysis. The alterations of mitochondria membrane potential and respiratory activity, downregulation of energy metabolism transcripts result in suppression of energy homeostasis and arrest of Mcf10A cells growth. The data obtained in this study impacts the recognition of direct control of mitochondria by cellular mechanisms associated with altered energy metabolism genes governing the breast tissue susceptibility and response to medication by danazol. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. The cytotoxic effect of oxybuprocaine on human corneal epithelial cells by inducing cell cycle arrest and mitochondria-dependent apoptosis.

    Science.gov (United States)

    Fan, W-Y; Wang, D-P; Wen, Q; Fan, T-J

    2017-08-01

    Oxybuprocaine (OBPC) is a widely used topical anesthetic in eye clinic, and prolonged and repeated usage of OBPC might be cytotoxic to the cornea, especially to the outmost corneal epithelium. In this study, we characterized the cytotoxic effect of OBPC on human corneal epithelial (HCEP) cells and investigated its possible cellular and molecular mechanisms using an in vitro model of non-transfected HCEP cells. Our results showed that OBPC at concentrations ranging from 0.025% to 0.4% had a dose- and time-dependent cytotoxicity to HCEP cells. Moreover, OBPC arrested the cells at S phase and induced apoptosis of these cells by inducing plasma membrane permeability, phosphatidylserine externalization, DNA fragmentation, and apoptotic body formation. Furthermore, OBPC could trigger the activation of caspase-2, -3, and -9, downregulate the expression of Bcl-xL, upregulate the expression of Bax along with the cytoplasmic amount of mitochondria-released apoptosis-inducing factor, and disrupt mitochondrial transmembrane potential. Our results suggest that OBPC has a dose- and time-dependent cytotoxicity to HCEP cells by inducing cell cycle arrest and cell apoptosis via a death receptor-mediated mitochondria-dependent proapoptotic pathway, and this novel finding provides new insights into the acute cytotoxicity and its toxic mechanisms of OBPC on HCEP cells.

  17. The anti-apoptotic effect of fluid mechanics preconditioning by cells membrane and mitochondria in rats brain microvascular endothelial cells.

    Science.gov (United States)

    Tian, Shan; Zhu, Fengping; Hu, Ruiping; Tian, Song; Chen, Xingxing; Lou, Dan; Cao, Bing; Chen, Qiulei; Li, Bai; Li, Fang; Bai, Yulong; Wu, Yi; Zhu, Yulian

    2018-01-01

    Exercise preconditioning is a simple and effective way to prevent ischemia. This paper further provided the mechanism in hemodynamic aspects at the cellular level. To study the anti-apoptotic effects of fluid mechanics preconditioning, Cultured rats brain microvascular endothelial cells were given fluid intervention in a parallel plate flow chamber before oxygen glucose deprivation. It showed that fluid mechanics preconditioning could inhibit the apoptosis of endothelial cells, and this process might be mediated by the shear stress activation of Tie-2 on cells membrane surface and Bcl-2 on the mitochondria surface. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Difference in membrane repair capacity between cancer cell lines and a normal cell line

    DEFF Research Database (Denmark)

    Frandsen, Stine Krog; McNeil, Anna K.; Novak, Ivana

    2016-01-01

    repair was investigated by disrupting the plasma membrane using laser followed by monitoring fluorescent dye entry over time in seven cancer cell lines, an immortalized cell line, and a normal primary cell line. The kinetics of repair in living cells can be directly recorded using this technique...... cancer cell lines (p immortalized cell line (p

  19. Repercussion of mitochondria deformity induced by anti-Hsp90 drug 17AAG in human tumor cells

    KAUST Repository

    Vishal, Chaturvedi

    2011-06-07

    Inhibiting Hsp90 chaperone roles using 17AAG induces cytostasis or apoptosis in tumor cells through destabilization of several mutated cancer promoting proteins. Although mitochondria are central in deciding the fate of cells, 17AAG induced effects on tumor cell mitochondria were largely unknown. Here, we show that Hsp90 inhibition with 17AAG first affects mitochondrial integrity in different human tumor cells, neuroblastoma, cervical cancer and glial cells. Using human neuroblastoma tumor cells, we found the early effects associated with a change in mitochondrial membrane potential, elongation and engorgement of mitochondria because of an increased matrix vacuolization. These effects are specific to Hsp90 inhibition as other chemotherapeutic drugs did not induce similar mitochondrial deformity. Further, the effects are independent of oxidative damage and cytoarchitecture destabilization since cytoskeletal disruptors and mitochondrial metabolic inhibitors also do not induce similar deformity induced by 17AAG. The 1D PAGE LC MS/ MS mitochondrial proteome analysis of 17AAG treated human neuroblastoma cells showed a loss of 61% proteins from membrane, metabolic, chaperone and ribonucleoprotein families. About 31 unmapped protein IDs were identified from proteolytic processing map using Swiss-Prot accession number, and converted to the matching gene name searching the ExPASy proteomics server. Our studies display that Hsp90 inhibition effects at first embark on mitochondria of tumor cells and compromise mitochondrial integrity. the author(s), publisher and licensee Libertas Academica Ltd.

  20. Ceramide-Induced Apoptosis in Renal Tubular Cells: A Role of Mitochondria and Sphingosine-1-Phoshate

    Science.gov (United States)

    Ueda, Norishi

    2015-01-01

    Ceramide is synthesized upon stimuli, and induces apoptosis in renal tubular cells (RTCs). Sphingosine-1 phosphate (S1P) functions as a survival factor. Thus, the balance of ceramide/S1P determines ceramide-induced apoptosis. Mitochondria play a key role for ceramide-induced apoptosis by altered mitochondrial outer membrane permeability (MOMP). Ceramide enhances oligomerization of pro-apoptotic Bcl-2 family proteins, ceramide channel, and reduces anti-apoptotic Bcl-2 proteins in the MOM. This process alters MOMP, resulting in generation of reactive oxygen species (ROS), cytochrome C release into the cytosol, caspase activation, and apoptosis. Ceramide regulates apoptosis through mitogen-activated protein kinases (MAPKs)-dependent and -independent pathways. Conversely, MAPKs alter ceramide generation by regulating the enzymes involving ceramide metabolism, affecting ceramide-induced apoptosis. Crosstalk between Bcl-2 family proteins, ROS, and many signaling pathways regulates ceramide-induced apoptosis. Growth factors rescue ceramide-induced apoptosis by regulating the enzymes involving ceramide metabolism, S1P, and signaling pathways including MAPKs. This article reviews evidence supporting a role of ceramide for apoptosis and discusses a role of mitochondria, including MOMP, Bcl-2 family proteins, ROS, and signaling pathways, and crosstalk between these factors in the regulation of ceramide-induced apoptosis of RTCs. A balancing role between ceramide and S1P and the strategy for preventing ceramide-induced apoptosis by growth factors are also discussed. PMID:25751724

  1. A newly identified essential complex, Dre2-Tah18, controls mitochondria integrity and cell death after oxidative stress in yeast.

    Directory of Open Access Journals (Sweden)

    Laurence Vernis

    Full Text Available A mutated allele of the essential gene TAH18 was previously identified in our laboratory in a genetic screen for new proteins interacting with the DNA polymerase delta in yeast [1]. The present work shows that Tah18 plays a role in response to oxidative stress. After exposure to lethal doses of H(2O(2, GFP-Tah18 relocalizes to the mitochondria and controls mitochondria integrity and cell death. Dre2, an essential Fe/S cluster protein and homologue of human anti-apoptotic Ciapin1, was identified as a molecular partner of Tah18 in the absence of stress. Moreover, Ciapin1 is able to replace yeast Dre2 in vivo and physically interacts with Tah18. Our results are in favour of an oxidative stress-induced cell death in yeast that involves mitochondria and is controlled by the newly identified Dre2-Tah18 complex.

  2. ER-mitochondria contacts control surface glycan expression and sensitivity to killer lymphocytes in glioma stem-like cells.

    Science.gov (United States)

    Bassoy, Esen Yonca; Kasahara, Atsuko; Chiusolo, Valentina; Jacquemin, Guillaume; Boydell, Emma; Zamorano, Sebastian; Riccadonna, Cristina; Pellegatta, Serena; Hulo, Nicolas; Dutoit, Valérie; Derouazi, Madiha; Dietrich, Pierre Yves; Walker, Paul R; Martinvalet, Denis

    2017-06-01

    Glioblastoma is a highly heterogeneous aggressive primary brain tumor, with the glioma stem-like cells (GSC) being more sensitive to cytotoxic lymphocyte-mediated killing than glioma differentiated cells (GDC). However, the mechanism behind this higher sensitivity is unclear. Here, we found that the mitochondrial morphology of GSCs modulates the ER-mitochondria contacts that regulate the surface expression of sialylated glycans and their recognition by cytotoxic T lymphocytes and natural killer cells. GSCs displayed diminished ER-mitochondria contacts compared to GDCs. Forced ER-mitochondria contacts in GSCs increased their cell surface expression of sialylated glycans and reduced their susceptibility to cytotoxic lymphocytes. Therefore, mitochondrial morphology and dynamism dictate the ER-mitochondria contacts in order to regulate the surface expression of certain glycans and thus play a role in GSC recognition and elimination by immune effector cells. Targeting the mitochondrial morphology, dynamism, and contacts with the ER could be an innovative strategy to deplete the cancer stem cell compartment to successfully treat glioblastoma. © 2017 The Authors.

  3. ERα36, a variant of estrogen receptor α, is predominantly localized in mitochondria of human uterine smooth muscle and leiomyoma cells.

    Directory of Open Access Journals (Sweden)

    Yitang Yan

    Full Text Available ERα36 is a naturally occurring, membrane-associated, isoform of estrogen receptor α. The expression of ERα36 is due to alternative splicing and different promoter usage. ERα36 is a dominant-negative effector of ERα66-mediated transactivational activities and has the potential to trigger membrane-initiated mitogenic, nongenomic, estrogen signaling; however, the subcellular localization of ERα36 remains controversial. To determine the cellular localization of ERα36 in estrogen-responsive human uterine smooth muscle (ht-UtSMC and leiomyoma (fibroid; ht-UtLM cells, we conducted systematic confocal microscopy and subcellular fractionation analysis using ERα36 antibodies. With Image J colocalizaton analysis plugin, confocal images were analyzed to obtain a Pearson's Correlation Coefficient (PCC to quantify signal colocalization of ERα36 with mitochondrial, endoplasmic reticulum, and cytoskeletal components in both cell lines. When cells were double-stained with an ERα36 antibody and a mitochondrial-specific dye, MitoTracker, the PCC for the two channel signals were both greater than 0.75, indicating strong correlation between ERα36 and mitochondrial signals in the two cell lines. A blocking peptide competition assay confirmed that the mitochondria-associated ERα36 signal detected by confocal analysis was specific for ERα36. In contrast, confocal images double-stained with an ERα36 antibody and endoplasmic reticulum or cytoskeletal markers, had PCCs that were all less than 0.4, indicating no or very weak signal correlation. Fractionation studies showed that ERα36 existed predominantly in membrane fractions, with minimal or undetected amounts in the cytosol, nuclear, chromatin, and cytoskeletal fractions. With isolated mitochondrial preparations, we confirmed that a known mitochondrial protein, prohibitin, was present in mitochondria, and by co-immunoprecipitation analysis that ERα36 was associated with prohibitin in ht-UtLM cells. The

  4. Overexpression of DNA ligase III in mitochondria protects cells against oxidative stress and improves mitochondrial DNA base excision repair

    DEFF Research Database (Denmark)

    Akbari, Mansour; Keijzers, Guido; Maynard, Scott

    2014-01-01

    slower than the preceding mitochondrial BER steps. Overexpression of DNA ligase III in mitochondria improved the rate of overall BER, increased cell survival after menadione induced oxidative stress and reduced autophagy following the inhibition of the mitochondrial electron transport chain complex I...

  5. Graphene Oxide Nanoribbons Induce Autophagic Vacuoles in Neuroblastoma Cell Lines

    Directory of Open Access Journals (Sweden)

    Emanuela Mari

    2016-11-01

    Full Text Available Since graphene nanoparticles are attracting increasing interest in relation to medical applications, it is important to understand their potential effects on humans. In the present study, we prepared graphene oxide (GO nanoribbons by oxidative unzipping of single-wall carbon nanotubes (SWCNTs and analyzed their toxicity in two human neuroblastoma cell lines. Neuroblastoma is the most common solid neoplasia in children. The hallmark of these tumors is the high number of different clinical variables, ranging from highly metastatic, rapid progression and resistance to therapy to spontaneous regression or change into benign ganglioneuromas. Patients with neuroblastoma are grouped into different risk groups that are characterized by different prognosis and different clinical behavior. Relapse and mortality in high risk patients is very high in spite of new advances in chemotherapy. Cell lines, obtained from neuroblastomas have different genotypic and phenotypic features. The cell lines SK-N-BE(2 and SH-SY5Y have different genetic mutations and tumorigenicity. Cells were exposed to low doses of GO for different times in order to investigate whether GO was a good vehicle for biological molecules delivering individualized therapy. Cytotoxicity in both cell lines was studied by measuring cellular oxidative stress (ROS, mitochondria membrane potential, expression of lysosomial proteins and cell growth. GO uptake and cytoplasmic distribution of particles were studied by Transmission Electron Microscopy (TEM for up to 72 h. The results show that GO at low concentrations increased ROS production and induced autophagy in both neuroblastoma cell lines within a few hours of exposure, events that, however, are not followed by growth arrest or death. For this reason, we suggest that the GO nanoparticle can be used for therapeutic delivery to the brain tissue with minimal effects on healthy cells.

  6. Novel mitochondria-targeted heat-soluble proteins identified in the anhydrobiotic Tardigrade improve osmotic tolerance of human cells.

    Directory of Open Access Journals (Sweden)

    Sae Tanaka

    Full Text Available Tardigrades are able to tolerate almost complete dehydration through transition to a metabolically inactive state, called "anhydrobiosis". Late Embryogenesis Abundant (LEA proteins are heat-soluble proteins involved in the desiccation tolerance of many anhydrobiotic organisms. Tardigrades, Ramazzottius varieornatus, however, express predominantly tardigrade-unique heat-soluble proteins: CAHS (Cytoplasmic Abundant Heat Soluble and SAHS (Secretory Abundant Heat Soluble proteins, which are secreted or localized in most intracellular compartments, except the mitochondria. Although mitochondrial integrity is crucial to ensure cellular survival, protective molecules for mitochondria have remained elusive. Here, we identified two novel mitochondrial heat-soluble proteins, RvLEAM and MAHS (Mitochondrial Abundant Heat Soluble, as potent mitochondrial protectants from Ramazzottius varieornatus. RvLEAM is a group3 LEA protein and immunohistochemistry confirmed its mitochondrial localization in tardigrade cells. MAHS-green fluorescent protein fusion protein localized in human mitochondria and was heat-soluble in vitro, though no sequence similarity with other known proteins was found, and one region was conserved among tardigrades. Furthermore, we demonstrated that RvLEAM protein as well as MAHS protein improved the hyperosmotic tolerance of human cells. The findings of the present study revealed that tardigrade mitochondria contain at least two types of heat-soluble proteins that might have protective roles in water-deficient environments.

  7. Mechanism for ginkgolic acid (15 : 1)-induced MDCK cell necrosis: Mitochondria and lysosomes damages and cell cycle arrest.

    Science.gov (United States)

    Yao, Qing-Qing; Liu, Zhen-Hua; Xu, Ming-Cheng; Hu, Hai-Hong; Zhou, Hui; Jiang, Hui-Di; Yu, Lu-Shan; Zeng, Su

    2017-05-01

    Ginkgolic acids (GAs), primarily found in the leaves, nuts, and testa of ginkgo biloba, have been identified with suspected allergenic, genotoxic and cytotoxic properties. However, little information is available about GAs toxicity in kidneys and the underlying mechanism has not been thoroughly elucidated so far. Instead of GAs extract, the renal cytotoxicity of GA (15 : 1), which was isolated from the testa of Ginkgo biloba, was assessed in vitro by using MDCK cells. The action of GA (15 : 1) on cell viability was evaluated by the MTT and neutral red uptake assays. Compared with the control, the cytotoxicity of GA (15 : 1) on MDCK cells displayed a time- and dose-dependent manner, suggesting the cells mitochondria and lysosomes were damaged. It was confirmed that GA (15 : 1) resulted in the loss of cells mitochondrial trans-membrane potential (ΔΨm). In propidium iodide (PI) staining analysis, GA (15 : 1) induced cell cycle arrest at the G0/G1 and G2/M phases, influencing on the DNA synthesis and cell mitosis. Characteristics of necrotic cell death were observed in MDCK cells at the experimental conditions, as a result of DNA agarose gel electrophoresis and morphological observation of MDCK cells. In conclusion, these findings might provide useful information for a better understanding of the GA (15 : 1) induced renal toxicity. Copyright © 2017 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

  8. Non-thermal plasma induces mitochondria-mediated apoptotic signaling pathway via ROS generation in HeLa cells.

    Science.gov (United States)

    Li, Wei; Yu, K N; Ma, Jie; Shen, Jie; Cheng, Cheng; Zhou, Fangjian; Cai, Zhiming; Han, Wei

    2017-11-01

    Non-thermal plasma (NTP) has been proposed as a novel therapeutic method for anticancer treatment. Although increasing evidence suggests that NTP selectively induces apoptosis in some types of tumor cells, the molecular mechanisms underlying this phenomenon remain unclear. In this study, we further investigated possible molecular mechanisms for NTP-induced apoptosis of HeLa cells. The results showed that NTP exposure significantly inhibited the growth and viability of HeLa cells. Morphological observation and flow cytometry analysis demonstrated that NTP exposure induced HeLa cell apoptosis. NTP exposure also activated caspase-9 and caspase-3, which subsequently cleaved poly (ADP- ribose) polymerase. Furthermore, NTP exposure suppressed Bcl-2 expression, enhanced Bax expression and translocation to mitochondria, activated mitochondria-mediated apoptotic pathway, followed by the release of cytochrome c. Further studies showed that NTP treatment led to ROS generation, whereas blockade of ROS generation by N-acetyl-l-cysteine (NAC, ROS scavengers) significantly prevented NTP-induced mitochondrial alteration and subsequent apoptosis of HeLa cells via suppressing Bax translocation, cytochrome c and caspase-3 activation. Taken together, our results indicated that NTP exposure induced mitochondria-mediated intrinsic apoptosis of HeLa cells was activated by ROS generation. These findings provide insights to the therapeutic potential and clinical research of NTP as a novel tool in cervical cancer treatment. Copyright © 2017. Published by Elsevier Inc.

  9. An antimicrobial peptidomimetic induces Mucorales cell death through mitochondria-mediated apoptosis.

    Directory of Open Access Journals (Sweden)

    E Magda Barbu

    Full Text Available The incidence of mucormycosis has dramatically increased in immunocompromised patients. Moreover, the array of cellular targets whose inhibition results in fungal cell death is rather limited. Mitochondria have been mechanistically identified as central regulators of detoxification and virulence in fungi. Our group has previously designed and developed a proteolytically-resistant peptidomimetic motif D(KLAKLAK2 with pleiotropic action ranging from targeted (i.e., ligand-directed activity against cancer and obesity to non-targeted activity against antibiotic resistant gram-negative rods. Here we evaluated whether this non-targeted peptidomimetic motif is active against Mucorales. We show that D(KLAKLAK2 has marked fungicidal action, inhibits germination, and reduces hyphal viability. We have also observed cellular changes characteristic of apoptosis in D(KLAKLAK2-treated Mucorales cells. Moreover, the fungicidal activity was directly correlated with vacuolar injury, mitochondrial swelling and mitochondrial membrane depolarization, intracellular reactive oxygen species accumulation (ROS, and increased caspase-like enzymatic activity. Finally, these apoptotic features were prevented by the addition of the ROS scavenger N-acetyl-cysteine indicating mechanistic pathway specificity. Together, these findings indicate that D(KLAKLAK2 makes Mucorales exquisitely susceptible via mitochondrial injury-induced apoptosis. This prototype may serve as a candidate drug for the development of translational applications against mucormycosis and perhaps other fungal infections.

  10. An antimicrobial peptidomimetic induces Mucorales cell death through mitochondria-mediated apoptosis.

    Science.gov (United States)

    Barbu, E Magda; Shirazi, Fazal; McGrath, Danielle M; Albert, Nathaniel; Sidman, Richard L; Pasqualini, Renata; Arap, Wadih; Kontoyiannis, Dimitrios P

    2013-01-01

    The incidence of mucormycosis has dramatically increased in immunocompromised patients. Moreover, the array of cellular targets whose inhibition results in fungal cell death is rather limited. Mitochondria have been mechanistically identified as central regulators of detoxification and virulence in fungi. Our group has previously designed and developed a proteolytically-resistant peptidomimetic motif D(KLAKLAK)2 with pleiotropic action ranging from targeted (i.e., ligand-directed) activity against cancer and obesity to non-targeted activity against antibiotic resistant gram-negative rods. Here we evaluated whether this non-targeted peptidomimetic motif is active against Mucorales. We show that D(KLAKLAK)2 has marked fungicidal action, inhibits germination, and reduces hyphal viability. We have also observed cellular changes characteristic of apoptosis in D(KLAKLAK)2-treated Mucorales cells. Moreover, the fungicidal activity was directly correlated with vacuolar injury, mitochondrial swelling and mitochondrial membrane depolarization, intracellular reactive oxygen species accumulation (ROS), and increased caspase-like enzymatic activity. Finally, these apoptotic features were prevented by the addition of the ROS scavenger N-acetyl-cysteine indicating mechanistic pathway specificity. Together, these findings indicate that D(KLAKLAK)2 makes Mucorales exquisitely susceptible via mitochondrial injury-induced apoptosis. This prototype may serve as a candidate drug for the development of translational applications against mucormycosis and perhaps other fungal infections.

  11. Mitochondria in Lung Diseases

    Science.gov (United States)

    Aravamudan, Bharathi; Thompson, Michael A.; Pabelick, Christina M.; Prakash, Y. S.

    2014-01-01

    Summary Mitochondria are autonomous cellular organelles that oversee a variety of functions such as metabolism, energy production, calcium buffering, and cell fate determination. Regulation of their morphology and diverse activities beyond energy production are being recognized as playing major roles in cellular health and dysfunction. This review is aimed at summarizing what is known regarding mitochondrial contributions to pathogenesis of lung diseases. Emphasis is given to understanding the importance of structural and functional aspects of mitochondria in both normal cellular function (based on knowledge from other cell types) and in development and modulation of lung diseases such as asthma, COPD, cystic fibrosis and cancer. Emerging techniques that allow examination of mitochondria, and potential strategies to target mitochondria in the treatment of lung diseases are also discussed. PMID:23978003

  12. Milestones and recent discoveries on cell death mediated by mitochondria and their interactions with biologically active amines.

    Science.gov (United States)

    Grancara, Silvia; Ohkubo, Shinji; Artico, Marco; Ciccariello, Mauro; Manente, Sabrina; Bragadin, Marcantonio; Toninello, Antonio; Agostinelli, Enzo

    2016-10-01

    Mitochondria represent cell "powerhouses," being involved in energy transduction from the electrochemical gradient to ATP synthesis. The morphology of their cell types may change, according to various metabolic processes or osmotic pressure. A new morphology of the inner membrane and mitochondrial cristae, significantly different from the previous one, has been proposed for the inner membrane and mitochondrial cristae, based on the technique of electron tomography. Mitochondrial Ca(2+) transport (the transporter has been isolated) generates reactive oxygen species and induces the mitochondrial permeability transition of both inner and outer mitochondrial membranes, leading to induction of necrosis and apoptosis. In the mitochondria of several cell types (liver, kidney, and heart), mitochondrial oxidative stress is an essential step in the induction of cell death, although not in brain, in which the phenomenon is caused by a different mechanism. Mitochondrial permeability transition drives both apoptosis and necrosis, whereas mitochondrial outer membrane permeability is characteristic of apoptosis. Adenine nucleotide translocase remains the most important component involved in membrane permeability, with the opening of the transition pore, although other proteins, such as ATP synthase or phosphate carriers, have been proposed. Intrinsic cell death is triggered by the release from mitochondria of proteic factors, such as cytochrome c, apoptosis inducing factor, and Smac/DIABLO, with the activation of caspases upon mitochondrial permeability transition or mitochondrial outer membrane permeability induction. Mitochondrial permeability transition induces the permeability of the inner membrane in sites in contact with the outer membrane; mitochondrial outer membrane permeability forms channels on the outer membrane by means of various stimuli involving Bcl-2 family proteins. The biologically active amines, spermine, and agmatine, have specific functions on mitochondria

  13. Tuft (caveolated) cells in two human colon carcinoma cell lines.

    OpenAIRE

    Barkla, D. H.; Whitehead, R. H.; Foster, H.; Tutton, P. J.

    1988-01-01

    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 f...

  14. A Novel Mitochondria-Dependent Apoptotic Pathway (MAP) in Prostate Cancer (Pca) Cells

    National Research Council Canada - National Science Library

    Chandra, Dhyan

    2004-01-01

    ...) are also up-regulated (Chandra et al., J. Biol. Chem., 277, 50842-54; 2002). Later, when the apoptotic machinery is activated, I notice that there is prominent localization of active caspase-9 and -3 in the mitochondria...

  15. Calcium Handling by Endoplasmic Reticulum and Mitochondria in a Cell Model of Huntington’s Disease

    Science.gov (United States)

    De Mario, Agnese; Scarlatti, Chiara; Costiniti, Veronica; Primerano, Simona; Lopreiato, Raffaele; Calì, Tito; Brini, Marisa; Giacomello, Marta; Carafoli, Ernesto

    2016-01-01

    Huntington disease (HD) is caused by the CAG (Q) expansion in exon 1 of the IT15 gene encoding a polyglutamine (poly-Q) stretch of the Huntingtin protein (Htt). In the wild type protein, the repeats specify a stretch of up 34 Q in the N-terminal portion of Htt. In the pathological protein (mHtt) the poly-Q tract is longer. Proteolytic cleavage of the protein liberates an N-terminal fragment containing the expanded poly-Q tract becomes harmful to cells, in particular to striatal neurons. The fragments cause the transcriptional dysfunction of genes that are essential for neuronal survival. Htt, however, could also have non-transcriptional effects, e.g. it could directly alter Ca2+ homeostasis and/or mitochondrial morphology and function. Ca2+ dyshomeostasis and mitochondrial dysfunction are considered important in the molecular aetiology of the disease. Here we have analyzed the effect of the overexpression of Htt fragments (18Q, wild type form, wtHtt and 150Q mutated form, mHtt) on Ca2+ homeostasis in striatal neuronal precursor cells (Q7/7). We have found that the transient overexpression of the Htt fragments increases Ca2+ transients in the mitochondria of cells stimulated with Ca2+-mobilizing agonists. The bulk Ca2+ transients in the cytosol were unaffected, but the Ca2+ content of the endoplasmic reticulum was significantly decreased in the case of mHtt expression. To rule out possible transcriptional effects due to the presence of mHtt, we have measured the mRNA level of a subunit of the respiratory chain complex II, whose expression is commonly altered in many HD models. No effects on the mRNA level was found suggesting that, in our experimental condition, transcriptional action of Htt is not occurring and that the effects on Ca2+ homeostasis were dependent to non-transcriptional mechanisms. PMID:26819834

  16. Calcium Handling by Endoplasmic Reticulum and Mitochondria in a Cell Model of Huntington's Disease.

    Science.gov (United States)

    De Mario, Agnese; Scarlatti, Chiara; Costiniti, Veronica; Primerano, Simona; Lopreiato, Raffaele; Calì, Tito; Brini, Marisa; Giacomello, Marta; Carafoli, Ernesto

    2016-01-06

    Huntington disease (HD) is caused by the CAG (Q) expansion in exon 1 of the IT15 gene encoding a polyglutamine (poly-Q) stretch of the Huntingtin protein (Htt). In the wild type protein, the repeats specify a stretch of up 34 Q in the N-terminal portion of Htt. In the pathological protein (mHtt) the poly-Q tract is longer. Proteolytic cleavage of the protein liberates an N-terminal fragment containing the expanded poly-Q tract becomes harmful to cells, in particular to striatal neurons. The fragments cause the transcriptional dysfunction of genes that are essential for neuronal survival. Htt, however, could also have non-transcriptional effects, e.g. it could directly alter Ca2+ homeostasis and/or mitochondrial morphology and function. Ca2+ dyshomeostasis and mitochondrial dysfunction are considered important in the molecular aetiology of the disease. Here we have analyzed the effect of the overexpression of Htt fragments (18Q, wild type form, wtHtt and 150Q mutated form, mHtt) on Ca2+ homeostasis in striatal neuronal precursor cells (Q7/7). We have found that the transient overexpression of the Htt fragments increases Ca2+ transients in the mitochondria of cells stimulated with Ca2+-mobilizing agonists. The bulk Ca2+ transients in the cytosol were unaffected, but the Ca2+ content of the endoplasmic reticulum was significantly decreased in the case of mHtt expression. To rule out possible transcriptional effects due to the presence of mHtt, we have measured the mRNA level of a subunit of the respiratory chain complex II, whose expression is commonly altered in many HD models. No effects on the mRNA level was found suggesting that, in our experimental condition, transcriptional action of Htt is not occurring and that the effects on Ca2+ homeostasis were dependent to non-transcriptional mechanisms.

  17. Cantharidin Induced Oral Squamous Cell Carcinoma Cell Apoptosis via the JNK-Regulated Mitochondria and Endoplasmic Reticulum Stress-Related Signaling Pathways.

    Directory of Open Access Journals (Sweden)

    Chin-Chuan Su

    Full Text Available Oral cancer is a subtype of head and neck cancer which represents 2.65% of all human malignancies. Most of oral cancer is histopathologically diagnosed as oral squamous cell carcinoma (OSCC. OSCC is characterized by a high degree of local invasion and a high rate of metastasis to the cervical lymph nodes. How to prevention and treatment of OSCC is important and imperative. Here, we investigated the therapeutic effect and molecular mechanism of cantharidin, an active compound isolated from blister beetles, on OSCC in vitro. Results showed that cantharidin significantly decreased cell viability in human tongue squamous carcinoma-derived SAS, CAL-27, and SCC-4 cell lines. The further mechanistic studies were carried out in SAS cells. Cantharidin also significantly increased apoptosis-related signals, including caspase-9, caspase-7 and caspase-3 proteins. Besides, cantharidin decreased mitochondrial transmembrane potential (MMP and induced cytochrome c and apoptosis inducing factor (AIF release. Cantharidin also increased Bax, Bid, and Bak protein expressions and decreased Bcl-2 protein expression. Cantharidin could also increase the endoplasmic reticulum (ER stress signals, including the expressions of phosphorylated eIF-2α and CHOP, but not Grp78 and Grp94. Furthermore, cantharidin reduced pro-caspase-12 protein expression. In signals of mitogen-activated protein kinases, cantharidin increased the phosphorylation of JNK, but not ERK and p38. Transfection of shRNA-JNK to OSCC cells effectively reversed the cantharidin-induced cell apoptotic signals, including the mitochondrial and ER stress-related signaling molecules. Taken together, these findings suggest that cantharidin induces apoptosis in OSCC cells via the JNK-regulated mitochondria and ER stress-related signaling pathways.

  18. The distribution of mitochondria-rich cells in the gills of air-breathing fishes.

    Science.gov (United States)

    Lin, Hui-Chen; Sung, Wen-Ting

    2003-01-01

    Respiration and ion regulation are the two principal functions of teleostean gills. Mainly found in the gill filaments of fish, mitochondria-rich cells (MRCs) proliferate to increase the ionoregulatory capacity of the gill in response to osmotic challenges. Gill lamellae consist mostly of pavement cells, which are the major site of gas exchange. Although lamellar MRCs have been reported in some fish species, there has been little discussion of which fish species are likely to have lamellar MRCs. In this study, we first compared the number of filament and lamellar MRCs in air-breathing and non-air-breathing fish species acclimated to freshwater and 5 g NaCl L(-1) conditions. An increase in filament MRCs was found in both air-breathing and non-air-breathing fish acclimated to freshwater. Lamellar MRCs were found only in air-breathing species, but the number of lamellar MRCs did not change significantly with water conditions, except in Periophthalmus cantonensis. Next, we surveyed the distribution of MRCs in the gills of 66 fish species (including 29 species from the previous literature) from 12 orders, 28 families, and 56 genera. Our hypothesis that lamellar MRCs are more likely to be found in air-breathing fishes was supported by a significant association between the presence of lamellar MRCs and the mode of breathing at three levels of systematic categories (species, genus, and family). Based on this integrative view of the multiple functions of fish gills, we should reexamine the role of MRCs in freshwater fish.

  19. Bioimaging of Fluorescence-Labeled Mitochondria in Subcutaneously Grafted Murine Melanoma Cells by the “In Vivo Cryotechnique”

    Science.gov (United States)

    Lei, Ting; Huang, Zheng; Ohno, Nobuhiko; Wu, Bao; Sakoh, Takashi; Saitoh, Yurika; Saiki, Ikuo

    2014-01-01

    The microenvironments of organs with blood flow affect the metabolic profiles of cancer cells, which are influenced by mitochondrial functions. However, histopathological analyses of these aspects have been hampered by technical artifacts of conventional fixation and dehydration, including ischemia/anoxia. The purpose of this study was to combine the in vivo cryotechnique (IVCT) with fluorescent protein expression, and examine fluorescently labeled mitochondria in grafted melanoma tumors. The intensity of fluorescent proteins was maintained well in cultured B16-BL6 cells after cryotechniques followed by freeze-substitution (FS). In the subcutaneous tumors of mitochondria-targeted DsRed2 (mitoDsRed)-expressing cells, a higher number of cancer cells were found surrounding the widely opened blood vessels that contained numerous erythrocytes. Such blood vessels were immunostained positively for immunoglobulin M and ensheathed by basement membranes. MitoDsRed fluorescence was detected in scattering melanoma cells using the IVCT-FS method, and the total mitoDsRed volume in individual cancer cells was significantly decreased with the expression of markers of hypoxia. MitoDsRed was frequently distributed throughout the cytoplasm and in processes extending along basement membranes. IVCT combined with fluorescent protein expression is a useful tool to examine the behavior of fluorescently labeled cells and organelles. We propose that the mitochondrial volume is dynamically regulated in the hypoxic microenvironment and that mitochondrial distribution is modulated by cancer cell interactions with basement membranes. PMID:24394469

  20. Intracellular position of mitochondria and chloroplasts in bundle sheath and mesophyll cells of C3 grasses in relation to photorespiratory CO2 loss

    Directory of Open Access Journals (Sweden)

    Yuto Hatakeyama

    2016-10-01

    Full Text Available In C3 plants, photosynthetic efficiency is reduced by photorespiration. A part of CO2 fixed during photosynthesis in chloroplasts is lost from mitochondria during photorespiration by decarboxylation of glycine by glycine decarboxylase (GDC. Thus, the intracellular position of mitochondria in photosynthetic cells is critical to the rate of photorespiratory CO2 loss. We investigated the intracellular position of mitochondria in parenchyma sheath (PS and mesophyll cells of 10 C3 grasses from 3 subfamilies (Ehrhartoideae, Panicoideae, and Pooideae by immunostaining for GDC and light and electron microscopic observation. Immunostaining suggested that many mitochondria were located in the inner half of PS cells and on the vacuole side of chloroplasts in mesophyll cells. Organelle quantification showed that 62–75% of PS mitochondria were located in the inner half of cells, and 62–78% of PS chloroplasts were in the outer half. In mesophyll cells, 61–92% of mitochondria were positioned on the vacuole side of chloroplasts and stromules. In PS cells, such location would reduce the loss of photorespiratory CO2 by lengthening the path of CO2 diffusion and allow more efficient fixation of CO2 from intercellular spaces. In mesophyll cells, it would facilitate scavenging by chloroplasts of photorespiratory CO2 released from mitochondria. Our data suggest that the PS cells of C3 grasses have already acquired an initial structure leading to proto-Kranz and further C3–C4 intermediate anatomy. We also found that in the Pooideae, organelle positioning in PS cells on the phloem side resembles that in mesophyll cells.

  1. Rhein Elicits In Vitro Cytotoxicity in Primary Human Liver HL-7702 Cells by Inducing Apoptosis through Mitochondria-Mediated Pathway

    Directory of Open Access Journals (Sweden)

    Guy-Armel Bounda

    2015-01-01

    Full Text Available Objective. To study rhein-induced apoptosis signaling pathway and to investigate its molecular mechanisms in primary human hepatic cells. Results. Cell viability of HL-7702 cells treated with rhein showed significant decrease in dose-dependent manner. Following rhein treatment (25 μM, 50 μM, and 100 μM for 12 h, the detection of apoptotic cells was significantly analyzed by flow cytometry and nuclear morphological changes by Hoechst 33258, respectively. Fatty degeneration studies showed upregulation level of the relevant hepatic markers (P < 0.01. Caspase activities expressed significant upregulation of caspase-3, caspase-9, and caspase-8. Moreover, apoptotic cells by rhein were significantly inhibited by Z-LEHD-FMK and Z-DEVD-FMK, caspase-9 inhibitor, and caspase-3 inhibitor, respectively. Overproduction of reactive oxygen species, lipid peroxidation, and loss of mitochondrial membrane potential were detected by fluorometry. Additionally, NAC, a ROS scavenger, significantly attenuated rhein-induced oxidative damage in HL-7702 cells. Furthermore, real-time qPCR results showed significant upregulation of p53, PUMA, Apaf-1, and Casp-9 and Casp-3 mRNA, with no significant changes of Fas and Cytochrome-c. Immunoblotting revealed significant Cytochrome-c release from mitochondria into cytosol and no change in Fas expression. Conclusion. Taken together, these observations suggested that rhein could induce apoptosis in HL-7702 cells via mitochondria-mediated signal pathway with involvement of oxidative stress mechanism.

  2. Nitric oxide protects the mitochondria of anterior pituitary cells and prevents cadmium-induced cell death by reducing oxidative stress.

    Science.gov (United States)

    Poliandri, Ariel H B; Machiavelli, Leticia I; Quinteros, Alnilan F; Cabilla, Jimena P; Duvilanski, Beatriz H

    2006-02-15

    Cadmium (Cd2+) is a highly toxic metal that affects the endocrine system. We have previously shown that Cd2+ induces caspase-3 activation and apoptosis of anterior pituitary cells and that endogenous nitric oxide (NO) protects these cells from Cd2+. Here we investigate the mechanisms by which NO exerts this protective role. Cd2+ (25 microM) reduced the mitochondrial membrane potential (MMP) as measured by flow cytometry. Cd2+-induced apoptosis was mitochondrial dependent since cyclosporin A protected the cells from this metal. Inhibition of NO synthesis with 0.5 mM L-NAME increased the effect of Cd2+ on MMP, whereas the NO donor DETANONOate (0.1 mM) reduced it. Cd2+ increased the production of reactive oxygen species (ROS) as measured by flow cytometry. This effect was electron-transfer-chain-dependent since it was inhibited by rotenone. In fact, rotenone reduced the cytotoxic effect of the metal. The action of Cd2+ on mitochondrial integrity was ROS dependent. Trolox, an antioxidant, inhibited the effect of the metal on the MMP. Cd2+-induced increase in ROS generation was reduced by DETANONOate. There are discrepancies concerning the role of NO in Cd2+ toxicity. Here we show that NO reduces Cd2+ toxicity by protecting the mitochondria from oxidative stress in a system where NO plays a regulatory role.

  3. Why translation counts for mitochondria - retrograde signalling links mitochondrial protein synthesis to mitochondrial biogenesis and cell proliferation.

    Science.gov (United States)

    Battersby, Brendan J; Richter, Uwe

    2013-10-01

    Organelle biosynthesis is a key requirement for cell growth and division. The regulation of mitochondrial biosynthesis exhibits additional layers of complexity compared with that of other organelles because they contain their own genome and dedicated ribosomes. Maintaining these components requires gene expression to be coordinated between the nucleo-cytoplasmic compartment and mitochondria in order to monitor organelle homeostasis and to integrate the responses to the physiological and developmental demands of the cell. Surprisingly, the parameters that are used to monitor or count mitochondrial abundance are not known, nor are the signalling pathways. Inhibiting the translation on mito-ribosomes genetically or with antibiotics can impair cell proliferation and has been attributed to defects in aerobic energy metabolism, even though proliferating cells rely primarily on glycolysis to fuel their metabolic demands. However, a recent study indicates that mitochondrial translational stress and the rescue mechanisms that relieve this stress cause the defect in cell proliferation and occur before any impairment of oxidative phosphorylation. Therefore, the process of mitochondrial translation in itself appears to be an important checkpoint for the monitoring of mitochondrial homeostasis and might have a role in establishing mitochondrial abundance within a cell. This hypothesis article will explore the evidence supporting a role for mito-ribosomes and translation in a mitochondria-counting mechanism.

  4. A new hypothesis of pathogenesis based on the divorce between mitochondria and their host cells: possible relevance for Alzheimer's disease.

    Science.gov (United States)

    Agnati, L F; Guidolin, D; Baluska, F; Leo, G; Barlow, P W; Carone, C; Genedani, S

    2010-06-01

    On the basis of not only the endosymbiotic theory of eukaryotic cell organization and evolution but also of observations of transcellular communication via Tunneling NanoTubes (TNTs), the hypothesis is put forward that when mitochondria, which were once independently living prokaryote-like organisms, are subjected to detrimental genetic, toxic, or environmental conditions, including age-related endogenous factors, they can regress towards their original independent state. At that point, they can become potentially pathogenic intruders within their eukaryotic host cell. Because of the protoplasmic disequilibrium caused by an altered, or mutated, mitochondral population, certain host cells with a minimal capacity for self-renewal, such as dopaminergic neurons, risk a loss of function and degenerate. It is also proposed that altered mitochondria, as well as their mutated mtDNA, can migrate, via TNTs, into adjacent cells. In this way, neurodegenerative states are propagated between cells (glia and/or neurons) of the Central Nervous System (CNS) and that this leads to conditions such as Alzheimer's and Parkinson's disease. This proposal finds indirect support from observations on rotenone-poisoned glioblastoma cells which have been co-cultured with non-poisoned cells. Immunocytochemical techniques revealed that mitochondria, moving along the TNTs, migrated from the poisoned cells towards the healthy cells. It has also been demonstrated by means of immunocytochemistry that, in glioblastoma cell cultures, Amyloid Precursor Protein (APP) is present in TNTs, hence it may migrate from one cell to neighbouring cells. This datum may be of high relevance for a better understanding of Alzheimer's Disease (AD) since molecular, cellular, and animal model studies have revealed that the formation of amyloid beta (Abeta) and other derivatives of the APP are key pathogenic factors in AD, causing mitochondrial dysfunction, free radical generation, oxidative damage, and inflammation

  5. Betulinic acid-induced mitochondria-dependent cell death is counterbalanced by an autophagic salvage response

    NARCIS (Netherlands)

    Potze, L.; Mullauer, F. B.; Colak, S.; Kessler, J. H.; Medema, J. P.

    2014-01-01

    Betulinic acid (BetA) is a plant-derived pentacyclic triterpenoid that exerts potent anti-cancer effects in vitro and in vivo. It was shown to induce apoptosis via a direct effect on mitochondria. This is largely independent of proapoptotic BAK and BAX, but can be inhibited by cyclosporin A (CsA),

  6. Light-induced translocation of Pyronine G from mitochondria to nucleoli in monkey kidney CV-1 cells

    Science.gov (United States)

    Geze, Marc; Dellinger, M.; Bazin, M.; Santus, Rene C.

    1996-12-01

    Pyronine G (3,6-bis-N,N-dimethylaminoxanthylium chloride; PG) is a cationic dye that concentrates in mitochondria of living cells due to the high membrane potential of these organelles, similarly to rhodamine 123 and many other cationic dyes. Pyronine G also shows a preferential affinity for RNA. Upon light irradiation PG has been shown to induce cell death, but the photosensitizing properties of this molecule and the mechanism of cell death are not well understood. Microfluorometry and most particularly microspectrofluorometry are now powerful non-invasive techniques for quantitative studies of single living cells in real time which allow, for example, knowing how living cells are affected by photosensitization. To demonstrate the usefulness of image acquisition with high resolution and high sensitive camera, we present data on photosensitizer relocalization during illumination leading to functional and structural damage in the cells.

  7. Physcion induces mitochondria-driven apoptosis in colorectal cancer cells via downregulating EMMPRIN.

    Science.gov (United States)

    Chen, Xuehong; Gao, Hui; Han, Yantao; Ye, Junli; Xie, Jing; Wang, Chunbo

    2015-10-05

    Physcion, an anthraquinone derivative widely isolated and characterized from both terrestrial and marine sources, has anti-tumor effects on a variety of carcinoma cells, mainly through inhibition of cell proliferation, apoptosis induction and cell cycle arrest. However, little is known about the mechanisms underlying its role in tumor progression. In the present study, we investigated the molecular mechanisms involved in physcion-induced apoptosis in human colorectal cancer (CRC) lines HCT116. Our results showed that physcion inhibited tumor cell viability in a dose- and time-dependent manner, and induced cell apoptosis via intrinsic mitochondrial pathway. Our results also revealed that physcion treatment significantly inhibited extracelluar matrix metalloproteinase inducer (EMMPRIN) expression in HCT116 cells in a dose-dependent manner and overexpression of EMMPRIN protein markedly reduced physcion-induced cell apoptosis. Furthermore, our results strongly indicated the modulating effect of physcion on EMMPRIN is correlated with AMP-activated protein kinase (AMPK)/Hypoxia-inducible factor 1α (HIF-1α) signaling pathway. Our data provide the first experimental evidence that physcion induces mitochondrial apoptosis in CRC cells by downregulating of EMMPRIN via AMPK/HIF-1α signaling pathway and suggest a new mechanism to explain its anti-tumor effects. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. Aluminium based adjuvants and their effects on mitochondria and lysosomes of phagocytosing cells.

    Science.gov (United States)

    Ohlsson, Lars; Exley, Christopher; Darabi, Anna; Sandén, Emma; Siesjö, Peter; Eriksson, Håkan

    2013-11-01

    Aluminium oxyhydroxide, Al(OH)3 is one of few compounds approved as an adjuvant in human vaccines. However, the mechanism behind its immune stimulating properties is still poorly understood. In vitro co-culture of an aluminium adjuvant and the human monocytic cell line THP-1 resulted in reduced cell proliferation. Inhibition occurred at concentrations of adjuvant several times lower than would be found at the injection site using a vaccine formulation containing an aluminium adjuvant. Based on evaluation of the mitochondrial membrane potential, THP-1 cells showed no mitochondrial rupture after co-culture with the aluminium adjuvant, instead an increase in mitochondrial activity was seen. The THP-1 cells are phagocytosing cells and after co-culture with the aluminium adjuvant the phagosomal pathway was obstructed. Primary or early phagosomes mature into phagolysosomes with an internal pH of 4.5 - 5 and carry a wide variety of hydrolysing enzymes. Co-culture with the aluminium adjuvant yielded a reduced level of acidic vesicles and cathepsin L activity, a proteolytic enzyme of the phagolysosomes, was almost completely inhibited. THP-1 cells are an appropriate in vitro model in order to investigate the mechanism behind the induction of a phagocytosing antigen presenting cell into an inflammatory cell by aluminium adjuvants. Much information will be gained by investigating the phagosomal pathway and what occurs inside the phagosomes and to elucidate the ultimate fate of phagocytosed aluminium particles. © 2013.

  9. Establishment of cell lines with rat spermatogonial stem cell characteristics

    NARCIS (Netherlands)

    van Pelt, Ans M. M.; Roepers-Gajadien, Hermien L.; Gademan, Iris S.; Creemers, Laura B.; de Rooij, Dirk G.; van Dissel-Emiliani, Federica M. F.

    2002-01-01

    Spermatogonial cell lines were established by transfecting a mixed population of purified rat A(s) (stem cells), A(pr) and A(al) spermatogonia with SV40 large T antigen. Two cell lines were characterized and found to express Hsp90alpha and oct-4, specific markers for germ cells and A spermatogonia,

  10. Bovine lactoferricin causes apoptosis in Jurkat T-leukemia cells by sequential permeabilization of the cell membrane and targeting of mitochondria

    International Nuclear Information System (INIS)

    Mader, Jamie S.; Richardson, Angela; Salsman, Jayme; Top, Deniz; Antueno, Roberto de; Duncan, Roy; Hoskin, David W.

    2007-01-01

    Bovine lactoferricin (LfcinB) is a cationic antimicrobial peptide that kills Jurkat T-leukemia cells by the mitochondrial pathway of apoptosis. However, the process by which LfcinB triggers mitochondria-dependent apoptosis is not well understood. Here, we show that LfcinB-induced apoptosis in Jurkat T-leukemia cells was preceded by LfcinB binding to, and progressive permeabilization of the cell membrane. Colloidal gold electron microscopy revealed that LfcinB entered the cytoplasm of Jurkat T-leukemia cells prior to the onset of mitochondrial depolarization. LfcinB was not internalized by endocytosis because endocytosis inhibitors did not prevent LfcinB-induced cytotoxicity. Furthermore, intracellular delivery of LfcinB via fusogenic liposomes caused the death of Jurkat T-leukemia cells, as well as normal human fibroblasts. Collectively, these findings suggest that LfcinB caused damage to the cell membrane that allowed LfcinB to enter the cytoplasm of Jurkat T-leukemia cells and mediate cytotoxicity. In addition, confocal microscopy showed that intracellular LfcinB co-localized with mitochondria in Jurkat T-leukemia cells, while flow cytometry and colloidal gold electron microscopy showed that LfcinB rapidly associated with purified mitochondria. Furthermore, purified mitochondria treated with LfcinB rapidly lost transmembrane potential and released cytochrome c. We conclude that LfcinB-induced apoptosis in Jurkat T-leukemia cells resulted from cell membrane damage and the subsequent disruption of mitochondrial membranes by internalized LfcinB

  11. Exogenous ether lipids predominantly target mitochondria

    DEFF Research Database (Denmark)

    Kuerschner, Lars; Richter, Doris; Hannibal-Bach, Hans Kristian

    2012-01-01

    Ether lipids are ubiquitous constituents of cellular membranes with no discrete cell biological function assigned yet. Using fluorescent polyene-ether lipids we analyzed their intracellular distribution in living cells by microscopy. Mitochondria and the endoplasmic reticulum accumulated high......, accumulated to mitochondria and induced morphological changes and cellular apoptosis. These data indicate that edelfosine could exert its pro-apoptotic power by targeting and damaging mitochondria and thereby inducing cellular apoptosis. In general, this study implies an important role of mitochondria...

  12. Taurine inhibits 2,5-hexanedione-induced oxidative stress and mitochondria-dependent apoptosis in PC12 cells.

    Science.gov (United States)

    Li, Shuangyue; Guan, Huai; Qian, Zhiqiang; Sun, Yijie; Gao, Chenxue; Li, Guixin; Yang, Yi; Piao, Fengyuan; Hu, Shuhai

    2017-04-07

    2,5-hexanedione (HD) is the ultimate neurotoxic metabolite of hexane, causing the progression of nerve diseases in human. It was reported that HD induced apoptosis and oxidative stress. Taurine has been shown to be a potent antioxidant. In the present study, we investigated the protection of taurine against HD-induced apoptosis in PC12 cells and the underlying mechanism. Our results showed the decreased viability and increased apoptosis in HD-exposed PC12 cells. HD also induced the disturbance of Bax and Bcl-2 expression, the loss of MMP, the release of mitochondrial cytochrome c and caspase-3 activation in PC12 cells. Moreover, HD resulted in an increase in reactive oxygen species (ROS) level and a decline in the activities of superoxidedismutase and catalase in PC12 cells. However, taurine pretreatment ameliorated the increased apoptosis and the alterations in key regulators of mitochondria-dependent pathway in PC12 exposed to HD. The increased ROS level and the decreased activities of the antioxidant enzymes in HD group were attenuated by taurine. These results indicate that pretreatment of taurine may, at least partly, prevent HD-induced apoptosis via inhibiting mitochondria-dependent pathway. It is also suggested that the potential of taurine against HD-induced apoptosis may benefit from its anti-oxidative property.

  13. Targeting mitochondria in cancer cells using gold nanoparticle-enhanced radiotherapy: A Monte Carlo study

    Energy Technology Data Exchange (ETDEWEB)

    Kirkby, Charles, E-mail: charles.kirkby@albertahealthservices.ca; Ghasroddashti, Esmaeel [Department of Medical Physics, Jack Ady Cancer Centre, Lethbridge, Alberta T1J 1W5 (Canada); Department of Physics and Astronomy, University of Calgary, Calgary, Alberta T2N 1N4 (Canada); Department of Oncology, University of Calgary, Calgary, Alberta T2N 4N2 (Canada)

    2015-02-15

    Purpose: Radiation damage to mitochondria has been shown to alter cellular processes and even lead to apoptosis. Gold nanoparticles (AuNPs) may be used to enhance these effects in scenarios where they collect on the outer membranes of mitochondria. A Monte Carlo (MC) approach is used to estimate mitochondrial dose enhancement under a variety of conditions. Methods: The PENELOPE MC code was used to generate dose distributions resulting from photons striking a 13 nm diameter AuNP with various thicknesses of water-equivalent coatings. Similar dose distributions were generated with the AuNP replaced by water so as to estimate the gain in dose on a microscopic scale due to the presence of AuNPs within an irradiated volume. Models of mitochondria with AuNPs affixed to their outer membrane were then generated—considering variation in mitochondrial size and shape, number of affixed AuNPs, and AuNP coating thickness—and exposed (in a dose calculation sense) to source spectra ranging from 6 MV to 90 kVp. Subsequently dose enhancement ratios (DERs), or the dose with the AuNPs present to that for no AuNPs, for the entire mitochondrion and its components were tallied under these scenarios. Results: For a representative case of a 1000 nm diameter mitochondrion affixed with 565 AuNPs, each with a 13 nm thick coating, the mean DER over the whole organelle ranged from roughly 1.1 to 1.6 for the kilovoltage sources, but was generally less than 1.01 for the megavoltage sources. The outer membrane DERs remained less than 1.01 for the megavoltage sources, but rose to 2.3 for 90 kVp. The voxel maximum DER values were as high as 8.2 for the 90 kVp source and increased further when the particles clustered together. The DER exhibited dependence on the mitochondrion dimensions, number of AuNPs, and the AuNP coating thickness. Conclusions: Substantial dose enhancement directly to the mitochondria can be achieved under the conditions modeled. If the mitochondrion dose can be directly

  14. Mitochondria-targeted antioxidant mitoquinone deactivates human and rat hepatic stellate cells and reduces portal hypertension in cirrhotic rats.

    Science.gov (United States)

    Vilaseca, Marina; García-Calderó, Héctor; Lafoz, Erica; Ruart, Maria; López-Sanjurjo, Cristina Isabel; Murphy, Michael P; Deulofeu, Ramon; Bosch, Jaume; Hernández-Gea, Virginia; Gracia-Sancho, Jordi; García-Pagán, Juan Carlos

    2017-07-01

    In cirrhosis, activated hepatic stellate cells (HSC) play a major role in increasing intrahepatic vascular resistance and developing portal hypertension. We have shown that cirrhotic livers have increased reactive oxygen species (ROS), and that antioxidant therapy decreases portal pressure. Considering that mitochondria produce many of these ROS, our aim was to assess the effects of the oral mitochondria-targeted antioxidant mitoquinone on hepatic oxidative stress, HSC phenotype, liver fibrosis and portal hypertension. Ex vivo: Hepatic stellate cells phenotype was analysed in human precision-cut liver slices in response to mitoquinone or vehicle. In vitro: Mitochondrial oxidative stress was analysed in different cell type of livers from control and cirrhotic rats. HSC phenotype, proliferation and viability were assessed in LX2, and in primary human and rat HSC treated with mitoquinone or vehicle. In vivo: CCl 4 - and thioacetamide-cirrhotic rats were treated with mitoquinone (5 mg/kg/day) or the vehicle compound, DecylTPP, for 2 weeks, followed by measurement of oxidative stress, systemic and hepatic haemodynamic, liver fibrosis, HSC phenotype and liver inflammation. Mitoquinone deactivated human and rat HSC, decreased their proliferation but with no effects on viability. In CCl 4 -cirrhotic rats, mitoquinone decreased hepatic oxidative stress, improved HSC phenotype, reduced intrahepatic vascular resistance and diminished liver fibrosis. These effects were associated with a significant reduction in portal pressure without changes in arterial pressure. These results were further confirmed in the thioacetamide-cirrhotic model. We propose mitochondria-targeted antioxidants as a novel treatment approach against portal hypertension and cirrhosis. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  15. Noscapine induces mitochondria-mediated apoptosis in human colon cancer cells in vivo and in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Zi-Rong; Liu, Meng; Peng, Xiu-Lan; Lei, Xiao-Fei; Zhang, Ji-Xiang [Department of Gastroenterology, Renmin Hospital of Wuhan University, Wuhan 430060, Hubei Province (China); Dong, Wei-Guo, E-mail: dongwg1966@yahoo.com.cn [Department of Gastroenterology, Renmin Hospital of Wuhan University, Wuhan 430060, Hubei Province (China)

    2012-05-11

    Highlights: Black-Right-Pointing-Pointer Noscapine inhibited cell viability of colon cancer in a time- and dose- dependent manner. Black-Right-Pointing-Pointer G{sub 2}/M phase arrest and chromatin condensation and nuclear fragmentation were induced. Black-Right-Pointing-Pointer Noscapine promoted apoptosis via mitochondrial pathways. Black-Right-Pointing-Pointer Tumorigenicity was inhibited by noscapine. -- Abstract: Noscapine, a phthalide isoquinoline alkaloid derived from opium, has been widely used as a cough suppressant for decades. Noscapine has recently been shown to potentiate the anti-cancer effects of several therapies by inducing apoptosis in various malignant cells without any detectable toxicity in cells or tissues. However, the mechanism by which noscapine induces apoptosis in colon cancer cells remains unclear. The signaling pathways by which noscapine induces apoptosis were investigated in colon cancer cell lines treated with various noscapine concentrations for 72 h, and a dose-dependent inhibition of cell viability was observed. Noscapine effectively inhibited the proliferation of LoVo cells in vitro (IC{sub 50} = 75 {mu}M). This cytotoxicity was reflected by cell cycle arrest at G{sub 2}/M and subsequent apoptosis, as indicated by increased chromatin condensation and fragmentation, the upregulation of Bax and cytochrome c (Cyt-c), the downregulation of survivin and Bcl-2, and the activation of caspase-3 and caspase-9. Moreover, in a xenograft tumor model in mice, noscapine injection clearly inhibited tumor growth via the induction of apoptosis, which was demonstrated using a TUNEL assay. These results suggest that noscapine induces apoptosis in colon cancer cells via mitochondrial pathways. Noscapine may be a safe and effective chemotherapeutic agent for the treatment of human colon cancer.

  16. Noscapine induces mitochondria-mediated apoptosis in human colon cancer cells in vivo and in vitro

    International Nuclear Information System (INIS)

    Yang, Zi-Rong; Liu, Meng; Peng, Xiu-Lan; Lei, Xiao-Fei; Zhang, Ji-Xiang; Dong, Wei-Guo

    2012-01-01

    Highlights: ► Noscapine inhibited cell viability of colon cancer in a time- and dose- dependent manner. ► G 2 /M phase arrest and chromatin condensation and nuclear fragmentation were induced. ► Noscapine promoted apoptosis via mitochondrial pathways. ► Tumorigenicity was inhibited by noscapine. -- Abstract: Noscapine, a phthalide isoquinoline alkaloid derived from opium, has been widely used as a cough suppressant for decades. Noscapine has recently been shown to potentiate the anti-cancer effects of several therapies by inducing apoptosis in various malignant cells without any detectable toxicity in cells or tissues. However, the mechanism by which noscapine induces apoptosis in colon cancer cells remains unclear. The signaling pathways by which noscapine induces apoptosis were investigated in colon cancer cell lines treated with various noscapine concentrations for 72 h, and a dose-dependent inhibition of cell viability was observed. Noscapine effectively inhibited the proliferation of LoVo cells in vitro (IC 50 = 75 μM). This cytotoxicity was reflected by cell cycle arrest at G 2 /M and subsequent apoptosis, as indicated by increased chromatin condensation and fragmentation, the upregulation of Bax and cytochrome c (Cyt-c), the downregulation of survivin and Bcl-2, and the activation of caspase-3 and caspase-9. Moreover, in a xenograft tumor model in mice, noscapine injection clearly inhibited tumor growth via the induction of apoptosis, which was demonstrated using a TUNEL assay. These results suggest that noscapine induces apoptosis in colon cancer cells via mitochondrial pathways. Noscapine may be a safe and effective chemotherapeutic agent for the treatment of human colon cancer.

  17. The Aging Mitochondria

    Directory of Open Access Journals (Sweden)

    Pierre Theurey

    2018-01-01

    Full Text Available Mitochondrial dysfunction is a central event in many pathologies and contributes as well to age-related processes. However, distinguishing between primary mitochondrial dysfunction driving aging and a secondary mitochondrial impairment resulting from other cell alterations remains challenging. Indeed, even though mitochondria undeniably play a crucial role in aging pathways at the cellular and organismal level, the original hypothesis in which mitochondrial dysfunction and production of free radicals represent the main driving force of cell degeneration has been strongly challenged. In this review, we will first describe mitochondrial dysfunctions observed in aged tissue, and how these features have been linked to mitochondrial reactive oxygen species (ROS–mediated cell damage and mitochondrial DNA (mtDNA mutations. We will also discuss the clues that led to consider mitochondria as the starting point in the aging process, and how recent research has showed that the mitochondria aging axis represents instead a more complex and multifactorial signaling pathway. New working hypothesis will be also presented in which mitochondria are considered at the center of a complex web of cell dysfunctions that eventually leads to cell senescence and death.

  18. Aspirin Induces Apoptosis through Release of Cytochrome c from Mitochondria

    Directory of Open Access Journals (Sweden)

    Katja C. Zimmermann

    2000-01-01

    Full Text Available Nonsteroidal anti-inflammatory drugs (NSAID reduce the risk for cancer, due to their anti proliferative and apoptosis-inducing effects. A critical pathway for apoptosis involves the release of cytochrome c from mitochondria, which then interacts with Apaf-1 to activate caspase proteases that orchestrate cell death. In this study we found that treatment of a human cancer cell line with aspirin induced caspase activation and the apoptotic cell morphology, which was blocked by the caspase inhibitor zVAD-fmk. Further analysis of the mechanism underlying this apoptotic event showed that aspirin induces translocation of Bax to the mitochondria and triggers release of cytochrome c into the cytosol. The release of cytochrome c from mitochondria was inhibited by overexpression of the antiapoptotic protein Bcl-2 and cells that lack Apaf-1 were resistant to aspirin-induced apoptosis. These data provide evidence that the release of cytochrome c is an important part of the apoptotic mechanism of aspirin.

  19. Design of mitochondria-targeted colorimetric and ratiometric fluorescent probes for rapid detection of SO2 derivatives in living cells

    Science.gov (United States)

    Yang, Yutao; Zhou, Tingting; Bai, Bozan; Yin, Caixia; Xu, Wenzhi; Li, Wei

    2018-05-01

    Two mitochondria-targeted colorimetric and ratiometric fluorescent probes for SO2 derivatives were constructed based on the SO2 derivatives-triggered Michael addition reaction. The probes exhibit high specificity toward HSO3-/SO32- by interrupting their conjugation system resulting in a large ratiometric blue shift of 46-121 nm in their emission spectrum. The two well-resolved emission bands can ensure accurate detection of HSO3-. The detection limits were calculated to be 1.09 and 1.35 μM. Importantly, probe 1 and probe 2 were successfully used to fluorescence ratiometric imaging of endogenous HSO3- in BT-474 cells.

  20. Characterization of Phospholipids in Insulin Secretory Granules and Mitochondria in Pancreatic Beta Cells and Their Changes with Glucose Stimulation*

    Science.gov (United States)

    MacDonald, Michael J.; Ade, Lacmbouh; Ntambi, James M.; Ansari, Israr-Ul H.; Stoker, Scott W.

    2015-01-01

    The lipid composition of insulin secretory granules (ISG) has never previously been thoroughly characterized. We characterized the phospholipid composition of ISG and mitochondria in pancreatic beta cells without and with glucose stimulation. The phospholipid/protein ratios of most phospholipids containing unsaturated fatty acids were higher in ISG than in whole cells and in mitochondria. The concentrations of negatively charged phospholipids, phosphatidylserine, and phosphatidylinositol in ISG were 5-fold higher than in the whole cell. In ISG phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, and sphingomyelin, fatty acids 12:0 and 14:0 were high, as were phosphatidylserine and phosphatidylinositol containing 18-carbon unsaturated FA. With glucose stimulation, the concentration of many ISG phosphatidylserines and phosphatidylinositols increased; unsaturated fatty acids in phosphatidylserine increased; and most phosphatidylethanolamines, phosphatidylcholines, sphingomyelins, and lysophosphatidylcholines were unchanged. Unsaturation and shorter fatty acid length in phospholipids facilitate curvature and fluidity of membranes, which favors fusion of membranes. Recent evidence suggests that negatively charged phospholipids, such as phosphatidylserine, act as coupling factors enhancing the interaction of positively charged regions in SNARE proteins in synaptic or secretory vesicle membrane lipid bilayers with positively charged regions in SNARE proteins in the plasma membrane lipid bilayer to facilitate docking of vesicles to the plasma membrane during exocytosis. The results indicate that ISG phospholipids are in a dynamic state and are consistent with the idea that changes in ISG phospholipids facilitate fusion of ISG with the plasma membrane-enhancing glucose-stimulated insulin exocytosis. PMID:25762724

  1. Characterization of phospholipids in insulin secretory granules and mitochondria in pancreatic beta cells and their changes with glucose stimulation.

    Science.gov (United States)

    MacDonald, Michael J; Ade, Lacmbouh; Ntambi, James M; Ansari, Israr-Ul H; Stoker, Scott W

    2015-04-24

    The lipid composition of insulin secretory granules (ISG) has never previously been thoroughly characterized. We characterized the phospholipid composition of ISG and mitochondria in pancreatic beta cells without and with glucose stimulation. The phospholipid/protein ratios of most phospholipids containing unsaturated fatty acids were higher in ISG than in whole cells and in mitochondria. The concentrations of negatively charged phospholipids, phosphatidylserine, and phosphatidylinositol in ISG were 5-fold higher than in the whole cell. In ISG phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, and sphingomyelin, fatty acids 12:0 and 14:0 were high, as were phosphatidylserine and phosphatidylinositol containing 18-carbon unsaturated FA. With glucose stimulation, the concentration of many ISG phosphatidylserines and phosphatidylinositols increased; unsaturated fatty acids in phosphatidylserine increased; and most phosphatidylethanolamines, phosphatidylcholines, sphingomyelins, and lysophosphatidylcholines were unchanged. Unsaturation and shorter fatty acid length in phospholipids facilitate curvature and fluidity of membranes, which favors fusion of membranes. Recent evidence suggests that negatively charged phospholipids, such as phosphatidylserine, act as coupling factors enhancing the interaction of positively charged regions in SNARE proteins in synaptic or secretory vesicle membrane lipid bilayers with positively charged regions in SNARE proteins in the plasma membrane lipid bilayer to facilitate docking of vesicles to the plasma membrane during exocytosis. The results indicate that ISG phospholipids are in a dynamic state and are consistent with the idea that changes in ISG phospholipids facilitate fusion of ISG with the plasma membrane-enhancing glucose-stimulated insulin exocytosis. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Inhibition of mitochondria- and endoplasmic reticulum stress-mediated autophagy augments temozolomide-induced apoptosis in glioma cells.

    Directory of Open Access Journals (Sweden)

    Chien-Ju Lin

    Full Text Available Autophagy is a crucial process for cells to maintain homeostasis and survival through degradation of cellular proteins and organelles, including mitochondria and endoplasmic reticula (ER. We previously demonstrated that temozolomide (TMZ, an alkylating agent for brain tumor chemotherapy, induced reactive oxygen species (ROS/extracellular signal-regulated kinase (ERK-mediated autophagy to protect glioma cells from apoptosis. In this study, we investigated the role of mitochondrial damage and ER stress in TMZ-induced cytotoxicity. Mitochondrial depolarization and mitochondrial permeability transition pore (MPTP opening were observed as a prelude to TMZ-induced autophagy, and these were followed by the loss of mitochondrial mass. Electron transport chain (ETC inhibitors, such as rotenone (a complex I inhibitor, sodium azide (a complex IV inhibitor, and oligomycin (a complex V inhibitor, or the MPTP inhibitor, cyclosporine A, decreased mitochondrial damage-mediated autophagy, and therefore increased TMZ-induced apoptosis. TMZ treatment triggered ER stress with increased expression of GADD153 and GRP78 proteins, and deceased pro-caspase 12 protein. ER stress consequently induced autophagy through c-Jun N-terminal kinases (JNK and Ca(2+ signaling pathways. Combination of TMZ with 4-phenylbutyrate (4-PBA, an ER stress inhibitor, augmented TMZ-induced cytotoxicity by inhibiting autophagy. Taken together, our data indicate that TMZ induced autophagy through mitochondrial damage- and ER stress-dependent mechanisms to protect glioma cells. This study provides evidence that agents targeting mitochondria or ER may be potential anticancer strategies.

  3. On Cellular Darwinism: Mitochondria.

    Science.gov (United States)

    Bull, Larry

    2016-01-01

    The significant role of mitochondria within cells is becoming increasingly clear. This letter uses the NKCS model of coupled fitness landscapes to explore aspects of organelle-nucleus coevolution. The phenomenon of mitochondrial diversity is allowed to emerge under a simple intracellular evolutionary process, including varying the relative rate of evolution by the organelle. It is shown how the conditions for the maintenance of more than one genetic variant of mitochondria are similar to those previously suggested as needed for the original symbiotic origins of the relationship using the NKCS model.

  4. Dynamic movement of cytochrome c from mitochondria into cytosol and peripheral circulation in massive hepatic cell injury.

    Science.gov (United States)

    Kobayashi, Yoshinori; Mori, Masaaki; Naruto, Takuya; Kobayashi, Naoki; Sugai, Toshiyuki; Imagawa, Tomoyuki; Yokota, Shumpei

    2004-12-01

    In the process of apoptosis, it is known that the transition of cytochrome c from mitochondria into the cytosol occurs, and tumor necrosis factor (TNF)-alpha is one of the molecules responsible for this event. But in the state of hypercytokine induced by D-galactosamine (D-GaIN)/Lipopolysaccharide (LPS), the localization of cytochrome c is little known. Rats were administrated with D-GaIN(700 mg/kg)/LPS(200 microg/kg). Blood and tissue samples were collected and examined for levels of pro-inflammatory cytokines, the apoptosis of liver cells, and the localization of cytochrome c. Before administration of D-GaIN/LPS, cytochrome c was definitely localized in the mitochondria. At 2 h after simultaneous administration of D-GaIN/LPS, cytochrome c had accumulated in the cytosol following abrupt increases of plasma TNF-alpha. Massive cell destruction due to apoptosis proved by Terminal deoxynucleo-tidyl transferase-mediated dUTP nick end labeling staining was observed in liver tissue 4 h later and markedly increased levels of cytochrome c were detected in the plasma 12 h after D-GaIN/LPS administration. Liver injury induced by simultaneous administration of D-GaIN/LPS was closely associated with the production of TNF-alpha, and also with the dynamic movement of cytochrome c from the mitochondria into the cytosol, and then into the systemic circulation. The detection of plasma cytochrome c levels may be a useful clinical tool for the detection of apoptosis in vivo.

  5. Silencing of Pokemon enhances caspase-dependent apoptosis via fas- and mitochondria-mediated pathways in hepatocellular carcinoma cells.

    Science.gov (United States)

    Zhang, Yu-Qin; Xiao, Chuan-Xing; Lin, Bi-Yun; Shi, Ying; Liu, Yun-Peng; Liu, Jing-Jing; Guleng, Bayasi; Ren, Jian-Lin

    2013-01-01

    The role of Pokemon (POK erythroid myeloid ontogenic actor), a recently identified POK transcription factor with proto-oncogenic activity, in hepatocellular carcinogenesis has only been assessed by a few studies. Our previous study revealed that Pokemon is overexpressed in hepatocellular carcinomas (HCC) and promotes HCC cell proliferation and migration via an AKT- and ERK- dependent manner. In the present study, we used the TUNEL assay and FACS analysis to demonstrate that oxaliplatin induced apoptosis was significantly increased in cells with silenced Pokemon. Western blots showed that p53 expression and phosphorylation were significantly increased in Pokemon defective cells, thereby initiating the mitochondria-mediated and death receptor-mediated apoptotic pathways. In the mitochondria-mediated pathway, expression of pro-apoptotic Bcl-2 family members (including Bad, Bid, Bim and Puma) as well as AIF was increased and decreasing the mitochondrial membrane potential resulted in cytochrome C released from mitochondrial in HepG2 si-Pokemon cells. In addition, upon oxaliplatin treatment of Pokemon-silenced cells, the FAS receptor, FADD and their downstream targets caspase-10 and caspase-8 were activated, causing increased release of caspase-8 active fragments p18 and p10. Increased activated caspase-8-mediated cleavage and activation of downstream effector caspases such as caspase-9 and caspase-3 was observed in HepG2 si-Pokemon cells as compared to control. Therefore, Pokemon might serve as an important mediator of crosstalk between intrinsic and extrinsic apoptotic pathways in HCC cells. Moreover, our findings suggest that Pokemon could be an attractive therapeutic target gene for human cancer therapy.

  6. Silencing of Pokemon enhances caspase-dependent apoptosis via fas- and mitochondria-mediated pathways in hepatocellular carcinoma cells.

    Directory of Open Access Journals (Sweden)

    Yu-Qin Zhang

    Full Text Available The role of Pokemon (POK erythroid myeloid ontogenic actor, a recently identified POK transcription factor with proto-oncogenic activity, in hepatocellular carcinogenesis has only been assessed by a few studies. Our previous study revealed that Pokemon is overexpressed in hepatocellular carcinomas (HCC and promotes HCC cell proliferation and migration via an AKT- and ERK- dependent manner. In the present study, we used the TUNEL assay and FACS analysis to demonstrate that oxaliplatin induced apoptosis was significantly increased in cells with silenced Pokemon. Western blots showed that p53 expression and phosphorylation were significantly increased in Pokemon defective cells, thereby initiating the mitochondria-mediated and death receptor-mediated apoptotic pathways. In the mitochondria-mediated pathway, expression of pro-apoptotic Bcl-2 family members (including Bad, Bid, Bim and Puma as well as AIF was increased and decreasing the mitochondrial membrane potential resulted in cytochrome C released from mitochondrial in HepG2 si-Pokemon cells. In addition, upon oxaliplatin treatment of Pokemon-silenced cells, the FAS receptor, FADD and their downstream targets caspase-10 and caspase-8 were activated, causing increased release of caspase-8 active fragments p18 and p10. Increased activated caspase-8-mediated cleavage and activation of downstream effector caspases such as caspase-9 and caspase-3 was observed in HepG2 si-Pokemon cells as compared to control. Therefore, Pokemon might serve as an important mediator of crosstalk between intrinsic and extrinsic apoptotic pathways in HCC cells. Moreover, our findings suggest that Pokemon could be an attractive therapeutic target gene for human cancer therapy.

  7. An off-on fluorescence probe targeting mitochondria based on oxidation-reduction response for tumor cell and tissue imaging

    Science.gov (United States)

    Yao, Hanchun; Cao, Li; Zhao, Weiwei; Zhang, Suge; Zeng, Man; Du, Bin

    2017-10-01

    In this study, a tumor-targeting poly( d, l-lactic-co-glycolic acid) (PLGA) loaded "off-on" fluorescent probe nanoparticle (PFN) delivery system was developed to evaluate the region of tumor by off-on fluorescence. The biodegradability of the nanosize PFN delivery system readily released the probe under tumor acidic conditions. The probe with good biocompatibility was used to monitor the intracellular glutathione (GSH) of cancer cells and selectively localize to mitochondria for tumor imaging. The incorporated tumor-targeting probe was based on the molecular photoinduced electron transfer (PET) mechanism preventing fluorescence ("off" state) and could be easily released under tumor acidic conditions. However, the released tumor-targeting fluorescence probe molecule was selective towards GSH with high selectivity and an ultra-sensitivity for the mitochondria of cancer cells and tissues significantly increasing the probe molecule fluorescence signal ("on" state). The tumor-targeting fluorescence probe showed sensitivity to GSH avoiding interference from cysteine and homocysteine. The PFNs could enable fluorescence-guided cancer imaging during cancer therapy. This work may expand the biological applications of PFNs as a diagnostic reagent, which will be beneficial for fundamental research in tumor imaging. [Figure not available: see fulltext.

  8. Increased mitochondrial mass in cells with functionally compromised mitochondria after exposure to both direct gamma radiation and bystander factors.

    LENUS (Irish Health Repository)

    Nugent, Sharon M E

    2007-07-01

    The bystander effect describes radiation-like damage in unirradiated cells either in the vicinity of irradiated cells or exposed to medium from irradiated cells. This study aimed to further characterize the poorly understood mitochondrial response to both direct irradiation and bystander factor(s) in human keratinocytes (HPV-G) and Chinese hamster ovarian cells (CHO-K1). Oxygen consumption rates were determined during periods of state 4, state 3 and uncoupled respiration. Mitochondrial mass was determined using MitoTracker FM. CHO-K1 cells showed significantly reduced oxygen consumption rates 4 h after exposure to 5 Gy direct radiation and irradiated cell conditioned medium (ICCM) and an apparent recovery 12-24 h later. The apparent recovery was likely due to the substantial increase in mitochondrial mass observed in these cells as soon as 4 h after exposure. HPV-G cells, on the other hand, showed a sustained increase in oxygen consumption rates after ICCM exposure and a transient increase 4 h after exposure to 5 Gy direct radiation. A significant increase in mitochondrial mass per HPV-G cell was observed after exposure to both direct radiation and ICCM. These findings are indicative of a stress response to mitochondrial dysfunction that increases the number of mitochondria per cell.

  9. Cell lines authentication and mycoplasma detection as minimun quality control of cell lines in biobanking.

    Science.gov (United States)

    Corral-Vázquez, C; Aguilar-Quesada, R; Catalina, P; Lucena-Aguilar, G; Ligero, G; Miranda, B; Carrillo-Ávila, J A

    2017-06-01

    Establishment of continuous cell lines from human normal and tumor tissues is an extended and useful methodology for molecular characterization of cancer pathophysiology and drug development in research laboratories. The exchange of these cell lines between different labs is a common practice that can compromise assays reliability due to contamination with microorganism such as mycoplasma or cells from different flasks that compromise experiment reproducibility and reliability. Great proportions of cell lines are contaminated with mycoplasma and/or are replaced by cells derived for a different origin during processing or distribution process. The scientific community has underestimated this problem and thousand of research experiment has been done with cell lines that are incorrectly identified and wrong scientific conclusions have been published. Regular contamination and authentication tests are necessary in order to avoid negative consequences of widespread misidentified and contaminated cell lines. Cell banks generate, store and distribute cell lines for research, being mandatory a consistent and continuous quality program. Methods implementation for guaranteeing both, the absence of mycoplasma and authentication in the supplied cell lines, has been performed in the Andalusian Health System Biobank. Specifically, precise results were obtained using real time PCR detection for mycoplasma and 10 STRs identification by capillary electrophoresis for cell line authentication. Advantages and disadvantages of these protocols are discussed.

  10. Fraction against Human Cancer Cell Lines

    African Journals Online (AJOL)

    fraction of A. sieberi against seven cancer cell lines (Colo20, HCT116, DLD, MCF7, Jurkat, HepG2 and ... The morphology of the HepG2 cell nucleus was investigated by Hoechst 33342, ..... Gong F, Liang Y, Xie P, Chau F. Information theory.

  11. Pollen mitochondria in cytoplasmically male sterile tobacco zygotic and embryonic cells

    International Nuclear Information System (INIS)

    Symillides, Y.

    1985-09-01

    An attempt is being made to establish cytoplasmic organelles transmission during the process of fertilization, by using tobacco grain pollen labelled with leucine 14 C and tritiated thymidine. Through autoradiography the fate of pollen germination and its entry into the embryo sac has been studied. A few days after fertilization, labelled cytoplasmic organelles - mainly mitochondria - were detected in the embryo sac. However, labelling was not observed in cytoplasmic organelles by using tritiated thymidine. For more conclusive results labelled DNA incorporated in cytoplasmic organelles have to be traced during the embryo and endosperm development

  12. Resveratrol protects against hyperglycemia-induced oxidative damage to mitochondria by activating SIRT1 in rat mesangial cells

    International Nuclear Information System (INIS)

    Xu, Ying; Nie, Ling; Yin, Yang-Guang; Tang, Jian-Lin; Zhou, Ji-Yin; Li, Dan-Dan; Zhou, Shi-Wen

    2012-01-01

    Oxidative stress and mitochondrial dysfunction are involved in the pathogenesis of diabetic nephropathy (DN). Resveratrol has potent protective effects on diabetes and diabetic complications including diabetic nephropathy. We aimed to investigate the protective effects of resveratrol on mitochondria and the underlying mechanisms by using an in vitro model of hyperglycemia. We exposed primary cultured rat mesangial cells to high glucose (30 mM) for 48 h. We found that pretreatment with resveratrol (10 μM) 6 h prior to high glucose treatment significantly reduced hyperglycemia-induced increase in reactive oxygen species (ROS) production and mitochondrial superoxide generation, as well as stimulated MnSOD activity. In addition, resveratrol pretreatment significantly reversed the decrease of mitochondrial complex III activity in glucose-treated mesangial cells, which is considered to be the major source of mitochondrial oxidative stress in glucose-treated cells. Furthermore, resveratrol pretreatment efficiently restored the hyperpolarization of ∆Ψm, increased ATP production and preserved the mtDNA content. All of these protective effects of resveratrol were successfully blocked by siRNA targeting SIRT1 and EX-527, a specific inhibitor of SIRT1 activity. Our results indicated that resveratrol efficiently reduced oxidative stress and maintained mitochondrial function related with activating SIRT1 in glucose-treated mesangial cells. It suggested that resveratrol is pharmacologically promising for treating diabetic nephropathy. -- Highlights: ► We treat mesangial cells with glucose as an in vitro model of diabetic nephropathy. ► We find that the nephroprotective effects of resveratrol relate with mitochondria. ► The beneficial effect of resveratrol was prevented by siRNA SIRT1 or its inhibitor.

  13. Resveratrol protects against hyperglycemia-induced oxidative damage to mitochondria by activating SIRT1 in rat mesangial cells

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Ying [Base for Drug Clinical Trial, Xinqiao Hospital, Third Military Medical University, Chongqing, 400037 (China); Nie, Ling [Department of Nephrology, Xinqiao Hospital, Third Military Medical University, Chongqing, 400037 (China); Yin, Yang-Guang [Emergency Department, Xinqiao Hospital, Third Military Medical University, Chongqing, 400037 (China); Tang, Jian-Lin; Zhou, Ji-Yin; Li, Dan-Dan [Base for Drug Clinical Trial, Xinqiao Hospital, Third Military Medical University, Chongqing, 400037 (China); Zhou, Shi-Wen, E-mail: Zhoushiwen1956@yahoo.cn [Base for Drug Clinical Trial, Xinqiao Hospital, Third Military Medical University, Chongqing, 400037 (China)

    2012-03-15

    Oxidative stress and mitochondrial dysfunction are involved in the pathogenesis of diabetic nephropathy (DN). Resveratrol has potent protective effects on diabetes and diabetic complications including diabetic nephropathy. We aimed to investigate the protective effects of resveratrol on mitochondria and the underlying mechanisms by using an in vitro model of hyperglycemia. We exposed primary cultured rat mesangial cells to high glucose (30 mM) for 48 h. We found that pretreatment with resveratrol (10 μM) 6 h prior to high glucose treatment significantly reduced hyperglycemia-induced increase in reactive oxygen species (ROS) production and mitochondrial superoxide generation, as well as stimulated MnSOD activity. In addition, resveratrol pretreatment significantly reversed the decrease of mitochondrial complex III activity in glucose-treated mesangial cells, which is considered to be the major source of mitochondrial oxidative stress in glucose-treated cells. Furthermore, resveratrol pretreatment efficiently restored the hyperpolarization of ∆Ψm, increased ATP production and preserved the mtDNA content. All of these protective effects of resveratrol were successfully blocked by siRNA targeting SIRT1 and EX-527, a specific inhibitor of SIRT1 activity. Our results indicated that resveratrol efficiently reduced oxidative stress and maintained mitochondrial function related with activating SIRT1 in glucose-treated mesangial cells. It suggested that resveratrol is pharmacologically promising for treating diabetic nephropathy. -- Highlights: ► We treat mesangial cells with glucose as an in vitro model of diabetic nephropathy. ► We find that the nephroprotective effects of resveratrol relate with mitochondria. ► The beneficial effect of resveratrol was prevented by siRNA SIRT1 or its inhibitor.

  14. Sinularin Induces Apoptosis through Mitochondria Dysfunction and Inactivation of the pI3K/Akt/mTOR Pathway in Gastric Carcinoma Cells

    Directory of Open Access Journals (Sweden)

    Yu-Jen Wu

    2016-07-01

    Full Text Available Sinularin is an active compound isolated from the cultured soft coral Sinularia flexibilis. In this study, we investigated the effects of sinularin on two human gastric cancer cell lines, AGS and NCI-N87. Our results demonstrated that sinularin suppressed the proliferation of gastric cancer cells in a dose-dependent manner and induced apoptosis. In addition, the loss of mitochondrial membrane potential, the release of cytochrome C, the activation of Bax, Bad and caspase-3/9, and the suppression of p-Bad, Bcl-xL and Bcl-2 were observed in the cells treated with sinularin. This finding suggests that sinularin-induced apoptosis is associated with mitochondria-mediated apoptosis and occurs through caspase-dependent pathways. Furthermore, sinularin inhibited the phosphoinositol 3-kinase/Akt/mechanistic target of the rapamycin signaling pathway. Taken together, our results show that sinularin-induced apoptosis is mediated by activation of the caspase cascade and mitochondrial dysfunction. Our findings suggest that sinularin merits further evaluation as a chemotherapeutic agent for human gastric cancer.

  15. Retinoic acid-induced differentiation increases the rate of oxygen consumption and enhances the spare respiratory capacity of mitochondria in SH-SY5Y cells.

    Science.gov (United States)

    Xun, Zhiyin; Lee, Do-Yup; Lim, James; Canaria, Christie A; Barnebey, Adam; Yanonne, Steven M; McMurray, Cynthia T

    2012-04-01

    Retinoic acid (RA) is used in differentiation therapy to treat a variety of cancers including neuroblastoma. The contributing factors for its therapeutic efficacy are poorly understood. However, mitochondria (MT) have been implicated as key effectors in RA-mediated differentiation process. Here we utilize the SH-SY5Y human neuroblastoma cell line as a model to examine how RA influences MT during the differentiation process. We find that RA confers an approximately sixfold increase in the oxygen consumption rate while the rate of glycolysis modestly increases. RA treatment does not increase the number of MT or cause measurable changes in the composition of the electron transport chain. Rather, RA treatment significantly increases the mitochondrial spare respiratory capacity. We propose a competition model for the therapeutic effects of RA. Specifically, the high metabolic rate in differentiated cells limits the availability of metabolic nutrients for use by the undifferentiated cells and suppresses their growth. Thus, RA treatment provides a selective advantage for the differentiated state. Published by Elsevier Ireland Ltd.

  16. Water-Soluble Triarylborane Chromophores for One- and Two-Photon Excited Fluorescence Imaging of Mitochondria in Cells.

    Science.gov (United States)

    Griesbeck, Stefanie; Zhang, Zuolun; Gutmann, Marcus; Lühmann, Tessa; Edkins, Robert M; Clermont, Guillaume; Lazar, Adina N; Haehnel, Martin; Edkins, Katharina; Eichhorn, Antonius; Blanchard-Desce, Mireille; Meinel, Lorenz; Marder, Todd B

    2016-10-04

    Three water-soluble tetracationic quadrupolar chromophores comprising two three-coordinate boron π-acceptor groups bridged by thiophene-containing moieties were synthesised for biological imaging applications. Compound 3 containing the bulkier 5-(3,5-Me2 C6 H2 )-2,2'-(C4 H2 S)2 -5'-(3,5-Me2 C6 H2 ) bridge is stable over a long period of time, exhibits a high fluorescence quantum yield and strong one- and two-photon absorption (TPA), and has a TPA cross section of 268 GM at 800 nm in water. Confocal laser scanning fluorescence microscopy studies in live cells indicated localisation of the chromophore at the mitochondria; moreover, cytotoxicity measurements proved biocompatibility. Thus, chromophore 3 has excellent potential for one- and two-photon-excited fluorescence imaging of mitochondrial function in cells. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Vitamin K2 Induces Mitochondria-Related Apoptosis in Human Bladder Cancer Cells via ROS and JNK/p38 MAPK Signal Pathways.

    Science.gov (United States)

    Duan, Fengsen; Yu, Yuejin; Guan, Rijian; Xu, Zhiliang; Liang, Huageng; Hong, Ling

    2016-01-01

    The effects of vitamin K2 on apoptosis in a variety of cancer cells have been well established in previous studies. However, the apoptotic effect of vitamin K2 on bladder cancer cells has not been evaluated. The aim of this study is to examine the apoptotic activity of Vitamin K2 in bladder cancer cells and investigate the underlying mechanism. In this study, Vitamin K2 induced apoptosis in bladder cancer cells through mitochondria pathway including loss of mitochondria membrane potential, cytochrome C release and caspase-3 cascade. Furthermore, the phosphorylation of c-Jun N-terminal kinase (JNK) and p38 MAPK was detected in Vitamin K2-treated cells and both SP600125 (an inhibitor of JNK) and SB203580 (an inhibitor of p38 MAPK) completely abolished the Vitamin K2-induced apoptosis and loss of mitochondria membrane potential. Moreover, the generation of reactive oxygen species (ROS) was detected in bladder cancer cells, upon treatment of vitamin K2 and the anti-oxidant N-acetyl cysteine (NAC) almost blocked the Vitamin K2-triggered apoptosis, loss of mitochondria membrane potential and activation of JNK and p38 MAPK. Taken together, these findings revealed that Vitamin K2 induces apoptosis in bladder cancer cells via ROS-mediated JNK/p38 MAPK and Mitochondrial pathways.

  18. Melatonin promotes Bax sequestration to mitochondria reducing cell susceptibility to apoptosis via the lipoxygenase metabolite 5-hydroxyeicosatetraenoic acid

    KAUST Repository

    Radogna, Flavia

    2015-03-01

    Extra-neurological functions of melatonin include control of the immune system and modulation of apoptosis. We previously showed that melatonin inhibits the intrinsic apoptotic pathway in leukocytes via stimulation of high affinity MT1/MT2 receptors, thereby promoting re-localization of the anti-apoptotic Bcl-2 protein to mitochondria. Here we show that Bcl-2 sequesters pro-apoptotic Bax into mitochondria in an inactive form after melatonin treatment, thus reducing cell propensity to apoptosis. Bax translocation and the anti-apoptotic effect of melatonin are strictly dependent on the presence of Bcl-2, and on the 5-lipoxygenase (5-LOX) metabolite 5-hydroxyeicosatetraenoic acid (5-HETE), which we have previously shown to be produced as a consequence of melatonin binding to its low affinity target calmodulin. Therefore, the anti-apoptotic effect of melatonin requires the simultaneous, independent interaction with high (MT1/MT2) and low (calmodulin) affinity targets, eliciting two independent signal transduction pathways converging into Bax sequestration and inactivation. MT1/MT2 vs. lipoxygenase pathways are activated by 10-9 vs. 10-5M melatonin, respectively; the anti-apoptotic effect of melatonin is achieved at 10-5M, but drops to 10-9M upon addition of exogenous 5-HETE, revealing that lipoxygenase activation is the rate-limiting pathway. Therefore, in areas of inflammation with increased 5-HETE levels, physiological nanomolar concentrations of melatonin may suffice to maintain leukocyte viability.

  19. Amplification of arsenic genotoxicity by TiO2 nanoparticles in mammalian cells: new insights from physicochemical interactions and mitochondria.

    Science.gov (United States)

    Wang, Xinan; Liu, Yun; Wang, Juan; Nie, Yaguang; Chen, Shaopeng; Hei, Tom K; Deng, Zhaoxiang; Wu, Lijun; Zhao, Guoping; Xu, An

    2017-10-01

    Titanium dioxide nanoparticles (TiO 2 NPs) have shown great adsorption capacity for arsenic (As); however, the potential impact of TiO 2 NPs on the behavior and toxic responses of As remains largely unexplored. In the present study, we focused on the physicochemical interaction between TiO 2 NPs and As(III) to clarify the underlying mechanisms involved in their synergistic genotoxic effect on mammalian cells. Our data showed that As(III) mainly interacted with TiO 2 NPs by competitively occupying the sites of hydroxyl groups on the surface of TiO 2 NP aggregates, resulting in more aggregation of TiO 2 NPs. Although TiO 2 NPs at concentrations used here had no cytotoxic or genotoxic effects on cells, they efficiently increased the genotoxicity of As(III) in human-hamster hybrid (A L ) cells. The synergistic genotoxicity of TiO 2 NPs and As(III) was partially inhibited by various endocytosis pathway inhibitors while it was completely blocked by an As(III)-specific chelator. Using a mitochondrial membrane potential fluorescence probe, a reactive oxygen species (ROS) probe together with mitochondrial DNA-depleted ρ 0 A L cells, we discovered that mitochondria were essential for mediating the synergistic DNA-damaging effects of TiO 2 NPs and As(III). These data provide novel mechanistic proof that TiO 2 NPs enhanced the genotoxicity of As(III) via physicochemical interactions, which were mediated by mitochondria-dependent ROS.

  20. Cell fusion induced by ionizing radiation in various cell lines

    International Nuclear Information System (INIS)

    Khair, M.B.

    1994-07-01

    Cell fusion induced by ionizing radiation has been studied in rat's hepatocytes in vivo and in different cell lines in vitro. These cell lines were: Hela cells, V-79 fibroblasts, human and rat lymphocytes. For irradiation, 0.85 MeV fission neutrons and 14 MeV fast neutrons were used. Cell analyses were performed by fluorescent dyes using immunofluorescent microscope and flow cytometre. Our results in vivo showed that, regardless the dose-rate, a dose of 1 Gy approximately was enough to induce a significant level of cell fusion depending on neutron energy and the age of rats. The level of cell fusion was also significant in Hela cells at a dose of 0.5 Gy. Similar effect, but to a lesser extent, was observed in V-79 cells. Whereas, in lymphocytes insignificant cell fusion was noticed. The varying levels of cell-fusion in different cell lines could be attributed to the type of cells and mutual contact between cells. Furthermore irradiation did not show any influence on cell division ability in both hepatocytes and Hela cells and that fused cells were also able to divide forming a new generation of cells. (author). 36 refs., 8 figs., 10 tabs

  1. Thioredoxin-2 (TRX-2) is an essential gene regulating mitochondria-dependent apoptosis.

    Science.gov (United States)

    Tanaka, Toru; Hosoi, Fumihito; Yamaguchi-Iwai, Yuko; Nakamura, Hajime; Masutani, Hiroshi; Ueda, Shugo; Nishiyama, Akira; Takeda, Shunichi; Wada, Hiromi; Spyrou, Giannis; Yodoi, Junji

    2002-04-02

    Thioredoxin-2 (Trx-2) is a mitochondria-specific member of the thioredoxin superfamily. Mitochondria have a crucial role in the signal transduction for apoptosis. To investigate the biological significance of Trx-2, we cloned chicken TRX-2 cDNA and generated clones of the conditional Trx-2-deficient cells using chicken B-cell line, DT40. Here we show that TRX-2 is an essential gene and that Trx-2-deficient cells undergo apoptosis upon repression of the TRX-2 transgene, showing an accumulation of intracellular reactive oxygen species (ROS). Cytochrome c is released from mitochondria, while caspase-9 and caspase-3, but not caspase-8, are activated upon inhibition of the TRX-2 transgene. In addition, Trx-2 and cytochrome c are co-immunoprecipitated in an in vitro assay. These results suggest that mitochondrial Trx-2 is essential for cell viability, playing a crucial role in the scavenging ROS in mitochondria and regulating the mitochondrial apoptosis signaling pathway.

  2. Cell Line Data Base: structure and recent improvements towards molecular authentication of human cell lines.

    Science.gov (United States)

    Romano, Paolo; Manniello, Assunta; Aresu, Ottavia; Armento, Massimiliano; Cesaro, Michela; Parodi, Barbara

    2009-01-01

    The Cell Line Data Base (CLDB) is a well-known reference information source on human and animal cell lines including information on more than 6000 cell lines. Main biological features are coded according to controlled vocabularies derived from international lists and taxonomies. HyperCLDB (http://bioinformatics.istge.it/hypercldb/) is a hypertext version of CLDB that improves data accessibility by also allowing information retrieval through web spiders. Access to HyperCLDB is provided through indexes of biological characteristics and navigation in the hypertext is granted by many internal links. HyperCLDB also includes links to external resources. Recently, an interest was raised for a reference nomenclature for cell lines and CLDB was seen as an authoritative system. Furthermore, to overcome the cell line misidentification problem, molecular authentication methods, such as fingerprinting, single-locus short tandem repeat (STR) profile and single nucleotide polymorphisms validation, were proposed. Since this data is distributed, a reference portal on authentication of human cell lines is needed. We present here the architecture and contents of CLDB, its recent enhancements and perspectives. We also present a new related database, the Cell Line Integrated Molecular Authentication (CLIMA) database (http://bioinformatics.istge.it/clima/), that allows to link authentication data to actual cell lines.

  3. [Effects of the maca extract on the ultrastructures of mitochondria in the spinal nerve cell and exercise endurance].

    Science.gov (United States)

    Yu, Fa-Rong; Yang, Bo; Li, Zuo-Ping; Lian, Xiu-Zhen; Xie, Ming-Ren; Li, Deng-Lou; Zhang, Shi-Shuang

    2017-06-08

    To investigate the effects of maca extract on the ultrastructures of mitochondria in the spinal nerve cell and exercise endurance. The Wistar rats were randomly divided into 5 groups, including the control group (no swimming), the swimming group (free swimming), and 3 treatment groups treated with the maca extract at the doses of 4.0, 5.3 and 8.0 g/kg body weight. The animals in swimming and treatment groups were then for free swimming in the circulating water flow daily for 15 days. On the 16 th day after swimming endurance, the spinal and muscular tissues were collected from all groups. The mitochondrial ultrastructures of the neurons of the spinal cells were observed with the projection electron microscope, and the levels of the glycogen, malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and Ca 2+ in muscle tissues were determined by the RIA method. When rats were treated with maca extract (at 4.0, 5.3, 8.0 g/kg body weight), the total swimming time and the swimming duration before sinking were increased by 19.83%, 60.28%, 77.55%, and 55.34%, 73.91%, 94.47%, respectively, compared with the simple swimming group( P maca extract (4.0, 5.3, 8.0 g/kg body weight) were reduced by 7.79%, 18.18%, 31.17%, 16.95%, 27.34%, 43.31% and 13.51%, 23.19%, 43.15%, respectively. Our results demonstrated the protective effects of maca extract on the mitochondria of spinal cell and suggested that maca extract could improve the muscle antioxidant activity by increasing the levels of SOD, GSH-Px, and muscle glycogen.

  4. Hsp105 family proteins suppress staurosporine-induced apoptosis by inhibiting the translocation of Bax to mitochondria in HeLa cells

    International Nuclear Information System (INIS)

    Yamagishi, Nobuyuki; Ishihara, Keiichi; Saito, Youhei; Hatayama, Takumi

    2006-01-01

    Hsp105 (Hsp105α and Hsp105β), major heat shock proteins in mammalian cells, belong to a subgroup of the HSP70 family, HSP105/110. Previously, we have shown that Hsp105α has completely different effects on stress-induced apoptosis depending on cell type. However, the molecular mechanisms by which Hsp105α regulates stress-induced apoptosis are not fully understood. Here, we established HeLa cells that overexpress either Hsp105α or Hsp105β by removing doxycycline and examined how Hsp105 modifies staurosporine (STS)-induced apoptosis in HeLa cells. Apoptotic features such as the externalization of phosphatidylserine on the plasma membrane and nuclear morphological changes were induced by the treatment with STS, and the STS-induced apoptosis was suppressed by overexpression of Hsp105α or Hsp105β. In addition, we found that overexpression of Hsp105α or Hsp105β suppressed the activation of caspase-3 and caspase-9 by preventing the release of cytochrome c from mitochondria. Furthermore, the translocation of Bax to mitochondria, which results in the release of cytochrome c from the mitochondria, was also suppressed by the overexpression of Hsp105α or Hsp105β. Thus, it is suggested that Hsp105 suppresses the stress-induced apoptosis at its initial step, the translocation of Bax to mitochondria in HeLa cells

  5. Tuft (caveolated) cells in two human colon carcinoma cell lines.

    Science.gov (United States)

    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.

  6. Gentamicin-induced apoptosis in LLC-PK1 cells: Involvement of lysosomes and mitochondria

    International Nuclear Information System (INIS)

    Servais, Helene; Van Der Smissen, Patrick; Thirion, Gaetan; Van der Essen, Gauthier; Van Bambeke, Francoise; Tulkens, Paul M.; Mingeot-Leclercq, Marie-Paule

    2005-01-01

    Gentamicin accumulates in lysosomes and induces apoptosis in kidney proximal tubules and renal cell lines. Using LLC-PK1 cells, we have examined the concentration- and time-dependency of the effects exerted by gentamicin (1-3 mM; 0-3 days) on (i) lysosomal stability; (ii) activation of mitochondrial pathway; (iii) occurrence of apoptosis (concentrations larger than 3 mM caused extensive necrosis as assessed by the measurement of lactate dehydrogenase release). Within 2 h, gentamicin induced a partial relocalization [from lysosomes to cytosol] of the weak organic base acridine orange. We thereafter observed (a) a loss of mitochondrial membrane potential (as from 10 h, based on spectrophotometric and confocal microscopy using JC1 probe) and (b) the release of cytochrome c from granules to cytosol, and the activation of caspase-9 (as from 12 h; evidenced by Western blot analysis). Increase in caspase-3 activity (assayed with Ac-DEVD-AFC in the presence of z-VAD-fmk]) and appearance of fragmented nuclei (DAPI staining) was then detected as from 16 to 24 h together with nuclear fragmentation. Gentamicin produces a fast (within 4 h) release of calcein from negatively-charged liposomes at pH 5.4, which was slowed down by raising the pH to 7.4, or when phosphatidylinositol was replaced by cardiolipin (to mimic the inner mitochondrial membrane). The present data provide temporal evidence that gentamicin causes apoptosis in LLC-PK1 with successive alteration of the permeability of lysosomes, triggering of the mitochondrial pathway, and activation of caspase-3

  7. Peptidomic analysis of human cell lines

    Science.gov (United States)

    Gelman, Julia S.; Sironi, Juan; Castro, Leandro M.; Ferro, Emer S.; Fricker, Lloyd D.

    2011-01-01

    Peptides have been proposed to function in intracellular signaling within the cytosol. Although cytosolic peptides are considered to be highly unstable, a large number of peptides have been detected in mouse brain and other biological samples. In the present study, we evaluated the peptidome of three diverse cell lines: SH-SY5Y, MCF7, and HEK293 cells. A comparison of the peptidomes revealed considerable overlap in the identity of the peptides found in each cell line. The majority of the observed peptides are not derived from the most abundant or least stable proteins in the cell, and approximately half of the cellular peptides correspond to the N- or C- termini of the precursor proteins. Cleavage site analysis revealed a preference for hydrophobic residues in the P1 position. Quantitative peptidomic analysis indicated that the levels of most cellular peptides are not altered in response to elevated intracellular calcium, suggesting that calpain is not responsible for their production. The similarity of the peptidomes of the three cell lines and the lack of correlation with the predicted cellular degradome implies the selective formation or retention of these peptides, consistent with the hypothesis that they are functional in the cells. PMID:21204522

  8. Mitochondria and Endothelial Function

    Science.gov (United States)

    Kluge, Matthew A.; Fetterman, Jessica L.; Vita, Joseph A.

    2013-01-01

    In contrast to their role in other cell types with higher energy demands, mitochondria in endothelial cells primarily function in signaling cellular responses to environmental cues. This article provides an overview of key aspects of mitochondrial biology in endothelial cells, including subcellular location, biogenesis, dynamics, autophagy, ROS production and signaling, calcium homeostasis, regulated cell death, and heme biosynthesis. In each section, we introduce key concepts and then review studies showing the importance of that mechanism to endothelial control of vasomotor tone, angiogenesis, and inflammatory activation. We particularly highlight the small number of clinical and translational studies that have investigated each mechanism in human subjects. Finally, we review interventions that target different aspects of mitochondrial function and their effects on endothelial function. The ultimate goal of such research is the identification of new approaches for therapy. The reviewed studies make it clear that mitochondria are important in endothelial physiology and pathophysiology. A great deal of work will be needed, however, before mitochondria-directed therapies are available for the prevention and treatment of cardiovascular disease. PMID:23580773

  9. Breast cancer cell lines: friend or foe?

    International Nuclear Information System (INIS)

    Burdall, Sarah E; Hanby, Andrew M; Lansdown, Mark RJ; Speirs, Valerie

    2003-01-01

    The majority of breast cancer research is conducted using established breast cancer cell lines as in vitro models. An alternative is to use cultures established from primary breast tumours. Here, we discuss the pros and cons of using both of these models in translational breast cancer research

  10. (Asteraceae) Fraction against Human Cancer Cell Lines

    African Journals Online (AJOL)

    Purpose: To investigate the anti-proliferative and apoptotic activity of crude and dichloromethane fraction of A. sieberi against seven cancer cell lines (Colo20, HCT116, DLD, MCF7, Jurkat, HepG2 and L929). Methods: A. sieberi was extracted with methanol and further purification was carried out using liquidliquid extraction ...

  11. Radiation sensitivity of Merkel cell carcinoma cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Leonard, J.H.; Ramsay, J.R.; Birrell, G.W. [Queensland Institute of Medical Research (Australia)] [and others

    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) 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.

  12. Radiation sensitivity of Merkell cell carcinoma cell lines

    International Nuclear Information System (INIS)

    Leonard, J. Helen; Ramsay, Jonathan R.; Kearsley, John H.; Birrell, Geoff W.

    1995-01-01

    Purpose: 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. Methods and Materials: 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) assay was used for these lines, to estimate cell growth after γ 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. Results: 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 γ 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. Conclusion: 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

  13. [Mitochondria inheritance in yeast saccharomyces cerevisiae].

    Science.gov (United States)

    Fizikova, A Iu

    2011-01-01

    The review is devoted to the main mechanisms of mitochondria inheritance in yeast Saccharonmyces cerevisiae. The genetic mechanisms of functionally active mitochondria inheritance in eukaryotic cells is one of the most relevant in modem researches. A great number of genetic diseases are associated with mitochondria dysfunction. Plasticity of eukaryotic cell metabolism according to the environmental changes is ensured by adequate mitochondria functioning by means of ATP synthesis coordination, reactive oxygen species accumulation, apoptosis regulation and is an important factor of cell adaptation to stress. Mitochondria participation in important for cell vitality processes masters the presence of accurate mechanisms of mitochondria functions regulation according to environment fluctuations. The mechanisms of mitochondria division and distribution are highly conserved. Baker yeast S. cerevisiae is an ideal model object for mitochondria researches due to energetic metabolism lability, ability to switch over respiration to fermentation, and petite-positive phenotype. Correction of metabolism according to the environmental changes is necessary for cell vitality. The influence of respiratory, carbon, amino acid and phosphate metabolism on mitochondria functions was shown. As far as the mechanisms that stabilize functions of mitochondria and mtDNA are highly conserve, we can project yeast regularities on higher eukaryotes systems. This makes it possible to approximate understanding the etiology and pathogenesis of a great number of human diseases.

  14. Alamethicin permeabilizes the plasma membrane and mitochondria but not the tonoplast in tobacco (Nicotiana tabacum L. cv Bright Yellow) suspension cells

    DEFF Research Database (Denmark)

    Matic, S.; Geisler, D.A.; Møller, I.M.

    2005-01-01

    remained intact, as indicated by an unaffected tonoplast proton gradient. Low-flux permeabilization of plasma membranes and mitochondria at moderate AlaM concentrations was reversible and did not affect cell vigour. Higher AlaM concentrations induced cell death. After the addition of catalase that removes...... concentrations. Possible uses and limitations of this method for plant cell research are discussed.......The ion channel-forming peptide AlaM (alamethicin) is known to permeabilize isolated mitochondria as well as animal cells. When intact tobacco (Nicotiana tabacum L.) Bright Yellow-2 cells were treated with AlaM, the cells became permeable for low-molecular-mass molecules as shown by induced leakage...

  15. Cadmium induced changes in cell organelles: An ultrastructural study using cadmium sensitive and resistant muntjac fibroblast cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Ord, M.J.; Chibber, R.; Bouffler, S.D.

    1988-09-01

    A detailed electron microscopy study of cadmium sensitive and resistant muntjac fibroblast cell lines has identified a wide range of intracellular damage following exposure to cadmium. Damaged organelles included cell membrane, mitochondria, Golgi cisternae and tubular network, chromatin, nucleoli, microfilaments and ribosomes. Although cell membrane damage was generally the earliest indication of adverse cadmium action, particularly with continuous cadmium exposures, cells could tolerate extensive membrane loss. Mitochondrial distortion and some damage to Golgi was also tolerated. The turning point at which cadmium became lethal was generally marked by a cascade of events which included damage to both nuclear and cytoplasmic components. These results for fibroblasts are discussed and compared with damage reported in other types of cells.

  16. Nanoparticle Delivery of Artesunate Enhances the Anti-tumor Efficiency by Activating Mitochondria-Mediated Cell Apoptosis

    Science.gov (United States)

    Liu, Rui; Yu, Xiwei; Su, Chang; Shi, Yijie; Zhao, Liang

    2017-06-01

    Artemisinin and its derivatives were considered to exert a broad spectrum of anti-cancer activities, and they induced significant anti-cancer effects in tumor cells. Artemisinin and its derivatives could be absorbed quickly, and they were widely distributed, selectively killing tumor cells. Since low concentrations of artesunate primarily depended on oncosis to induce cell death in tumor cells, its anti-tumor effects were undesirable and limited. To obtain better anti-tumor effects, in this study, we took advantage of a new nanotechnology to design novel artesunate-loaded bovine serum albumin nanoparticles to achieve the mitochondrial accumulation of artesunate and induce mitochondrial-mediated apoptosis. The results showed that when compared with free artesunate's reliance on oncotic death, artesunate-loaded bovine serum albumin nanoparticles showed higher cytotoxicity and their significant apoptotic effects were induced through the distribution of artesunate in the mitochondria. This finding indicated that artesunate-loaded bovine serum albumin nanoparticles damaged the mitochondrial integrity and activated mitochondrial-mediated cell apoptosis by upregulating apoptosis-related proteins and facilitating the rapid release of cytochrome C.

  17. Tissue kinetics, ion transport, and recruitment of mitochondria-rich cells in the skin of the toad (Bufo bufo) in response to exposure to distilled water

    DEFF Research Database (Denmark)

    Budtz, Poul Egede; Christoffersen, Betina C.; Johansen, Jesper S.

    1995-01-01

    Mitochondria-rich cells (MRC) of the amphibian epidermis are responsible for active chloride uptake at low external salinity, and new MRCs are recruited in response to exposure to distilled (deionized) water. The time-course of this recruitment, the tissue kinetics and ion transport have been...

  18. A universal mammalian vaccine cell line substrate.

    Directory of Open Access Journals (Sweden)

    Jackelyn Murray

    Full Text Available Using genome-wide small interfering RNA (siRNA screens for poliovirus, influenza A virus and rotavirus, we validated the top 6 gene hits PV, RV or IAV to search for host genes that when knocked-down (KD enhanced virus permissiveness and replication over wild type Vero cells or HEp-2 cells. The enhanced virus replication was tested for 12 viruses and ranged from 2-fold to >1000-fold. There were variations in virus-specific replication (strain differences across the cell lines examined. Some host genes (CNTD2, COQ9, GCGR, NDUFA9, NEU2, PYCR1, SEC16G, SVOPL, ZFYVE9, and ZNF205 showed that KD resulted in enhanced virus replication. These findings advance platform-enabling vaccine technology, the creation of diagnostic cells substrates, and are informative about the host mechanisms that affect virus replication in mammalian cells.

  19. Immunolocation of mitochondria-rich cells in epidermis of the common toad, Bufo bufo L

    DEFF Research Database (Denmark)

    Spies, Ingrid B.M.

    1997-01-01

    Adult toads, skin, flask-shaped cells, cytoskeleton, keratin expression, immunohistochemistry, differentiation.......Adult toads, skin, flask-shaped cells, cytoskeleton, keratin expression, immunohistochemistry, differentiation....

  20. Induction of Apoptosis in Human Multiple Myeloma Cell Lines by Ebselen via Enhancing the Endogenous Reactive Oxygen Species Production

    Directory of Open Access Journals (Sweden)

    Liang Zhang

    2014-01-01

    Full Text Available Ebselen a selenoorganic compound showing glutathione peroxidase like activity is an anti-inflammatory and antioxidative agent. Its cytoprotective activity has been investigated in recent years. However, experimental evidence also shows that ebselen causes cell death in several cancer cell types whose mechanism has not yet been elucidated. In this study, we examined the effect of ebselen on multiple myeloma (MM cell lines in vitro. The results showed that ebselen significantly enhanced the production of reactive oxygen species (ROS accompanied by cell viability decrease and apoptosis rate increase. Further studies revealed that ebselen can induce Bax redistribution from the cytosol to mitochondria leading to mitochondrial membrane potential ΔΨm changes and cytochrome C release from the mitochondria to cytosol. Furtherly, we found that exogenous addition of N-acetyl cysteine (NAC completely diminished the cell damage induced by ebselen. This result suggests that relatively high concentration of ebselen can induce MM cells apoptosis in culture by enhancing the production of endogenous ROS and triggering mitochondria mediated apoptotic pathway.

  1. Induction of apoptosis in human multiple myeloma cell lines by ebselen via enhancing the endogenous reactive oxygen species production.

    Science.gov (United States)

    Zhang, Liang; Zhou, Liwei; Du, Jia; Li, Mengxia; Qian, Chengyuan; Cheng, Yi; Peng, Yang; Xie, Jiayin; Wang, Dong

    2014-01-01

    Ebselen a selenoorganic compound showing glutathione peroxidase like activity is an anti-inflammatory and antioxidative agent. Its cytoprotective activity has been investigated in recent years. However, experimental evidence also shows that ebselen causes cell death in several cancer cell types whose mechanism has not yet been elucidated. In this study, we examined the effect of ebselen on multiple myeloma (MM) cell lines in vitro. The results showed that ebselen significantly enhanced the production of reactive oxygen species (ROS) accompanied by cell viability decrease and apoptosis rate increase. Further studies revealed that ebselen can induce Bax redistribution from the cytosol to mitochondria leading to mitochondrial membrane potential ΔΨm changes and cytochrome C release from the mitochondria to cytosol. Furtherly, we found that exogenous addition of N-acetyl cysteine (NAC) completely diminished the cell damage induced by ebselen. This result suggests that relatively high concentration of ebselen can induce MM cells apoptosis in culture by enhancing the production of endogenous ROS and triggering mitochondria mediated apoptotic pathway.

  2. Effects of the antitumoural dequalinium on NB4 and K562 human leukemia cell lines. Mitochondrial implication in cell death.

    Science.gov (United States)

    Galeano, Eva; Nieto, Elena; García-Pérez, Ana Isabel; Delgado, M Dolores; Pinilla, Montserrat; Sancho, Pilar

    2005-10-01

    Dequalinium (DQA) is a delocalized lipophylic cation that selectively targets the mitochondria of carcinoma cells. However, the underlying mechanisms of DQA action are not yet well understood. We have studied the effects of DQA on two different leukemia cell lines: NB4, derived from acute promyelocytic leukemia, and K562, derived from chronic myeloid leukemia. We found that DQA displays differential cytotoxic activity in these cell lines. In NB4 cells, a low DQA concentration (2microM) induces a mixture of apoptosis and necrosis, whereas a high DQA concentration (20microM) induces mainly necrosis. However, K562 cell death was always by necrosis as the cells showed a resistance to apoptosis at all time-periods and DQA concentrations assayed. In both cell lines, the cell death seems to be mediated by alterations of mitochondrial function as evidenced by loss of mitochondrial transmembrane potential, O2*- accumulation and ATP depletion. The current study improves the knowledge on DQA as a novel anticancer agent with a potential application in human acute promyelocytic leukemia chemotherapy.

  3. Interaction theory of mammalian mitochondria.

    Science.gov (United States)

    Nakada, K; Inoue, K; Hayashi, J

    2001-11-09

    We generated mice with deletion mutant mtDNA by its introduction from somatic cells into mouse zygotes. Expressions of disease phenotypes are limited to tissues expressing mitochondrial dysfunction. Considering that all these mice share the same nuclear background, these observations suggest that accumulation of the mutant mtDNA and resultant expressions of mitochondrial dysfunction are responsible for expression of disease phenotypes. On the other hand, mitochondrial dysfunction and expression of clinical abnormalities were not observed until the mutant mtDNA accumulated predominantly. This protection is due to the presence of extensive and continuous interaction between exogenous mitochondria from cybrids and recipient mitochondria from embryos. Thus, we would like to propose a new hypothesis on mitochondrial biogenesis, interaction theory of mitochondria: mammalian mitochondria exchange genetic contents, and thus lost the individuality and function as a single dynamic cellular unit. Copyright 2001 Academic Press.

  4. Cellular radiosensitivity of small-cell lung cancer cell lines

    DEFF Research Database (Denmark)

    Krarup, M; Poulsen, H S; Spang-Thomsen, M

    1997-01-01

    PURPOSE: The objective of this study was to determine the radiobiological characteristics of a panel of small-cell lung cancer (SCLC) cell lines by use of a clonogenic assay. In addition, we tested whether comparable results could be obtained by employing a growth extrapolation method based...

  5. Antioxidant mechanism of mitochondria-targeted plastoquinone SkQ1 is suppressed in aglycemic HepG2 cells dependent on oxidative phosphorylation

    Czech Academy of Sciences Publication Activity Database

    Ježek, J.; Engstová, Hana; Ježek, Petr

    2017-01-01

    Roč. 1858, č. 9 (2017), s. 750-762 ISSN 0005-2728 R&D Projects: GA ČR(CZ) GA17-01813S Institutional support: RVO:67985823 Keywords : mitochondria-targeted antioxidant SkQ1 * mitochondrial Complex I superoxide formation * mitochondrial Complex III superoxide formation * HepG2 cell s * NAD(P)H fluorescence lifetime imaging microscopy Subject RIV: ED - Physiology OBOR OECD: Cell biology Impact factor: 4.932, year: 2016

  6. Cytotoxic effects and apoptosis induction of enrofloxacin in hepatic cell line of grass carp (Ctenopharyngodon idellus).

    Science.gov (United States)

    Liu, Bo; Cui, Yanting; Brown, Paul B; Ge, Xianping; Xie, Jun; Xu, Pao

    2015-12-01

    We determined the effect of enrofloxacin on the lactate dehydrogenase (LDH) release, reactive oxygen species (ROS), superoxide dismutase (SOD), total antioxidant capacity (T-AOC), malondialdehyde (MDA), mitochondria membrane potential (ΔΨm) and apoptosis in the hepatic cell line of grass carp (Ctenopharyngodon idellus). Cultured cells were treated with different concentrations of enrofloxacin (12.5-200 ug/mL) for 24 h. We found that the cytotoxic effect of enrofloxacin was mediated by apoptosis, and that this apoptosis occurred in a dose-dependent manner. The doses of 50,100 and 200 μg/mL enrofloxacin increased the LDH release and MDA concentration, induced cell apoptosis and reduced the ΔΨm compared to the control. The highest dose of 200 ug/mL enrofloxacin also significantly induced apoptosis accompanied by ΔΨm disruption and ROS generation and significantly reduced T-AOC and increased MDA concentration compared to the control. Our results suggest that the dose of 200 ug/mL enrofloxacin exerts its cytotoxic effect and produced ROS via apoptosis by affecting the mitochondria of the hepatic cells of grass carp. Copyright © 2015 Elsevier Ltd. All rights reserved.

  7. PDE2A2 regulates mitochondria morphology and apoptotic cell death via local modulation of cAMP/PKA signalling.

    Science.gov (United States)

    Monterisi, Stefania; Lobo, Miguel J; Livie, Craig; Castle, John C; Weinberger, Michael; Baillie, George; Surdo, Nicoletta C; Musheshe, Nshunge; Stangherlin, Alessandra; Gottlieb, Eyal; Maizels, Rory; Bortolozzi, Mario; Micaroni, Massimo; Zaccolo, Manuela

    2017-05-02

    cAMP/PKA signalling is compartmentalised with tight spatial and temporal control of signal propagation underpinning specificity of response. The cAMP-degrading enzymes, phosphodiesterases (PDEs), localise to specific subcellular domains within which they control local cAMP levels and are key regulators of signal compartmentalisation. Several components of the cAMP/PKA cascade are located to different mitochondrial sub-compartments, suggesting the presence of multiple cAMP/PKA signalling domains within the organelle. The function and regulation of these domains remain largely unknown. Here, we describe a novel cAMP/PKA signalling domain localised at mitochondrial membranes and regulated by PDE2A2. Using pharmacological and genetic approaches combined with real-time FRET imaging and high resolution microscopy, we demonstrate that in rat cardiac myocytes and other cell types mitochondrial PDE2A2 regulates local cAMP levels and PKA-dependent phosphorylation of Drp1. We further demonstrate that inhibition of PDE2A, by enhancing the hormone-dependent cAMP response locally, affects mitochondria dynamics and protects from apoptotic cell death.

  8. Monoamine oxidase-dependent endoplasmic reticulum-mitochondria dysfunction and mast cell degranulation lead to adverse cardiac remodeling in diabetes.

    Science.gov (United States)

    Deshwal, Soni; Forkink, Marleen; Hu, Chou-Hui; Buonincontri, Guido; Antonucci, Salvatore; Di Sante, Moises; Murphy, Michael P; Paolocci, Nazareno; Mochly-Rosen, Daria; Krieg, Thomas; Di Lisa, Fabio; Kaludercic, Nina

    2018-02-19

    Monoamine oxidase (MAO) inhibitors ameliorate contractile function in diabetic animals, but the mechanisms remain unknown. Equally elusive is the interplay between the cardiomyocyte alterations induced by hyperglycemia and the accompanying inflammation. Here we show that exposure of primary cardiomyocytes to high glucose and pro-inflammatory stimuli leads to MAO-dependent increase in reactive oxygen species that causes permeability transition pore opening and mitochondrial dysfunction. These events occur upstream of endoplasmic reticulum (ER) stress and are abolished by the MAO inhibitor pargyline, highlighting the role of these flavoenzymes in the ER/mitochondria cross-talk. In vivo, streptozotocin administration to mice induced oxidative changes and ER stress in the heart, events that were abolished by pargyline. Moreover, MAO inhibition prevented both mast cell degranulation and altered collagen deposition, thereby normalizing diastolic function. Taken together, these results elucidate the mechanisms underlying MAO-induced damage in diabetic cardiomyopathy and provide novel evidence for the role of MAOs in inflammation and inter-organelle communication. MAO inhibitors may be considered as a therapeutic option for diabetic complications as well as for other disorders in which mast cell degranulation is a dominant phenomenon.

  9. Salvianic acid A sodium protects HUVEC cells against tert-butyl hydroperoxide induced oxidative injury via mitochondria-dependent pathway.

    Science.gov (United States)

    Jia, Dan; Li, Tian; Chen, Xiaofei; Ding, Xuan; Chai, Yifeng; Chen, Alex F; Zhu, Zhenyu; Zhang, Chuan

    2018-01-05

    Salvianic acid A (Danshensu) is a major water-soluble component extracted from Salvia miltiorrhiza (Danshen), which has been widely used in clinic in China for treatment of cardiovascular diseases (CVDs). This study aimed to investigate the protective effects of salvianic acid A sodium (SAAS) against tert-butyl hydroperoxide (t-BHP) induced human umbilical vein endothelial cell (HUVEC) oxidative injury and the underlying molecular mechanisms. In the antioxidant activity-assessing model, SAAS pretreatment significantly ameliorated the cell growth inhibition and apoptosis induced by t-BHP. An ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) based-metabolic profiling was developed to investigate the metabolic changes of HUVEC cells in response to t-BHP and SAAS. The results revealed that t-BHP injury upregulated 13 metabolites mainly involved in tryptophan metabolism and phenylalanine metabolism which were highly correlated with mitochondrial function and oxidative stress, and 50 μM SAAS pretreatment effectively reversed these metabolic changes. Further biomedical research indicated that SAAS pretreatment reduced the t-BHP induced increase of lactate dehydrogenase (LDH), intracellular reactive oxygen species (ROS), malondialdehyde (MDA) and mitochondrial membrane potential (MMP), and the decrease of key antioxidant enzymes through mitochondria antioxidative pathways via JAK2/STAT3 and PI3K/Akt/GSK-3β signalings. Taken together, our results suggested that SAAS may protect HUVEC cells against t-BHP induced oxidative injury via mitochondrial antioxidative defense system. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Diversity in host clone performance within a Chinese hamster ovary cell line.

    Science.gov (United States)

    O'Callaghan, Peter M; Berthelot, Maud E; Young, Robert J; Graham, James W A; Racher, Andrew J; Aldana, Dulce

    2015-01-01

    Much effort has been expended to improve the capabilities of individual Chinese hamster ovary (CHO) host cell lines to synthesize recombinant therapeutic proteins (rPs). However, given the increasing variety in rP molecular types and formats it may be advantageous to employ a toolbox of CHO host cell lines in biomanufacturing. Such a toolbox would contain a panel of hosts with specific capabilities to synthesize certain molecular types at high volumetric concentrations and with the correct product quality (PQ). In this work, we examine a panel of clonally derived host cell lines isolated from CHOK1SV for the ability to manufacture two model proteins, an IgG4 monoclonal antibody (Mab) and an Fc-fusion protein (etanercept). We show that these host cell lines vary in their relative ability to synthesize these proteins in transient and stable pool production format. Furthermore, we examined the PQ attributes of the stable pool-produced Mab and etanercept (by N-glycan ultra performance liquid chromatography (UPLC) and liquid chromatography - tandem mass spectrometry (LC-MS/MS), respectively), and uncovered substantial variation between the host cell lines in Mab N-glycan micro-heterogeneity and etanercept N and O-linked macro-heterogeneity. To further investigate the capabilities of these hosts to act as cell factories, we examined the glycosylation pathway gene expression profiles as well as the levels of endoplasmic reticulum (ER) and mitochondria in the untransfected hosts. We uncovered a moderate correlation between ER mass and the volumetric product concentration in transient and stable pool Mab production. This work demonstrates the utility of leveraging diversity within the CHOK1SV pool to identify new host cell lines with different performance characteristics. © 2015 American Institute of Chemical Engineers.

  11. [Linezolid-induced Apoptosis through Mitochondrial Damage and Role of Superoxide Dismutase-1 in Human Monocytic Cell Line U937].

    Science.gov (United States)

    Fujii, Satoshi; Muraoka, Sanae; Miyamoto, Atsushi; Sakurai, Koichi

    2018-01-01

     Cytopenia is a major adverse event associated with linezolid therapy. The objective of this study was to examine whether the cytotoxicity of linezolid to eukaryotic cells was associated with mitochondrial dysfunction and apoptosis-like cell death in human leukemic monocyte lymphoma cell line U937. Apoptosis-like cell death was clearly observed when cells were incubated with linezolid, depending on the duration and linezolid concentration. Mitochondrial membrane potential of cells treated with linezolid collapsed in a short period of time, but the number of mitochondria did not decrease. Cytotoxicity of linezolid was relieved by the knockdown of superoxide dismutase-1 in U937 cells. On the other hand, no autophagy was observed in cells treated with linezolid. These results suggest that mitochondrial damages would be linked to the induction of apoptosis in U937 cells treated with linezolid and that its mechanism does not involve autophagy.

  12. Targeting mitochondria by Zn(II)N-alkylpyridylporphyrins: the impact of compound sub-mitochondrial partition on cell respiration and overall photodynamic efficacy.

    Science.gov (United States)

    Odeh, Ahmad M; Craik, James D; Ezzeddine, Rima; Tovmasyan, Artak; Batinic-Haberle, Ines; Benov, Ludmil T

    2014-01-01

    Mitochondria play a key role in aerobic ATP production and redox control. They harness crucial metabolic pathways and control cell death mechanisms, properties that make these organelles essential for survival of most eukaryotic cells. Cancer cells have altered cell death pathways and typically show a shift towards anaerobic glycolysis for energy production, factors which point to mitochondria as potential culprits in cancer development. Targeting mitochondria is an attractive approach to tumor control, but design of pharmaceutical agents based on rational approaches is still not well established. The aim of this study was to investigate which structural features of specially designed Zn(II)N-alkylpyridylporphyrins would direct them to mitochondria and to particular mitochondrial targets. Since Zn(II)N-alkylpyridylporphyrins can act as highly efficient photosensitizers, their localization can be confirmed by photodamage to particular mitochondrial components. Using cultured LS174T adenocarcinoma cells, we found that subcellular distribution of Zn-porphyrins is directed by the nature of the substituents attached to the meso pyridyl nitrogens at the porphyrin ring. Increasing the length of the aliphatic chain from one carbon (methyl) to six carbons (hexyl) increased mitochondrial uptake of the compounds. Such modifications also affected sub-mitochondrial distribution of the Zn-porphyrins. The amphiphilic hexyl derivative (ZnTnHex-2-PyP) localized in the vicinity of cytochrome c oxidase complex, causing its inactivation during illumination. Photoinactivation of critical cellular targets explains the superior efficiency of the hexyl derivative in causing mitochondrial photodamage, and suppressing cellular respiration and survival. Design of potent photosensitizers and redox-active scavengers of free radicals should take into consideration not only selective organelle uptake and localization, but also selective targeting of critical macromolecular structures.

  13. Mitochondria as determinant of nucleotide pools and chromosomal stability

    DEFF Research Database (Denmark)

    Madsen, Claus Desler; Munch-Petersen, Birgitte; Stevnsner, Tinna

    2007-01-01

    Mitochondrial function plays an important role in multiple human diseases and mutations in the mitochondrial genome have been detected in nearly every type of cancer investigated to date. However, the mechanism underlying the interrelation is unknown. We used human cell lines depleted of mitochon...... mitochondrial activity. Our results suggest that mitochondria are central players in maintaining genomic stability and in controlling essential nuclear processes such as upholding a balanced supply of nucleotides....

  14. Analysis of cell line variation in biochemical production of protoporphyrin IX

    Science.gov (United States)

    Gibbs, Summer L.; Chen, Bin; O'Hara, Julia A.; Hoopes, P. Jack; Hasan, Tayyaba; Pogue, Brian W.

    2006-02-01

    Protoporphyrin IX (PpIX) is produced via the heme synthesis pathway by the cell following administration of aminolevulinic acid (ALA). ALA synthase, the enzyme that produces ALA in the cell from glycine and succinyl-coenzyme A, is inhibited in a feedback mechanism by heme and thus is the rate limiting enzyme in the heme synthesis pathway. Since ALA is administered systemically, the rate limiting step that naturally exists in the cells is bypassed, however it is currently unclear why cells have different rate limiting steps in the ALA-PpIX synthesis pathway, and more specifically which types of cancer cells are most productive. It has been determined that when the same amount of ALA is administered to a wide panel of cancer cells in vitro that vastly differing amounts of PpIX are produced. The steps for the ALA-PpIX pathway occur in and around the mitochondria of the cell, but interestingly no correlation is seen between PpIX production and mitochondrial content of the cell, following ALA administration. However, total cell area shows positive correlation with PpIX production. Administration of the iron chelator, 1,2-dimethyl-3-hydroxy-4-pyridone (L1) in combination with ALA allows the final step in the heme synthesis pathway, conversion of PpIX to heme, to be delayed and thus increases the detectable amount of PpIX in each cell line. The cell lines that have the lowest PpIX production following administration of ALA alone show the largest increase in production following the combined administration of ALA and L1. PpIX fluorescence is thought to be a measure of cellular activity and the goal of the current study was to determine which cell lines would be the most promising targets for fluorescence detection or monitoring response to therapy. The results indicate that the cells with larger size and larger numbers of mitochondria may be good potential targets for this therapy. While this conclusion may appear obvious, it is not universally true, and cellular specific

  15. Morphological and quantitative changes in mitochondria, plastids, and peroxisomes during the log-to-stationary transition of the growth phase in cultured tobacco BY-2 cells.

    Science.gov (United States)

    Toyooka, Kiminori; Sato, Mayuko; Wakazaki, Mayumi; Matsuoka, Ken

    2016-01-01

    We developed a wide-range and high-resolution transmission electron microscope acquisition system and obtained giga-pixel images of tobacco BY-2 cells during the log and stationary phases of cell growth. We demonstrated that the distribution and ultrastructure of compartments involved in membrane traffic (i.e., Golgi apparatus, multivesicular body, and vesicle cluster) change during the log-to-stationary transition. Mitochondria, peroxisomes, and plastids were also enumerated. Electron densities of mitochondria and peroxisomes were altered during the growth-phase shift, while their numbers were reduced by nearly half. Plastid structure dramatically changed from atypical to spherical with starch granules. Nearly the same number of plastids was observed in both log and stationary phases. These results indicate that mechanisms regulating organelle populations differ from organelle to organelle.

  16. Glutathione and Mitochondria

    Directory of Open Access Journals (Sweden)

    Vicent eRibas

    2014-07-01

    Full Text Available Glutathione (GSH is the main nonprotein thiol in cells whose functions are dependent on the redox-active thiol of its cysteine moiety that serves as a cofactor for a number of antioxidant and detoxifying enzymes. While synthesized exclusively in the cytosol from its constituent amino acids, GSH is distributed in different compartments, including mitochondria where its concentration in the matrix equals that of the cytosol. This feature and its negative charge at physiological pH imply the existence of specific carriers to import GSH from the cytosol to the mitochondrial matrix, where it plays a key role in defense against respiration-induced reactive oxygen species and in the detoxification of lipid hydroperoxides and electrophiles. Moreover, as mitochondria play a central strategic role in the activation and mode of cell death, mitochondrial GSH has been shown to critically regulate the level of sensitization to secondary hits that induce mitochondrial membrane permeabilization and release of proteins confined in the intermembrane space that once in the cytosol engage the molecular machinery of cell death. In this review, we summarize recent data on the regulation of mitochondrial GSH and its role in cell death and prevalent human diseases, such as cancer, fatty liver disease and Alzheimer’s disease.

  17. Fisetin inhibits cellular proliferation and induces mitochondria-dependent apoptosis in human gastric cancer cells.

    Science.gov (United States)

    Sabarwal, Akash; Agarwal, Rajesh; Singh, Rana P

    2017-02-01

    The anticancer effects of fisetin, a dietary agent, are largely unknown against human gastric cancer. Herein, we investigated the mechanisms of fisetin-induced inhibition of growth and survival of human gastric carcinoma AGS and SNU-1 cells. Fisetin (25-100 μM) caused significant decrease in the levels of G1 phase cyclins and CDKs, and increased the levels of p53 and its S15 phosphorylation in gastric cancer cells. We also observed that growth suppression and death of non-neoplastic human intestinal FHs74int cells were minimally affected by fisetin. Fisetin strongly increased apoptotic cells and showed mitochondrial membrane depolarization in gastric cancer cells. DNA damage was observed as early as 3 h after fisetin treatment which was accompanied with gamma-H2A.X(S139) phosphorylation and cleavage of PARP. Fisetin-induced apoptosis was observed to be independent of p53. DCFDA and MitoSOX analyses showed an increase in mitochondrial ROS generation in time- and dose-dependent fashion. It also increased cellular nitrite and superoxide generation. Pre-treatment with N-acetyl cysteine (NAC) inhibited ROS generation and also caused protection from fisetin-induced DNA damage. The formation of comets were observed in only fisetin treated cells which was blocked by NAC pre-treatment. Further investigation of the source of ROS, using mitochondrial respiratory chain (MRC) complex inhibitors, suggested that fisetin caused ROS generation specifically through complex I. Collectively, these results for the first time demonstrated that fisetin possesses anticancer potential through ROS production most likely via MRC complex I leading to apoptosis in human gastric carcinoma cells. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  18. Nano Copper Induces Apoptosis in PK-15 Cells via a Mitochondria-Mediated Pathway.

    Science.gov (United States)

    Zhang, Hui; Chang, Zhenyu; Mehmood, Khalid; Abbas, Rao Zahid; Nabi, Fazul; Rehman, Mujeeb Ur; Wu, Xiaoxing; Tian, Xinxin; Yuan, Xiaodan; Li, Zhaoyang; Zhou, Donghai

    2018-01-01

    Nano-sized copper particles are widely used in various chemical, physical, and biological fields. However, earlier studies have shown that nano copper particles (40-100 μg/mL) can induce cell toxicity and apoptosis. Therefore, this study was conducted to investigate the role of nano copper in mitochondrion-mediated apoptosis in PK-15 cells. The cells were treated with different doses of nano copper (20, 40, 60, and 80 μg/mL) to determine the effects of apoptosis using acridine orange/ethidium bromide (AO/EB) fluorescence staining and a flow cytometry assay. The levels of malondialdehyde (MDA) and superoxide dismutase (SOD) in the PK-15 cells were examined using commercially available kits. Moreover, the mRNA levels of the Bax, Bid, Caspase-3, and CYCS genes were assessed by real-time PCR. The results revealed that nano copper exposure induced apoptosis and changed the mitochondrial membrane potential. In addition, nano copper significantly altered the levels of the Bax, Bid, Caspase-3, and CYCS genes at a concentration of 40 μg/mL. To summarize, nano copper significantly (P nano copper can play an important role in inducing the apoptotic pathway in PK-15 cells, which may be the mechanism by which nano copper induces nephrotoxicity.

  19. Menadione inhibits MIBG uptake in two neuroendocrine cell lines

    NARCIS (Netherlands)

    Cornelissen, J.; Tytgat, G. A.; van den Brug, M.; van Kuilenburg, A. B.; Voûte, P. A.; van Gennip, A. H.

    1997-01-01

    In this paper we report on our studies of the effect of menadione on the uptake of MIBG in the neuroendocrine cell lines PC12 and SK-N-SH. Menadione inhibits the uptake of MIBG in both cell lines in a dose-dependent manner. Inhibition of MIBG uptake is most pronounced in the PC12 cell line.

  20. A non-toxic fluorogenic dye for mitochondria labeling.

    Science.gov (United States)

    Han, Junyan; Han, Myung Shin; Tung, Ching-Hsuan

    2013-11-01

    Mitochondria, powerhouses of cells, are responsible for many critical cellular functions, such as cell energy metabolism, reactive oxygen species production, and apoptosis regulation. Monitoring mitochondria morphology in live cells temporally and spatially could help with the understanding of the mechanisms of mitochondrial functional regulation and the pathogenesis of mitochondria-related diseases. A novel non-cytotoxic fluorogenic compound, AcQCy7, was developed as a mitochondria-specific dye. AcQCy7 emitted no fluorescent signal outside of cells, but it became fluorescent after intracellular hydrolysis of the acetyl group. The hydrolyzed fluorescent product was well retained in mitochondria, enabling long-lasting fluorescence imaging of mitochondria without cell washing. A 2-day culture study using AcQCy7 showed no sign of cytotoxicity, whereas a commonly used mitochondria-staining probe, Mitochondria Tracker Green, caused significant cell death even at a much lower concentration. Apoptosis-causing mitochondria fission was monitored clearly in real time by AcQCy7. A simple add-and-read mitochondria specific dye AcQCy7 has been validated in various cell models. Bright mitochondria specific fluorescent signal in treated cells lasted several days without noticeable toxicity. The probe AcQCy7 has been proofed to be a non-toxic agent for long-term mitochondria imaging. © 2013.

  1. Fe-S Cluster Biogenesis in Isolated Mammalian Mitochondria

    Science.gov (United States)

    Pandey, Alok; Pain, Jayashree; Ghosh, Arnab K.; Dancis, Andrew; Pain, Debkumar

    2015-01-01

    Iron-sulfur (Fe-S) clusters are essential cofactors, and mitochondria contain several Fe-S proteins, including the [4Fe-4S] protein aconitase and the [2Fe-2S] protein ferredoxin. Fe-S cluster assembly of these proteins occurs within mitochondria. Although considerable data exist for yeast mitochondria, this biosynthetic process has never been directly demonstrated in mammalian mitochondria. Using [35S]cysteine as the source of sulfur, here we show that mitochondria isolated from Cath.A-derived cells, a murine neuronal cell line, can synthesize and insert new Fe-35S clusters into aconitase and ferredoxins. The process requires GTP, NADH, ATP, and iron, and hydrolysis of both GTP and ATP is necessary. Importantly, we have identified the 35S-labeled persulfide on the NFS1 cysteine desulfurase as a genuine intermediate en route to Fe-S cluster synthesis. In physiological settings, the persulfide sulfur is released from NFS1 and transferred to a scaffold protein, where it combines with iron to form an Fe-S cluster intermediate. We found that the release of persulfide sulfur from NFS1 requires iron, showing that the use of iron and sulfur for the synthesis of Fe-S cluster intermediates is a highly coordinated process. The release of persulfide sulfur also requires GTP and NADH, probably mediated by a GTPase and a reductase, respectively. ATP, a cofactor for a multifunctional Hsp70 chaperone, is not required at this step. The experimental system described here may help to define the biochemical basis of diseases that are associated with impaired Fe-S cluster biogenesis in mitochondria, such as Friedreich ataxia. PMID:25398879

  2. Cancer cell metabolism and mitochondria: Nutrient plasticity for TCA cycle fueling.

    Science.gov (United States)

    Corbet, Cyril; Feron, Olivier

    2017-08-01

    Warburg's hypothesis that cancer cells take up a lot of glucose in the presence of ambient oxygen but convert pyruvate into lactate due to impaired mitochondrial function led to the misconception that cancer cells rely on glycolysis as their major source of energy. Most recent 13 C-based metabolomic studies, including in cancer patients, indicate that cancer cells may also fully oxidize glucose. In addition to glucose-derived pyruvate, lactate, fatty acids and amino acids supply substrates to the TCA cycle to sustain mitochondrial metabolism. Here, we discuss how the metabolic flexibility afforded by these multiple mitochondrial inputs allows cancer cells to adapt according to the availability of the different fuels and the microenvironmental conditions such as hypoxia and acidosis. In particular, we focused on the role of the TCA cycle in interconnecting numerous metabolic routes in order to highlight metabolic vulnerabilities that represent attractive targets for a new generation of anticancer drugs. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Horizontal transfer of mitochondria between mammalian cells: beyond co-culture approaches

    Czech Academy of Sciences Publication Activity Database

    Berridge, M.V.; McConnell, M. J.; Grasso, C.; Bajziková, Martina; Kovářová, Jaromíra; Neužil, Jiří

    2016-01-01

    Roč. 38, June (2016), s. 75-82 ISSN 0959-437X R&D Projects: GA ČR(CZ) GAP301/10/1937; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:86652036 Keywords : MESENCHYMAL STEM-CELLS * GENE-TRANSFER * SUPPORTING ASPARTATE BIOSYNTHESIS Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.825, year: 2016

  4. Role of mitochondria in modulation of spontaneous Ca2+ waves in freshly dispersed interstitial cells of Cajal from the rabbit urethra.

    Science.gov (United States)

    Sergeant, Gerard P; Bradley, Eamonn; Thornbury, Keith D; McHale, Noel G; Hollywood, Mark A

    2008-10-01

    Interstitial cells of Cajal (ICC) isolated from the rabbit urethra exhibit pacemaker activity that results from spontaneous Ca(2+) waves. The purpose of this study was to investigate if this activity was influenced by Ca(2+) uptake into mitochondria. Spontaneous Ca(2+) waves were recorded using a Nipkow spinning disk confocal microscope and spontaneous transient inward currents (STICs) were recorded using the whole-cell patch clamp technique. Disruption of the mitochondrial membrane potential with the electron transport chain inhibitors rotenone (10 microm) and antimycin A (5 microm) abolished Ca(2+) waves and increased basal Ca(2+) levels. Similar results were achieved when mitochondria membrane potential was collapsed using the protonophores FCCP (0.2 microm) and CCCP (1 microm). Spontaneous Ca(2+) waves were not inhibited by the ATP synthase inhibitor oligomycin (1 microm), suggesting that these effects were not attributable to an effect on ATP levels. STICs recorded under voltage clamp at -60 mV were also inhibited by CCCP and antimycin A. Dialysis of cells with the mitochondrial uniporter inhibitor RU360 (10 microm) also inhibited STICS. Stimulation of Ca(2+) uptake into mitochondria using the plant flavonoid kaempferol (10 microm) induced a series of propagating Ca(2+) waves. The kaempferol-induced activity was inhibited by application of caffeine (10 mm) or removal of extracellular Ca(2+), but was not significantly affected by the IP(3) receptor blocker 2-APB (100 microm). These data suggest that spontaneous Ca(2+) waves in urethral ICC are regulated by buffering of cytoplasmic Ca(2+) by mitochondria.

  5. Polycyclic aromatic hydrocarbon components contribute to the mitochondria-antiapoptotic effect of fine particulate matter on human bronchial epithelial cells via the aryl hydrocarbon receptor

    Directory of Open Access Journals (Sweden)

    Baeza-Squiban Armelle

    2010-07-01

    Full Text Available Abstract Background Nowadays, effects of fine particulate matter (PM2.5 are well-documented and related to oxidative stress and pro-inflammatory response. Nevertheless, epidemiological studies show that PM2.5 exposure is correlated with an increase of pulmonary cancers and the remodeling of the airway epithelium involving the regulation of cell death processes. Here, we investigated the components of Parisian PM2.5 involved in either the induction or the inhibition of cell death quantified by different parameters of apoptosis and delineated the mechanism underlying this effect. Results In this study, we showed that low levels of Parisian PM2.5 are not cytotoxic for three different cell lines and primary cultures of human bronchial epithelial cells. Conversely, a 4 hour-pretreatment with PM2.5 prevent mitochondria-driven apoptosis triggered by broad spectrum inducers (A23187, staurosporine and oligomycin by reducing the mitochondrial transmembrane potential loss, the subsequent ROS production, phosphatidylserine externalization, plasma membrane permeabilization and typical morphological outcomes (cell size decrease, massive chromatin and nuclear condensation, formation of apoptotic bodies. The use of recombinant EGF and specific inhibitor led us to rule out the involvement of the classical EGFR signaling pathway as well as the proinflammatory cytokines secretion. Experiments performed with different compounds of PM2.5 suggest that endotoxins as well as carbon black do not participate to the antiapoptotic effect of PM2.5. Instead, the water-soluble fraction, washed particles and organic compounds such as polycyclic aromatic hydrocarbons (PAH could mimic this antiapoptotic activity. Finally, the activation or silencing of the aryl hydrocarbon receptor (AhR showed that it is involved into the molecular mechanism of the antiapoptotic effect of PM2.5 at the mitochondrial checkpoint of apoptosis. Conclusions The PM2.5-antiapoptotic effect in addition

  6. Mechanistic studies of cancer cell mitochondria- and NQO1-mediated redox activation of beta-lapachone, a potentially novel anticancer agent

    International Nuclear Information System (INIS)

    Li, Jason Z.; Ke, Yuebin; Misra, Hara P.; Trush, Michael A.; Li, Y. Robert; Zhu, Hong; Jia, Zhenquan

    2014-01-01

    Beta-lapachone (beta-Lp) derived from the Lapacho tree is a potentially novel anticancer agent currently under clinical trials. Previous studies suggested that redox activation of beta-Lp catalyzed by NAD(P)H:quinone oxidoreductase 1 (NQO1) accounted for its killing of cancer cells. However, the exact mechanisms of this effect remain largely unknown. Using chemiluminescence and electron paramagnetic resonance (EPR) spin-trapping techniques, this study for the first time demonstrated the real-time formation of ROS in the redox activation of beta-lapachone from cancer cells mediated by mitochondria and NQO1 in melanoma B16–F10 and hepatocellular carcinoma HepG2 cancer cells. ES936, a highly selective NQO1 inhibitor, and rotenone, a selective inhibitor of mitochondrial electron transport chain (METC) complex I were found to significantly block beta-Lp meditated redox activation in B16–F10 cells. In HepG2 cells ES936 inhibited beta-Lp-mediated oxygen radical formation by ∼ 80% while rotenone exerted no significant effect. These results revealed the differential contribution of METC and NQO1 to beta-lapachone-induced ROS formation and cancer cell killing. In melanoma B16–F10 cells that do not express high NQO1 activity, both NOQ1 and METC play a critical role in beta-Lp redox activation. In contrast, in hepatocellular carcinoma HepG2 cells expressing extremely high NQO1 activity, redox activation of beta-Lp is primarily mediated by NQO1 (METC plays a minor role). These findings will contribute to our understanding of how cancer cells are selectively killed by beta-lapachone and increase our ability to devise strategies to enhance the anticancer efficacy of this potentially novel drug while minimizing its possible adverse effects on normal cells. - Highlights: • Both isolated mitochondria and purified NQO1 are able to generate ROS by beta-Lp. • The differential roles of mitochondria and NQO1 in mediating redox activation of beta-Lp • In cancer cells with

  7. Mechanistic studies of cancer cell mitochondria- and NQO1-mediated redox activation of beta-lapachone, a potentially novel anticancer agent

    Energy Technology Data Exchange (ETDEWEB)

    Li, Jason Z. [Virginia Tech CRC, Blacksburg, VA (United States); Ke, Yuebin [Shenzhen Center for Disease Control and Prevention, Shenzhen 518055 (China); Misra, Hara P. [Virginia Tech CRC, Blacksburg, VA (United States); Trush, Michael A. [Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD (United States); Li, Y. Robert [Campbell University School of Osteopathic Medicine, Buies Creek, NC (United States); Virginia Tech-Wake Forest University SBES, Blacksburg, VA (United States); Department of Biology, University of North Carolina at Greensboro, NC (United States); Zhu, Hong, E-mail: zhu@campbell.edu [Campbell University School of Osteopathic Medicine, Buies Creek, NC (United States); Jia, Zhenquan, E-mail: z_jia@uncg.edu [Department of Biology, University of North Carolina at Greensboro, NC (United States)

    2014-12-15

    Beta-lapachone (beta-Lp) derived from the Lapacho tree is a potentially novel anticancer agent currently under clinical trials. Previous studies suggested that redox activation of beta-Lp catalyzed by NAD(P)H:quinone oxidoreductase 1 (NQO1) accounted for its killing of cancer cells. However, the exact mechanisms of this effect remain largely unknown. Using chemiluminescence and electron paramagnetic resonance (EPR) spin-trapping techniques, this study for the first time demonstrated the real-time formation of ROS in the redox activation of beta-lapachone from cancer cells mediated by mitochondria and NQO1 in melanoma B16–F10 and hepatocellular carcinoma HepG2 cancer cells. ES936, a highly selective NQO1 inhibitor, and rotenone, a selective inhibitor of mitochondrial electron transport chain (METC) complex I were found to significantly block beta-Lp meditated redox activation in B16–F10 cells. In HepG2 cells ES936 inhibited beta-Lp-mediated oxygen radical formation by ∼ 80% while rotenone exerted no significant effect. These results revealed the differential contribution of METC and NQO1 to beta-lapachone-induced ROS formation and cancer cell killing. In melanoma B16–F10 cells that do not express high NQO1 activity, both NOQ1 and METC play a critical role in beta-Lp redox activation. In contrast, in hepatocellular carcinoma HepG2 cells expressing extremely high NQO1 activity, redox activation of beta-Lp is primarily mediated by NQO1 (METC plays a minor role). These findings will contribute to our understanding of how cancer cells are selectively killed by beta-lapachone and increase our ability to devise strategies to enhance the anticancer efficacy of this potentially novel drug while minimizing its possible adverse effects on normal cells. - Highlights: • Both isolated mitochondria and purified NQO1 are able to generate ROS by beta-Lp. • The differential roles of mitochondria and NQO1 in mediating redox activation of beta-Lp • In cancer cells with

  8. The Cellosaurus, a Cell-Line Knowledge Resource

    Science.gov (United States)

    Bairoch, Amos

    2018-01-01

    The Cellosaurus is a knowledge resource on cell lines. It aims to describe all cell lines used in biomedical research. Its scope encompasses both vertebrates and invertebrates. Currently, information for >100,000 cell lines is provided. For each cell line, it provides a wealth of information, cross-references, and literature citations. The Cellosaurus is available on the ExPASy server (https://web.expasy.org/cellosaurus/) and can be downloaded in a variety of formats. Among its many uses, the Cellosaurus is a key resource to help researchers identify potentially contaminated/misidentified cell lines, thus contributing to improving the quality of research in the life sciences. PMID:29805321

  9. Reactive oxygen species regulated mitochondria-mediated apoptosis in PC12 cells exposed to chlorpyrifos

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Jeong Eun [Department of Pharmacology, College of Medicine, Hanyang University, Seoul (Korea, Republic of); Hanyang Biomedical Research Institute, Seoul (Korea, Republic of); Park, Jae Hyeon [Hanyang Biomedical Research Institute, Seoul (Korea, Republic of); Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul (Korea, Republic of); Shin, In Chul [Department of Pharmacology, College of Medicine, Hanyang University, Seoul (Korea, Republic of); Koh, Hyun Chul, E-mail: hckoh@hanyang.ac.kr [Department of Pharmacology, College of Medicine, Hanyang University, Seoul (Korea, Republic of); Hanyang Biomedical Research Institute, Seoul (Korea, Republic of); Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul (Korea, Republic of)

    2012-09-01

    Reactive oxidative species (ROS) generated by environmental toxicants including pesticides could be one of the factors underlying the neuronal cell damage in neurodegenerative diseases. In this study we found that chlorpyrifos (CPF) induced apoptosis in dopaminergic neuronal components of PC12 cells as demonstrated by the activation of caspases and nuclear condensation. Furthermore, CPF also reduced the tyrosine hydroxylase-positive immunoreactivity in substantia nigra of the rat. In addition, CPF induced inhibition of mitochondrial complex I activity. Importantly, N-acetyl cysteine (NAC) treatment effectively blocked apoptosis via the caspase-9 and caspase-3 pathways while NAC attenuated the inhibition of mitochondrial complex I activity as well as the oxidative metabolism of dopamine (DA). These results demonstrated that CPF-induced apoptosis was involved in mitochondrial dysfunction through the production of ROS. In the response of cellular antioxidant systems to CPF, we found that CPF treatment increased HO-1 expression while the expression of CuZnSOD and MnSOD was reduced. In addition, we found that CPF treatment activated MAPK pathways, including ERK 1/2, the JNK, and the p38 MAP kinase in a time-dependent manner. NAC treatment abolished MAPK phosphorylation caused by CPF, indicating that ROS are upstream signals of MAPK. Interestingly, MAPK inhibitors abolished cytotoxicity and reduced ROS generation by CPF treatment. Our results demonstrate that CPF induced neuronal cell death in part through MAPK activation via ROS generation, suggesting its potential to generate oxidative stress via mitochondrial damage and its involvement in oxidative stress-related neurodegenerative disease. -- Highlights: ► Chlorpyrifos induces apoptosis. ► Chlorpyrifos inhibits mitochondrial complex I activity. ► ROS is involved in chlorpyrifos-induced apoptosis. ► Chlorpyrifos affects cellular antioxidant systems. ► Chlorpyrifos-induced apoptosis mediates activation of MAPK.

  10. Reactive oxygen species regulated mitochondria-mediated apoptosis in PC12 cells exposed to chlorpyrifos

    International Nuclear Information System (INIS)

    Lee, Jeong Eun; Park, Jae Hyeon; Shin, In Chul; Koh, Hyun Chul

    2012-01-01

    Reactive oxidative species (ROS) generated by environmental toxicants including pesticides could be one of the factors underlying the neuronal cell damage in neurodegenerative diseases. In this study we found that chlorpyrifos (CPF) induced apoptosis in dopaminergic neuronal components of PC12 cells as demonstrated by the activation of caspases and nuclear condensation. Furthermore, CPF also reduced the tyrosine hydroxylase-positive immunoreactivity in substantia nigra of the rat. In addition, CPF induced inhibition of mitochondrial complex I activity. Importantly, N-acetyl cysteine (NAC) treatment effectively blocked apoptosis via the caspase-9 and caspase-3 pathways while NAC attenuated the inhibition of mitochondrial complex I activity as well as the oxidative metabolism of dopamine (DA). These results demonstrated that CPF-induced apoptosis was involved in mitochondrial dysfunction through the production of ROS. In the response of cellular antioxidant systems to CPF, we found that CPF treatment increased HO-1 expression while the expression of CuZnSOD and MnSOD was reduced. In addition, we found that CPF treatment activated MAPK pathways, including ERK 1/2, the JNK, and the p38 MAP kinase in a time-dependent manner. NAC treatment abolished MAPK phosphorylation caused by CPF, indicating that ROS are upstream signals of MAPK. Interestingly, MAPK inhibitors abolished cytotoxicity and reduced ROS generation by CPF treatment. Our results demonstrate that CPF induced neuronal cell death in part through MAPK activation via ROS generation, suggesting its potential to generate oxidative stress via mitochondrial damage and its involvement in oxidative stress-related neurodegenerative disease. -- Highlights: ► Chlorpyrifos induces apoptosis. ► Chlorpyrifos inhibits mitochondrial complex I activity. ► ROS is involved in chlorpyrifos-induced apoptosis. ► Chlorpyrifos affects cellular antioxidant systems. ► Chlorpyrifos-induced apoptosis mediates activation of MAPK.

  11. TSA-induced cell death in prostate cancer cell lines is caspase-2 dependent and involves the PIDDosome.

    Science.gov (United States)

    Taghiyev, Agshin F; Guseva, Natalya V; Glover, Rebecca A; Rokhlin, Oskar W; Cohen, Michael B

    2006-09-01

    The histone deacetylase inhibitor Trichostatin A (TSA) has previously been found to induce caspase activity in the human prostate cancer cell lines DU145 and LNCaP. TSA treatment resulted in the release of cytochrome c and Smac/DIABLO from mitochondria in DU145, and activation of caspase-9 in both cell lines. We concluded that TSA mediated its effect via the mitochondrial pathway. The aim of the current study was to determine how TSA initiated the caspase cascade. The results revealed that caspase-2 plays an important role in TSA-induced apoptosis. Inhibition of caspase-2 by siRNA or expression of caspase-2dn substantially decreased caspase activity after TSA treatment in both cell lines, siRNA caspase-2 also inhibited TSA-induced cell death. Caspase-2 acts upstream of caspase-8 and -9 and mediates mitochondrial cytochrome c release. Coimmunoprecipitation experiments show that caspase-2 formed protein complexes with RADD/RAIDD and PIDD. Together, these data indicate that caspase-2 initiates caspase cascade after TSA treatment and involves the formation of the PIDDosome.

  12. LW6, a hypoxia-inducible factor 1 inhibitor, selectively induces apoptosis in hypoxic cells through depolarization of mitochondria in A549 human lung cancer cells.

    Science.gov (United States)

    Sato, Mariko; Hirose, Katsumi; Kashiwakura, Ikuo; Aoki, Masahiko; Kawaguchi, Hideo; Hatayama, Yoshiomi; Akimoto, Hiroyoshi; Narita, Yuichiro; Takai, Yoshihiro

    2015-09-01

    Hypoxia‑inducible factor 1 (HIF‑1) activates the transcription of genes that act upon the adaptation of cancer cells to hypoxia. LW6, an HIF‑1 inhibitor, was hypothesized to improve resistance to cancer therapy in hypoxic tumors by inhibiting the accumulation of HIF‑1α. A clear anti‑tumor effect under low oxygen conditions would indicate that LW6 may be an improved treatment strategy for cancer in hypoxia. In the present study, the HIF‑1 inhibition potential of LW6 on the growth and apoptosis of A549 lung cancer cells in association with oxygen availability was evaluated. LW6 was observed to inhibit the expression of HIF‑1α induced by hypoxia in A549 cells at 20 mM, independently of the von Hippel‑Lindau protein. In addition, at this concentration, LW6 induced hypoxia‑selective apoptosis together with a reduction in the mitochondrial membrane potential. The intracellular reactive oxygen species levels increased in LW6‑treated hypoxic A549 cells and LW6 induced a hypoxia‑selective increase of mitochondrial O2•‑. In conclusion, LW6 inhibited the growth of hypoxic A549 cells by affecting the mitochondria. The inhibition of the mitochondrial respiratory chain is suggested as a potentially effective strategy to target apoptosis in cancer cells.

  13. Atorvastatin affects negatively respiratory function of isolated endothelial mitochondria.

    Science.gov (United States)

    Broniarek, Izabela; Jarmuszkiewicz, Wieslawa

    2018-01-01

    The purpose of this research was to elucidate the direct effects of two popular blood cholesterol-lowering drugs used to treat cardiovascular diseases, atorvastatin and pravastatin, on respiratory function, membrane potential, and reactive oxygen species formation in mitochondria isolated from human umbilical vein endothelial cells (EA.hy926 cell line). Hydrophilic pravastatin did not significantly affect endothelial mitochondria function. In contrast, hydrophobic calcium-containing atorvastatin induced a loss of outer mitochondrial membrane integrity, an increase in hydrogen peroxide formation, and reductions in maximal (phosphorylating or uncoupled) respiratory rate, membrane potential and oxidative phosphorylation efficiency. The atorvastatin-induced changes indicate an impairment of mitochondrial function at the level of ATP synthesis and at the level of the respiratory chain, likely at complex I and complex III. The atorvastatin action on endothelial mitochondria was highly dependent on calcium ions and led to a disturbance in mitochondrial calcium homeostasis. Uptake of calcium ions included in atorvastatin molecule induced mitochondrial uncoupling that enhanced the inhibition of the mitochondrial respiratory chain by atorvastatin. Our results indicate that hydrophobic calcium-containing atorvastatin, widely used as anti-atherosclerotic agent, has a direct negative action on isolated endothelial mitochondria. Copyright © 2017. Published by Elsevier Inc.

  14. Cellular radiosensitivity of small-cell lung cancer cell lines

    International Nuclear Information System (INIS)

    Krarup, Marianne; Poulsen, Hans Skovgaard; Spang-Thomsen, Mogens

    1997-01-01

    Purpose: The objective of this study was to determine the radiobiological characteristics of a panel of small-cell lung cancer (SCLC) cell lines by use of a clonogenic assay. In addition, we tested whether comparable results could be obtained by employing a growth extrapolation method based on the construction of continuous exponential growth curves. Methods and Materials: Fifteen SCLC cell lines were studied, applying a slightly modified clonogenic assay and a growth extrapolation method. A dose-survival curve was obtained for each experiment and used for calculating several survival parameters. The multitarget single hit model was applied to calculate the cellular radiosensitivity (D 0 ), the capacity for sublethal damage repair (D q ), and the extrapolation number (n). Values for α and β were determined from best-fit curves according to the linear-quadratic model and these values were applied to calculate the surviving fraction after 2-Gy irradiation (SF 2 ). Results: In our investigation, the extrapolation method proved to be inappropriate for the study of in vitro cellular radiosensitivity due to lack of reproducibility. The results obtained by the clonogenic assay showed that the cell lines studied were radiobiologically heterogeneous with no discrete features of the examined parameters including the repair capacity. Conclusion: The results indicate that SCLC tumors per se are not generally candidates for hyperfractionated radiotherapy

  15. Absolute quantification of superoxide dismutase in cytosol and mitochondria of mice hepatic cells exposed to mercury by a novel metallomic approach

    Energy Technology Data Exchange (ETDEWEB)

    García-Sevillano, M.A.; García-Barrera, T. [Department of Chemistry and Materials Science, Faculty of Experimental Sciences, University of Huelva, Campus de El Carmen, Huelva 21007 (Spain); Research Center on Health and Environment (CYSMA), University of Huelva (Spain); International Campus of Excellence on Agrofood (ceiA3), University of Huelva (Spain); Navarro, F. [International Campus of Excellence on Agrofood (ceiA3), University of Huelva (Spain); Department of Environmental Biology and Public Health, Cell Biology, Faculty of Experimental Sciences, University of Huelva, Campus El Carmen, Huelva 21007 (Spain); Gómez-Ariza, J.L., E-mail: ariza@uhu.es [Department of Chemistry and Materials Science, Faculty of Experimental Sciences, University of Huelva, Campus de El Carmen, Huelva 21007 (Spain); Research Center on Health and Environment (CYSMA), University of Huelva (Spain); International Campus of Excellence on Agrofood (ceiA3), University of Huelva (Spain)

    2014-09-09

    Highlights: • Identification and quantification of Cu,Zn-superoxide dismutase in mice hepatic cells. • IDA-ICP-MSis applied to obtain a high degree of accuracy, precision and sensibility. • This methodology reduces the time of analysis and avoids clean-up procedures. • The application of this method to Hg-exposed mice reveals perturbations in Cu,Zn-SOD. - Abstract: In the last years, the development of new methods for analyzing accurate and precise individual metalloproteins is of increasing importance, since numerous metalloproteins are excellent biomarkers of oxidative stress and diseases. In that way, methods based on the use of post column isotopic dilution analysis (IDA) or enriched protein standards are required to obtain a sufficient degree of accuracy, precision and high limits of detection. This paper reports the identification and absolute quantification of Cu,Zn-superoxide dismutase (Cu,Zn-SOD) in cytosol and mitochondria from mice hepatic cells using a innovative column switching analytical approach. The method consisted of orthogonal chromatographic systems coupled to inductively coupling plasma-mass spectrometry equipped with a octopole reaction systems (ICP-ORS-MS) and UV detectors: size exclusion fractionation (SEC) of the cytosolic and mitochondrial extracts followed by online anion exchange chromatographic (AEC) separation of Cu/Zn containing species. After purification, Cu,Zn-SOD was identified after tryptic digestion by molecular mass spectrometry (MS). The MS/MS spectrum of a doubly charged peptide was used to obtain the sequence of the protein using the MASCOT searching engine. This optimized methodology reduces the time of analysis and avoids the use of sample preconcentration and clean-up procedures, such as cut-off centrifuged filters, solid phase extraction (SPE), precipitation procedures, off-line fractions insolates, etc. In this sense, the method is robust, reliable and fast with typical chromatographic run time less than 20 min

  16. Susceptibility testing of fish cell lines for virus isolation

    DEFF Research Database (Denmark)

    Ariel, Ellen; Skall, Helle Frank; Olesen, Niels Jørgen

    2009-01-01

    and laboratories, but also between lineages of the same cell line. To minimise the occurrence of false negatives in a cell culture based surveillance system, we have investigated methods, to select cell lineages that are relatively superior in their susceptibility to a panel of virus isolates. The procedures...... cell lineages, we increased the number of isolates of each virus, propagated stocks in a given cell line and tested all lineages of that line in use in the laboratory. Testing of relative cell line susceptibility between laboratories is carried out annually via the Inter-laboratory Proficiency Test...... sensitivity for surveillance purposes within a cell line and between laboratories.In terms of economic and practical considerations as well as attempting to approach a realistic test system, we suggest the optimal procedure for susceptibility testing of fish cell lines for virus isolation to be a combination...

  17. Induction of G2/M arrest and apoptosis through mitochondria pathway by a dimer sesquiterpene lactone from Smallanthus sonchifolius in HeLa cells.

    Science.gov (United States)

    Kitai, Yurika; Zhang, Xia; Hayashida, Yushi; Kakehi, Yoshiyuki; Tamura, Hirotoshi

    2017-07-01

    Dimer sesquiterpene lactones (SLs), uvedafolin and enhydrofolin, against four monomer SLs isolated from yacon, Smallanthus sonchifolius, leaf were the most cytotoxic substances on HeLa cells (IC 50 values 2.96-3.17 μM at 24 hours). However, the cytotoxic mechanism of dimer SL has not been elucidated yet. Therefore, in this study, we clarified the in vitro cytotoxic mechanism of uvedafolin on the HeLa cells, and evaluated the cytotoxicity against NIH/3T3 cells which were used as normal cells. In consequence, the dimer SLs had low toxicity for the NIH/3T3 cells (IC 50 4.81-4.98 μM at 24 hours) and then the uvedafolin mediated cell cycle arrest at the G 2 /M phase and induced apoptosis on the HeLa cells evidenced by appearance of a subG1 peak. Uvedafolin induced apoptosis was attributed to caspase-9 and caspase-3/7 activities. An effectively induced apoptosis pathway was demonstrated from mitochondria membrane potential change and cytochrome c release to cytosol. These results reveal that uvedafolin induced apoptosis via the mitochondria pathway. The present results indicate the potential of uvedafolin as a leading compound of new anticancer agents. Copyright © 2016. Published by Elsevier B.V.

  18. Bcl-2 prevents loss of mitochondria in CCCP-induced apoptosis

    International Nuclear Information System (INIS)

    Graaf, Aniek O. de; Heuvel, Lambert P. van den; Dijkman, Henry B.P.M.; Abreu, Ronney A. de; Birkenkamp, Kim U.; Witte, Theo de; Reijden, Bert A. van der; Smeitink, Jan A.M.; Jansen, Joop H.

    2004-01-01

    Bcl-2 family proteins regulate apoptosis at the level of mitochondria. To examine the mechanism of Bcl-2 function, we investigated the effects of the protonophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP) on two hematopoietic cell lines and Bcl-2 overexpressing transfectants. CCCP directly interferes with mitochondrial function and induces apoptosis. We show that Bcl-2 inhibits apoptosis and that the antiapoptotic effect of Bcl-2 takes place upstream of caspase activation and nuclear changes associated with apoptosis, since these were markedly inhibited in cells overexpressing Bcl-2. Bcl-2 does not prevent the decrease in mitochondrial membrane potential nor the alterations in cellular ATP content induced by CCCP in FL5.12 and Jurkat cells. A higher number of mitochondria was observed in untreated Bcl-2 transfected cells compared to parental cells, as shown by electron microscopy. Exposure to CCCP induced a dramatic decrease in the number of mitochondria and severely disrupted mitochondrial ultrastructure, with apparent swelling and loss of cristae in parental cells. Bcl-2 clearly diminished the disruption of mitochondrial structure and preserved a higher number of mitochondria. These data suggest that CCCP induces apoptosis by structural disruption of mitochondria and that Bcl-2 prevents apoptosis and mitochondrial degeneration by preserving mitochondrial integrity

  19. Bcl-2 prevents loss of mitochondria in CCCP-induced apoptosis.

    Science.gov (United States)

    de Graaf, Aniek O; van den Heuvel, Lambert P; Dijkman, Henry B P M; de Abreu, Ronney A; Birkenkamp, Kim U; de Witte, Theo; van der Reijden, Bert A; Smeitink, Jan A M; Jansen, Joop H

    2004-10-01

    Bcl-2 family proteins regulate apoptosis at the level of mitochondria. To examine the mechanism of Bcl-2 function, we investigated the effects of the protonophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP) on two hematopoietic cell lines and Bcl-2 overexpressing transfectants. CCCP directly interferes with mitochondrial function and induces apoptosis. We show that Bcl-2 inhibits apoptosis and that the antiapoptotic effect of Bcl-2 takes place upstream of caspase activation and nuclear changes associated with apoptosis, since these were markedly inhibited in cells overexpressing Bcl-2. Bcl-2 does not prevent the decrease in mitochondrial membrane potential nor the alterations in cellular ATP content induced by CCCP in FL5.12 and Jurkat cells. A higher number of mitochondria was observed in untreated Bcl-2 transfected cells compared to parental cells, as shown by electron microscopy. Exposure to CCCP induced a dramatic decrease in the number of mitochondria and severely disrupted mitochondrial ultrastructure, with apparent swelling and loss of cristae in parental cells. Bcl-2 clearly diminished the disruption of mitochondrial structure and preserved a higher number of mitochondria. These data suggest that CCCP induces apoptosis by structural disruption of mitochondria and that Bcl-2 prevents apoptosis and mitochondrial degeneration by preserving mitochondrial integrity.

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

    Science.gov (United States)

    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.

  1. ERKs and mitochondria-related pathways are essential for glycyrrhizic acid-mediated neuroprotection against glutamate-induced toxicity in differentiated PC12 cells

    International Nuclear Information System (INIS)

    Wang, D.; Guo, T.Q.; Wang, Z.Y.; Lu, J.H.; Liu, D.P.; Meng, Q.F.; Xie, J.; Zhang, X.L.; Liu, Y.; Teng, L.S.

    2014-01-01

    The present study focuses on the neuroprotective effect of glycyrrhizic acid (GA, a major compound separated from Glycyrrhiza Radix, which is a crude Chinese traditional drug) against glutamate-induced cytotoxicity in differentiated PC12 (DPC12) cells. The results showed that GA treatment improved cell viability and ameliorated abnormal glutamate-induced alterations in mitochondria in DPC12 cells. GA reversed glutamate-suppressed B-cell lymphoma 2 levels, inhibited glutamate-enhanced expressions of Bax and cleaved caspase 3, and reduced cytochrome C (Cyto C) release. Exposure to glutamate strongly inhibited phosphorylation of AKT (protein kinase B) and extracellular signal-regulated kinases (ERKs); however, GA pretreatment enhanced activation of ERKs but not AKT. The presence of PD98059 (a mitogen-activated protein/extracellular signal-regulated kinase kinase [MEK] inhibitor) but not LY294002 (a phosphoinositide 3-kinase [PI3K] inhibitor) diminished the potency of GA for improving viability of glutamate-exposed DPC12 cells. These results indicated that ERKs and mitochondria-related pathways are essential for the neuroprotective effect of GA against glutamate-induced toxicity in DPC12 cells. The present study provides experimental evidence supporting GA as a potential therapeutic agent for use in the treatment of neurodegenerative diseases

  2. ERKs and mitochondria-related pathways are essential for glycyrrhizic acid-mediated neuroprotection against glutamate-induced toxicity in differentiated PC12 cells

    Energy Technology Data Exchange (ETDEWEB)

    Wang, D. [School of Life Sciences, Jilin University, Changchun (China); The State Engineering Laboratory of AIDS Vaccine, Jilin University, Changchun (China); Guo, T.Q. [School of Life Sciences, Jilin University, Changchun (China); Wang, Z.Y. [State Key Laboratory of Theoretical and Computational Chemistry, Jilin University, Changchun (China); Lu, J.H.; Liu, D.P.; Meng, Q.F.; Xie, J. [School of Life Sciences, Jilin University, Changchun (China); Zhang, X.L. [Faculty of ScienceNational University of Singapore (Singapore); Liu, Y. [School of Life Sciences, Jilin University, Changchun (China); Teng, L.S. [School of Life Sciences, Jilin University, Changchun (China); The State Engineering Laboratory of AIDS Vaccine, Jilin University, Changchun (China)

    2014-07-25

    The present study focuses on the neuroprotective effect of glycyrrhizic acid (GA, a major compound separated from Glycyrrhiza Radix, which is a crude Chinese traditional drug) against glutamate-induced cytotoxicity in differentiated PC12 (DPC12) cells. The results showed that GA treatment improved cell viability and ameliorated abnormal glutamate-induced alterations in mitochondria in DPC12 cells. GA reversed glutamate-suppressed B-cell lymphoma 2 levels, inhibited glutamate-enhanced expressions of Bax and cleaved caspase 3, and reduced cytochrome C (Cyto C) release. Exposure to glutamate strongly inhibited phosphorylation of AKT (protein kinase B) and extracellular signal-regulated kinases (ERKs); however, GA pretreatment enhanced activation of ERKs but not AKT. The presence of PD98059 (a mitogen-activated protein/extracellular signal-regulated kinase kinase [MEK] inhibitor) but not LY294002 (a phosphoinositide 3-kinase [PI3K] inhibitor) diminished the potency of GA for improving viability of glutamate-exposed DPC12 cells. These results indicated that ERKs and mitochondria-related pathways are essential for the neuroprotective effect of GA against glutamate-induced toxicity in DPC12 cells. The present study provides experimental evidence supporting GA as a potential therapeutic agent for use in the treatment of neurodegenerative diseases.

  3. Electroacupuncture inhibits apoptosis in annulus fibrosis cells through suppression of the mitochondria-dependent pathway in a rat model of cervical intervertebral disc degradation

    Directory of Open Access Journals (Sweden)

    Jun Liao

    2012-01-01

    Full Text Available The purpose of this study was to investigate whether treatment with electroacupuncture (EA inhibited mitochondria-dependent apoptosis in annulus fibrosis (AF cells in a rat model of cervical intervertebral disc degradation induced by unbalanced dynamic and static forces. Forty Sprague-Dawley rats were used in this study, of which 30 underwent surgery to induce cervical intervertebral disc degradation, 10 rats received EA at acupoints Dazhui (DU 14 and Shousanli (LI 10. TUNEL staining was measured to assess apoptosis in AF cells, immunohistochemistry was used to examine Bcl-2 and Bax expression, colorimetric assays were used to determine caspase 9 and caspase 3 activities and RT-PCR and western blotting were used to assess the mRNA and protein expression of Crk and ERK2. Treatment with EA reduced the number of AF-positive cells in TUNEL staining, increased Bcl-2-positive cells and decreased Bax-positive cells in immunohistochemical staining, significantly inhibited the activation of caspases-9 and -3, and enhanced the mRNA and protein expression of Crk and ERK2. Our data show that EA inhibits AF cell apoptosis via the mitochondria-dependent pathway and up-regulates Crk and ERK2 expression. These results suggest that treatment with may be a good alternative therapy for preventing cervical spondylosis.

  4. Pregnenolone biosynthesis in C6-2B glioma cell mitochondria: regulation by a mitochondrial diazepam binding inhibitor receptor.

    OpenAIRE

    Papadopoulos, V; Guarneri, P; Kreuger, K E; Guidotti, A; Costa, E

    1992-01-01

    The C6-2B glioma cell line, rich in mitochondrial receptors that bind with high affinity to benzodiazepines, imidazopyridines, and isoquinolinecarboxamides (previously called peripheral-type benzodiazepine receptors), was investigated as a model to study the significance of the polypeptide diazepam binding inhibitor (DBI) and the putative DBI processing products on mitochondrial receptor-regulated steroidogenesis. DBI and its naturally occurring fragments have been found to be present in high...

  5. Mitochondria, Energy and Cancer: The Relationship with Ascorbic Acid

    Science.gov (United States)

    González, Michael J.; Rosario-Pérez, Glorivee; Guzmán, Angélica M.; Miranda-Massari, Jorge R.; Duconge, Jorge; Lavergne, Julio; Fernandez, Nadia; Ortiz, Norma; Quintero, Ana; Mikirova, Nina; Riordan, Neil H.; Ricart, Carlos M.

    2012-01-01

    Ascorbic Acid (AA) has been used in the prevention and treatment of cancer with reported effectiveness. Mitochondria may be one of the principal targets of ascorbate's cellular activity and it may play an important role in the development and progression of cancer. Mitochondria, besides generating adenosine triphosphate (ATP), has a role in apoptosis regulation and in the production of regulatory oxidative species that may be relevant in gene expression. At higher concentrations AA may increase ATP production by increasing mitochondrial electron flux, also may induce apoptotic cell death in tumor cell lines, probably via its pro-oxidant action In contrast, at lower concentrations AA displays antioxidant properties that may prevent the activation of oxidant-induced apoptosis. These concentration dependent activities of ascorbate may explain in part the seemingly contradictory results that have been reported previously. PMID:23565030

  6. Proteomic analysis of mitochondria in respiratory epithelial cells infected with human respiratory syncytial virus and functional implications for virus and cell biology.

    Science.gov (United States)

    Munday, Diane C; Howell, Gareth; Barr, John N; Hiscox, Julian A

    2015-03-01

    The aim of this study was to quantitatively characterise the mitochondrial proteome of airway epithelial cells infected with human respiratory syncytial virus (HRSV), a major cause of paediatric illness. Quantitative proteomics, underpinned by stable isotope labelling with amino acids in cell culture, coupled to LC-MS/MS, was applied to mitochondrial fractions prepared from HRSV-infected and mock-infected cells 12 and 24 h post-infection. Datasets were analysed using ingenuity pathway analysis, and the results were validated and characterised using bioimaging, targeted inhibition and gene depletion. The data quantitatively indicated that antiviral signalling proteins converged on mitochondria during HRSV infection. The mitochondrial receptor protein Tom70 was found to act in an antiviral manner, while its chaperone, Hsp90, was confirmed to be a positive viral factor. Proteins associated with different organelles were also co-enriched in the mitochondrial fractions from HRSV-infected cells, suggesting that alterations in organelle dynamics and membrane associations occur during virus infection. Protein and pathway-specific alterations occur to the mitochondrial proteome in a spatial and temporal manner during HRSV infection, suggesting that this organelle may have altered functions. These could be targeted as part of potential therapeutic strategies to disrupt virus biology. © 2014 Royal Pharmaceutical Society.

  7. Titanium Dioxide Nanoparticles Induce Endoplasmic Reticulum Stress-Mediated Autophagic Cell Death via Mitochondria-Associated Endoplasmic Reticulum Membrane Disruption in Normal Lung Cells

    Science.gov (United States)

    Yu, Kyeong-Nam; Chang, Seung-Hee; Park, Soo Jin; Lim, Joohyun; Lee, Jinkyu; Yoon, Tae-Jong; Kim, Jun-Sung; Cho, Myung-Haing

    2015-01-01

    Nanomaterials are used in diverse fields including food, cosmetic, and medical industries. Titanium dioxide nanoparticles (TiO2-NP) are widely used, but their effects on biological systems and mechanism of toxicity have not been elucidated fully. Here, we report the toxicological mechanism of TiO2-NP in cell organelles. Human bronchial epithelial cells (16HBE14o-) were exposed to 50 and 100 μg/mL TiO2-NP for 24 and 48 h. Our results showed that TiO2-NP induced endoplasmic reticulum (ER) stress in the cells and disrupted the mitochondria-associated endoplasmic reticulum membranes (MAMs) and calcium ion balance, thereby increasing autophagy. In contrast, an inhibitor of ER stress, tauroursodeoxycholic acid (TUDCA), mitigated the cellular toxic response, suggesting that TiO2-NP promoted toxicity via ER stress. This novel mechanism of TiO2-NP toxicity in human bronchial epithelial cells suggests that further exhaustive research on the harmful effects of these nanoparticles in relevant organisms is needed for their safe application. PMID:26121477

  8. MODERATE CYTOTOXICITY OF PROANTHOCYANIDINS TO HUMAN TUMOR-CELL LINES

    NARCIS (Netherlands)

    KOLODZIEJ, H; HABERLAND, C; WOERDENBAG, HJ; KONINGS, AWT

    In the present study the cytotoxicity of 16 proanthocyanidins was evaluated in GLC(4), a human small cell lung carcinoma cell line, and in COLO 320, a human colorectal cancer cell line, using the microculture tetrazolium (MTT) assay. With IC50 values ranging from 18 to >200 mu m following continuous

  9. CORSEN, a new software dedicated to microscope-based 3D distance measurements: mRNA-mitochondria distance, from single-cell to population analyses.

    Science.gov (United States)

    Jourdren, Laurent; Delaveau, Thierry; Marquenet, Emelie; Jacq, Claude; Garcia, Mathilde

    2010-07-01

    Recent improvements in microscopy technology allow detection of single molecules of RNA, but tools for large-scale automatic analyses of particle distributions are lacking. An increasing number of imaging studies emphasize the importance of mRNA localization in the definition of cell territory or the biogenesis of cell compartments. CORSEN is a new tool dedicated to three-dimensional (3D) distance measurements from imaging experiments especially developed to access the minimal distance between RNA molecules and cellular compartment markers. CORSEN includes a 3D segmentation algorithm allowing the extraction and the characterization of the cellular objects to be processed--surface determination, aggregate decomposition--for minimal distance calculations. CORSEN's main contribution lies in exploratory statistical analysis, cell population characterization, and high-throughput assays that are made possible by the implementation of a batch process analysis. We highlighted CORSEN's utility for the study of relative positions of mRNA molecules and mitochondria: CORSEN clearly discriminates mRNA localized to the vicinity of mitochondria from those that are translated on free cytoplasmic polysomes. Moreover, it quantifies the cell-to-cell variations of mRNA localization and emphasizes the necessity for statistical approaches. This method can be extended to assess the evolution of the distance between specific mRNAs and other cellular structures in different cellular contexts. CORSEN was designed for the biologist community with the concern to provide an easy-to-use and highly flexible tool that can be applied for diverse distance quantification issues.

  10. Investigation of the selenium metabolism in cancer cell lines

    DEFF Research Database (Denmark)

    Lunøe, Kristoffer; Gabel-Jensen, Charlotte; Stürup, Stefan

    2011-01-01

    The aim of this work was to compare different selenium species for their ability to induce cell death in different cancer cell lines, while investigating the underlying chemistry by speciation analysis. A prostate cancer cell line (PC-3), a colon cancer cell line (HT-29) and a leukaemia cell line...... (Jurkat E6-1) were incubated with five selenium compounds representing inorganic as well as organic Se compounds in different oxidation states. Selenomethionine (SeMet), Se-methylselenocysteine (MeSeCys), methylseleninic acid (MeSeA), selenite and selenate in the concentration range 5-100 mu M were...... incubated with cells for 24 h and the induction of cell death was measured using flow cytometry. The amounts of total selenium in cell medium, cell lysate and the insoluble fractions was determined by ICP-MS. Speciation analysis of cellular fractions was performed by reversed phase, anion exchange and size...

  11. Derivation and characterization of a pig embryonic stem cell-derived exocrine pancreatic cell line

    Science.gov (United States)

    The establishment and initial characterization of a pig embryonic stem cell-derived pancreatic cell line, PICM-31, and a colony-cloned derivative cell line, PICM-31A, is described. The cell lines were propagated for several months at split ratios of 1:3 or 1:5 at each passage on STO feeder cells af...

  12. Calcium plays a key role in paraoxon-induced apoptosis in EL4 cells by regulating both endoplasmic reticulum- and mitochondria-associated pathways.

    Science.gov (United States)

    Li, Lan; Du, Yi; Ju, Furong; Ma, Shunxiang; Zhang, Shengxiang

    2016-01-01

    Paraoxon (POX) is one of the most toxic organophosphorus pesticides, but its toxic mechanisms associated with apoptosis remain unclear. The aim of this study was to investigate calcium-associated mechanisms in POX-induced apoptosis in EL4 cells. EL4 cells were exposed to POX for 0-16 h. EGTA was used to chelate Ca(2+ ) in extracellular medium, and heparin and procaine were used to inhibit Ca(2+ )efflux from the endoplasmic reticulum (ER). Z-ATAD-FMK was used to inhibit caspase-12 activity. The apoptotic rate assay, western blotting and immunocytochemistry (ICC) were used to reveal the mechanisms of POX-induced apoptosis. POX significantly increased the expression and activation of caspase-12 and caspase-3, enhanced expression of calpain 1 and calpain 2, and induced the release of cyt c, but did not change the expression of Grp 78. Inhibiting caspase-12 activity alleviated POX-induced upregulation of calpain 1 and caspase-3, promoted POX-induced upregulation of calpain 2, and reduced POX-induced cyt c release, suggesting that there was a cross-talk between the ER-associated pathway and mitochondria-associated apoptotic signals. Attenuating intracellular calcium concentration with EGTA, heparin or procaine decreased POX-induced upregulation of calpain 1, calpain 2, caspase-12 and caspase-3, and reduced POX-induced cyt c release. After pretreatment with EGTA or procaine, POX significantly promoted expression of Grp 78. Calcium played a key role in POX-induced apoptosis in EL4 cells by regulating both ER- and mitochondria-associated pathways. The cross-talk of ER- and mitochondria-associated pathways was accomplished through calcium signal.

  13. Feeder-cell-independent culture of the pig-embryonic-stem-cell-derived exocrine pancreatic cell line, PICM-31

    Science.gov (United States)

    The adaptation to feeder-independent growth of a pig embryonic stem cell-derived pancreatic cell line is described. The parental PICM-31 cell line, previously characterized as an exocrine pancreas cell line, was colony-cloned two times in succession resulting in the subclonal cell line, PICM-31A1. P...

  14. Fisetin Induces Apoptosis of HSC3 Human Oral Cancer Cells Through Endoplasmic Reticulum Stress and Dysfunction of Mitochondria-mediated Signaling Pathways.

    Science.gov (United States)

    Shih, Yung-Luen; Hung, Fang-Ming; Lee, Ching-Hsiao; Yeh, Ming-Yang; Lee, Mei-Hui; Lu, Hsu-Feng; Chen, Yung-Liang; Liu, Jia-You; Chung, Jing-Gung

    2017-01-01

    Oral cancer has been reported to be one of the major cancer-related diseases in human populations and the treatment of oral cancer is still unsatisfied. Fisetin, is a flavonoid from plants and has several biological activities such as antioxidant, anti-inflammatory and anticancer function, but its cytotoxicity in human oral cancer cells is unknown. In the present study, we investigated fisetin-induced cytotoxic effects on HSC3 human oral cancer cells in vitro. Materials and Methods/Results: We used flow cytometric assay to show fisetin induced apoptotic cell death through increased reactive oxygen species and Ca 2+ , but reduced the mitochondrial membrane potential and increased caspase-8, -9 and -3 activities in HSC3 cells. Furthermore, we also used 4' 6-diamidino-2-phenylindole staining to show that fisetin induced chromatin condensation (apoptotic cell death), and Comet assay to show that fisetin induced DNA damage in HSC3 cells. Western blotting was used to examine the levels of apoptotic-associated protein and results indicated that fisetin increased expression of pro-apoptotic proteins such as B-cell lymphoma 2 (BCL2) antagonist/killer (BAK) and BCL2-associated X (BAX) but reduced that of anti-apoptotic protein such as BCL2 and BCL-x, and increased the cleaved forms of caspase-3, -8 and -9, and cytochrome c, apoptosis-inducing factor (AIF) and endonuclease G (ENDO G) in HSC3 cells. Confocal microscopy showed that fisetin increased the release of cytochrome c, AIF and ENDO G from mitochondria into the cytoplasm. Based on these observations, we suggest that fisetin induces apoptotic cell death through endoplasmic reticulum stress- and mitochondria-dependent pathways. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  15. Peroxisomal abnormalities in the immortalized human hepatocyte (IHH) cell line.

    Science.gov (United States)

    Klouwer, Femke C C; Koster, Janet; Ferdinandusse, Sacha; Waterham, Hans R

    2017-04-01

    The immortalized human hepatocyte (IHH) cell line is increasingly used for studies related to liver metabolism, including hepatic glucose, lipid, lipoprotein and triglyceride metabolism, and the effect of therapeutic interventions. To determine whether the IHH cell line is a good model to investigate hepatic peroxisomal metabolism, we measured several peroxisomal parameters in IHH cells and, for comparison, HepG2 cells and primary skin fibroblasts. This revealed a marked plasmalogen deficiency and a deficient fatty acid α-oxidation in the IHH cells, due to a defect of PEX7, a cytosolic receptor protein required for peroxisomal import of a subset of peroxisomal proteins. These abnormalities have consequences for the lipid homeostasis of these cells and thus should be taken into account for the interpretation of data previously generated by using this cell line and when considering using this cell line for future research.

  16. Radiation response of haematopoietic cell lines of human origin

    International Nuclear Information System (INIS)

    Lehnert, S.; Rybka, W.B.; Suissa, S.; Giambattisto, D.

    1986-01-01

    Six human haematopoietic cell lines, five of leukaemic origin, including cells with myeloid, lymphoid and undifferentiated phenotype have been studied with respect to radiation response. The intrinsic radio-sensitivity of the cells varied widely, the D 0 s ranging from 0.53 to 1.39 Gy. Five of the cell lines showed some capacity to accumulate sublethal damage; in three of these, enhanced survival was demonstrated in split-dose experiments. One cell line (HL-60) was anomalous in that although little accumulation of sublethal damage was demonstrable, survival was enhanced by fractionation of the dose. Five of the six cell lines studied were of leukaemic origin. The results support the belief that, in contrast to the almost constant radiosensitivity of normal haematopoietic cell progenitors, leukaemic cell progenitors may show a wide range of radiosensitivities. (author)

  17. Biochemistry of Mitochondria

    Directory of Open Access Journals (Sweden)

    Filiz Koc

    2003-02-01

    Full Text Available Mitochondria are energy source of cells. They have external and internal membranes, cristas and matrix. External membranes consist of specialized transport proteins. They have monoamine oxidase and citokrome-c reductase which both play role in KREBS cycle as catalyst and many enzymes which are necessary for phospholipid and phosphoric acid synthesis. Enzymes of electron transport chain and oxidative phosphorylation are located in the internal membranes. Also, here, there are transport systems for specific substances, such as ATP, ADP, P1, pyruvate, succinate, malate, citrate, and -ketoglutarate . Matrix; having gel-like consistency, contains a large number of enzymes. [Archives Medical Review Journal 2003; 12(0.100: 1-13

  18. Modulation of mitochondrial bioenergetics in a skeletal muscle cell line model of mitochondrial toxicity

    Directory of Open Access Journals (Sweden)

    William Dott

    2014-01-01

    Full Text Available Mitochondrial toxicity is increasingly being implicated as a contributing factor to many xenobiotic-induced organ toxicities, including skeletal muscle toxicity. This has necessitated the need for predictive in vitro models that are able to sensitively detect mitochondrial toxicity of chemical entities early in the research and development process. One such cell model involves substituting galactose for glucose in the culture media. Since cells cultured in galactose are unable to generate sufficient ATP from glycolysis they are forced to rely on mitochondrial oxidative phosphorylation for ATP generation and consequently are more sensitive to mitochondrial perturbation than cells grown in glucose. The aim of this study was to characterise cellular growth, bioenergetics and mitochondrial toxicity of the L6 rat skeletal muscle cell line cultured in either high glucose or galactose media. L6 myoblasts proliferated more slowly when cultured in galactose media, although they maintained similar levels of ATP. Galactose cultured L6 cells were significantly more sensitive to classical mitochondrial toxicants than glucose-cultured cells, confirming the cells had adapted to galactose media. Analysis of bioenergetic function with the XF Seahorse extracellular flux analyser demonstrated that oxygen consumption rate (OCR was significantly increased whereas extracellular acidification rate (ECAR, a measure of glycolysis, was decreased in cells grown in galactose. Mitochondria operated closer to state 3 respiration and had a lower mitochondrial membrane potential and basal mitochondrial O2·– level compared to cells in the glucose model. An antimycin A (AA dose response revealed that there was no difference in the sensitivity of OCR to AA inhibition between glucose and galactose cells. Importantly, cells in glucose were able to up-regulate glycolysis, while galactose cells were not. These results confirm that L6 cells are able to adapt to growth in a

  19. Human rhabdomyosarcoma cell lines for rhabdomyosarcoma research: Utility and pitfalls

    Directory of Open Access Journals (Sweden)

    Ashley R.P. Hinson

    2013-07-01

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

  20. Human Rhabdomyosarcoma Cell Lines for Rhabdomyosarcoma Research: Utility and Pitfalls

    Science.gov (United States)

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

    2013-01-01

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

  1. Csseverin inhibits apoptosis through mitochondria-mediated pathways triggered by Ca2 + dyshomeostasis in hepatocarcinoma PLC cells.

    Directory of Open Access Journals (Sweden)

    Mengchen Shi

    2017-11-01

    Full Text Available Numerous experimental and epidemiological studies have demonstrated a link between Clonorchis sinensis (C. sinensis infestation and cholangiocarcinoma (CCA as well as hepatocellular carcinoma (HCC. The underlying molecular mechanism involved in the malignancy of CCA and HCC has not yet been addressed. Csseverin, a component of the excretory/secretory products of C. sinensis (CsESPs, was confirmed to cause obvious apoptotic inhibition in the human HCC cell line PLC. However, the antiapoptotic mechanism is unclear. In the present study, we investigated the cellular features of the antiapoptotic mechanism upon transfection of the Csseverin gene.In the present study, we evaluated the effects of Csseverin gene overexpression on the apoptosis of PLC cells using an Annexin PE/7-AAD assay. Western blotting was applied to quantify the activation of caspase-3 and caspase-9, the mitochondrial translocation of Bax and the release of Cyt c upon Csseverin overexpression in PLC cells. Laser scanning confocal microscopy was used to analyze the changes of intracellular calcium. Fluorescence assay and immunofluorescence assays were performed to observe the changes of the mitochondrial permeability transition pore (MPTP.The overexpression of Csseverin in PLC cells showed apoptosis resistance after the induction of apoptosis. Additionally, the activation of caspase-3 and caspase-9 was specifically weakened in Csseverin overexpression PLC cells. The overexpression of Csseverin reduced the increase in intracellular free Ca2+, thereby inhibiting MPTP opening in PLC cells. Moreover, Bax mitochondrial translocation and the subsequent release of Cyt c were downregulated in apoptotic Csseverin overexpression PLC cells.The present findings suggest that Csseverin, a component of CsESPs, confers protection from human HCC cell apoptosis via the inactivation of membranous Ca2+ channels. Csseverin might be involved in the process of HCC through C. sinensis infestation in

  2. PERK silence inhibits glioma cell growth under low glucose stress by blockage of p-AKT and subsequent HK2's mitochondria translocation

    KAUST Repository

    Hou, Xu

    2015-03-12

    Glioma relies on glycolysis to obtain energy and sustain its survival under low glucose microenvironment in vivo. The mechanisms on glioma cell glycolysis regulation are still unclear. Signaling mediated by Double-stranded RNA-activated protein kinase (PKR) - like ER kinase (PERK) is one of the important pathways of unfolded protein response (UPR) which is comprehensively activated in cancer cells upon the hypoxic and low glucose stress. Here we show that PERK is significantly activated in human glioma tissues. PERK silencing results in decreased glioma cell viability and ATP/lactate production upon low glucose stress, which is mediated by partially blocked AKT activation and subsequent inhibition of Hexokinase II (HK2)\\'s mitochondria translocation. More importantly, PERK silenced glioma cells show decreased tumor formation capacity. Our results reveal that PERK activation is involved in glioma glycolysis regulation and may be a potential molecular target for glioma treatment.

  3. PERK silence inhibits glioma cell growth under low glucose stress by blockage of p-AKT and subsequent HK2's mitochondria translocation

    KAUST Repository

    Hou, Xu; Liu, Yaohua; Liu, Huailei; Chen, Xin; Liu, Min; Che, Hui; Guo, Fei; Wang, Chunlei; Zhang, Daming; Wu, Jianing; Chen, Xiaofeng; Shen, Chen; Li, Chenguang; Peng, Fei; Bi, Yunke; Yang, Zhuowen; Yang, Guang; Ai, Jing; Gao, Xin; Zhao, Shiguang

    2015-01-01

    Glioma relies on glycolysis to obtain energy and sustain its survival under low glucose microenvironment in vivo. The mechanisms on glioma cell glycolysis regulation are still unclear. Signaling mediated by Double-stranded RNA-activated protein kinase (PKR) - like ER kinase (PERK) is one of the important pathways of unfolded protein response (UPR) which is comprehensively activated in cancer cells upon the hypoxic and low glucose stress. Here we show that PERK is significantly activated in human glioma tissues. PERK silencing results in decreased glioma cell viability and ATP/lactate production upon low glucose stress, which is mediated by partially blocked AKT activation and subsequent inhibition of Hexokinase II (HK2)'s mitochondria translocation. More importantly, PERK silenced glioma cells show decreased tumor formation capacity. Our results reveal that PERK activation is involved in glioma glycolysis regulation and may be a potential molecular target for glioma treatment.

  4. Erythroid precursors from patients with low-risk myelodysplasia demonstrate ultrastructural features of enhanced autophagy of mitochondria

    NARCIS (Netherlands)

    Houwerzijl, E. J.; Pol, H-W D.; Blom, N. R.; van der Want, J. J. L.; de Wolf, J. Thm; Vellenga, E.

    Recent studies in erythroid cells have shown that autophagy is an important process for the physiological clearance of mitochondria during terminal differentiation. However, autophagy also plays an important role in removing damaged and dysfunctional mitochondria. Defective mitochondria and impaired

  5. Ursodeoxycholic acid suppresses mitochondria-dependent programmed cell death induced by sodium nitroprusside in SH-SY5Y cells

    International Nuclear Information System (INIS)

    Chun, Hong Sung; Low, Walter C.

    2012-01-01

    Although ursodeoxycholic acid (UDCA) and its highly water-soluble formula (Yoo's solution; YS) have been shown to prevent neuronal damage, the effects of UDCA or YS against Parkinson's disease (PD)-related dopaminergic cell death has not been studied. This study investigated the protective effects of UDCA and YS on sodium nitroprusside (SNP)-induced cytotoxicity in human dopaminergic SH-SY5Y cells. Both UDCA (50–200 μM) and YS (100–200 μM) dose-dependently prevented SNP (1 mM)-induced cell death. Results showed that both UDCA and YS effectively attenuated the production of total reactive oxygen species (ROS), peroxynitrite (ONOO − ) and nitric oxide (NO), and markedly inhibited the mitochondrial membrane potential (MMP) loss and intracellular reduced glutathione (GSH) depletion. SNP-induced programmed cell death events, such as nuclear fragmentation, caspase-3/7 and -9 activation, Bcl-2/Bax ratio decrease, and cytochrome c release, were significantly attenuated by both UDCA and YS. Furthermore, selective inhibitor of phosphatidylinositiol-3-kinase (PI3K), LY294002, and Akt/PKB inhibitor, triciribine, reversed the preventive effects of UDCA on the SNP-induced cytotoxicity and Bax translocation. These results suggest that UDCA can protect SH-SY5Y cells under programmed cell death process by regulating PI3K-Akt/PKB pathways.

  6. Cytotoxic Effect on Cancerous Cell Lines by Biologically Synthesized Silver Nanoparticles

    Directory of Open Access Journals (Sweden)

    Balaji Kulandaivelu

    Full Text Available The biosynthesis of nanoparticles has been proposed as an environmental friendly and cost effective alternative to chemical and physical methods. Silver nanoparticles are biologically synthesized and characterized were used in the study. The invitro cytotoxic effect of biologically synthesized silver nanoparticles against MCF-7 cancer cell lines were assessed. The cytotoxic effects of the silver nanoparticles could significantly inhibited MCF-7 cancer cell lines proliferation in a time and concentration-dependent manner by MTT assay. Acridine orange, ethidium bromide (AO/EB dual staining, caspase-3 and DNA fragmentation assays were carried out using various concentrations of silver nanoparticles ranging from 1 to 100 μg/mL. At 100 μg/mL concentration, the silver nanoparticles exhibited significant cytotoxic effects and the apoptotic features were confirmed through caspase-3 activation and DNA fragmentation assays. Western blot analysis has revealed that nanoparticle was able to induce cytochrome c release from the mitochondria, which was initiated by the inhibition of Bcl-2 and activation of Bax. Thus, the results of the present study indicate that biologically synthesized silver nanoparticles might be used to treat breast cancer. The present studies suggest that these nanoparticles could be a new potential adjuvant chemotherapeutic and chemo preventive agent against cytotoxic cells. However, it necessitates clinical studies to ascertain their potential as anticancer agents.

  7. Authentication of M14 melanoma cell line proves misidentification of MDA‐MB‐435 breast cancer cell line

    Science.gov (United States)

    Korch, Christopher; Hall, Erin M.; Dirks, Wilhelm G.; Ewing, Margaret; Faries, Mark; Varella‐Garcia, Marileila; Robinson, Steven; Storts, Douglas; Turner, Jacqueline A.; Wang, Ying; Burnett, Edward C.; Healy, Lyn; Kniss, Douglas; Neve, Richard M.; Nims, Raymond W.; Reid, Yvonne A.; Robinson, William A.

    2017-01-01

    A variety of analytical approaches have indicated that melanoma cell line UCLA‐SO‐M14 (M14) and breast carcinoma cell line MDA‐MB‐435 originate from a common donor. This indicates that at some point in the past, one of these cell lines became misidentified, meaning that it ceased to correspond to the reported donor and instead became falsely identified (through cross‐contamination or other means) as a cell line from a different donor. Initial studies concluded that MDA‐MB‐435 was the misidentified cell line and M14 was the authentic cell line, although contradictory evidence has been published, resulting in further confusion. To address this question, we obtained early samples of the melanoma cell line (M14), a lymphoblastoid cell line from the same donor (ML14), and donor serum preserved at the originator's institution. M14 samples were cryopreserved in December 1975, before MDA‐MB‐435 cells were established in culture. Through a series of molecular characterizations, including short tandem repeat (STR) profiling and cytogenetic analysis, we demonstrated that later samples of M14 and MDA‐MB‐435 correspond to samples of M14 frozen in 1975, to the lymphoblastoid cell line ML14, and to the melanoma donor's STR profile, sex and blood type. This work demonstrates conclusively that M14 is the authentic cell line and MDA‐MB‐435 is misidentified. With clear provenance information and authentication testing of early samples, it is possible to resolve debates regarding the origins of problematic cell lines that are widely used in cancer research. PMID:28940260

  8. Trichloroethylene toxicity in a human hepatoma cell line

    Energy Technology Data Exchange (ETDEWEB)

    Thevenin, E.; McMillian, J. [Medical Univ. of Charleston South Carolina, SC (United States)

    1994-12-31

    The experiments conducted in this study were designed to determine the usefullness of hepatocyte cultures and a human hepatoma cell line as model systems for assessing human susceptibility to hepatocellular carcinoma due to exposure to trichloroethylene. The results from these studies will then be analyzed to determine if human cell lines can be used to conduct future experiments of this nature.

  9. Derivation of the human embryonic stem cell line RCM1

    Directory of Open Access Journals (Sweden)

    P.A. De Sousa

    2016-03-01

    Full Text Available The human embryonic stem cell line RCM-1 was derived from a failed to fertilise egg undergoing parthenogenetic stimulation. The cell line shows normal pluripotency marker expression and differentiation to three germ layers in vitro and in vivo. It has a normal 46XX female karyotype and microsatellite PCR identity, HLA and blood group typing data is available.

  10. Isoflavones extracted from chickpea Cicer arietinum L. sprouts induce mitochondria-dependent apoptosis in human breast cancer cells.

    Science.gov (United States)

    Chen, Hua; Ma, Hai-Rong; Gao, Yan-Hua; Zhang, Xue; Habasi, Madina; Hu, Rui; Aisa, Haji Akber

    2015-02-01

    Isoflavones are important chemical components of the seeds and sprouts of chickpeas. We systematically investigated the effects of isoflavones extracted from chickpea sprouts (ICS) on the human breast cancer cell lines SKBr3 and Michigan Cancer Foundation-7 (MCF-7). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays showed that ICS (10-60 µg/mL) significantly inhibited the proliferation of both cell lines in a time-dependent and dose-dependent fashion. Wright-Giemsa staining as well as annexin V-fluorescein isothiocyanate and propidium iodide (Annexin V/PI) staining showed that ICS significantly increased cytoclasis and apoptotic body formation. Quantitative Annexin V/PI assays further showed that the number of apoptotic cells increased in a dose-dependent manner following ICS treatment. Semiquantitative reverse transcription PCR showed that ICS increased the expression of the apoptosis-promoting gene Bcl-2-associated X protein and decreased the expression of the antiapoptotic gene Bcl-2. Western blot analysis showed that treatment of SKBr3 and MCF-7 cells with ICS increased the expression of caspase 7, caspase 9, P53, and P21 in a dose-dependent manner. Flow cytometry assays using the fluorescent probe 3,3'-dihexyloxacarbocyanine iodide showed a dose-dependent decrease in mitochondrial membrane potential following ICS treatment. Treatment using ICS also induced a dose-dependent increase in reactive oxygen species production. This is the first study to demonstrate that ICS may be a chemopreventive or therapeutic agent against breast cancer. Copyright © 2014 John Wiley & Sons, Ltd.

  11. Comparison of steroid receptors from the androgen responsive DDT1 cell line and the nonresponsive HVP cell line.

    Science.gov (United States)

    Norris, J S; Kohler, P O

    1978-01-01

    Two hamster cell lines have been isolated from androgen target tissue. The DDT1 cells derived from ductus deferens tissue exhibit a growth response to androgens, while the HVP cells derived from ventral prostate are androgen unresponsive. Both cell lines contain androgen receptors, that are similar when compared by kinetic methods, sedimentation velocity, chromatographic procedures or nuclear translocation ability. The forms of the high salt extracted nuclear receptors are indistinguishable chromatographically. Therefore, we postulate that the lesion preventing androgen induced growth in the HVP cell line is subseqent to nuclear translocation of the steroid receptor complex.

  12. Mitochondria and redox homoeostasis as chemotherapeutic targets of Araucaria angustifolia (Bert.) O. Kuntze in human larynx HEp-2 cancer cells.

    Science.gov (United States)

    Branco, Cátia dos Santos; de Lima, Émilin Dreher; Rodrigues, Tiago Selau; Scheffel, Thamiris Becker; Scola, Gustavo; Laurino, Claudia Cilene Fernandes Correia; Moura, Sidnei; Salvador, Mirian

    2015-04-25

    Natural products are among one of the most promising fields in finding new molecular targets in cancer therapy. Laryngeal carcinoma is one of the most common cancers affecting the head and neck regions, and is associated with high morbidity rate if left untreated. The aim of this study was to examine the antiproliferative effect of Araucaria angustifolia on laryngeal carcinoma HEp-2 cells. The results showed that A. angustifolia extract (AAE) induced a significant cytotoxicity in HEp-2 cells compared to the non-tumor human epithelial (HEK-293) cells, indicating a selective activity of AAE for the cancer cells. A. angustifolia extract was able to increase oxidative damage to lipids and proteins, and the production of nitric oxide, along with the depletion of enzymatic antioxidant defenses (superoxide dismutase and catalase) in the tumor cell line. Moreover, AAE was able to induce DNA damage, nuclear fragmentation and chromatin condensation. A significant increase in the Apoptosis Inducing Factor (AIF), Bax, poly-(ADP-ribose) polymerase (PARP) and caspase-3 cleavage expression were also found. These effects could be related to the ability of AAE to increase the production of reactive oxygen species through inhibition of the mitochondrial electron transport chain complex I activity and ATP production by the tumor cells. The phytochemical analysis of A. angustifolia, performed using High Resolution Mass Spectrometry (HRMS) in MS and MS/MS mode, showed the presence of dodecanoic and hexadecanoic acids, and phenolic compounds, which may be associated with the chemotherapeutic effect observed in this study. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  13. Identification of a novel rhabdovirus in Spodoptera frugiperda cell lines.

    Science.gov (United States)

    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

  14. Lipid droplets interact with mitochondria using SNAP23

    DEFF Research Database (Denmark)

    Jägerström, Sara; Polesie, Sam; Wickström, Ylva

    2009-01-01

    peroxisomes and the endoplasmic reticulum. We have used electron and confocal microscopy to demonstrate that LD form complexes with mitochondria in NIH 3T3 fibroblasts. Using an in vitro system of purified LD and mitochondria, we also show the formation of the LD-mitochondria complex, in which cytosolic...... factors are involved. Moreover, the presence of LD markers in mitochondria isolated by subcellular fractionations is demonstrated. Finally, ablation of SNAP23 using siRNA reduced complex formation and beta oxidation, which suggests that the LD-mitochondria complex is functional in the cell....

  15. Amyloid β induces NLRP3 inflammasome activation in retinal pigment epithelial cells via NADPH oxidase- and mitochondria-dependent ROS production.

    Science.gov (United States)

    Wang, Ke; Yao, Yong; Zhu, Xue; Zhang, Kai; Zhou, Fanfan; Zhu, Ling

    2017-06-01

    Amyloid β (Aβ)-induced chronic inflammation is believed to be a key pathogenic process in early-stage age-related macular degeneration (AMD). Nucleotide oligomerization domain (NOD)-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome activation triggered by Aβ is responsible for retinal pigment epithelium (RPE) dysfunction in the onset of AMD; however, the detailed molecular mechanism remains unclear. In this study, we investigated the involvement of NADPH oxidase- and mitochondria-derived reactive oxygen species (ROS) in the process of Aβ 1-40 -induced NLRP3 inflammasome activation in LPS-primed ARPE-19 cells. The results showed that Aβ 1-40 could induce excessive ROS generation, MAPK/NF-κB signaling activation and subsequently NLRP3 inflammasome activation in LPS-primed ARPE-19 cells. Furthermore, the inductive effect of Aβ 1-40 on NLRP3 inflammasome activation was mediated in a manner dependent on NADPH oxidase- and mitochondria-derived ROS. Our findings may provide a novel insight into the molecular mechanism by which Aβ contributes to the early-stage AMD. © 2016 Wiley Periodicals, Inc.

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

    Science.gov (United States)

    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)

  17. Recombinant protein production from stable mammalian cell lines and pools.

    Science.gov (United States)

    Hacker, David L; Balasubramanian, Sowmya

    2016-06-01

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

  18. Susceptibility of various cell lines to Neospora caninum tachyzoites cultivation

    Directory of Open Access Journals (Sweden)

    Khordadmehr, M.,

    2014-05-01

    Full Text Available Neospora caninum is a coccidian protozoan parasite which is a major cause of bovine abortions and neonatal mortality in cattle, sheep, goat and horse. Occasionally, cultured cells are used for isolation and multiplication of the agent in vitro with several purposes. In this study the tachyzoite yields of N. caninum were compared in various cell cultures as the host cell lines. Among the cell cultures tested, two presented good susceptibility to the agent: cell lines Vero and MA-104. SW742 and TLI (in vitro suspension culture of lymphoid cells infected with Theileria lestoquardi showed moderate sensitivity. No viable tachyzoite were detected in the culture of MDCK and McCoy cell lines. These results demonstrate that MA-104 and SW742 cells present adequate susceptibility to N. caninum compared to Vero cells, which have been largely used to multiply the parasite in vitro. Moreover, these have easy manipulation, fast multiplication and relatively low nutritional requirements. In addition, the result of this study showed that TLI cell line as a suspension cell culture is susceptible to Nc-1 tachyzoites infection and could be used as an alternative host cell line for tachyzoites culture in vitro studies.

  19. Mitochondria in neutrophil apoptosis

    NARCIS (Netherlands)

    van Raam, B. J.; Verhoeven, A. J.; Kuijpers, T. W.

    2006-01-01

    Central in the regulation of the short life span of neutrophils are their mitochondria. These organelles hardly contribute to the energy status of neutrophils but play a vital role in the apoptotic process. Not only do the mitochondria contain cytotoxic proteins that are released during apoptosis

  20. Natural killer cells for immunotherapy – Advantages of cell lines over blood NK cells

    Directory of Open Access Journals (Sweden)

    Hans eKlingemann

    2016-03-01

    Full Text Available Natural killer cells are potent cytotoxic effector cells for cancer therapy and potentially for severe viral infections. However, there are technical challenges to obtain sufficient numbers of functionally active NK cells form a patient’s blood since they represent only 10% of the lymphocytes. Especially, cancer patients are known to have dysfunctional NK cells. The alternative is to obtain cells from a healthy donor, which requires depletion of the allogeneic T-cells. Establishing cell lines from donor blood NK cells have not been successful, in contrast to blood NK cells obtained from patients with a clonal NK cell lymphoma. Those cells can be expanded in culture in the presence of IL-2. However, except for the NK-92 cell line none of the other six known cell lines has consistent and reproducibly high anti-tumor cytotoxicity, nor can they be easily genetically manipulated to recognize specific tumor antigens or to augment monoclonal antibody activity through ADCC. NK-92 is also the only cell line product that has been widely given to patients with advanced cancer with demonstrated efficiency and minimal side effects.

  1. Application of DNA fingerprints for cell-line individualization.

    Science.gov (United States)

    Gilbert, D A; Reid, Y A; Gail, M H; Pee, D; White, C; Hay, R J; O'Brien, S J

    1990-09-01

    DNA fingerprints of 46 human cell lines were derived using minisatellite probes for hypervariable genetic loci. The incidence of 121 HaeIII DNA fragments among 33 cell lines derived from unrelated individuals was used to estimate allelic and genotypic frequencies for each fragment and for composite individual DNA fingerprints. We present a quantitative estimate of the extent of genetic difference between individuals, an estimate based on the percentage of restriction fragments at which they differ. The average percent difference (APD) among pairwise combinations from the population of 33 unrelated cell lines was 76.9%, compared with the APD in band sharing among cell lines derived from the same individual (less than or equal to 1.2%). Included in this survey were nine additional cell lines previously implicated as HeLa cell derivatives, and these lines were clearly confirmed as such by DNA fingerprints (APD less than or equal to 0.6%). On the basis of fragment frequencies in the tested cell line population, a simple genetic model was developed to estimate the frequencies of each DNA fingerprint in the population. The median incidence was 2.9 X 10(-17), and the range was 2.4 X 10(-21) to 6.6 X 10(-15). This value approximates the probability that a second cell line selected at random from unrelated individuals will match a given DNA fingerprint. Related calculations address the chance that any two DNA fingerprints would be identical among a large group of cell lines. This estimate is still very slight; for example, the chance of two or more common DNA fingerprints among 1 million distinct individuals is less than .001. The procedure provides a straightforward, easily interpreted, and statistically robust method for identification and individualization of human cells.

  2. Exogenous ether lipids predominantly target mitochondria.

    Directory of Open Access Journals (Sweden)

    Lars Kuerschner

    Full Text Available Ether lipids are ubiquitous constituents of cellular membranes with no discrete cell biological function assigned yet. Using fluorescent polyene-ether lipids we analyzed their intracellular distribution in living cells by microscopy. Mitochondria and the endoplasmic reticulum accumulated high amounts of ether-phosphatidylcholine and ether-phosphatidylethanolamine. Both lipids were specifically labeled using the corresponding lyso-ether lipids, which we established as supreme precursors for lipid tagging. Polyfosine, a fluorescent analogue of the anti-neoplastic ether lipid edelfosine, accumulated to mitochondria and induced morphological changes and cellular apoptosis. These data indicate that edelfosine could exert its pro-apoptotic power by targeting and damaging mitochondria and thereby inducing cellular apoptosis. In general, this study implies an important role of mitochondria in ether lipid metabolism and intracellular ether lipid trafficking.

  3. Mitochondria and Mitochondrial ROS in Cancer: Novel Targets for Anticancer Therapy.

    Science.gov (United States)

    Yang, Yuhui; Karakhanova, Svetlana; Hartwig, Werner; D'Haese, Jan G; Philippov, Pavel P; Werner, Jens; Bazhin, Alexandr V

    2016-12-01

    Mitochondria are indispensable for energy metabolism, apoptosis regulation, and cell signaling. Mitochondria in malignant cells differ structurally and functionally from those in normal cells and participate actively in metabolic reprogramming. Mitochondria in cancer cells are characterized by reactive oxygen species (ROS) overproduction, which promotes cancer development by inducing genomic instability, modifying gene expression, and participating in signaling pathways. Mitochondrial and nuclear DNA mutations caused by oxidative damage that impair the oxidative phosphorylation process will result in further mitochondrial ROS production, completing the "vicious cycle" between mitochondria, ROS, genomic instability, and cancer development. The multiple essential roles of mitochondria have been utilized for designing novel mitochondria-targeted anticancer agents. Selective drug delivery to mitochondria helps to increase specificity and reduce toxicity of these agents. In order to reduce mitochondrial ROS production, mitochondria-targeted antioxidants can specifically accumulate in mitochondria by affiliating to a lipophilic penetrating cation and prevent mitochondria from oxidative damage. In consistence with the oncogenic role of ROS, mitochondria-targeted antioxidants are found to be effective in cancer prevention and anticancer therapy. A better understanding of the role played by mitochondria in cancer development will help to reveal more therapeutic targets, and will help to increase the activity and selectivity of mitochondria-targeted anticancer drugs. In this review we summarized the impact of mitochondria on cancer and gave summary about the possibilities to target mitochondria for anticancer therapies. J. Cell. Physiol. 231: 2570-2581, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  4. Radiation sensitivity of human lung cancer cell lines

    International Nuclear Information System (INIS)

    Carmichael, J.; Degraff, W.G.; Gamson, J.; Russo, G.; Mitchell, J.B.; Gazdar, A.F.; Minna, J.D.; Levitt, M.L.

    1989-01-01

    X-Ray survival curves were determined using a panel of 17 human lung cancer cell lines, with emphasis on non-small cell lung cancer (NSCLC). In contrast to classic small cell lung cancer (SCLC) cell lines, NSCLC cell lines were generally less sensitive to radiation as evidenced by higher radiation survival curve extrapolation numbers, surviving fraction values following a 2Gy dose (SF2) and the mean inactivation dose values (D) values. The spectrum of in vitro radiation responses observed was similar to that expected in clinical practice, although mesothelioma was unexpectedly sensitive in vitro. Differences in radiosensitivity were best distinguished by comparison of SF2 values. Some NSCLC lines were relatively sensitive, and in view of this demonstrable variability in radiation sensitivity, the SF2 value may be useful for in vitro predictive assay testing of clinical specimens. (author)

  5. Differential heat shock response of primary human cell cultures and established cell lines

    DEFF Research Database (Denmark)

    Richter, W W; Issinger, O G

    1986-01-01

    degrees C treatment, whereas in immortalized cell lines usually 90% of the cells were found in suspension. Enhanced expression of the major heat shock protein (hsp 70) was found in all heat-treated cells. In contrast to the primary cell cultures, established and transformed cell lines synthesized...

  6. Antiproliferative effect of isopentenylated coumarins on several cancer cell lines.

    Science.gov (United States)

    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.

  7. Establishment and characterization of rat portal myofibroblast cell lines.

    Directory of Open Access Journals (Sweden)

    Michel Fausther

    Full Text Available The major sources of scar-forming myofibroblasts during liver fibrosis are activated hepatic stellate cells (HSC and portal fibroblasts (PF. In contrast to well-characterized HSC, PF remain understudied and poorly defined. This is largely due to the facts that isolation of rodent PF for functional studies is technically challenging and that PF cell lines had not been established. To address this, we have generated two polyclonal portal myofibroblast cell lines, RGF and RGF-N2. RGF and RGF-N2 were established from primary PF isolated from adult rat livers that underwent culture activation and subsequent SV40-mediated immortalization. Specifically, Ntpdase2/Cd39l1-sorted primary PF were used to generate the RGF-N2 cell line. Both cell lines were functionally characterized by RT-PCR, immunofluorescence, immunoblot and bromodeoxyuridine-based proliferation assay. First, immortalized RGF and RGF-N2 cells are positive for phenotypic myofibroblast markers alpha smooth muscle actin, type I collagen alpha-1, tissue inhibitor of metalloproteinases-1, PF-specific markers elastin, type XV collagen alpha-1 and Ntpdase2/Cd39l1, and mesenchymal cell marker ecto-5'-nucleotidase/Cd73, while negative for HSC-specific markers desmin and lecithin retinol acyltransferase. Second, both RGF and RGF-N2 cell lines are readily transfectable using standard methods. Finally, RGF and RGF-N2 cells attenuate the growth of Mz-ChA-1 cholangiocarcinoma cells in co-culture, as previously demonstrated for primary PF. Immortalized rat portal myofibroblast RGF and RGF-N2 cell lines express typical markers of activated PF-derived myofibroblasts, are suitable for DNA transfection, and can effectively inhibit cholangiocyte proliferation. Both RGF and RGF-N2 cell lines represent novel in vitro cellular models for the functional studies of portal (myofibroblasts and their contribution to the progression of liver fibrosis.

  8. Lycopene protects human SH-SY5Y neuroblastoma cells against hydrogen peroxide-induced death via inhibition of oxidative stress and mitochondria-associated apoptotic pathways

    Science.gov (United States)

    FENG, CHUNSHENG; LUO, TIANFEI; ZHANG, SHUYAN; LIU, KAI; ZHANG, YANHONG; LUO, YINAN; GE, PENGFEI

    2016-01-01

    Oxidative stress, which is characterized by excessive production of reactive oxygen species (ROS), is a common pathway that results in neuronal injury or death due to various types of pathological stress. Although lycopene has been identified as a potent antioxidant, its effect on hydrogen peroxide (H2O2)-induced neuronal damage remains unclear. In the present study, pretreatment with lycopene was observed to protect SH-SY5Y neuroblastoma cells against H2O2-induced death via inhibition of apoptosis resulting from activation of caspase-3 and translocation of apoptosis inducing factor (AIF) to the nucleus. Furthermore, the over-produced ROS, as well as the reduced activities of anti-oxidative enzymes, superoxide dismutase and catalase, were demonstrated to be alleviated by lycopene. Additionally, lycopene counteracted H2O2-induced mitochondrial dysfunction, which was evidenced by suppression of mitochondrial permeability transition pore opening, attenuation of the decline of the mitochondrial membrane potential, and inhibition of the increase of Bax and decrease of Bcl-2 levels within the mitochondria. The release of cytochrome c and AIF from the mitochondria was also reduced. These results indicate that lycopene is a potent neuroprotectant against apoptosis, oxidative stress and mitochondrial dysfunction, and could be administered to prevent neuronal injury or death. PMID:27035331

  9. A stromal myoid cell line provokes thymic erythropoiesis between ...

    African Journals Online (AJOL)

    Background: The thymus provides an optimal cellular and humoral microenvironment for cell line committed differentiation of haematopoietic stem cells. The immigration process requires the secretion of at least one peptide called thymotaxine by cells of the reticulo-epithelial (RE) network of the thymic stromal cellular ...

  10. Cytotoxicity against MCF-7 breast cancer cell line and interaction ...

    African Journals Online (AJOL)

    N6-furfuryladenine (kinetin) is a cytokinin growth factor with several biological effects observed in human cells and fruit flies. Kinetin exists naturally in the DNA of almost all organisms tested so far, including human cells and various plants. The cytotoxicity effect of kinetin on MCF-7 breast cancer cell lines was measured by ...

  11. A ginseng saponin metabolite-induced apoptosis in HepG2 cells involves a mitochondria-mediated pathway and its downstream caspase-8 activation and Bid cleavage

    International Nuclear Information System (INIS)

    Oh, Seon-Hee; Lee, Byung-Hoon

    2004-01-01

    20-O-(β-D-Glucopyranosyl)-20(S)-protopanaxadiol (IH901), an intestinal bacterial metabolite of ginseng saponin formed from ginsenosides Rb1, Rb2, and Rc, is suggested to be a potential chemopreventive agent. Here, we show that IH901 induces apoptosis in human hepatoblastoma HepG2 cells. IH901 led to an early activation of procaspase-3 (12 h posttreatment), and the activation of caspase-8 became evident only later (18 h posttreatment). Caspase activation was a necessary requirement for apoptosis because caspase inhibitors significantly inhibited cell death by IH901. Treatment of HepG2 cells with IH901 also induced the cleavage of cytosolic factors such as Bid and Bax and translocation of truncated Bid (tBid) to mitochondria. A time-dependent release of cytochrome c from mitochondria was observed, which was accompanied by activation of caspase-9. A broad-spectrum caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk), and a specific inhibitor for caspase-8, N-benzyloxycarbonyl-Ile-Glu-Thr-Asp-fluoromethylketone (zIETD-fmk), abrogated Bid processing and translocation, and caspase-3 activation. Cytochrome c release was inhibited by zVAD-fmk, however, the inhibition by zIETD-fmk was not complete. The activation of caspase-8 was inhibited not only by zIETD-fmk but also by zVAD-fmk. The results, together with the kinetic change of caspase activation, indicate that activation of caspase-8 occurred downstream of caspase-3 and -9. Our data suggest that the activation of caspase-8 after early caspase-3 activation might act as an amplification loop necessary for successful apoptosis. Primary hepatocytes isolated from normal Sprague-Dawley rats were not affected by IH901 (0-60 μM). The very low toxicity in normal hepatocytes and high activity in hepatoblastoma HepG2 cells suggest that IH901 is a promising experimental cancer chemopreventive agent

  12. Pathway-specific differences between tumor cell lines and normal and tumor tissue cells

    Directory of Open Access Journals (Sweden)

    Tozeren Aydin

    2006-11-01

    Full Text Available Abstract Background Cell lines are used in experimental investigation of cancer but their capacity to represent tumor cells has yet to be quantified. The aim of the study was to identify significant alterations in pathway usage in cell lines in comparison with normal and tumor tissue. Methods This study utilized a pathway-specific enrichment analysis of publicly accessible microarray data and quantified the gene expression differences between cell lines, tumor, and normal tissue cells for six different tissue types. KEGG pathways that are significantly different between cell lines and tumors, cell lines and normal tissues and tumor and normal tissue were identified through enrichment tests on gene lists obtained using Significance Analysis of Microarrays (SAM. Results Cellular pathways that were significantly upregulated in cell lines compared to tumor cells and normal cells of the same tissue type included ATP synthesis, cell communication, cell cycle, oxidative phosphorylation, purine, pyrimidine and pyruvate metabolism, and proteasome. Results on metabolic pathways suggested an increase in the velocity nucleotide metabolism and RNA production. Pathways that were downregulated in cell lines compared to tumor and normal tissue included cell communication, cell adhesion molecules (CAMs, and ECM-receptor interaction. Only a fraction of the significantly altered genes in tumor-to-normal comparison had similar expressions in cancer cell lines and tumor cells. These genes were tissue-specific and were distributed sparsely among multiple pathways. Conclusion Significantly altered genes in tumors compared to normal tissue were largely tissue specific. Among these genes downregulation was a major trend. In contrast, cell lines contained large sets of significantly upregulated genes that were common to multiple tissue types. Pathway upregulation in cell lines was most pronounced over metabolic pathways including cell nucleotide metabolism and oxidative

  13. Global Conservation of Protein Status between Cell Lines and Xenografts

    Directory of Open Access Journals (Sweden)

    Julian Biau

    2016-08-01

    Full Text Available Common preclinical models for testing anticancer treatment include cultured human tumor cell lines in monolayer, and xenografts derived from these cell lines in immunodeficient mice. Our goal was to determine how similar the xenografts are compared with their original cell line and to determine whether it is possible to predict the stability of a xenograft model beforehand. We studied a selection of 89 protein markers of interest in 14 human cell cultures and respective subcutaneous xenografts using the reverse-phase protein array technology. We specifically focused on proteins and posttranslational modifications involved in DNA repair, PI3K pathway, apoptosis, tyrosine kinase signaling, stress, cell cycle, MAPK/ERK signaling, SAPK/JNK signaling, NFκB signaling, and adhesion/cytoskeleton. Using hierarchical clustering, most cell culture-xenograft pairs cluster together, suggesting a global conservation of protein signature. Particularly, Akt, NFkB, EGFR, and Vimentin showed very stable protein expression and phosphorylation levels highlighting that 4 of 10 pathways were highly correlated whatever the model. Other proteins were heterogeneously conserved depending on the cell line. Finally, cell line models with low Akt pathway activation and low levels of Vimentin gave rise to more reliable xenograft models. These results may be useful for the extrapolation of cell culture experiments to in vivo models in novel targeted drug discovery.

  14. Induction of apoptosis by opium in some tumor cell lines.

    Science.gov (United States)

    Khaleghi, M; Farsinejad, A; Dabiri, S; Asadikaram, G

    2016-09-30

    The current study is aimed at investigation of the opium effects on the apoptosis of different cell lines in culture medium and compares such effects with one another. The study is carried out on over 8 cell lines (AA8, AGS, Hela, HepG2, MCF7, N2a, PC12, WEHI). A 2.86 x 10-4 g/ml opium concentration was prepared and added to the culture medium of the cell lines for 48 hours. Cytotoxicity was tested by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. The apoptotic effect of opium on the cell lines was analyzed by Annexin-PI test. Opium with concentration of 2.86 x 10-4 g/ml in 48 hours significantly induces apoptosis in certain cell lines (i.e. AA8, N2a, WEHI), apoptosis and necrosis in some others (i.e. Hela, HepG2, MCF7, and PC12), and also solely necrosis in the AGS cell line. One could infer that the usage of opium with different levels in different tissues leads to certain disorders in some tissues and may have therapeutic effects under distinctive conditions (i.e. unchecked growth of cells) as confirmed by the results.

  15. SU-F-T-678: Clotrimazole Sensitizes MCF-7 Breast Cancer Cell Line to Radiation

    Energy Technology Data Exchange (ETDEWEB)

    Garcia, L; Tambasco, M [San Diego State University, San Diego, CA (United States)

    2016-06-15

    Purpose: To study the effects of Clotrimazole (CLT) on radiosensitivity of MCF-7 Cells in correlation to detachment of Hexokinase II from the Voltage Dependent Anion Channel on the outer membrane of the mitochondria. Apoptotic fractions were also analyzed in relation to the detachment of Hexokinase. Methods: This study focused on the mammary adenocarcinoma cell line, MCF-7. Colony forming assays were used to analyze radiosensitization by CLT. Flow cytometry methods were used to analyze apoptotic vs necrotic fractions after treatment with CLT. Spectrophotometery was used to analyze the mitochondrial bound and soluble fraction of Hexokinase by means of relative enzymatic activity. Results: Our preliminary data have shown that CLT sensitizes MCF-7 cells to radiation in a dose and incubation time dependent manner up. We have also demonstrated that there are two radiosensitizing periods in MCF-7 cells with the first corresponding to the cycle arrest after 24 hours observed in other cell lines. The second radiosensitizing period occurs with incubation in CLT after irradiation which reaches maximum effect around 24 hours of incubation time. Preliminary data from our Hexokinase detachment assay show a factor of two increase in the ratio of unbound to bound Hexokinase when comparing incubation for 24 hours in media containing 0 and 20 µM CLT. Conclusion: This study and others indicate CLT as a possible radiosensitizing agent in cancer therapies. While CLT itself shows toxicity to the liver in high doses, this study further demonstrates that disruption of the Warburg Effect and unbinding of mitochondrial bound Hexokinase as a possible pathway for cancer treatment.

  16. SU-F-T-678: Clotrimazole Sensitizes MCF-7 Breast Cancer Cell Line to Radiation

    International Nuclear Information System (INIS)

    Garcia, L; Tambasco, M

    2016-01-01

    Purpose: To study the effects of Clotrimazole (CLT) on radiosensitivity of MCF-7 Cells in correlation to detachment of Hexokinase II from the Voltage Dependent Anion Channel on the outer membrane of the mitochondria. Apoptotic fractions were also analyzed in relation to the detachment of Hexokinase. Methods: This study focused on the mammary adenocarcinoma cell line, MCF-7. Colony forming assays were used to analyze radiosensitization by CLT. Flow cytometry methods were used to analyze apoptotic vs necrotic fractions after treatment with CLT. Spectrophotometery was used to analyze the mitochondrial bound and soluble fraction of Hexokinase by means of relative enzymatic activity. Results: Our preliminary data have shown that CLT sensitizes MCF-7 cells to radiation in a dose and incubation time dependent manner up. We have also demonstrated that there are two radiosensitizing periods in MCF-7 cells with the first corresponding to the cycle arrest after 24 hours observed in other cell lines. The second radiosensitizing period occurs with incubation in CLT after irradiation which reaches maximum effect around 24 hours of incubation time. Preliminary data from our Hexokinase detachment assay show a factor of two increase in the ratio of unbound to bound Hexokinase when comparing incubation for 24 hours in media containing 0 and 20 µM CLT. Conclusion: This study and others indicate CLT as a possible radiosensitizing agent in cancer therapies. While CLT itself shows toxicity to the liver in high doses, this study further demonstrates that disruption of the Warburg Effect and unbinding of mitochondrial bound Hexokinase as a possible pathway for cancer treatment.

  17. Lining cells on normal human vertebral bone surfaces

    International Nuclear Information System (INIS)

    Henning, C.B.; Lloyd, E.L.

    1982-01-01

    Thoracic vertebrae from two individuals with no bone disease were studied with the electron microscope to determine cell morphology in relation to bone mineral. The work was undertaken to determine if cell morphology or spatial relationships between the bone lining cells and bone mineral could account for the relative infrequency of bone tumors which arise at this site following radium intake, when compared with other sites, such as the head of the femur. Cells lining the vertebral mineral were found to be generally rounded in appearance with varied numbers of cytoplasmic granules, and they appeared to have a high density per unit of surface area. These features contrasted with the single layer of flattened cells characteristic of the bone lining cells of the femur. A tentative discussion of the reasons for the relative infrequency of tumors in the vertebrae following radium acquisition is presented

  18. Molybdenum induces pancreatic β-cell dysfunction and apoptosis via interdependent of JNK and AMPK activation-regulated mitochondria-dependent and ER stress-triggered pathways

    International Nuclear Information System (INIS)

    Yang, Tsung-Yuan; Yen, Cheng-Chieh; Lee, Kuan-I; Su, Chin-Chuan; Yang, Ching-Yao; Wu, Chin-Ching; Hsieh, Shang-Shu; Ueng, Kwo-Chang; Huang, Chun-Fa

    2016-01-01

    Molybdenum (Mo), a well-known toxic environmental and industrial pollutant, causes adverse health effects and diseases in humans and has received attention as a potential risk factor for DM. However, the roles of Mo in the mechanisms of the toxicological effects in pancreatic β-cells are mostly unclear. In this study, the results revealed dysfunction of insulin secretion and apoptosis in the pancreatic β-cell-derived RIN-m5F cells and the isolated mouse islets in response to Mo. These effects were accompanied by a mitochondria-dependent apoptotic signals including a decreased in the MMP, an increase in cytochrome c release, and the activation of caspase cascades and PARP. In addition, ER stress was triggered as indicated by several key molecules of the UPR. Furthermore, exposure to Mo induced the activation of ERK1/2, JNK, AMPKα, and GSK3-α/β. Pretreatment with specific pharmacological inhibitors (in RIN-m5F cells and isolated mouse islets) of JNK (SP600125) and AMPK (Compound C) or transfection with si-RNAs (in RIN-m5F cells) specific to JNK and AMPKα effectively prevented the Mo-induced apoptosis and related signals, but inhibitors of ERK1/2 and GSK3-α/β (PD98059 and LiCl, respectively) did not reverse the Mo-induced effects. Additionally, both the inhibitors and specific si-RNAs could suppress the Mo-induced phosphorylation of JNK and AMPKα each other. Taken together, these results suggest that Mo exerts its cytotoxicity on pancreatic β-cells by inducing dysfunction and apoptosis via interdependent JNK and AMPK activation downstream-regulated mitochondrial-dependent and ER stress-triggered apoptosis pathways. - Highlights: • Molybdenum (Mo) induces pancreatic β-cell dysfunction and apoptosis. • Mo causes β-cell death via mitochondria-dependent caspase cascades signals. • ER stress-triggered apoptotic pathway also regulates Mo-induced β-cell death. • Interdependent of JNK and AMPK activation involves in Mo-induced β-cell apoptosis.

  19. Molybdenum induces pancreatic β-cell dysfunction and apoptosis via interdependent of JNK and AMPK activation-regulated mitochondria-dependent and ER stress-triggered pathways

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Tsung-Yuan [Institute of Medicine, Chung Shan Medical University, Taichung 402, Taiwan (China); Department of Internal Medicine, Chung Shan Medical University Hospital, Taichung 402, Taiwan (China); Yen, Cheng-Chieh [Department of Occupational Safety and Health, College of Health Care and Management, Chung Shan Medical University, Taichung 402, Taiwan (China); Department of Occupational Medicine, Chung Shan Medical University Hospital, Taichung 402, Taiwan (China); Lee, Kuan-I [Department of Emergency, Taichung Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Taichung 427, Taiwan (China); Su, Chin-Chuan [Department of Otorhinolaryngology, Head and Neck Surgery, Changhua Christian Hospital, Changhua County 500, Taiwan (China); Graduate Institute of Basic Medical Science, School of Medicine, College of Medicine, China Medical University, Taichung 404, Taiwan (China); Yang, Ching-Yao [Department of Surgery, National Taiwan University Hospital, Taipei 100, Taiwan (China); Department of Surgery, College of Medicine, National Taiwan University, Taipei 100, Taiwan (China); Wu, Chin-Ching [Department of Public Health, China Medical University, Taichung 404, Taiwan (China); Hsieh, Shang-Shu, E-mail: gile1123@yahoo.com.tw [Department of Emergency, Taichung Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Taichung 427, Taiwan (China); Ueng, Kwo-Chang, E-mail: kcueng@gmail.com [Department of Internal Medicine, Chung Shan Medical University Hospital, Taichung 402, Taiwan (China); School of Medicine, Chung Shan Medical University, Taichung 402, Taiwan (China); Huang, Chun-Fa, E-mail: cfhuang@mail.cmu.edu.tw [School of Chinese Medicine, College of Chinese Medicine, China Medical University, Taichung 404, Taiwan (China)

    2016-03-01

    Molybdenum (Mo), a well-known toxic environmental and industrial pollutant, causes adverse health effects and diseases in humans and has received attention as a potential risk factor for DM. However, the roles of Mo in the mechanisms of the toxicological effects in pancreatic β-cells are mostly unclear. In this study, the results revealed dysfunction of insulin secretion and apoptosis in the pancreatic β-cell-derived RIN-m5F cells and the isolated mouse islets in response to Mo. These effects were accompanied by a mitochondria-dependent apoptotic signals including a decreased in the MMP, an increase in cytochrome c release, and the activation of caspase cascades and PARP. In addition, ER stress was triggered as indicated by several key molecules of the UPR. Furthermore, exposure to Mo induced the activation of ERK1/2, JNK, AMPKα, and GSK3-α/β. Pretreatment with specific pharmacological inhibitors (in RIN-m5F cells and isolated mouse islets) of JNK (SP600125) and AMPK (Compound C) or transfection with si-RNAs (in RIN-m5F cells) specific to JNK and AMPKα effectively prevented the Mo-induced apoptosis and related signals, but inhibitors of ERK1/2 and GSK3-α/β (PD98059 and LiCl, respectively) did not reverse the Mo-induced effects. Additionally, both the inhibitors and specific si-RNAs could suppress the Mo-induced phosphorylation of JNK and AMPKα each other. Taken together, these results suggest that Mo exerts its cytotoxicity on pancreatic β-cells by inducing dysfunction and apoptosis via interdependent JNK and AMPK activation downstream-regulated mitochondrial-dependent and ER stress-triggered apoptosis pathways. - Highlights: • Molybdenum (Mo) induces pancreatic β-cell dysfunction and apoptosis. • Mo causes β-cell death via mitochondria-dependent caspase cascades signals. • ER stress-triggered apoptotic pathway also regulates Mo-induced β-cell death. • Interdependent of JNK and AMPK activation involves in Mo-induced β-cell apoptosis.

  20. DNA fingerprinting of the NCI-60 cell line panel.

    Science.gov (United States)

    Lorenzi, Philip L; Reinhold, William C; Varma, Sudhir; Hutchinson, Amy A; Pommier, Yves; Chanock, Stephen J; Weinstein, John N

    2009-04-01

    The National Cancer Institute's NCI-60 cell line panel, the most extensively characterized set of cells in existence and a public resource, is frequently used as a screening tool for drug discovery. Because many laboratories around the world rely on data from the NCI-60 cells, confirmation of their genetic identities represents an essential step in validating results from them. Given the consequences of cell line contamination or misidentification, quality control measures should routinely include DNA fingerprinting. We have, therefore, used standard DNA microsatellite short tandem repeats to profile the NCI-60, and the resulting DNA fingerprints are provided here as a reference. Consistent with previous reports, the fingerprints suggest that several NCI-60 lines have common origins: the melanoma lines MDA-MB-435, MDA-N, and M14; the central nervous system lines U251 and SNB-19; the ovarian lines OVCAR-8 and OVCAR-8/ADR (also called NCI/ADR); and the prostate lines DU-145, DU-145 (ATCC), and RC0.1. Those lines also show that the ability to connect two fingerprints to the same origin is not affected by stable transfection or by the development of multidrug resistance. As expected, DNA fingerprints were not able to distinguish different tissues-of-origin. The fingerprints serve principally as a barcodes.

  1. Mitochondria: more than just a powerhouse.

    Science.gov (United States)

    McBride, Heidi M; Neuspiel, Margaret; Wasiak, Sylwia

    2006-07-25

    Pioneering biochemical studies have long forged the concept that the mitochondria are the 'energy powerhouse of the cell'. These studies, combined with the unique evolutionary origin of the mitochondria, led the way to decades of research focusing on the organelle as an essential, yet independent, functional component of the cell. Recently, however, our conceptual view of this isolated organelle has been profoundly altered with the discovery that mitochondria function within an integrated reticulum that is continually remodeled by both fusion and fission events. The identification of a number of proteins that regulate these activities is beginning to provide mechanistic details of mitochondrial membrane remodeling. However, the broader question remains regarding the underlying purpose of mitochondrial dynamics and the translation of these morphological transitions into altered functional output. One hypothesis has been that mitochondrial respiration and metabolism may be spatially and temporally regulated by the architecture and positioning of the organelle. Recent evidence supports and expands this idea by demonstrating that mitochondria are an integral part of multiple cell signaling cascades. Interestingly, proteins such as GTPases, kinases and phosphatases are involved in bi-directional communication between the mitochondrial reticulum and the rest of the cell. These proteins link mitochondrial function and dynamics to the regulation of metabolism, cell-cycle control, development, antiviral responses and cell death. In this review we will highlight the emerging evidence that provides molecular definition to mitochondria as a central platform in the execution of diverse cellular events.

  2. Functional Mitochondria in Health and Disease.

    Science.gov (United States)

    Herst, Patries M; Rowe, Matthew R; Carson, Georgia M; Berridge, Michael V

    2017-01-01

    The ability to rapidly adapt cellular bioenergetic capabilities to meet rapidly changing environmental conditions is mandatory for normal cellular function and for cancer progression. Any loss of this adaptive response has the potential to compromise cellular function and render the cell more susceptible to external stressors such as oxidative stress, radiation, chemotherapeutic drugs, and hypoxia. Mitochondria play a vital role in bioenergetic and biosynthetic pathways and can rapidly adjust to meet the metabolic needs of the cell. Increased demand is met by mitochondrial biogenesis and fusion of individual mitochondria into dynamic networks, whereas a decrease in demand results in the removal of superfluous mitochondria through fission and mitophagy. Effective communication between nucleus and mitochondria (mito-nuclear cross talk), involving the generation of different mitochondrial stress signals as well as the nuclear stress response pathways to deal with these stressors, maintains bioenergetic homeostasis under most conditions. However, when mitochondrial DNA (mtDNA) mutations accumulate and mito-nuclear cross talk falters, mitochondria fail to deliver critical functional outputs. Mutations in mtDNA have been implicated in neuromuscular and neurodegenerative mitochondriopathies and complex diseases such as diabetes, cardiovascular diseases, gastrointestinal disorders, skin disorders, aging, and cancer. In some cases, drastic measures such as acquisition of new mitochondria from donor cells occurs to ensure cell survival. This review starts with a brief discussion of the evolutionary origin of mitochondria and summarizes how mutations in mtDNA lead to mitochondriopathies and other degenerative diseases. Mito-nuclear cross talk, including various stress signals generated by mitochondria and corresponding stress response pathways activated by the nucleus are summarized. We also introduce and discuss a small family of recently discovered hormone-like mitopeptides

  3. Functional Mitochondria in Health and Disease

    Directory of Open Access Journals (Sweden)

    Patries M. Herst

    2017-11-01

    Full Text Available The ability to rapidly adapt cellular bioenergetic capabilities to meet rapidly changing environmental conditions is mandatory for normal cellular function and for cancer progression. Any loss of this adaptive response has the potential to compromise cellular function and render the cell more susceptible to external stressors such as oxidative stress, radiation, chemotherapeutic drugs, and hypoxia. Mitochondria play a vital role in bioenergetic and biosynthetic pathways and can rapidly adjust to meet the metabolic needs of the cell. Increased demand is met by mitochondrial biogenesis and fusion of individual mitochondria into dynamic networks, whereas a decrease in demand results in the removal of superfluous mitochondria through fission and mitophagy. Effective communication between nucleus and mitochondria (mito-nuclear cross talk, involving the generation of different mitochondrial stress signals as well as the nuclear stress response pathways to deal with these stressors, maintains bioenergetic homeostasis under most conditions. However, when mitochondrial DNA (mtDNA mutations accumulate and mito-nuclear cross talk falters, mitochondria fail to deliver critical functional outputs. Mutations in mtDNA have been implicated in neuromuscular and neurodegenerative mitochondriopathies and complex diseases such as diabetes, cardiovascular diseases, gastrointestinal disorders, skin disorders, aging, and cancer. In some cases, drastic measures such as acquisition of new mitochondria from donor cells occurs to ensure cell survival. This review starts with a brief discussion of the evolutionary origin of mitochondria and summarizes how mutations in mtDNA lead to mitochondriopathies and other degenerative diseases. Mito-nuclear cross talk, including various stress signals generated by mitochondria and corresponding stress response pathways activated by the nucleus are summarized. We also introduce and discuss a small family of recently discovered hormone

  4. Application of DNA fingerprints for cell-line individualization.

    OpenAIRE

    Gilbert, D A; Reid, Y A; Gail, M H; Pee, D; White, C; Hay, R J; O'Brien, S J

    1990-01-01

    DNA fingerprints of 46 human cell lines were derived using minisatellite probes for hypervariable genetic loci. The incidence of 121 HaeIII DNA fragments among 33 cell lines derived from unrelated individuals was used to estimate allelic and genotypic frequencies for each fragment and for composite individual DNA fingerprints. We present a quantitative estimate of the extent of genetic difference between individuals, an estimate based on the percentage of restriction fragments at which they d...

  5. Peroxisome-mitochondria interplay and disease.

    Science.gov (United States)

    Schrader, Michael; Costello, Joseph; Godinho, Luis F; Islinger, Markus

    2015-07-01

    Peroxisomes and mitochondria are ubiquitous, highly dynamic organelles with an oxidative type of metabolism in eukaryotic cells. Over the years, substantial evidence has been provided that peroxisomes and mitochondria exhibit a close functional interplay which impacts on human health and development. The so-called "peroxisome-mitochondria connection" includes metabolic cooperation in the degradation of fatty acids, a redox-sensitive relationship, an overlap in key components of the membrane fission machineries and cooperation in anti-viral signalling and defence. Furthermore, combined peroxisome-mitochondria disorders with defects in organelle division have been revealed. In this review, we present the latest progress in the emerging field of peroxisomal and mitochondrial interplay in mammals with a particular emphasis on cooperative fatty acid β-oxidation, redox interplay, organelle dynamics, cooperation in anti-viral signalling and the resulting implications for disease.

  6. Mechanisms of communication between mitochondria and lysosomes.

    Science.gov (United States)

    Raimundo, Nuno; Fernández-Mosquera, Lorena; Yambire, King Faisal; Diogo, Cátia V

    2016-10-01

    Mitochondria and lysosomes have long been studied in the context of their classic functions: energy factory and recycle bin, respectively. In the last twenty years, it became evident that these organelles are much more than simple industrial units, and are indeed in charge of many of cellular processes. Both mitochondria and lysosomes are now recognized as far-reaching signaling platforms, regulating many key aspects of cell and tissue physiology. It has furthermore become clear that mitochondria and lysosomes impact each other. The mechanisms underlying the cross-talk between these organelles are only now starting to be addressed. In this review, we briefly summarize how mitochondria, lysosomes and the lysosome-related process of autophagy affect each other in physiology and pathology. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. Isolation of Mitochondria from Potato Tubers

    DEFF Research Database (Denmark)

    Havelund, Jesper F.; Salvato, Fernanda; Chen, Mingjie

    2014-01-01

    One way to study the function of plant mitochondria is to extract them from plant tissues in an uncontaminated, intact and functional form. The reductionist assumption is that the components present in such a preparation and the in vitro measurable functions or activities reliably reflect...... the in vivo properties of the organelle inside the plant cell. Here, we describe a method to isolate mitochondria from a relatively homogeneous plant tissue, the dormant potato tuber. The homogenization is done using a juice extractor, which is a relatively gentle homogenization procedure where...... the mitochondria are only exposed to strong shearing forces once. After removal of starch and large tissue pieces by filtration, differential centrifugation is used to remove residual starch as well as larger organelles. The crude mitochondria are then first purified by using a step Percoll gradient...

  8. Establishment of cell lines from adult T-cell leukemia cells dependent on negatively charged polymers.

    Science.gov (United States)

    Kagami, Yoshitoyo; Uchiyama, Susumu; Kato, Harumi; Okada, Yasutaka; Seto, Masao; Kinoshita, Tomohiro

    2017-07-05

    Growing adult T-cell leukemia/lymphoma (ATLL) cells in vitro is difficult. Here, we examined the effects of static electricity in the culture medium on the proliferation of ATLL cells. Six out of 10 ATLL cells did not proliferate in vitro and thus had to be cultured in a medium containing negatively charged polymers. In the presence of poly-γ-glutamic acid (PGA) or chondroitin sulfate (CDR), cell lines (HKOX3-PGA, HKOX3-CDR) were established from the same single ATLL case using interleukin (IL)-2, IL-4, and feeder cells expressing OX40L (OX40L + HK). Dextran sulfate inhibited growth in both HKOX3 cell lines. Both PGA and OX40L + HK were indispensable for HKOX3-PGA growth, but HKOX3-CDR could proliferate in the presence of CDR or OX40L + HK alone. Thus, the specific action of each negatively charged polymer promoted the growth of specific ATLL cells in vitro.

  9. Isolation of two chloroethylnitrosourea-sensitive Chinese hamster cell lines

    International Nuclear Information System (INIS)

    Hata, H.; Numata, M.; Tohda, H.; Yasui, A.; Oikawa, A.

    1991-01-01

    1-[(4-Amino-2-methylpyrimidin-5-yl)methyl]-3-(2-chloroethyl)-3- nitrosourea hydrochloride (ACNU), a cancer chemotherapeutic bifunctional alkylating agent, causes chloroethylation of DNA and subsequent DNA strand cross-linking through an ethylene bridge. We isolated and characterized two ACNU-sensitive mutants from mutagenized Chinese hamster ovary cells and found them to be new drug-sensitive recessive Chinese hamster mutants. Both mutants were sensitive to various monofunctional alkylating agents in a way similar to that of the parental cell lines CHO9. One mutant (UVS1) was cross-sensitive to UV and complemented the UV sensitivity of all Chinese hamster cell lines of 7 established complementation groups. Since UV-induced unscheduled DNA synthesis was very low, a new locus related to excision repair is thought to be defective in this cell line. Another ACNU-sensitive mutant, CNU1, was slightly more sensitive to UV than the parent cell line. CNU1 was cross-sensitive to 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea and slightly more sensitive to mitomycin C. No increased accumulation of ACNU and a low level of UV-induced unscheduled DNA synthesis in this cell as compared with the parental cell line suggest that there is abnormality in a repair response of this mutant cell to some types of DNA cross-links

  10. In vitro Rb-1 gene transfer to retinoblastoma cell lines

    International Nuclear Information System (INIS)

    Choi, Sang Wook; Ham, Yong Hoh; Kim, Mee Heui

    1994-04-01

    After transfection of Rb-vector to packaging cell line (CRIP) by Ca-P precipitation method, we could select nineteen colonies of G-418 resistant clone by ring cloning. Each colony was transduced to NIH3T3 cells to select the one which produces high titer virus. After NIH3T3 cells transduction, we could get 28 colony counts for the high, 127 for the middle, and 6 for the low viral titer. With the supernatant of the high viral titer colony (CRIPRb 2-5). We transduct retinoblastoma cell lines. 5 figs, 11 refs. (Author)

  11. Airborne urban particles (Milan winter-PM2.5) cause mitotic arrest and cell death: Effects on DNA, mitochondria, AhR binding and spindle organization

    Energy Technology Data Exchange (ETDEWEB)

    Gualtieri, Maurizio [Applied Cell Biology and Particles Effects, Department of Environmental Science, University Milano-Bicocca, Piazza della Scienza 1, 20126 Milano (Italy); Division of Environmental Medicine, Norwegian Institute of Public Health, P.O. Box 4404 Nydalen, N-0403 Oslo (Norway); Ovrevik, Johan [Division of Environmental Medicine, Norwegian Institute of Public Health, P.O. Box 4404 Nydalen, N-0403 Oslo (Norway); Mollerup, Steen [Section for Toxicology, National Institute of Occupational Health, N-0033 Oslo (Norway); Asare, Nana [Division of Environmental Medicine, Norwegian Institute of Public Health, P.O. Box 4404 Nydalen, N-0403 Oslo (Norway); Longhin, Eleonora [Applied Cell Biology and Particles Effects, Department of Environmental Science, University Milano-Bicocca, Piazza della Scienza 1, 20126 Milano (Italy); Dahlman, Hans-Jorgen [Division of Environmental Medicine, Norwegian Institute of Public Health, P.O. Box 4404 Nydalen, N-0403 Oslo (Norway); Camatini, Marina [Applied Cell Biology and Particles Effects, Department of Environmental Science, University Milano-Bicocca, Piazza della Scienza 1, 20126 Milano (Italy); Centre Research POLARIS, Department of Environmental Science, University Milano-Bicocca, Piazza della Scienza 1, 20126 Milano (Italy); Holme, Jorn A., E-mail: jorn.holme@fhi.no [Division of Environmental Medicine, Norwegian Institute of Public Health, P.O. Box 4404 Nydalen, N-0403 Oslo (Norway)

    2011-08-01

    Highlights: {yields} PM2.5 induces mitotic arrest in BEAS-2B cells. {yields} PM2.5 induces DNA damage and activates DNA damage response. {yields} AhR regulated genes (Cyp1A1, Cyp1B1 and AhRR) are upregulated after PM exposure. {yields} Mitotic spindle assembly is perturbed in PM exposed cells. - Abstract: Airborne particulate matter (PM) is considered to be an important contributor to lung diseases. In the present study we report that Milan winter-PM2.5 inhibited proliferation in human bronchial epithelial cells (BEAS-2B) by inducing mitotic arrest. The cell cycle arrest was followed by an increase in mitotic-apoptotic cells, mitotic slippage and finally an increase in 'classical' apoptotic cells. Exposure to winter-PM10 induced only a slight effect which may be due to the presence of PM2.5 in this fraction while pure combustion particles failed to disturb mitosis. Fewer cells expressing the mitosis marker phospho-histone H3 compared to cells with condensed chromosomes, suggest that PM2.5 induced premature mitosis. PM2.5 was internalized into the cells and often localized in laminar organelles, although particles without apparent plasma membrane covering were also seen. In PM-containing cells mitochondria and lysosomes were often damaged, and in mitotic cells fragmented chromosomes often appeared. PM2.5 induced DNA strands breaks and triggered a DNA-damage response characterized by increased phosphorylation of ATM, Chk2 and H2AX; as well as induced a marked increase in expression of the aryl hydrocarbon receptor (AhR)-regulated genes, CYP1A1, CYP1B1 and AhRR. Furthermore, some disturbance of the organization of microtubules was indicated. It is hypothesized that the induced mitotic arrest and following cell death was due to a premature chromosome condensation caused by a combination of DNA, mitochondrial and spindle damage.

  12. Establishment and characterization of a novel osteosarcoma cell line: CHOS.

    Science.gov (United States)

    Liu, Yunlu; Feng, Xiaobo; Zhang, Yukun; Jiang, Hongyan; Cai, Xianyi; Yan, Xinxin; Huang, Zengfa; Mo, Fengbo; Yang, Wen; Yang, Cao; Yang, Shuhua; Liu, Xianzhe

    2016-12-01

    Osteosarcoma has a well-recognized bimodal distribution, with the first peak in adolescence and another in the elderly age-group. The elderly patients have different clinical features and a poorer prognosis as compared to adolescents. To better understand the biological features of osteosarcoma in the elderly population, we established a new human osteosarcoma cell line from a 58-year-old man with primary chondroblastic osteosarcoma. After 6 months of continuous culture in vitro for over 50 passages, an immortalized cell line CHOS was established. The cell line was well-characterized by cytogenetic, biomarker, functional, and histological analyses. The CHOS cells exhibited a spindle-shaped morphology and a doubling time of 36 h. Cytogenetic analysis of CHOS cells revealed the loss of chromosome Y and the gain of chromosome 12. Quantitative real-time polymerase chain reaction (RT-PCR), Western blotting and/or immunofluorescence revealed the expression of chondroblastic, mesenchymal and tumor metastasis markers in the CHOS cells. Compared with the osteosarcoma cell line, the CHOS cells were found to be more sensitive to cisplatin and doxorubicin, but were resistant to methotrexate. The cell line was highly tumorigenic and maintained the histological characteristics and invasive nature of the original tumor. Furthermore, on immunohistochemical analysis, the xenografts and metastases were found to co-express collagen II, aggrecan, vimentin and S100A4 that resembled the original tumor cells. Our results indicate, the potential of CHOS cell line to serve as a useful tool for further studies on the molecular biology of osteosarcoma, especially in the elderly patients. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:2116-2125, 2016. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  13. Membrane lipidome of an epithelial cell line

    DEFF Research Database (Denmark)

    Sampaio, Julio L; Gerl, Mathias J; Klose, Christian

    2011-01-01

    Tissue differentiation is an important process that involves major cellular membrane remodeling. We used Madin-Darby canine kidney cells as a model for epithelium formation and investigated the remodeling of the total cell membrane lipidome during the transition from a nonpolarized morphology...... to an epithelial morphology and vice versa. To achieve this, we developed a shotgun-based lipidomics workflow that enabled the absolute quantification of mammalian membrane lipidomes with minimal sample processing from low sample amounts. Epithelial morphogenesis was accompanied by a major shift from sphingomyelin...... to generate an apical membrane domain that serves as a protective barrier for the epithelial sheet....

  14. Antiapoptotic effect of novel compound from Herba leonuri - leonurine (SCM-198): a mechanism through inhibition of mitochondria dysfunction in H9c2 cells.

    Science.gov (United States)

    Liu, Xin Hua; Pan, Li Long; Gong, Qi Hai; Zhu, Yi Zhun

    2010-12-01

    Apoptosis of cardiomyocytes induced by oxidative stress play a critical role in cardiac dysfunction associated with ventricular remodeling and heart failure. We recently reported that leonurine attenuated hypoxia-induced cardiomyocyte damage. In this study, we investigated the mechanism of leonurine (originally from Herba leonuri but we synthesized it chemically it as also called SCM-198) (H₂O₂)-induced rat embryonic heart-derived H9c2 cells from apoptosis. Exposing H9c2 cells to H₂O₂ significantly decreased cell viability, and this was attenuated by pretreatment with leonurine for 4 h in a concentration-dependent manner. Meanwhile, leonurine was found to reduce intracellular reactive oxygen species (ROS) generation in H₂O₂-stimulated cell. Moreover, H9c2 cells stimulated by H₂O₂ was accompanied with apparent apoptotic characteristics, including fragmentation of DNA, apoptotic body formation, release of cytochrome c, translocation of Bax to mitochondria, loss of mitochondrial membrane potential (ΔΨ(m)) and activation of caspase 3. Furthermore, H₂O₂ also induced rapid and significant phosphorylation of the c-Jun-N-terminal kinase 1/2 (JNK1/2), which was inhibited SP600125 (a JNK1/2 inhibitor). All of these events were attenuated by leonurine pretreatment. Taken together, these results demonstrated that leonurine could protect H9c2 cells from H₂O₂-induced apoptosis via modulation of mitochondrial dysfunction associated with blocking the activation of JNK1/2.

  15. Effect of failures and repairs on multiple cell production lines

    Energy Technology Data Exchange (ETDEWEB)

    Legato, P.; Bobbio, A.; Roberti, L.

    1989-01-01

    This paper examines a production line composed of multiple stages, or cells, which are passed in sequential order to arrive to the final product. Two possible coordination disciplines are considered, namely: the classical tandem arrangement of sequential working centers with input buffer and the kanban scheme, considered the Japanese shop floor realization of the Just-In-Time (JIT) manifacturing approach. The production line is modelled and analysed by means of Stochastic Petri Nets (SPN). Finally an analysis is made of the possibility that the working cells can incur failure/repair cycles perturbing the production flow of the line and thus reduce performance indices.

  16. Tumor-Selective Cytotoxicity of Nitidine Results from Its Rapid Accumulation into Mitochondria

    Directory of Open Access Journals (Sweden)

    Hironori Iwasaki

    2017-01-01

    Full Text Available We identified a nitidine- (NTD- accumulating organelle and evaluated the net cytotoxicity of accumulated NTD. To evaluate tumor cell selectivity of the drug, we evaluated its selective cytotoxicity against 39 human cancer cell lines (JFCR39 panel, and the profile was compared with those of known anticancer drugs. Organelle specificity of NTD was visualized using organelle-targeted fluorescent proteins. Real-time analysis of cell growth, proliferation, and cytotoxicity was performed using the xCELLigence system. Selectivity of NTD in the JFCR39 panel was evaluated. Mitochondria-specific accumulation of NTD was observed. Real-time cytotoxicity analysis suggested that the mechanism of NTD-induced cell death is independent of the cell cycle. Short-term treatment indicated that this cytotoxicity only resulted from the accumulation of NTD into the mitochondria. The results from the JFCR39 panel indicated that NTD-mediated cytotoxicity resulted from unique mechanisms compared with those of other known anticancer drugs. These results suggested that the cytotoxicity of NTD is only induced by its accumulation in mitochondria. The drug triggered mitochondrial dysfunction in less than 2 h. Similarity analysis of the selectivity of NTD in 39 tumor cell lines strongly supported the unique tumor cell specificity of NTD. Thus, these features indicate that NTD may be a promising antitumor drug for new combination chemotherapies.

  17. Mitochondrial modulation of oxygen-dependent radiosensitivity in some human tumour cell lines.

    LENUS (Irish Health Repository)

    Anoopkumar-Dukie, S

    2009-10-01

    Oxygen-dependent radiosensitivity of tumour cells reflects direct oxidative damage to DNA, but non-nuclear mechanisms including signalling pathways may also contribute. Mitochondria are likely candidates because not only do they integrate signals from each of the main kinase pathways but mitochondrial kinases responsive to oxidative stress communicate to the rest of the cell. Using pharmacological and immunochemical methods, we tested the role of mitochondrial permeability transition (MPT) and the Bcl-2 proteins in oxygen-dependent radiosensitivity. Drug-treated or untreated cervical cancer HeLa, breast cancer MCF-7 and melanoma MeWo cell lines were irradiated at 6.2 Gy under normoxic and hypoxic conditions then allowed to proliferate for 7 days. The MPT blocker cyclosporin A (2 microM) strongly protected HeLa but not the other two lines against oxygen-dependent radiosensitivity. By contrast, bongkrekic acid (50 microM), which blocks MPT by targeting the adenine nucleotide transporter, had only marginal effect and calcineurin inhibitor FK-506 (0.1 microM) had none. Nor was evidence found for the modulation of oxygen-dependent radiosensitivity by Bax\\/Bcl-2 signalling, mitochondrial ATP-dependent potassium (mitoK(ATP)) channels or mitochondrial Ca(2+) uptake. In conclusion, calcineurin-independent protection by cyclosporin A suggests that MPT but not mitoK(ATP) or the mitochondrial apoptosis pathway plays a causal role in oxygen-dependent radiosensitivity of HeLa cells. Targeting MPT may therefore improve the effectiveness of radiotherapy in some solid tumours.

  18. Selection of radioresistant cells by vitamin A deficiency in a small cell lung cancer cell line

    International Nuclear Information System (INIS)

    Terasaki, Takeo; Shimosato, Yukio; Wada, Makio; Yokota, Jun; Terada, Masaaki

    1990-01-01

    Radiation sensitivity of a human small cell lung cancer cell line, Lu-134-B cells, cultured in serum-supplemented medium and of cells transferred to and cultured in delipidized serum-supplemented (vitamin A-deficient) medium was studied. The cells cultured in serum-supplemented medium showed the phenotype of classic small cell lung cancer sensitive to radiation, while cells transferred to delipidized serum-supplemented medium showed partial squamous cell differentiation and became resistant to radiation. These results suggest that some small cell lung cancer cells in vitro change their morphology and radiosensitivity depending on the culture conditions. The change in radiosensitivity was reproducible, and was not reversible by culture of the radioresistant cells in delipidized serum-supplemented medium with addition of retinoic acid (vitamin A-sufficient medium) for two months, although squamous cells disappeared. Acquisition of radioresistancy was considered to occur as the result of clonal selective growth in delipidized medium of a minor cell population in the original cell culture, based on a study of chromosome number. It was also found that there was no association of myc-family oncogenes with the changes of radiosensitivity in this cell line. (author)

  19. Intra- and Intercellular Quality Control Mechanisms of Mitochondria

    Directory of Open Access Journals (Sweden)

    Yoshimitsu Kiriyama

    2017-12-01

    Full Text Available Mitochondria function to generate ATP and also play important roles in cellular homeostasis, signaling, apoptosis, autophagy, and metabolism. The loss of mitochondrial function results in cell death and various types of diseases. Therefore, quality control of mitochondria via intra- and intercellular pathways is crucial. Intracellular quality control consists of biogenesis, fusion and fission, and degradation of mitochondria in the cell, whereas intercellular quality control involves tunneling nanotubes and extracellular vesicles. In this review, we outline the current knowledge on the intra- and intercellular quality control mechanisms of mitochondria.

  20. The toxicity of rifampicin polylactic acid nanoparticles against Mycobacterium bovis BCG and human macrophage THP-1 cell line

    International Nuclear Information System (INIS)

    Erokhina, M; Rybalkina, E; Lepekha, L; Barsegyan, G; Onishchenko, G

    2015-01-01

    Tuberculosis is rapidly becoming a major health problem. The rise in tuberculosis incidence stimulates efforts to develop more effective delivery systems for the existing antituberculous drugs while decreasing the side effects. The nanotechnology may provide novel drug delivery tools allowing controlled drug release. Rifampicin is one of the main antituberculous drugs, characterized by high toxicity, and Poly (L-lactic acid) (PLLA) is a biodegradable polymer used for the preparation of encapsulated drugs. The aim of our work was to evaluate the toxicity of rifampicin-PLLA nanoparticles against Mycobacterium bovis BCG using human macrophage THP-1 cell line. Our data demonstrate that rifampicin-PLLA is effective against M. bovis BCG in the infected macrophages. The drug is inducing the dysfunction of mitochondria and apoptosis in the macrophages and is acting as a potential substrate of Pgp thereby modulating cell chemosensitivity. The severity of the toxic effects of the rifampicin-PLLA nanoparticles is increasing in a dose-dependent manner. We suggest that free rifampicin induces death of M. bovis BCG after PLLA degradation and diffusion from phago-lysosomes to cytoplasm causing mitochondria dysfunction and affecting the Pgp activity. (paper)

  1. Solid Oxide Fuel Cell Systems PVL Line

    International Nuclear Information System (INIS)

    Shearer, Susan; Rush, Gregory

    2012-01-01

    In July 2010, Stark State College (SSC), received Grant DE-EE0003229 from the U.S. Department of Energy (DOE), Golden Field Office, for the development of the electrical and control systems, and mechanical commissioning of a unique 20kW scale high-pressure, high temperature, natural gas fueled Stack Block Test System (SBTS). SSC worked closely with subcontractor, Rolls-Royce Fuel Cell Systems (US) Inc. (RRFCS) over a 13 month period to successfully complete the project activities. This system will be utilized by RRFCS for pre-commercial technology development and training of SSC student interns. In the longer term, when RRFCS is producing commercial products, SSC will utilize the equipment for workforce training. In addition to DOE Hydrogen, Fuel Cells, and Infrastructure Technologies program funding, RRFCS internal funds, funds from the state of Ohio, and funding from the DOE Solid State Energy Conversion Alliance (SECA) program have been utilized to design, develop and commission this equipment. Construction of the SBTS (mechanical components) was performed under a Grant from the State of Ohio through Ohio's Third Frontier program (Grant TECH 08-053). This Ohio program supported development of a system that uses natural gas as a fuel. Funding was provided under the Department of Energy (DOE) Solid-state Energy Conversion Alliance (SECA) program for modifications required to test on coal synthesis gas. The subject DOE program provided funding for the electrical build, control system development and mechanical commissioning. Performance testing, which includes electrical commissioning, was subsequently performed under the DOE SECA program. Rolls-Royce Fuel Cell Systems is developing a megawatt-scale solid oxide fuel cell (SOFC) stationary power generation system. This system, based on RRFCS proprietary technology, is fueled with natural gas, and operates at elevated pressure. A critical success factor for development of the full scale system is the capability to

  2. Mitochondria and ageing: role in heart, skeletal muscle and adipose tissue

    Science.gov (United States)

    Boengler, Kerstin; Kosiol, Maik; Mayr, Manuel; Schulz, Rainer

    2017-01-01

    Abstract Age is the most important risk factor for most diseases. Mitochondria play a central role in bioenergetics and metabolism. In addition, several lines of evidence indicate the impact of mitochondria in lifespan determination and ageing. The best‐known hypothesis to explain ageing is the free radical theory, which proposes that cells, organs, and organisms age because they accumulate reactive oxygen species (ROS) damage over time. Mitochondria play a central role as the principle source of intracellular ROS, which are mainly formed at the level of complex I and III of the respiratory chain. Dysfunctional mitochondria generating less ATP have been observed in various aged organs. Mitochondrial dysfunction comprises different features including reduced mitochondrial content, altered mitochondrial morphology, reduced activity of the complexes of the electron transport chain, opening of the mitochondrial permeability transition pore, and increased ROS formation. Furthermore, abnormalities in mitochondrial quality control or defects in mitochondrial dynamics have also been linked to senescence. Among the tissues affected by mitochondrial dysfunction are those with a high‐energy demand and thus high mitochondrial content. Therefore, the present review focuses on the impact of mitochondria in the ageing process of heart and skeletal muscle. In this article, we review different aspects of mitochondrial dysfunction and discuss potential therapeutic strategies to improve mitochondrial function. Finally, novel aspects of adipose tissue biology and their involvement in the ageing process are discussed. PMID:28432755

  3. The Rice Mitochondria Proteome and its Response During Development and to the Environment

    Directory of Open Access Journals (Sweden)

    Shaobai eHuang

    2013-02-01

    Full Text Available Rice (Oryza sativa L. is both a major crop species and the key model grass for molecular and physiological research. Mitochondria are important in rice, as in all crops, as the main source of ATP for cell maintenance and growth. However, the practical significance of understanding the function of mitochondria in rice is increased by the widespread farming practice of using hybrids to boost rice production. This relies on cytoplasmic male-sterile (CMS lines with abortive pollen caused by dysfunctional mitochondria. We provide an overview of what is known about the mitochondrial proteome of rice seedlings. To date, more than 320 proteins have been identified in purified rice mitochondria using mass spectrometry. The insights from this work include a broad understanding of the major subunits of mitochondrial respiratory complexes and TCA cycle enzymes, carbon and nitrogen metabolism enzymes as well as details of the supporting machinery for biogenesis and the subset of stress-responsive mitochondrial proteins. Many proteins with unknown functions have also been found in rice mitochondria. Proteomic analysis has also revealed the features of rice mitochondrial protein presequences required for mitochondrial targeting, as well as cleavage site features for processing of precursors after import. Changes in the abundance of rice mitochondrial proteins in response to different stresses, especially anoxia and light, are summarized. Future research on quantitative analysis of the rice mitochondrial proteomes at the spatial and developmental level, its response to environmental stresses and recent advances in understanding of basis of rice CMS systems are highlighted.

  4. Ambient salinity modifies the action of triiodothyronine in the air-breathing fish Anabas testudineus Bloch: effects on mitochondria-rich cell distribution, osmotic and metabolic regulations.

    Science.gov (United States)

    Peter, M C Subhash; Leji, J; Peter, Valsa S

    2011-04-01

    The hydromineral and metabolic actions of thyroid hormone on osmotic acclimation in fish is less understood. We, therefore, studied the short-term action of triiodothyronine (T(3)), the potent thyroid hormone, on the distribution and the function of gill mitochondria-rich (MR) cells and on the whole body hydromineral and metabolic regulations of air-breathing fish (Anabas testudineus) adapted to either freshwater (FW) or acclimated to seawater (SA; 30 g L(-1)). As expected, 24 h T(3) injection (100 ng g(-1)) elevated (Pfish, suggest an action of T(3) on gill MR cell migration, though the density of these cells remained unchanged after T(3) treatment. The ouabain-sensitive Na(+), K(+)-ATPase activity, a measure of hydromineral competence, showed increases (Pfish after T(3) administration, but inhibited (Pfish and not in the SA fish. Exogenous T(3) reduced glucose (Pfish, whereas these metabolites were elevated (Pfish, suggesting a modulatory effect of ambient salinity on the T(3)-driven metabolic actions. Our data identify gill MR cell as a target for T(3) action as it promotes the spatial distribution and the osmotic function of these cells in both fresh water and in seawater. The results besides confirming the metabolic and osmotic actions of T(3) in fish support the hypothesis that the differential actions of T(3) may be due to the direct influence of ambient salinity, a major environmental determinant that alters the osmotic and metabolic strategies of fish. Copyright © 2011 Elsevier Inc. All rights reserved.

  5. Proteomic analysis of cell lines to identify the irinotecan resistance ...

    Indian Academy of Sciences (India)

    MADHU

    was selected from the wild-type LoVo cell line by chronic exposure to irinotecan ... dose–effect curves of anticancer drugs were drawn on semilogarithm .... alcohol metabolites daunorubicinol (Forrest and Gonzalez. 2000; Mordente et al. ..... Chen L, Huang C and Wei Y 2007 Proteomic analysis of liver cancer cells treated ...

  6. Novel human multiple myeloma cell line UHKT-893

    Czech Academy of Sciences Publication Activity Database

    Uherková, L.; Vančurová, I.; Vyhlídalová, I.; Pleschnerová, M.; Špička, I.; Mihalová, R.; Březinová, J.; Hodný, Zdeněk; Čermáková, K.; Polanská, V.; Marinov, I.; Jedelský, P.L.; Kuželová, K.; Stöckbauer, P.

    2013-01-01

    Roč. 37, č. 3 (2013), s. 320-326 ISSN 0145-2126 Institutional support: RVO:68378050 Keywords : human myeloma cell line * human multiple myeloma * plasma cell * IL-6 dependence * immunoglobulin * free light chain Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.692, year: 2013

  7. Frequency and distribution of Notch mutations in tumor cell lines

    International Nuclear Information System (INIS)

    Mutvei, Anders Peter; Fredlund, Erik; Lendahl, Urban

    2015-01-01

    Deregulated Notch signaling is linked to a variety of tumors and it is therefore important to learn more about the frequency and distribution of Notch mutations in a tumor context. In this report, we use data from the recently developed Cancer Cell Line Encyclopedia to assess the frequency and distribution of Notch mutations in a large panel of cancer cell lines in silico. Our results show that the mutation frequency of Notch receptor and ligand genes is at par with that for established oncogenes and higher than for a set of house-keeping genes. Mutations were found across all four Notch receptor genes, but with notable differences between protein domains, mutations were for example more prevalent in the regions encoding the LNR and PEST domains in the Notch intracellular domain. Furthermore, an in silico estimation of functional impact showed that deleterious mutations cluster to the ligand-binding and the intracellular domains of NOTCH1. For most cell line groups, the mutation frequency of Notch genes is higher than in associated primary tumors. Our results shed new light on the spectrum of Notch mutations after in vitro culturing of tumor cells. The higher mutation frequency in tumor cell lines indicates that Notch mutations are associated with a growth advantage in vitro, and thus may be considered to be driver mutations in a tumor cell line context. The online version of this article (doi:10.1186/s12885-015-1278-x) contains supplementary material, which is available to authorized users

  8. Clonogenic cell line survival of a human liver cancer cell line SMMC-7721 after carbon ion irradiation with different LET

    International Nuclear Information System (INIS)

    Lei Suwen; Su Xu; Wang Jifang; Li Wenjian

    2003-01-01

    Objective: To investigate the survival fraction of a human liver cancer cell line SMMC-7721 following irradiation with carbon ions with different LET. Methods: cells of the human liver cancer cell line SMMC-7721 were irradiated with carbon ions (LET=30 and 70 keV/μm). The survival fraction was determined with clonogenic assay after 9 days incubation in a 5% CO 2 incubator at 37 degree C. Results: When the survival fractions of 70 keV/μm were D s = 0.1 and D s=0.01 absorption dose were 2.94 and 5.88 Gy respectively, and those of 30 keV/μm were 4.00 and 8.00 Gy respectively. Conclusion: For the SMMC-7721 cell line, 70 keV/μm is more effective for cell killing than 30 keV/μm

  9. Guidelines for the use of cell lines in biomedical research.

    Science.gov (United States)

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

    2014-09-09

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

  10. Plant cyclopeptide RA-V kills human breast cancer cells by inducing mitochondria-mediated apoptosis through blocking PDK1–AKT interaction

    International Nuclear Information System (INIS)

    Fang, Xian-Ying; Chen, Wei; Fan, Jun-Ting; Song, Ran; Wang, Lu; Gu, Yan-Hong; Zeng, Guang-Zhi; Shen, Yan; Wu, Xue-Feng; Tan, Ning-Hua; Xu, Qiang; Sun, Yang

    2013-01-01

    In the present paper, we examined the effects of a natural cyclopeptide RA-V on human breast cancer cells and the underlying mechanisms. RA-V significantly inhibited the growth of human breast cancer MCF-7, MDA-MB-231 cells and murine breast cancer 4T1 cells. In addition, RA-V triggered mitochondrial apoptotic pathway which was indicated by the loss of mitochondrial membrane potential, the release of cytochrome c, and the activation of caspase cascade. Further study showed that RA-V dramatically inhibited phosphorylation of AKT and 3-phosphoinositide dependent protein kinase 1 (PDK1) in MCF-7 cells. Moreover, RA-V disrupted the interaction between PDK1 and AKT in MCF-7 cells. Furthermore, RA-V-induced apoptosis could be enhanced by phosphatidylinositol 3-kinase inhibitor or attenuated by over-expression of AKT in all the three kinds of breast cancer cells. Taken together, this study shows that RA-V, which can induce mitochondria-mediated apoptosis, exerts strong anti-tumor activity against human breast cancer. The underlying anti-cancer mechanism of RA-V is related to the blockage of the interaction between PDK1 and AKT. - Highlights: ► Plant cyclopeptide RA-V kills human breast cancer cells. ► RA-V triggered mitochondrial apoptotic pathway in human breast cancer cells. ► RA-V inhibited phosphorylation of AKT and PDK1 in breast cancer MCF-7 cells. ► Its mechanism is related to the blockage of the interaction between PDK1 and AKT

  11. Plant cyclopeptide RA-V kills human breast cancer cells by inducing mitochondria-mediated apoptosis through blocking PDK1–AKT interaction

    Energy Technology Data Exchange (ETDEWEB)

    Fang, Xian-Ying; Chen, Wei [State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, 22 Han Kou Road, Nanjing (China); Fan, Jun-Ting [State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming (China); Song, Ran; Wang, Lu [State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, 22 Han Kou Road, Nanjing (China); Gu, Yan-Hong [Department of Clinical Oncology, The First Affiliated Hospital of Nanjing Medical University, Nanjing (China); Zeng, Guang-Zhi [State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming (China); Shen, Yan; Wu, Xue-Feng [State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, 22 Han Kou Road, Nanjing (China); Tan, Ning-Hua, E-mail: nhtan@mail.kib.ac.cn [State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming (China); Xu, Qiang, E-mail: molpharm@163.com [State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, 22 Han Kou Road, Nanjing (China); Sun, Yang, E-mail: yangsun@nju.edu.cn [State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, 22 Han Kou Road, Nanjing (China)

    2013-02-15

    In the present paper, we examined the effects of a natural cyclopeptide RA-V on human breast cancer cells and the underlying mechanisms. RA-V significantly inhibited the growth of human breast cancer MCF-7, MDA-MB-231 cells and murine breast cancer 4T1 cells. In addition, RA-V triggered mitochondrial apoptotic pathway which was indicated by the loss of mitochondrial membrane potential, the release of cytochrome c, and the activation of caspase cascade. Further study showed that RA-V dramatically inhibited phosphorylation of AKT and 3-phosphoinositide dependent protein kinase 1 (PDK1) in MCF-7 cells. Moreover, RA-V disrupted the interaction between PDK1 and AKT in MCF-7 cells. Furthermore, RA-V-induced apoptosis could be enhanced by phosphatidylinositol 3-kinase inhibitor or attenuated by over-expression of AKT in all the three kinds of breast cancer cells. Taken together, this study shows that RA-V, which can induce mitochondria-mediated apoptosis, exerts strong anti-tumor activity against human breast cancer. The underlying anti-cancer mechanism of RA-V is related to the blockage of the interaction between PDK1 and AKT. - Highlights: ► Plant cyclopeptide RA-V kills human breast cancer cells. ► RA-V triggered mitochondrial apoptotic pathway in human breast cancer cells. ► RA-V inhibited phosphorylation of AKT and PDK1 in breast cancer MCF-7 cells. ► Its mechanism is related to the blockage of the interaction between PDK1 and AKT.

  12. Translocation of p53 to Mitochondria Is Regulated by Its Lipid Binding Property to Anionic Phospholipids and It Participates in Cell Death Control

    Directory of Open Access Journals (Sweden)

    Ching-Hao Li

    2010-02-01

    Full Text Available p53, can regulate cell apoptosis in both transcription-dependent and -independent manners. The transcription-independent pathway was demonstrated by the translocation of p53 to mitochondria. Our study showed that p53 mitochondrial translocation was found in mitomycin C (MMC-treated HepG2. The p53 C-terminal domain is clustered with potential nuclear leading sequences and showed strong electrostatic ion-ion interactions with cardiolipin, phosphatidylglycerol and phosphatidic acid in vitro. Disruption of cardiolipin biosynthesis by phosphatidylglycero-phosphate synthase (PGS or CDP-diacylglycerol synthase 2 (CDS-2 short hairpin RNA (shRNA transfection eliminated the MMC-induced translocation of mitochondrial p53. The elimination of mitochondrial p53 translocation also reduced Bcl-xL and Bcl-2 mitochondrial distribution. In HEK 293T models with saturated p53 expression, the mitochondrial partition of p53, Bcl-xL, and Bcl-2 obviously decreased in their PGS shRNA- or CDS-2 shRNA-expressing stable clones. In p53-null H1299 models, both the mitochondrial partitions of Bcl-xL and Bcl-2 were strongly reduced in relation to the HEK 293T models. The Bcl-xL mitochondrial partition was elevated in H1299 models expressing pCEP4-p53wt suggesting the direct carrier role of p53 in transporting Bcl-xL to the mitochondria. We also found that the cytosolic pool of Bcl-xL and Bcl-2 remained unaffected in the low-dose MMC treatment but decreased in the high-dose MMC treatment. The cytosolic pool of Bcl-2 and Bcl-xL directly regulated their amounts in p53-dependent mitochondrial distribution. In the low-dose MMC treatment, the increased mitochondrial p53, Bcl-xL, and Bcl-2 could attenuate apoptosis. However, in the high-dose MMC treatment, only the p53 translocated to the mitochondria and resulted in apoptosis progression. On the basis of this study, we thought mitochondrial p53 might regulate apoptosis in a biphasic manner.

  13. Bifenthrin activates homotypic aggregation in human T-cell lines.

    Science.gov (United States)

    Hoffman, Nataly; Tran, Van; Daniyan, Anthony; Ojugbele, Olutosin; Pryor, Stephen C; Bonventre, Josephine A; Flynn, Katherine; Weeks, Benjamin S

    2006-03-01

    Here, we addressed the concern that, despite the lack of overt toxicity, exposure to low levels of the common household pyrethroid pesticide, bifenthrin, could cause harm to the immune system. To do this, we measure the effect of bifenthrin on phytohemagglutinin (PHA) activation of homotypic aggregation in human T-cell lines. The human CD4+ H9, and Jurkat cell lines and the human promonocyte U937 cell line, were exposed to varying concentrations of bifenthrin. Cell viability was determined using the AlmarBlue Toxicity Assay. Concentrations of bifenthrin which did not reduce cell viability were determined and these concentrations were tested for the effect of bifenthrin on PHA-mediated homotypic aggregation. Blocking antibodies to ICAM and LFA-1 were used to disrupt aggregation and a nonspecific IgG was used as a control. Bifenthrin was found to be nontoxic at concentrations ranging from 10(-4) to 10(-13) M. Bifenthrin did not inhibit PHA induced cell aggregation in all cell lines tested. However, at 10(-4) M, bifenthrin to form aggregates stimulated homotypic aggregation in the H9 and Jurkat T-cell lines. The bifenthrin-induced aggregate formation, like that seen with PHA, was blocked by treating the cells with antibodies to either LFA-1 or ICAM. The results here show that bifenthrin activates T-cell function by stimulating ICAM/LFA-1 mediated homotypic aggregation. This data suggests that exposure to bifenthrin, even at "acceptable" limits, can increase the risk for and frequency of inflammatory responses and diseases such as asthma.

  14. Establishment, immortalisation and characterisation of pteropid bat cell lines.

    Directory of Open Access Journals (Sweden)

    Gary Crameri

    Full Text Available BACKGROUND: Bats are the suspected natural reservoir hosts for a number of new and emerging zoonotic viruses including Nipah virus, Hendra virus, severe acute respiratory syndrome coronavirus and Ebola virus. Since the discovery of SARS-like coronaviruses in Chinese horseshoe bats, attempts to isolate a SL-CoV from bats have failed and attempts to isolate other bat-borne viruses in various mammalian cell lines have been similarly unsuccessful. New stable bat cell lines are needed to help with these investigations and as tools to assist in the study of bat immunology and virus-host interactions. METHODOLOGY/FINDINGS: Black flying foxes (Pteropus alecto were captured from the wild and transported live to the laboratory for primary cell culture preparation using a variety of different methods and culture media. Primary cells were successfully cultured from 20 different organs. Cell immortalisation can occur spontaneously, however we used a retroviral system to immortalise cells via the transfer and stable production of the Simian virus 40 Large T antigen and the human telomerase reverse transcriptase protein. Initial infection experiments with both cloned and uncloned cell lines using Hendra and Nipah viruses demonstrated varying degrees of infection efficiency between the different cell lines, although it was possible to infect cells in all tissue types. CONCLUSIONS/SIGNIFICANCE: The approaches developed and optimised in this study should be applicable to bats of other species. We are in the process of generating further cell lines from a number of different bat species using the methodology established in this study.

  15. Mechanism(s of Toxic Action of Zn2+ and Selenite: A Study on AS-30D Hepatoma Cells and Isolated Mitochondria

    Directory of Open Access Journals (Sweden)

    Elena A. Belyaeva

    2011-01-01

    Full Text Available Mitochondria of AS-30D rat ascites hepatoma cells are found to be the main target for Zn2+ and sodium selenite (Na2SeO3. High [mu]M concentrations of Zn2+ or selenite were strongly cytotoxic, killing the AS-30D cells by both apoptotic and necrotic ways. Both Zn2+ and selenite produced strong changes in intracellular generation of reactive oxygen species (ROS and the mitochondrial dysfunction via the mitochondrial electron transport chain (mtETC disturbance, the membrane potential dissipation, and the mitochondrial permeability transition pore opening. The significant distinctions in toxic action of Zn2+ and selenite on AS-30D cells were found. Selenite induced a much higher intracellular ROS level (the early event compared to Zn2+ but a lower membrane potential loss and a lower decrease of the uncoupled respiration rate of the cells, whereas the mtETC disturbance was the early and critical event in the mechanism of Zn2+ cytotoxicity. Sequences of events manifested in the mitochondrial dysfunction produced by the metal/metalloid under test are compared with those obtained earlier for Cd2+, Hg2+, and Cu2+ on the same model system.

  16. Terpenoids Isolated From the Shoot of Plectranthus hadiensis Induces Apoptosis in Human Colon Cancer Cells Via the Mitochondria-Dependent Pathway.

    Science.gov (United States)

    Menon, Darsan B; Gopalakrishnan, V K

    2015-01-01

    The plant Plectranthus hadiensis is a rich source of many bioactive phytochemicals, especially terpenoids. The terpenoid fraction was isolated and phytochemical characterization was done using GC-MS. The aim of the present study was to find out the antiproliferative activity and the mechanism of cell death induction by the terpenoid fraction on human colon cancer cells (HCT-15). MTT assay was performed with different concentrations of the fraction (10, 20, and 50 µg/mL) to obtain IC50 value for 24 h to induce cell death. The induction of apoptosis were studied by Hoechst staining, acridine orange/ethidium bromide staining, Comet assay, DNA fragmentation, and caspase-3 activity assays. The mechanism of apoptosis induction was studied by expression analysis of antiapoptotic Bcl-2 and proapoptotic Bax using RT-PCR and also by Western blot analysis of proteins involved in the apoptotic pathway. The terpenoid fraction induced significant morphological changes and DNA fragmentation in the cells. Positive Hoechst staining and acridine orange/ethidium bromide staining indicated apoptosis induction by the fraction. DNA fragmentation, which is a characteristic feature of apoptosis, was also observed. Upregulation of caspase-3 activity and proapoptotic Bax, and the downregulation of antiapoptotic Bcl-2 and COX-2 confirmed that the apoptosis induction was via the mitochondria-dependent pathway.

  17. Mitochondria, Cybrids, Aging, and Alzheimer’s Disease

    Science.gov (United States)

    Swerdlow, Russell H.; Koppel, Scott; Weidling, Ian; Hayley, Clay; Ji, Yan; Wilkins, Heather M.

    2018-01-01

    Mitochondrial and bioenergetic function change with advancing age and may drive aging phenotypes. Mitochondrial and bioenergetic changes are also documented in various age-related neurodegenerative diseases, including Alzheimer’s disease (AD). In some instances AD mitochondrial and bioenergetic changes are reminiscent of those observed with advancing age, but are greater in magnitude. Mitochondrial and bioenergetic dysfunction could, therefore, link neurodegeneration to brain aging. Interestingly, mitochondrial defects in AD patients are not brain-limited, and mitochondrial function can be linked to classic AD histologic changes including amyloid precursor protein processing to beta amyloid. Also, transferring mitochondria from AD subjects to cell lines depleted of endogenous mitochondrial DNA (mtDNA) creates cytoplasmic hybrid (cybrid) cell lines that recapitulate specific biochemical, molecular, and histologic AD features. Such findings have led to the formulation of a “mitochondrial cascade hypothesis” that places mitochondrial dysfunction at the apex of the AD pathology pyramid. Data pertinent to this premise are reviewed. PMID:28253988

  18. Isolation of a primate embryonic stem cell line.

    OpenAIRE

    Thomson, J A; Kalishman, J; Golos, T G; Durning, M; Harris, C P; Becker, R A; Hearn, J P

    1995-01-01

    Embryonic stem cells have the ability to remain undifferentiated and proliferate indefinitely in vitro while maintaining the potential to differentiate into derivatives of all three embryonic germ layers. Here we report the derivation of a cloned cell line (R278.5) from a rhesus monkey blastocyst that remains undifferentiated in continuous passage for > 1 year, maintains a normal XY karyotype, and expresses the cell surface markers (alkaline phosphatase, stage-specific embryonic antigen 3, st...

  19. Cellular Glycolysis and The Differential Survival of Lung Fibroblast and Lung Carcinoma Cell Lines.

    Science.gov (United States)

    Farah, Ibrahim O

    2016-04-01

    Tumor growth and abnormal cell survival were shown to be associated with a number of cellular metabolic abnormalities revealed by impaired oral glucose tolerance, depressed lipoprotein lipase activity leading to hypertriglyceridemia, and changes in amino acid profile as evidenced by increased plasma free tryptophan levels in patients with breast, lung, colon, stomach, and other cancers from various origins. The above findings seem to relate to or indicate a shift to non-oxidative metabolic pathways in cancer. In contrast to normal cells, cancer cells may lose the ability to utilize aerobic respiration due to either defective mitochondria or hypoxia within the tumor microenvironments. Glucose was shown to be the major energy source in cancer cells where it utilizes aerobic /anaerobic glycolysis with the resultant lactic acid formation. The role of energetic modulations and use of glycolytic inhibitors on cancer/normal cell survival is not clearly established in the literature. We hypothesize that natural intermediates of glycolysis and the citric acid cycle will differentially and negatively impact the cancer phenotype in contrast to their no effects on the normal cell phenotype. Therefore, the purpose of this study was to evaluate six potential glycolytic modulators namely, Pyruvic acid, oxalic acid, Zn acetate, sodium citrate, fructose diphosphate (FDP) and sodium bicarbonate at μM concentrations on growing A549 (lung cancer) and MRC-5 (normal; human lung fibroblast) cell lines with the objective of determining their influence on visual impact, cell metabolic activity, cell viability and end-point cell survival. Exposed and non-exposed cells were tested with phase-contrast micro-scanning, survival/death and metabolic activity trends through MTT-assays, as well as death end-point determinations by testing re-growth on complete media and T4 cellometer counts. Results showed that oxalic acid and Zn acetate both influenced the pH of the medium and resulted in

  20. Sensory hair cell regeneration in the zebrafish lateral line.

    Science.gov (United States)

    Lush, Mark E; Piotrowski, Tatjana

    2014-10-01

    Damage or destruction of sensory hair cells in the inner ear leads to hearing or balance deficits that can be debilitating, especially in older adults. Unfortunately, the damage is permanent, as regeneration of the inner ear sensory epithelia does not occur in mammals. Zebrafish and other non-mammalian vertebrates have the remarkable ability to regenerate sensory hair cells and understanding the molecular and cellular basis for this regenerative ability will hopefully aid us in designing therapies to induce regeneration in mammals. Zebrafish not only possess hair cells in the ear but also in the sensory lateral line system. Hair cells in both organs are functionally analogous to hair cells in the inner ear of mammals. The lateral line is a mechanosensory system found in most aquatic vertebrates that detects water motion and aids in predator avoidance, prey capture, schooling, and mating. Although hair cell regeneration occurs in both the ear and lateral line, most research to date has focused on the lateral line due to its relatively simple structure and accessibility. Here we review the recent discoveries made during the characterization of hair cell regeneration in zebrafish. Copyright © 2014 Wiley Periodicals, Inc.

  1. Nestin expression in the cell lines derived from glioblastoma multiforme

    International Nuclear Information System (INIS)

    Veselska, Renata; Kuglik, Petr; Cejpek, Pavel; Svachova, Hana; Neradil, Jakub; Loja, Tomas; Relichova, Jirina

    2006-01-01

    Nestin is a protein belonging to class VI of intermediate filaments that is produced in stem/progenitor cells in the mammalian CNS during development and is consecutively replaced by other intermediate filament proteins (neurofilaments, GFAP). Down-regulated nestin may be re-expressed in the adult organism under certain pathological conditions (brain injury, ischemia, inflammation, neoplastic transformation). Our work focused on a detailed study of the nestin cytoskeleton in cell lines derived from glioblastoma multiforme, because re-expression of nestin together with down-regulation of GFAP has been previously reported in this type of brain tumor. Two cell lines were derived from the tumor tissue of patients treated for glioblastoma multiforme. Nestin and other cytoskeletal proteins were visualized using imunocytochemical methods: indirect immunofluorescence and immunogold-labelling. Using epifluorescence and confocal microscopy, we described the morphology of nestin-positive intermediate filaments in glioblastoma cells of both primary cultures and the derived cell lines, as well as the reorganization of nestin during mitosis. Our most important result came through transmission electron microscopy and provided clear evidence that nestin is present in the cell nucleus. Detailed information concerning the pattern of the nestin cytoskeleton in glioblastoma cell lines and especially the demonstration of nestin in the nucleus represent an important background for further studies of nestin re-expression in relationship to tumor malignancy and invasive potential

  2. SENSORY HAIR CELL REGENERATION IN THE ZEBRAFISH LATERAL LINE

    Science.gov (United States)

    Lush, Mark E.; Piotrowski, Tatjana

    2014-01-01

    Damage or destruction of sensory hair cells in the inner ear leads to hearing or balance deficits that can be debilitating, especially in older adults. Unfortunately, the damage is permanent, as regeneration of the inner ear sensory epithelia does not occur in mammals. Zebrafish and other non-mammalian vertebrates have the remarkable ability to regenerate sensory hair cells and understanding the molecular and cellular basis for this regenerative ability will hopefully aid us in designing therapies to induce regeneration in mammals. Zebrafish not only possess hair cells in the ear but also in the sensory lateral line system. Hair cells in both organs are functionally analogous to hair cells in the inner ear of mammals. The lateral line is a mechanosensory system found in most aquatic vertebrates that detects water motion and aids in predator avoidance, prey capture, schooling and mating. Although hair cell regeneration occurs in both the ear and lateral line, most research to date has focused on the lateral line due to its relatively simple structure and accessibility. Here we review the recent discoveries made during the characterization of hair cell regeneration in zebrafish. PMID:25045019

  3. Hyperthermia-enhanced TRAIL- and mapatumumab-induced apoptotic death is mediated through mitochondria in human colon cancer cells.

    Science.gov (United States)

    Song, Xinxin; Kim, Han-Cheon; Kim, Seog-Young; Basse, Per; Park, Bae-Hang; Lee, Byeong-Chel; Lee, Yong J

    2012-05-01

    Colorectal cancer is the third leading cause of cancer-related mortality in the world; death usually results from uncontrolled metastatic disease. Previously, we developed a novel strategy of TNF-related apoptosis-inducing ligand (Apo2L/TRAIL) in combination with hyperthermia to treat hepatic colorectal metastases. However, previous studies suggest a potential hepatocyte cytotoxicity with TRAIL. Unlike TRAIL, anti-human TRAIL receptor antibody induces apoptosis without hepatocyte toxicity. In this study, we evaluated the anti-tumor efficacy of humanized anti-death receptor 4 (DR4) antibody mapatumumab (Mapa) by comparing it with TRAIL in combination with hyperthermia. TRAIL, which binds to both DR4 and death receptor 5 (DR5), was approximately tenfold more effective than Mapa in inducing apoptosis. However, hyperthermia enhances apoptosis induced by either agent. We observed that the synergistic effect was mediated through elevation of reactive oxygen species, c-Jun N-terminal kinase activation, Bax oligomerization, and translocalization to the mitochondria, loss of mitochondrial membrane potential, release of cytochrome c to cytosol, activation of caspases, and increase in poly(ADP-ribose) polymerase cleavage. We believe that the successful outcome of this study will support the application of Mapa in combination with hyperthermia to colorectal hepatic metastases. Copyright © 2011 Wiley Periodicals, Inc.

  4. Differential protection by wildtype vs. organelle-specific Bcl-2 suggests a combined requirement of both the ER and mitochondria in ceramide-mediated caspase-independent programmed cell death

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    Belka Claus

    2009-10-01

    Full Text Available Abstract Background Programmed cell death (PCD is essential for development and homeostasis of multicellular organisms and can occur by caspase-dependent apoptosis or alternatively, by caspase-independent PCD (ciPCD. Bcl-2, a central regulator of apoptosis, localizes to both mitochondria and the endoplasmic reticulum (ER. Whereas a function of mitochondrial and ER-specific Bcl-2 in apoptosis has been established in multiple studies, corresponding data for ciPCD do not exist. Methods We utilized Bcl-2 constructs specifically localizing to mitochondria (Bcl-2 ActA, the ER (Bcl-2 cb5, both (Bcl-2 WT or the cytosol/nucleus (Bcl-2 ΔTM and determined their protective effect on ceramide-mediated ciPCD in transiently and stably transfected Jurkat cells. Expression of the constructs was verified by immunoblots. Ceramide-mediated ciPCD was induced by treatment with human recombinant tumor necrosis factor and determined by flow cytometric measurement of propidium iodide uptake as well as by optical analysis of cell morphology. Results Only wildtype Bcl-2 had the ability to efficiently protect from ceramide-mediated ciPCD, whereas expression of Bcl-2 solely at mitochondria, the ER, or the cytosol/nucleus did not prevent ceramide-mediated ciPCD. Conclusion Our data suggest a combined requirement for both mitochondria and the ER in the induction and the signaling pathways of ciPCD mediated by ceramide.

  5. Differential protection by wildtype vs. organelle-specific Bcl-2 suggests a combined requirement of both the ER and mitochondria in ceramide-mediated caspase-independent programmed cell death

    International Nuclear Information System (INIS)

    Deerberg, Andrea; Sosna, Justyna; Thon, Lutz; Belka, Claus; Adam, Dieter

    2009-01-01

    Programmed cell death (PCD) is essential for development and homeostasis of multicellular organisms and can occur by caspase-dependent apoptosis or alternatively, by caspase-independent PCD (ciPCD). Bcl-2, a central regulator of apoptosis, localizes to both mitochondria and the endoplasmic reticulum (ER). Whereas a function of mitochondrial and ER-specific Bcl-2 in apoptosis has been established in multiple studies, corresponding data for ciPCD do not exist. We utilized Bcl-2 constructs specifically localizing to mitochondria (Bcl-2 ActA), the ER (Bcl-2 cb5), both (Bcl-2 WT) or the cytosol/nucleus (Bcl-2 ΔTM) and determined their protective effect on ceramide-mediated ciPCD in transiently and stably transfected Jurkat cells. Expression of the constructs was verified by immunoblots. Ceramide-mediated ciPCD was induced by treatment with human recombinant tumor necrosis factor and determined by flow cytometric measurement of propidium iodide uptake as well as by optical analysis of cell morphology. Only wildtype Bcl-2 had the ability to efficiently protect from ceramide-mediated ciPCD, whereas expression of Bcl-2 solely at mitochondria, the ER, or the cytosol/nucleus did not prevent ceramide-mediated ciPCD. Our data suggest a combined requirement for both mitochondria and the ER in the induction and the signaling pathways of ciPCD mediated by ceramide

  6. Glycoproteins and sialyl transferase of human B lymphoblastoid cell lines

    International Nuclear Information System (INIS)

    Lui, S.W.L.; Ng, M.H.

    1980-01-01

    We used two radiolabeling methods to study glycoproteins on the surface of lymphoblastoid cells. One of the methods affects tritiation of residues which are oxidized with galactose oxidase and the other causes tritiation of neuraminic acid residues. This approach was shown to allow a better resolution of cell surface glycoproteins than if either method were used alone. Glycoproteins of B 1 - 19 cells which harbor the Epstein-Barr virus genomes were compared with those of its parental cell line, BJAB, which does not harbor the viral genomes. These studies did not reveal a unique viral protein. A 28,000 mol. wt. glycoprotein was found to be the most prominent neuraminic acidlabeled product of B 1 - 19 cells and also of the two other cell lines, Raji and Ly38, which harbor the EBV genomes. A similar molecular weight species from BJAB cells identified by galactose oxidase labeling might be deficient in neuraminic acid residues as it was poorly labeled by the periodate oxidation method. The neuraminic acid content and level of sialyl transferase of BJAB cells were found to be lower than those of the other cell lines studied. (auth.)

  7. Mitochondria-meditated pathways of organ failure upon inflammation

    Directory of Open Access Journals (Sweden)

    Andrey V. Kozlov

    2017-10-01

    Full Text Available Liver failure induced by systemic inflammatory response (SIRS is often associated with mitochondrial dysfunction but the mechanism linking SIRS and mitochondria-mediated liver failure is still a matter of discussion. Current hypotheses suggest that causative events could be a drop in ATP synthesis, opening of mitochondrial permeability transition pore, specific changes in mitochondrial morphology, impaired Ca2+ uptake, generation of mitochondrial reactive oxygen species (mtROS, turnover of mitochondria and imbalance in electron supply to the respiratory chain. The aim of this review is to critically analyze existing hypotheses, in order to highlight the most promising research lines helping to prevent liver failure induced by SIRS. Evaluation of the literature shows that there is no consistent support that impaired Ca++ metabolism, electron transport chain function and ultrastructure of mitochondria substantially contribute to liver failure. Moreover, our analysis suggests that the drop in ATP levels has protective rather than a deleterious character. Recent data suggest that the most critical mitochondrial event occurring upon SIRS is the release of mtROS in cytoplasm, which can activate two specific intracellular signaling cascades. The first is the mtROS-mediated activation of NADPH-oxidase in liver macrophages and endothelial cells; the second is the acceleration of the expression of inflammatory genes in hepatocytes. The signaling action of mtROS is strictly controlled in mitochondria at three points, (i at the site of ROS generation at complex I, (ii the site of mtROS release in cytoplasm via permeability transition pore, and (iii interaction with specific kinases in cytoplasm. The systems controlling mtROS-signaling include pro- and anti-inflammatory mediators, nitric oxide, Ca2+ and NADPH-oxidase. Analysis of the literature suggests that further research should be focused on the impact of mtROS on organ failure induced by inflammation

  8. Casein gene expression in mouse mammary epithelial cell lines: Dependence upon extracellular matrix and cell type

    International Nuclear Information System (INIS)

    Medina, D.; Oborn, C.J.; Li, M.L.; Bissell, M.J.

    1987-01-01

    The COMMA-D mammary cell line exhibits mammary-specific functional differentiation under appropriate conditions in cell culture. The cytologically heterogeneous COMMA-D parental line and the clonal lines DB-1, TA-5, and FA-1 derived from the COMMA-D parent were examined for similar properties of functional differentiation. In monolayer cell culture, the cell lines DB-1, TA-5, FA-1, and MA-4 were examined for expression of mammary-specific and epithelial-specific proteins by an indirect immunofluorescence assay. The clonal cell lines were relatively homogeneous in their respective staining properties and seemed to represent three subpopulations found in the heterogeneous parental COMMA-D lines. None of the four clonal lines appeared to represent myoepithelial cells. The cell lines were examined for expression of β-casein mRNA in the presence or absence of prolactin. The inducibility of β-casein in the COMMA-D cell line was further enhanced by a reconstituted basement membrane preparation enriched in laminin, collagen IV, and proteoglycans. These results support the hypothesis that the functional response of inducible mammary cell populations is a result of interaction among hormones, multiple extracellular matrix components, and specific cell types

  9. The neem limonoids azadirachtin and nimbolide induce cell cycle arrest and mitochondria-mediated apoptosis in human cervical cancer (HeLa) cells.

    Science.gov (United States)

    Priyadarsini, R Vidya; Murugan, R Senthil; Sripriya, P; Karunagaran, D; Nagini, S

    2010-06-01

    Limonoids from the neem tree (Azadirachta indica) have attracted considerable research attention in recent years owing to their potent antioxidant and anti-proliferative effects. The present study was designed to investigate the cellular and molecular mechanisms by which azadirachtin and nimbolide exert cytotoxic effects in the human cervical cancer (HeLa) cell line. Both azadirachtin and nimbolide significantly suppressed the viability of HeLa cells in a dose-dependent manner by inducing cell cycle arrest at G0/G1 phase accompanied by p53-dependent p21 accumulation and down-regulation of the cell cycle regulatory proteins cyclin B, cyclin D1 and PCNA. Characteristic changes in nuclear morphology, presence of a subdiploid peak and annexin-V staining pointed to apoptosis as the mode of cell death. Increased generation of reactive oxygen species with decline in the mitochondrial transmembrane potential and release of cytochrome c confirmed that the neem limonoids transduced the apoptotic signal via the mitochondrial pathway. Altered expression of the Bcl-2 family of proteins, inhibition of NF-kappaB activation and over-expression of caspases and survivin provide compelling evidence that azadirachtin and nimbolide induce a shift of balance toward a pro-apoptotic phenotype. Antioxidants such as azadirachtin and nimbolide that can simultaneously arrest the cell cycle and target multiple molecules involved in mitochondrial apoptosis offer immense potential as anti-cancer therapeutic drugs.

  10. Identification of genes associated with cisplatin resistance in human oral squamous cell carcinoma cell line

    OpenAIRE

    Zhang Ping; Zhang Zhiyuan; Zhou Xiaojian; Qiu Weiliu; Chen Fangan; Chen Wantao

    2006-01-01

    Abstract Background Cisplatin is widely used for chemotherapy of head and neck squamous cell carcinoma. However, details of the molecular mechanism responsible for cisplatin resistance are still unclear. The aim of this study was to identify the expression of genes related to cisplatin resistance in oral squamous cell carcinoma cells. Methods A cisplatin-resistant cell line, Tca/cisplatin, was established from a cisplatin-sensitive cell line, Tca8113, which was derived from moderately-differe...

  11. Apoptosis of murine melanoma B16-BL6 cells induced by quercetin targeting mitochondria, inhibiting expression of PKC-alpha and translocating PKC-delta.

    Science.gov (United States)

    Zhang, Xian-Ming; Chen, Jia; Xia, Yu-Gui; Xu, Qiang

    2005-03-01

    In our previous study, quercetin was found to induce apoptosis of murine melanoma B16-BL6 cells. The cellular and molecular mechanism of quercetin-induced apoptosis was investigated in the present study. Nuclear morphology was determined by fluorescence microscopy. DNA fragmentation was analyzed by electrophoresis and quantified by the diphenylamine method. The transmembrane potential of mitochondria was measured by flow cytometry. Bcl-2, Bcl-X(L), PKC-alpha, PKC-beta, and PKC-delta were detected by Western blotting. Caspase activity was determined spectrophotometrically. Quercetin induced the condensation of nuclei of B16-BL6 cells in a dose-dependent pattern as visualized by Hoechst 33258 and propidium iodide dying. Phorbol 12-myristate 13-acetate (PMA), a PKC activator, significantly enhanced apoptosis induced by quercetin, while doxorubicin, a PKC inhibitor, markedly decreased it. Both PMA and doxorubicin showed a consistent effect on the fragmentation of nuclear DNA caused by various dosages of quercetin. Quercetin dose-dependently led to loss of the mitochondrial membrane potential, which was also significantly reinforced or antagonized by PMA and doxorubicin, respectively. Moreover, PMA showed reinforcement, while doxorubicin showed significant antagonization, of the quercetin-mediated decrease in the expression of Bcl-2. Quercetin promoted caspase-3 activity in a dose-dependent manner, which was also regulated by PMA and doxorubicin with a pattern similar to that seen in their effect on apoptosis, mitochondrial membrane potential and Bcl-2 expression, but none of these were directly affected by PMA and doxorubicin. Free fatty acid and chlorpromazine, a PKC activator and inhibitor, respectively, did not interfere with these effects of quercetin. B16-BL6 cells expressed PKC-alpha, PKC-beta, and PKC-delta. Quercetin dose-dependently inhibited the expression of PKC-alpha but not that of PKC-beta and PKC-delta. Doxorubicin almost completely blocked the effect of

  12. Antiproliferative activity of flavonoids on several cancer cell lines.

    Science.gov (United States)

    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.

  13. DNA double strand break repair in a radioresistant cell line

    International Nuclear Information System (INIS)

    Koval, T.M.; Kazmar, E.R.

    1987-01-01

    TN-368 lepidopteran insect cells are on the order of 100 times more resistant to the lethal effects of ionizing radiation than cultured mammalian cells. DNA double strand breaks (DSB) are believed by many to be the critical molecular lesion leading to cell death. The authors therefore measured the rejoining of DSB in TN-368 and V79 Chinese hamster cells. Cells were irradiated on ice with /sup 137/Cs γ rays at a dose rate of 2.5 Gy/min, incubated for various periods of time, and assayed for DNA DSB using the method of neutral elution. The kinetics of DSB rejoining following a dose of 90.2 Gy are similar for both cell lines. Approximately 80% of the DSB are rejoined in both lines by 1 hr postirradiation. However, no further rejoining occurs in the TN-368 cells through at least 6 hr postirradiation, whereas 90% of the DSB are rejoined in the V79 cells by 2 hr postirradiation. Other studies (from 22.6 to 226 Gy) demonstrate that the amount of rejoining of DSB varies inversely with dose for the V79 cells but remains constant for the TN-368 cells. These findings do not support the hypothesis that unrejoined DNA DSB represent the major lesion resulting in cell death

  14. Dipeptidyl peptidase IV in two human glioma cell lines

    Directory of Open Access Journals (Sweden)

    A Sedo

    2009-12-01

    Full Text Available There is growing evidence that dipeptidyl peptidase IV [DPP-IV, EC 3.4.14.5] takes part in the metabolism of biologically active peptides participating in the regulation of growth and transformation of glial cells. However, the knowledge on the DPP-IV expression in human glial and glioma cells is still very limited. In this study, using histochemical and biochemical techniques, the DPP-IV activity was demonstrated in two commercially available human glioma cell lines of different transformation degree, as represented by U373 astrocytoma (Grade III and U87 glioblastoma multiforme (Grade IV lines. Higher total activity of the enzyme, as well as its preferential localisation in the plasma membrane, was observed in U87 cells. Compared to U373 population, U87 cells were morphologically more pleiomorphic, they were cycling at lower rate and expressing less Glial Fibrillary Acidic Protein. The data revealed positive correlation between the degree of transformation of cells and activity of DPP-IV. Great difference in expression of this enzyme, together with the phenotypic differences of cells, makes these lines a suitable standard model for further 57 studies of function of this enzyme in human glioma cells.

  15. Characterization of cloned cells from an immortalized fetal pulmonary type II cell line

    Energy Technology Data Exchange (ETDEWEB)

    Henderson, R.F.; Waide, J.J.; Lechner, J.F.

    1995-12-01

    A cultured cell line that maintained expression of pulmonary type II cell markers of differentiation would be advantageous to generate a large number of homogenous cells in which to study the biochemical functions of type II cells. Type II epithelial cells are the source of pulmonary surfactant and a cell of origin for pulmonary adenomas. Last year our laboratory reported the induction of expression of two phenotypic markers of pulmonary type II cells (alkaline phosphatase activity and surfactant lipid synthesis) in cultured fetal rat lung epithelial (FRLE) cells, a spontaneously immortalized cell line of fetal rat lung type II cell origin. Subsequently, the induction of the ability to synthesize surfactant lipid became difficult to repeat. We hypothesized that the cell line was heterogenuous and some cells were more like type II cells than others. The purpose of this study was to test this hypothesis and to obtain a cultured cell line with type II cell phenotypic markers by cloning several FRLE cells and characterizing them for phenotypic markers of type II cells (alkaline phosphatase activity and presence of surfactant lipids). Thirty cloned cell lines were analyzed for induced alkaline phosphatase activity (on x-axis) and for percent of phospholipids that were disaturated (i.e., surfactant).

  16. Identification of the TaBTF3 gene in wheat (Triticum aestivum L.) and the effect of its silencing on wheat chloroplast, mitochondria and mesophyll cell development.

    Science.gov (United States)

    Ma, Hong-Zhen; Liu, Guo-Qin; Li, Cheng-Wei; Kang, Guo-Zhang; Guo, Tian-Cai

    2012-10-05

    The full-length cDNA (882bp) and DNA (1742bp) sequences encoding a basic transcription factor 3, designated as TaBTF3, were first isolated from common wheat (Triticum aestivum L.). Subcellular localization studies revealed that the TaBTF3 protein was mainly located in the cytoplasm and nucleus. In TaBTF3-silenced transgenic wheat seedlings obtained using the Virus-induced gene silencing (VIGS) method, the chlorophyll pigment content was markedly reduced. However, the malonaldehyde (MDA) and H(2)O(2) contents were enhanced, and the structure of the wheat mesophyll cell was seriously damaged. Furthermore, transcripts of the chloroplast- and mitochondrial-encoded genes were significantly reduced in TaBTF3-silenced transgenic wheat plants. These results suggest that the TaBTF3 gene might function in the development of the wheat chloroplast, mitochondria and mesophyll cell. This paper is the first report to describe the involvement of TaBTF3 in maintaining the normal plant mesophyll cell structure. Copyright © 2012 Elsevier Inc. All rights reserved.

  17. Antiproliferative effect of Tualang honey on oral squamous cell carcinoma and osteosarcoma cell lines

    Directory of Open Access Journals (Sweden)

    Ismail Noorliza M

    2010-09-01

    Full Text Available Abstract Background The treatment of oral squamous cell carcinomas (OSCC and human osteosarcoma (HOS includes surgery and/or radiotherapy which often lead to reduced quality of life. This study was aimed to study the antiproliferative activity of local honey (Tualang on OSCC and HOS cell lines. Methods Several concentrations of Tualang honey (1% - 20% were applied on OSCC and HOS cell lines for 3, 6, 12, 24, 48 and 72 hours. Morphological characteristics were observed under light and fluorescent microscope. Cell viability was assessed using MTT assay and the optical density for absorbance values in each experiment was measured at 570 nm by an ELISA reader. Detection of cellular apoptosis was done using the Annexin V-FITC Apoptosis Detection Kit. Results Morphological appearance showed apoptotic cellular changes like becoming rounded, reduction in cell number, blebbed membrane and apoptotic nuclear changes like nuclear shrinkage, chromatin condensation and fragmented nucleus on OSCC and HOS cell lines. Cell viability assay showed a time and dose-dependent inhibitory effect of honey on both cell lines. The 50% inhibitory concentration (IC50 for OSCC and HOS cell lines was found to be 4% and 3.5% respectively. The maximum inhibition of cell growth of ≥80% was obtained at 15% for both cell lines. Early apoptosis was evident by flow cytometry where percentage of early apoptotic cells increased in dose and time dependent manner. Conclusion Tualang honey showed antiproliferative effect on OSCC and HOS cell lines by inducing early apoptosis.

  18. Microtubule-dependent changes in morphology and localization of chloride transport proteins in gill mitochondria-rich cells of the tilapia, Oreochromis mossambicus.

    Science.gov (United States)

    Yang, Wen-Kai; Wu, Yu-Ching; Tang, Cheng-Hao; Lee, Tsung-Han

    2016-08-01

    The tilapia (Oreochromis mossambicus) is a euryhaline fish exhibiting adaptive changes in cell size, phenotype, and ionoregulatory functions upon salinity challenge. Na(+) /Cl(-) cotransporter (NCC) and Na(+) /K(+) /2Cl(-) cotransporter (NKCC) are localized in the apical and basolateral membranes of mitochondria-rich (MR) cells of the gills. These cells are responsible for chloride absorption (NCC) and secretion (NKCC), respectively, thus, the switch of gill NCC and NKCC expression is a crucial regulatory mechanism for salinity adaptation in tilapia. However, little is known about the interaction of cytoskeleton and these adaptive changes. In this study, we examined the time-course of changes in the localization of NKCC/NCC in the gills of tilapia transferred from fresh water (FW) to brackish water (20‰) and from seawater (SW; 35‰) to FW. The results showed that basolateral NKCC disappeared and NCC was expressed in the apical membrane of MR cells. To further clarify the process of these adaptive changes, colchicine, a specific inhibitor of microtubule-dependent cellular regulating processes was used. SW-acclimated tilapia were transferred to SW, FW, and FW with colchicine (colchicine-FW) for 96 h. Compared with the FW-treatment group, in the MR cells of colchicine-FW-treatment group, (1) the average size was significantly larger, (2) only wavy-convex-subtype apical surfaces were found, and (3) the basolateral (cytoplasmic) NKCC signals were still exhibited. Taken together, our results suggest that changes in size, phenotype, as well as the expression of NCC and NKCC cotransporters of MR cells in the tilapia are microtubule-dependent. J. Morphol. 277:1113-1122, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  19. Maslinic acid inhibits proliferation of renal cell carcinoma cell lines and suppresses angiogenesis of endothelial cells

    Directory of Open Access Journals (Sweden)

    Parth Thakor

    2017-03-01

    Full Text Available Despite the introduction of many novel therapeutics in clinical practice, metastatic renal cell carcinoma (RCC remains a treatment-re-sistant cancer. As red and processed meat are considered risk factors for RCC, and a vegetable-rich diet is thought to reduce this risk, research into plant-based therapeutics may provide valuable complementary or alternative therapeutics for the management of RCC. Herein, we present the antiproliferative and antiangiogenic effects of maslinic acid, which occurs naturally in edible plants, particularly in olive fruits, and also in a variety of medicinal plants. Human RCC cell lines (ACHN, Caki-1, and SN12K1, endothelial cells (human umbilical vein endothelial cell line [HUVEC], and primary cultures of kidney proximal tubular epithelial cells (PTEC were treated with maslinic acid. Maslinic acid was relatively less toxic to PTEC when compared with RCC under similar experimental conditions. In RCC cell lines, maslinic acid induced a significant reduction in proliferation, proliferating cell nuclear antigen, and colony formation. In HUVEC, maslinic acid induced a significant reduction in capillary tube formation in vitro and vascular endothelial growth factor. This study provides a rationale for incorporating a maslinic acid–rich diet either to reduce the risk of developing kidney cancer or as an adjunct to existing antiangiogenic therapy to improve efficacy.

  20. THE ISLAMIC ETHICS OF MITOCHONDRIA TRANSPLANTATION

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    Anke Iman Bouzenita

    2017-12-01

    Full Text Available ABSTRACT: Biotechnology has opened a new chapter with the advent of mitochondria transplantation for cell-based therapy. Mitochondrial transplantation was successfully led to birth; however, cytoplasmic transplantation has caused apprehension, since the mixing of human ooplasm from two different maternal sources may generate mitochondrial DNA (mtDNA heteroplasmy in the offspring. Islamic legal verdicts on human cloning and somatic cell transfer have been overweighing explicit as to its prohibition, due to the change of creation, mixing of lineage and other evaluations. Is mitochondria transplantation equivalent to human cloning in that genetic information is proliferated and does it, therefore, take the same legal rule? Are there possible benefits (masalih for medical treatment that may render mitochondria transplantation permissible, or are possible harms (mafasid overweighing? Or is it a completely different procedure, taking a different rule? The paper will investigate into these questions and discuss the dimensions of Islamic ethics on the issue.

  1. Cellular and Phenotypic Characterization of Canine Osteosarcoma Cell Lines

    Directory of Open Access Journals (Sweden)

    Marie E. Legare, Jamie Bush, Amanda K. Ashley, Taka Kato, William H. Hanneman

    2011-01-01

    Full Text Available Canine and human osteosarcoma (OSA have many similarities, with the majority of reported cases occurring in the appendicular skeleton, gender predominance noted, high rate of metastasis at the time of presentation, and a lack of known etiology for this devastating disease. Due to poor understanding of the molecular mechanisms underlying OSA, we have characterized seven different OSA canine cell lines: Abrams, D17, Grey, Hughes, Ingles, Jarques, and Marisco and compared them to U2, a human OSA cell line, for the following parameters: morphology, growth, contact inhibition, migrational tendencies, alkaline phosphatase staining, heterologous tumor growth, double-strand DNA breaks, and oxidative damage. All results demonstrated the positive characteristics of the Abrams cell line for use in future studies of OSA. Of particular interest, the robust growth of a subcutaneous tumor and rapid pulmonary metastasis of the Abrams cell line in an immunocompromised mouse shows incredible potential for the future use of Abrams as a canine OSA model. Further investigations utilizing a canine cell model of OSA, such as Abrams, will be invaluable to understanding the molecular events underlying OSA, pharmaceutical inhibition of metastasis, and eventual prevention of this devastating disease.

  2. Mouse DRG Cell Line with Properties of Nociceptors.

    Science.gov (United States)

    Doran, Ciara; Chetrit, Jonathan; Holley, Matthew C; Grundy, David; Nassar, Mohammed A

    2015-01-01

    In vitro cell lines from DRG neurons aid drug discovery because they can be used for early stage, high-throughput screens for drugs targeting pain pathways, with minimal dependence on animals. We have established a conditionally immortal DRG cell line from the Immortomouse. Using immunocytochemistry, RT-PCR and calcium microfluorimetry, we demonstrate that the cell line MED17.11 expresses markers of cells committed to the sensory neuron lineage. Within a few hours under differentiating conditions, MED17.11 cells extend processes and following seven days of differentiation, express markers of more mature DRG neurons, such as NaV1.7 and Piezo2. However, at least at this time-point, the nociceptive marker NaV1.8 is not expressed, but the cells respond to compounds known to excite nociceptors, including the TRPV1 agonist capsaicin, the purinergic receptor agonist ATP and the voltage gated sodium channel agonist, veratridine. Robust calcium transients are observed in the presence of the inflammatory mediators bradykinin, histamine and norepinephrine. MED17.11 cells have the potential to replace or reduce the use of primary DRG culture in sensory, pain and developmental research by providing a simple model to study acute nociception, neurite outgrowth and the developmental specification of DRG neurons.

  3. Effect of selected insecticides on SF9 insect cell line

    International Nuclear Information System (INIS)

    Saleh, M.; Rahmo, A.; Hajjar, J.

    2013-01-01

    The toxic effect of three insecticides: dimethoate (organophosphate insecticide), acetamiprid (neonicotinoid insecticide) and deltamethrin (pyrethroid insecticide) were evaluated in vitro on cultured Sf9 cell line. Cell growth inhibition was measured by the 3- (4,5- dimethylthiazol - 2-yl) - 2,5 - diphenyl tetrazolium bromide (MTT) assay. Regression Analysis was used to estimate the 20% inhibition of cells growth (IC 20). The IC 20 values obtained for deltamethrin, acetamipridand dimethoate were: 46.8, 61.6 and 68.9 μM, respectively. The proportion of phagocytic cells was positively correlated with the applied concentrations of the insecticides. (author)

  4. The antiproliferative effect of coumarins on several cancer cell lines.

    Science.gov (United States)

    Kawaii, S; Tomono, Y; Ogawa, K; Sugiura, M; Yano, M; Yoshizawa, Y

    2001-01-01

    Twenty-one coumarins were examined for their antiproliferative activity towards several cancer cell lines, namely lung carcinoma (A549), melanin pigment producing mouse melanoma (B16 melanoma 4A5), human T-cell leukemia (CCRF-HSB-2), and human gastric cancer, lymph node metastasized (TGBC11TKB). The structure-activity relationship established from the results revealed that the 6,7-dihydroxy moiety had an important role for their antiproliferative activity. Analysis of cell cycle distribution indicated that esculetin-treated cells accumulated in the G1 (at 400 microM) or in S phase (at 100 microM).

  5. Characterization of stem-like cells in a new astroblastoma cell line

    Energy Technology Data Exchange (ETDEWEB)

    Coban, Esra Aydemir; Kasikci, Ezgi [Department of Genetics and Bioengineering, Faculty of Engineering and Architecture, Yeditepe University, Istanbul (Turkey); Karatas, Omer Faruk [Molecular Biology and Genetics Department, Erzurum Technical University, Erzurum (Turkey); Suakar, Oznur; Kuskucu, Aysegul [Department of Medical Genetics, Yeditepe University Medical School and Yeditepe University Hospital, Istanbul (Turkey); Altunbek, Mine [Department of Genetics and Bioengineering, Faculty of Engineering and Architecture, Yeditepe University, Istanbul (Turkey); Türe, Uğur [Department of Neurosurgery, Yeditepe University School of Medicine, Istanbul (Turkey); Sahin, Fikrettin [Department of Genetics and Bioengineering, Faculty of Engineering and Architecture, Yeditepe University, Istanbul (Turkey); Bayrak, Omer Faruk, E-mail: ofbayrak@yeditepe.edu.tr [Department of Medical Genetics, Yeditepe University Medical School and Yeditepe University Hospital, Istanbul (Turkey)

    2017-03-15

    Cell lines established from tumors are the most commonly used models in cancer research, and their use in recent years has enabled a greater understanding of the biology of cancer and the means to develop effective treatment strategies. Astroblastomas are uncommon neuroepithelial tumors of glial origin, predominantly affecting young people, mainly teenagers and children, predominantly females. To date, only a single study has reported that astroblastomas contain a large number of neural stem-like cells, which had only a partial proliferation capacity and differentiation. Our objective was to establish an astroblastoma cell line to investigate the presence of astroblastic cells and cancer stem-like cells. The migratory and invasion abilities of the cells were quantified with invasion and migration assays and compared to a glioblastoma cell line. The presence of stem cells was detected with surface-marker analysis by using flow cytometry, and measuring the differentiation ability with a differentiation assay and the self-renewal capacity with a sphere-forming assay. These characteristics may determine whether this novel cell line is a model for astroblastomas that may have stem-cell characteristics. With this novel cell line, scientists can investigate the molecular pathways underlying astroblastomas and develop new therapeutic strategies for patients with these tumors. - Highlights: • An establishment of a novel astroblastoma cell line was proposed. • The presence of astroblastic cells and cancer stem-like cells was investigated. • The molecular pathways underlying astroblastomas may be investigated. • New therapeutic strategies for patients with astroblastoma may be developed.

  6. The Predominant Pathway of Apoptosis in THP-1 Macrophage-Derived Foam Cells Induced by 5-Aminolevulinic Acid-Mediated Sonodynamic Therapy is the Mitochondria-Caspase Pathway Despite the Participation of Endoplasmic Reticulum Stress

    Directory of Open Access Journals (Sweden)

    Huan Wang

    2014-05-01

    Full Text Available Background: In advanced atherosclerosis, chronic endoplasmic reticulum (ER stress induces foam cells apoptosis and generates inflammatory reactions. Methods: THP-1 macrophage-derived foam cells (FC were incubated with 1 mM 5-aminolevulinic acid (ALA. After ALA mediated sonodynamic therapy (ALA-SDT, apoptosis of FC was assayed by Annexin V-PI staining. Intracellular reactive oxygen species (ROS and mitochondrial membrane potential were detected by staining with CellROX® Green Reagent and jc-1. Pretreatment of FC with N-acetylcysteine (NAC, Z-VAD-FMK or 4-phenylbutyrate (4-PBA, mitochondria apoptotic pathway associated proteins and C/EBP-homologous (CHOP expressions were assayed by wertern blotting. Results: Burst of apoptosis of FC was observed at 5-hour after ALA-SDT with 6-hour incubation of ALA and 0.4 W/cm2 ultrasound. After ALA-SDT, intracellular ROS level increased and mitochondrial membrane potential collapsed. Translocations of cytochrome c from mitochondria into cytosol and Bax from cytosol into mitochondria, cleaved caspase 9, cleaved caspase 3, upregulation of CHOP, as well as downregulation of Bcl-2 after ALA-SDT were detected, which could be suppressed by NAC. Activation of mitochondria-caspase pathway could not be inhibited by 4-PBA. Cleaved caspase 9 and caspase 3 as well as apoptosis induced by ALA-SDT could be inhibited by Z-VAD-FMK. Conclusion: The mitochondria-caspase pathway is predominant in the apoptosis of FC induced by ALA-SDT though ER stress participates in.

  7. In vitro invasion of small-cell lung cancer cell lines correlates with expression of epidermal growth factor receptor

    DEFF Research Database (Denmark)

    Damstrup, L; Rude Voldborg, B; Spang-Thomsen, M

    1998-01-01

    receptor (EGFR) in a panel of 21 small-cell lung cancer (SCLC) cell lines. We have previously reported that ten of these cell lines expressed EGFR protein detected by radioreceptor and affinity labelling assays. In 11 small-cell lung cancer (SCLC) cell lines, EGFR mRNA was detected by Northern blot...... analysis. In vitro invasion in a Boyden chamber assay was found in all EGFR-positive cell lines, whereas no invasion was detected in the EGFR-negative cell lines. Quantification of the in vitro invasion in 12 selected SCLC cell lines demonstrated that, in the EGFR-positive cell lines, between 5% and 16......-PCR). However, in vitro invasive SCLC cell lines could not be distinguished from non-invasive cell lines based on the expression pattern of these molecules. In six SCLC cell lines, in vitro invasion was also determined in the presence of the EGFR-neutralizing monoclonal antibody mAb528. The addition...

  8. Induction of Mitochondria Mediated Apoptosis in Human Breast Cancer Cells (T-47D) by Annona reticulata L. Leaves Methanolic Extracts.

    Science.gov (United States)

    Roham, Pratiksha H; Kharat, Kiran R; Mungde, Priyanka; Jadhav, Mahadev A; Makhija, Surinder J

    2016-01-01

    Annona reticulata Linn. (Common name: Bullock's-heart) (Annonaceae family) is a semi-evergreen and small deciduous tree. The extracts of various parts of Annona reticulata L. have been reported as cytotoxic to many cancer cells. Annona reticulata L. leaves' methanolic extract (ARME) was prepared and used against the breast cancer cells. The breast cancer cells (T-47D) viability and IC50 were evaluated by Vybrant® MTT Cell Proliferation Assay Kit. Detection of phosphatidylserine on membranes of apoptotic cells was done by Attune flow cytometer. RNA transcripts were quantified in ARME treated and untreated cells. Finally, the Vybrant® FAM Poly Caspases assay kit was used for analysis of polycaspases activity in T-47D cells. The IC50 (5 ± 0.5 µg/mL) of the ARME was found against breast cancer cells (T-47D). The Paclitaxel was used as a control standard drug for the study. The downregulation of Bcl-2 and upregulation of Bax and Bak, and caspases activation suggested induction of apoptosis in T-47D cells by ARME through mitochondrial pathway. The cell cycle halted at G2/M phase in the ARME treated cells. The ARME was found to be effective against Breast cancer cells (T-47D).

  9. Hypoxic cell turnover in different solid tumor lines

    International Nuclear Information System (INIS)

    Ljungkvist, Anna S.E.; Bussink, Johan; Kaanders, Johannes H.A.M.; Rijken, Paulus F.J.W.; Begg, Adrian C.; Raleigh, James A.; Kogel, Albert J. van der

    2005-01-01

    Purpose: Most solid tumors contain hypoxic cells, and the amount of tumor hypoxia has been shown to have a negative impact on the outcome of radiotherapy. The efficacy of combined modality treatments depends both on the sequence and timing of the treatments. Hypoxic cell turnover in tumors may be important for optimal scheduling of combined modality treatments, especially when hypoxic cell targeting is involved. Methods and Materials: Previously we have shown that a double bioreductive hypoxic marker assay could be used to detect changes of tumor hypoxia in relation to the tumor vasculature after carbogen and hydralazine treatments. This assay was used in the current study to establish the turnover rate of hypoxic cells in three different tumor models. The first hypoxic marker, pimonidazole, was administered at variable times before tumor harvest, and the second hypoxic marker, CCI-103F, was injected at a fixed time before harvest. Hypoxic cell turnover was defined as loss of pimonidazole (first marker) relative to CCI-103F (second marker). Results: The half-life of hypoxic cell turnover was 17 h in the murine C38 colon carcinoma line, 23 h and 49 h in the human xenograft lines MEC82 and SCCNij3, respectively. Within 24 h, loss of pimonidazole-stained areas in C38 and MEC82 occurred concurrent with the appearance of pimonidazole positive cell debris in necrotic regions. In C38 and MEC82, most of the hypoxic cells had disappeared after 48 h, whereas in SCCNij3, viable cells that had been labeled with pimonidazole were still observed after 5 days. Conclusions: The present study demonstrates that the double hypoxia marker assay can be used to study changes in both the proportion of hypoxic tumor cells and their lifespan at the same time. The present study shows that large differences in hypoxic cell turnover rates may exist among tumor lines, with half-lives ranging from 17-49 h

  10. Establishment of clinically relevant radioresistant cell lines and their characteristics

    International Nuclear Information System (INIS)

    Fukumoto, Manabu; Kuwahara, Yoshikazu; Suzuki, Masatoshi

    2014-01-01

    Although radiotherapy is one of the major therapeutic modalities for eradicating malignant tumors, the existence of radioresistant cells remains one of the most critical obstacles. Standard radiotherapy consists of fractionated radiation (FR) of 2-Gy X-rays once a day, 5 days a week, over 60 Gy in total. To understand the characteristics of radioresistant cells and to develop more effective radiotherapy, we have established novel radioresistant cell lines by long-term (> 5 years) exposure to moderate doses of fractionated X-rays. While all the parental human cancer cells ceased, their radioresistant derivatives continue to proliferate with daily exposure to 2-Gy FR for more than 30 days. We have coined those cells as 'clinically relevant radioresistant' (CRR) cells. Transplanted tumors into nude mice were also CRR, indicating that CRR cell lines are powerful tools to improve cancer radiotherapy. We have shown that the suppression of autophagic cell death but not apoptosis was mainly involved in cellular radioresistance. An inhibitor of the mTOR pathway which enhances autophagy was effective to overcome CRR tumors induced in nude mice. But the underlined mechanism was not through the inhibition of autophagy. Guanine nucleotide-binding protein 1 (GBP1) over expression was necessary for maintaining the CRR phenotype, but radioresistant cells were not necessarily cancer stem cells (CSCs). Targeting GBP1 positive cancer cells may be a more efficient method in conquering cancer than targeting CSCs. Slight but significant radioresistance was acquired by 0.5 Gy/12 hrs of long-term FR exposures to parental cells for more than 31 days in accordance with cyclinD1 over expression. This acquired radioresistance (ARR) was stably maintained in the tumor cells even on 31 days after the cessation of 0.5-Gy FR. Present observations give a mechanistic insight for ARR of tumor cells through long-term FR exposure, and provide novel therapeutic targets for radiosensitization

  11. Incorrect strain information for mouse cell lines: sequential influence of misidentification on sublines

    OpenAIRE

    Uchio-Yamada, Kozue; Kasai, Fumio; Ozawa, Midori; Kohara, Arihiro

    2016-01-01

    Misidentification or cross-contamination of cell lines can cause serious issues. Human cell lines have been authenticated by short tandem repeat profiling; however, mouse cell lines have not been adequately assessed. In this study, mouse cell lines registered with the JCRB cell bank were examined by simple sequence length polymorphism (SSLP) analysis to identify their strains. Based on comparisons with 7 major inbred strains, our results revealed their strains in 80 of 90 cell lines. However,...

  12. Cysteine modified polyaniline films improve biocompatibility for two cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Yslas, Edith I., E-mail: eyslas@exa.unrc.edu.ar [Departamento de Biología Molecular, Universidad Nacional de Río Cuarto, Agencia Postal Nro3, X580BYA Río Cuarto (Argentina); Cavallo, Pablo; Acevedo, Diego F.; Barbero, César A. [Departamento de Química, Universidad Nacional de Río Cuarto, Agencia Postal Nro3, X580BYA Río Cuarto (Argentina); Rivarola, Viviana A. [Departamento de Biología Molecular, Universidad Nacional de Río Cuarto, Agencia Postal Nro3, X580BYA Río Cuarto (Argentina)

    2015-06-01

    This work focuses on one of the most exciting application areas of conjugated conducting polymers, which is cell culture and tissue engineering. To improve the biocompatibility of conducting polymers we present an easy method that involves the modification of the polymer backbone using L-cysteine. In this publication, we show the synthesis of polyaniline (PANI) films supported onto Polyethylene terephthalate (PET) films, and modified using cysteine (PANI-Cys) in order to generate a biocompatible substrate for cell culture. The PANI-Cys films are characterized by Fourier Transform infrared and UV–visible spectroscopy. The changes in the hydrophilicity of the polymer films after and before the modification were tested using contact angle measurements. After modification the contact angle changes from 86° ± 1 to 90° ± 1, suggesting a more hydrophylic surface. The adhesion properties of LM2 and HaCaT cell lines on the surface of PANI-Cys films in comparison with tissue culture plastic (TCP) are studied. The PANI-Cys film shows better biocompatibility than PANI film for both cell lines. The cell morphologies on the TCP and PANI-Cys film were examined by florescence and Atomic Force Microscopy (AFM). Microscopic observations show normal cellular behavior when PANI-Cys is used as a substrate of both cell lines (HaCaT and LM2) as when they are cultured on TCP. The ability of these PANI-Cys films to support cell attachment and growth indicates their potential use as biocompatible surfaces and in tissue engineering. - Highlights: • A new surface PANI-Cys was produced on films of polyethylene terephthalate. • The relationship between surface characteristics and biocompatibility is analyzed. • The PANI-Cys film presents good biocompatibility for two cell lines.

  13. Cell lines radiosensitization of thyroid cancer by histone deacetylase inhibitors

    International Nuclear Information System (INIS)

    Perona, M; Dagrosa, M A; Rossich, L; Casal, M; Pisarev, M A; Thomasz, L; Juvenal G J

    2012-01-01

    Introduction: Thyroid cancer is the most common endocrine neoplasia. Surgical resection and radioactive iodine is an effective treatment for well-differentiated tumors. Histone deacetylase inhibitors (HDAC-I) are agents that cause hyperacetylation of histone proteins and as a consequence remodeling of chromatin structure. They can induce growth arrest, differentiation and apoptotic cell death in different tumor cells. The use of HDAC-I agents could be of utility to enhance the response to external radiation therapy of those thyroid cancers that are refractory to most conventional therapeutic treatments. Objective: To study the effect of HDAC-I as radiosensitizers for the treatment of thyroid cancer and their ability to induce differentiation of thyroid cancer cells. Materials and methods: The human thyroid follicular (WRO) and papillary (TPC-1) carcinoma cell lines were seeded and incubated with increasing doses (0, 0.3, 0.5, 1 and 1.5 mM) of the HDAC-I sodium butirate (NaB) and valproic acid (VA) to evaluate cell proliferation and iodide uptake. Cells were irradiated with a 60 Co γ-ray source (1 ± 5% Gy/min) and postirradiation survival was quantified with the colony formation assay. Survival fraction at 2 Gy (SF2) was calculated for each cell line. Cell cycle and cell death were evaluated at a dose of 3 Gy. Iodide uptake, PCR analysis and transient transfection studies were performed. Results: Cell proliferation was not significantly suppressed after 24 hours of incubation with both drugs at all assayed doses. Iodide uptake was not modified after incubation with HDAC-I of both cell lines. SF2 was reduced from 68 ± 1.6 % in the control WRO cells to 42 ± 3.8 % (P<0.001) in NaB-treated cells. In TPC-1 SF2 was reduced from 32 ± 1.1 % in the control cells to 24 ± 0.8 % (P<0.01). In VA-treated cells SF2 was reduced from 69 ± 0.02 % in control WRO cells to 56 ± 0.01 % (P<0.01) and from 31 ± 2 % in control TPC-1 cells to 11 ± 1 % (P<0.01). There was an arrest

  14. Microbeam X-ray fluorescence mapping of Cu and Fe in human prostatic carcinoma cell lines using synchrotron radiation

    Energy Technology Data Exchange (ETDEWEB)

    Rocha, K.M.J.; Leitao, R.G.; Oliveira-Barros, E.G.; Oliveira, M.A.; Canellas, C.G.L.; Anjos, M.J.; Nasciutti, L.E.; Lopes, R.T., E-mail: kjose@nuclear.ufrj.br, E-mail: marcelin@lin.ufrj.br, E-mail: ricardo@lin.ufrj.br, E-mail: roberta@lin.ufrj.br, E-mail: eligouveab@gmail.com, E-mail: maria_aparecida_ufrj@yahoo.com.br, E-mail: luiz.nasciutti@histo.ufrj.br, E-mail: roberta.leitao@uerj.br, E-mail: marcelin@uerj.br [Universidade Federal do Rio de Janeiro (UFRJ), RJ (Brazil). Laboratorio de Instrumentacao Nuclear; Universidade Federal do Rio de Janeiro (UFRJ), RJ (Brazil). Instituto de Ciencias Biomedicas; Universidade do Estado do Rio de Janeiro (UERJ), RJ (Brazil). Instituto de Fisica

    2017-11-01

    Cancer is a worldwide public health problem and prostate cancer continues to be one of the most common fatal cancers in men. Copper plays an important role in the aetiology and growth of tumours however, whether intratumoral copper is actually elevated in prostate cancer patients has not been established. Iron, an important trace element, plays a vital function in oxygen metabolism, oxygen uptake, and electron transport in mitochondria, energy metabolism, muscle function, and hematopoiesis. The X-ray microfluorescence technique (μXRF) is a rapid and non-destructive method of elemental analysis that provides useful elemental information about samples without causing damage or requiring extra sample preparations. This study investigated the behavior of cells in spheroids of human prostate cells, tumour cell line (DU145) and normal cell line (RWPE-1), after supplementation with zinc chloride by 24 hours using synchrotron X-ray microfluorescence (μSRXRF). The measurements were performed with a standard geometry of 45 deg of incidence, excited by a white beam using a pixel of 25 μm and a time of 300 ms/pixel at the XRF beamline at the Synchrotron Light National Laboratory (Campinas, Brazil). The results by SRμXRF showed non-uniform Cu and Fe distributions in all the spheroids analyzed. (author)

  15. Microbeam X-ray fluorescence mapping of Cu and Fe in human prostatic carcinoma cell lines using synchrotron radiation

    International Nuclear Information System (INIS)

    Rocha, K.M.J.; Leitao, R.G.; Oliveira-Barros, E.G.; Oliveira, M.A.; Canellas, C.G.L.; Anjos, M.J.; Nasciutti, L.E.; Lopes, R.T.; Universidade Federal do Rio de Janeiro; Universidade do Estado do Rio de Janeiro

    2017-01-01

    Cancer is a worldwide public health problem and prostate cancer continues to be one of the most common fatal cancers in men. Copper plays an important role in the aetiology and growth of tumours however, whether intratumoral copper is actually elevated in prostate cancer patients has not been established. Iron, an important trace element, plays a vital function in oxygen metabolism, oxygen uptake, and electron transport in mitochondria, energy metabolism, muscle function, and hematopoiesis. The X-ray microfluorescence technique (μXRF) is a rapid and non-destructive method of elemental analysis that provides useful elemental information about samples without causing damage or requiring extra sample preparations. This study investigated the behavior of cells in spheroids of human prostate cells, tumour cell line (DU145) and normal cell line (RWPE-1), after supplementation with zinc chloride by 24 hours using synchrotron X-ray microfluorescence (μSRXRF). The measurements were performed with a standard geometry of 45 deg of incidence, excited by a white beam using a pixel of 25 μm and a time of 300 ms/pixel at the XRF beamline at the Synchrotron Light National Laboratory (Campinas, Brazil). The results by SRμXRF showed non-uniform Cu and Fe distributions in all the spheroids analyzed. (author)

  16. Monitoring cell line identity in collections of human induced pluripotent stem cells

    Directory of Open Access Journals (Sweden)

    Raquel Sarafian

    2018-04-01

    Full Text Available The ability to reprogram somatic cells into induced pluripotent stem cells (hiPSCs has led to the generation of large collections of cell lines from thousands of individuals with specific phenotypes, many of which will be shared among different research groups as invaluable tools for biomedical research. As hiPSC-based research involves extensive culture of many cell lines, the issue periodic cell line identification is particularly important to ensure that cell line identity remains accurate. Here we analyzed the different commercially available genotyping methods considering ease of in-house genotyping, cost and informativeness, and applied one of them in our workflow for hiPSC generation. We show that the chosen STR method was able to establish a unique DNA profile for each of the 35 individuals/hiPSC lines at the examined sites, as well as identify two discrepancies resulting from inadvertently exchanged samples. Our results highlight the importance of hiPSC line genotyping by an in-house method that allows periodic cell line identification and demonstrate that STR is a useful approach to supplement less frequent karyotyping and epigenetic evaluations. Keywords: Induced pluripotent stem cells, Genotyping, Cell line identification, Short tandem repeats, Quality control

  17. Detection of immunotoxicity using T-cell based cytokine reporter cell lines ('Cell Chip')

    International Nuclear Information System (INIS)

    Ringerike, Tove; Ulleraas, Erik; Voelker, Rene; Verlaan, Bert; Eikeset, Aase; Trzaska, Dominika; Adamczewska, Violetta; Olszewski, Maciej; Walczak-Drzewiecka, Aurelia; Arkusz, Joanna; Loveren, Henk van; Nilsson, Gunnar; Lovik, Martinus; Dastych, Jaroslaw; Vandebriel, Rob J.

    2005-01-01

    Safety assessment of chemicals and drugs is an important regulatory issue. The evaluation of potential adverse effects of compounds on the immune system depends today on animal experiments. An increasing demand, however, exists for in vitro alternatives. Cytokine measurement is a promising tool to evaluate chemical exposure effects on the immune system. Fortunately, this type of measurement can be performed in conjunction with in vitro exposure models. We have taken these considerations as the starting point to develop an in vitro method to efficiently screen compounds for potential immunotoxicity. The T-cell lymphoma cell line EL-4 was transfected with the regulatory sequences of interleukin (IL)-2, IL-4, IL-10, interferon (IFN)-γ or actin fused to the gene for enhanced green fluorescent protein (EGFP) in either a stabile or a destabilised form. Consequently, changes in fluorescence intensity represent changes in cytokine expression with one cell line per cytokine. We used this prototype 'Cell Chip' to test, by means of flow cytometry, the immunomodulatory potential of 13 substances and were able to detect changes in cytokine expression in 12 cases (successful for cyclosporine, rapamycin, pentamidine, thalidomide, bis(tri-n-butyltin)oxide, house dust mite allergen (Der p I), 1-chloro-2,4-dinitrobenzene, benzocaine, tolylene 2,4-diisocyanate, potassium tetrachloroplatinate, sodium dodecyl sulphate and mercuric chloride; unsuccessful for penicillin G). In conclusion, this approach seems promising for in vitro screening for potential immunotoxicity, especially when additional cell lines besides T-cells are included

  18. Sphere-forming cell subpopulations with cancer stem cell properties in human hepatoma cell lines

    Directory of Open Access Journals (Sweden)

    Chen Lei

    2011-06-01

    Full Text Available Abstract Background Cancer stem cells (CSCs are regarded as the cause of tumor formation and recurrence. The isolation and identification of CSCs could help to develop novel therapeutic strategies specifically targeting CSCs. Methods Human hepatoma cell lines were plated in stem cell conditioned culture system allowed for sphere forming. To evaluate the stemness characteristics of spheres, the self-renewal, proliferation, chemoresistance, tumorigenicity of the PLC/PRF/5 sphere-forming cells, and the expression levels of stem cell related proteins in the PLC/PRF/5 sphere-forming cells were assessed, comparing with the parental cells. The stem cell RT-PCR array was performed to further explore the biological properties of liver CSCs. Results The PLC/PRF/5, MHCC97H and HepG2 cells could form clonal nonadherent 3-D spheres and be serially passaged. The PLC/PRF/5 sphere-forming cells possessed a key criteria that define CSCs: persistent self-renewal, extensive proliferation, drug resistance, overexpression of liver CSCs related proteins (Oct3/4, OV6, EpCAM, CD133 and CD44. Even 500 sphere-forming cells were able to form tumors in NOD/SCID mice, and the tumor initiating capability was not decreased when spheres were passaged. Besides, downstream proteins DTX1 and Ep300 of the CSL (CBF1 in humans, Suppressor of hairless in Drosophila and LAG1 in C. elegans -independent Notch signaling pathway were highly expressed in the spheres, and a gamma-secretase inhibitor MRK003 could significantly inhibit the sphere formation ability. Conclusions Nonadherent tumor spheres from hepatoma cell lines cultured in stem cell conditioned medium possess liver CSC properties, and the CSL-independent Notch signaling pathway may play a role in liver CSCs.

  19. Apoptosis induction of epifriedelinol on human cervical cancer cell line

    African Journals Online (AJOL)

    Background: Present investigation evaluates the antitumor activity of epifriedelinol for the management of cervical cancer by inducing process of apoptosis. Methods: Human Cervical Cancer Cell Line, C33A and HeLa were selected for study and treated with epifriedelinol at a concentration of (50-1000 μg/ml). Cytotoxicity of ...

  20. 77 FR 5489 - Identification of Human Cell Lines Project

    Science.gov (United States)

    2012-02-03

    ... individual or species. With the advent of standardized, simple, and rapid methods for human cell line... project will undergo STR profiling, a DNA profiling method that examines/screens for STRs (DNA elements 2... distinct DNA profile and when the STR DNA fragment sizes are converted to numeric values, the DNA profiles...

  1. Antibacterial and anti-breast cancer cell line activities of ...

    African Journals Online (AJOL)

    Purpose: To evaluate the activity of extracts of Sanghuangporus sp.1 fungus against pathogenic bacteria and a breast cancer cell line. Methods: The wild fruiting body and mycelium of Sanghuangporus sp.1 were extracted with water and ethanol by ultrasonication extraction. The activity of the extracts against pathogenic ...

  2. Discovery of a new mitochondria permeability transition pore (mPTP) inhibitor based on gallic acid.

    Science.gov (United States)

    Teixeira, José; Oliveira, Catarina; Cagide, Fernando; Amorim, Ricardo; Garrido, Jorge; Borges, Fernanda; Oliveira, Paulo J

    2018-12-01

    Pharmacological interventions targeting mitochondria present several barriers for a complete efficacy. Therefore, a new mitochondriotropic antioxidant (AntiOxBEN 3 ) based on the dietary antioxidant gallic acid was developed. AntiOxBEN 3 accumulated several thousand-fold inside isolated rat liver mitochondria, without causing disruption of the oxidative phosphorylation apparatus, as seen by the unchanged respiratory control ratio, phosphorylation efficiency, and transmembrane electric potential. AntiOxBEN 3 showed also limited toxicity on human hepatocarcinoma cells. Moreover, AntiOxBEN 3 presented robust iron-chelation and antioxidant properties in both isolated liver mitochondria and cultured rat and human cell lines. Along with its low toxicity profile and high antioxidant activity, AntiOxBEN 3 strongly inhibited the calcium-dependent mitochondrial permeability transition pore (mPTP) opening. From our data, AntiOxBEN 3 can be considered as a lead compound for the development of a new class of mPTP inhibitors and be used as mPTP de-sensitiser for basic research or clinical applications or emerge as a therapeutic application in mitochondria dysfunction-related disorders.

  3. AAVS1-Targeted Plasmid Integration in AAV Producer Cell Lines.

    Science.gov (United States)

    Luo, Yuxia; Frederick, Amy; Martin, John M; Scaria, Abraham; Cheng, Seng H; Armentano, Donna; Wadsworth, Samuel C; Vincent, Karen A

    2017-06-01

    Adeno-associated virus (AAV) producer cell lines are created via transfection of HeLaS3 cells with a single plasmid containing three components (the vector sequence, the AAV rep and cap genes, and a selectable marker gene). As this plasmid contains both the cis (Rep binding sites) and trans (Rep protein encoded by the rep gene) elements required for site-specific integration, it was predicted that plasmid integration might occur within the AAVS1 locus on human chromosome 19 (chr19). The objective of this study was to investigate whether integration in AAVS1 might be correlated with vector yield. Plasmid integration sites within several independent cell lines were assessed via Southern, fluorescence in situ hybridization (FISH) and PCR analyses. In the Southern analyses, the presence of fragments detected by both rep- and AAVS1-specific probes suggested that for several mid- and high-producing lines, plasmid DNA had integrated into the AAVS1 locus. Analysis with puroR and AAVS1-specific probes suggested that integration in AAVS1 was a more widespread phenomenon. High-producing AAV2-secreted alkaline phosphatase (SEAP) lines (masterwell 82 [MW82] and MW278) were evaluated via FISH using probes specific for the plasmid, AAVS1, and a chr19 marker. FISH analysis detected two plasmid integration sites in MW278 (neither in AAVS1), while a total of three sites were identified in MW82 (two in AAVS1). An inverse PCR assay confirmed integration within AAVS1 for several mid- and high-producing lines. In summary, the FISH, Southern, and PCR data provide evidence of site-specific integration of the plasmid within AAVS1 in several AAV producer cell lines. The data also suggest that integration in AAVS1 is a general phenomenon that is not necessarily restricted to high producers. The results also suggest that plasmid integration within the AAVS1 locus is not an absolute requirement for a high vector yield.

  4. Evaluating the uptake and intracellular fate of polystyrene nanoparticles by primary and hepatocyte cell lines in vitro

    International Nuclear Information System (INIS)

    Johnston, Helinor J.; Semmler-Behnke, Manuela; Brown, David M.; Kreyling, Wolfgang; Tran, Lang; Stone, Vicki

    2010-01-01

    Nanoparticles (NPs) are being used within diverse applications such as medicines, clothing, cosmetics and food. In order to promote the safe development of such nanotechnologies it is essential to assess the potential adverse health consequences associated with human exposure. The liver is recognised as a target site for NP toxicity, due to NP accumulation within this organ subsequent to injection, inhalation or instillation. The uptake of fluorescent polystyrene carboxylated particles (20 nm or 200 nm diameter) by hepatocytes was determined using confocal microscopy; with cells imaged 'live' during particle exposure or after exposure within fixed cells. Comparisons between the uptake of polystyrene particles by primary rat hepatocytes, and human hepatocyte cell lines (C3A and HepG2) were made. Uptake of particles by hepatocytes was size, time, and serum dependent. Specifically, the uptake of 200 nm particles was limited, but 20 nm NPs were internalised by all cell types from 10 min onwards. At 10 min, 20 nm NP fluorescence co-localised with the tubulin cytoskeleton staining; after 30 min NP fluorescence compartmentalised into structures located within and/or between cells. The fate of internalised NPs was considered and they were not contained within early endosomes or lysosomes, but within mitochondria of cell lines. NPs accumulated within bile canaliculi to a limited extent, which suggests that NPs can be eliminated within bile. This is in keeping with the finding that gold NPs were eliminated in bile following intravenous injection into rats. The findings were, in the main, comparable between primary rat hepatocytes and the different human hepatocyte cell lines.

  5. Effects of hypoxia on human cancer cell line chemosensitivity

    Science.gov (United States)

    2013-01-01

    Background Environment inside even a small tumor is characterized by total (anoxia) or partial oxygen deprivation, (hypoxia). It has been shown that radiotherapy and some conventional chemotherapies may be less effective in hypoxia, and therefore it is important to investigate how different drugs act in different microenvironments. In this study we perform a large screening of the effects of 19 clinically used or experimental chemotherapeutic drugs on five different cell lines in conditions of normoxia, hypoxia and anoxia. Methods A panel of 19 commercially available drugs: 5-fluorouracil, acriflavine, bortezomib, cisplatin, digitoxin, digoxin, docetaxel, doxorubicin, etoposide, gemcitabine, irinotecan, melphalan, mitomycin c, rapamycin, sorafenib, thalidomide, tirapazamine, topotecan and vincristine were tested for cytotoxic activity on the cancer cell lines A2780 (ovarian), ACHN (renal), MCF-7 (breast), H69 (SCLC) and U-937 (lymphoma). Parallel aliquots of the cells were grown at different oxygen pressures and after 72 hours of drug exposure viability was measured with the fluorometric microculture cytotoxicity assay (FMCA). Results Sorafenib, irinotecan and docetaxel were in general more effective in an oxygenated environment, while cisplatin, mitomycin c and tirapazamine were more effective in a low oxygen environment. Surprisingly, hypoxia in H69 and MCF-7 cells mostly rendered higher drug sensitivity. In contrast ACHN appeared more sensitive to hypoxia, giving slower proliferating cells, and consequently, was more resistant to most drugs. Conclusions A panel of standard cytotoxic agents was tested against five different human cancer cell lines cultivated at normoxic, hypoxic and anoxic conditions. Results show that impaired chemosensitivity is not universal, in contrast different cell lines behave different and some drugs appear even less effective in normoxia than hypoxia. PMID:23829203

  6. BH3-only protein Bim is associated with the degree of Helicobacter pylori-induced gastritis and is localized to the mitochondria of inflammatory cells in the gastric mucosa.

    Science.gov (United States)

    Akazawa, Yuko; Matsuda, Katsuya; Isomoto, Hajime; Matsushima, Kayoko; Kido, Yoko; Urabe, Shigetoshi; Yamaghchi, Naoyuki; Ohnita, Ken; Takeshima, Fuminao; Kondo, Hisayoshi; Tsugawa, Hitoshi; Suzuki, Hidekazu; Moss, Joel; Nakao, Kazuhiko; Nakashima, Masahiro

    2015-09-01

    BH3-only protein, Bim, is a pro-apoptotic protein that mediates mitochondria-dependent cell death. However, the role of Bim in Helicobacter pylori-associated gastritis remains unclear. This study aimed to assess the cellular localization of Bim and its possible role in H. pylori-induced gastritis. The study was conducted on biopsy specimens obtained from 80 patients who underwent upper gastrointestinal endoscopy (H. pylori-negative: n=30, positive: n=50). Association between Bim mRNA expression and severity of gastritis was evaluated and the localization of Bim was examined by immunofluorescence. Bim mRNA expression was positively correlated with the degree of gastritis, as defined by the Sydney system. Immunohistochemical analysis confirmed increased Bim expression in H. pylori-infected gastric mucosa compared with uninfected mucosa in both humans and mice. Bim localized in myeloperoxidase- and CD138-positive cells of H. pylori-infected lamina propria and submucosa of the gastric tract, indicating that this protein is predominantly expressed in neutrophils and plasma cells. In contrast, Bim did not localize in CD20-, CD3-, or CD68-positive cells. Bim was expressed in the mitochondria, where it was partially co-localized with activated Bax and cleaved-PARP. In conclusion, Bim is expressed in neutrophils and plasma cells in H. pylori-associated gastritis, where it may participate in the termination of inflammatory response by causing mitochondria-mediated apoptosis in specific leucocytes. Copyright © 2015 Elsevier GmbH. All rights reserved.

  7. Characterization of the camel skin cell line Dubca.

    Science.gov (United States)

    Klopries, M; Wernery, U; Kaaden, O R

    1995-01-01

    A skin fibroblast cell culture was established from a 2-month-old dromedary foetus. The cells were transformed by infection with SV40 and cloned in soft agar. The established cell line is now designated Dubca cells (Dubai camel) and has been in permanent culture for 95 passages. The cell culture was examined morphologically, chromosome preparations made and DNA fingerprinting performed by hybridization with the oligonucleotide probe (GTG)5. SV40 large T antigen was detected by western blotting. The viral host range was determined by infection with viruses of different families. Camelpox virus (CaPV) bovine herpesvirus-1 (BHV-1), vesicular stomatitis virus (VSV) and border disease virus (BDV) could be propagated in these cells.

  8. 9-β-arabinofuranosyladenine preferentially sensitizes radioresistant squamous cell carcinoma cell lines to x-rays

    International Nuclear Information System (INIS)

    Heaton, D.

    1992-06-01

    The effect of 9-β-arabinofuranosyladenine (ara-A) on sensitivity to the deleterious effects of x-rays was studied in six squamous cell carcinoma cell lines. Three lines were relatively radioresistant, having D 0 values of 2.31 to 2.89 Gy, and the other three lines were relatively radiosensitive, having D 0 values of between 1.07 and 1.45 Gy. Ara-A (50 or 500 μM) was added to cultures 30 min prior to irradiation and removed 30 min after irradiation, and sensitivity was measured in terms of cell survival. The radiosensitizing effect of ara-A was very dependent on the inherent radiosensitivity of the tumor cell line. Fifty micromolar concentrations of ara-A sensitized only the two most radioresistant lines, SCC-12B.2 and JSQ-3. Five hundred micromolar concentrations of ara-A sensitized the more sensitive cell lines, SQ-20B and SQ-9G, but failed to have any effect on the radiation response of the two most sensitive cell lines, SQ-38 and SCC-61. Concentrations of ara-A as low as 10 μM were equally efficient in inhibiting DNA synthesis in all six cell lines. These results suggest that the target for the radiosensitizing effect of ara-A is probably related to the factor controlling the inherent radiosensitivity of human tumor cells. Therefore, ara-A might be useful in overcoming radiation resistance in vivo

  9. 9-{beta}-arabinofuranosyladenine preferentially sensitizes radioresistant squamous cell carcinoma cell lines to x-rays

    Energy Technology Data Exchange (ETDEWEB)

    Heaton, D. [Rush Univ. Medical Center, Chicago, IL (United States). Therapeutic Radiology; Mustafi, R. [Chicago Univ., IL (United States). Dept. of Radiation and Cellular Oncology; Schwartz, J.L. [Chicago Univ., IL (United States). Dept. of Radiation and Cellular Oncology]|[Argonne National Lab., IL (United States)

    1992-06-01

    The effect of 9-{beta}-arabinofuranosyladenine (ara-A) on sensitivity to the deleterious effects of x-rays was studied in six squamous cell carcinoma cell lines. Three lines were relatively radioresistant, having D{sub 0} values of 2.31 to 2.89 Gy, and the other three lines were relatively radiosensitive, having D{sub 0} values of between 1.07 and 1.45 Gy. Ara-A (50 or 500 {mu}M) was added to cultures 30 min prior to irradiation and removed 30 min after irradiation, and sensitivity was measured in terms of cell survival. The radiosensitizing effect of ara-A was very dependent on the inherent radiosensitivity of the tumor cell line. Fifty micromolar concentrations of ara-A sensitized only the two most radioresistant lines, SCC-12B.2 and JSQ-3. Five hundred micromolar concentrations of ara-A sensitized the more sensitive cell lines, SQ-20B and SQ-9G, but failed to have any effect on the radiation response of the two most sensitive cell lines, SQ-38 and SCC-61. Concentrations of ara-A as low as 10 {mu}M were equally efficient in inhibiting DNA synthesis in all six cell lines. These results suggest that the target for the radiosensitizing effect of ara-A is probably related to the factor controlling the inherent radiosensitivity of human tumor cells. Therefore, ara-A might be useful in overcoming radiation resistance in vivo.

  10. Effects of cholera toxin on human colon carcinoma cell lines.

    Science.gov (United States)

    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)

  11. Differences in radiosensitivity between three HER2 overexpressing cell lines

    International Nuclear Information System (INIS)

    Steffen, Ann-Charlott; Tolmachev, Vladimir; Stenerloew, Bo; Goestring, Lovisa; Palm, Stig; Carlsson, Joergen

    2008-01-01

    HER2 is a potential target for radionuclide therapy, especially when HER2 overexpressing breast cancer cells are resistant to Herceptin registered treatment. Therefore, it is of interest to analyse whether HER2 overexpressing tumour cells have different inherent radiosensitivity. The radiosensitivity of three often used HER2 overexpressing cell lines, SKOV-3, SKBR-3 and BT-474, was analysed. The cells were exposed to conventional photon irradiation, low linear energy transfer (LET), to characterise their inherent radiosensitivity. The analysis was made with clonogenic survival and growth extrapolation assays. The cells were also exposed to alpha particles, high LET, from 211 At decays using the HER2-binding affibody molecule 211 At-(Z HER2:4 ) 2 as targeting agent. Assays for studies of internalisation of the affibody molecule were applied. SKOV-3 cells were most radioresistant, SKBR-3 cells were intermediate and BT-474 cells were most sensitive as measured with the clonogenic and growth extrapolation assays after photon irradiation. The HER2 dependent cellular uptake of 211 At was qualitatively similar for all three cell lines. However, the sensitivity to the alpha particles from 211 At differed; SKOV-3 was most resistant, SKBR-3 intermediate and BT-474 most sensitive. These differences were unexpected because it is assumed that all types of cells should have similar sensitivity to high-LET radiation. The sensitivity to alpha particle exposure correlated with internalisation of the affibody molecule and with size of the cell nucleus. There can be differences in radiosensitivity, which, if they also exist between patient breast cancer cells, are important to consider for both conventional radiotherapy and for HER2-targeted radionuclide therapy. (orig.)

  12. Radiosensitization of colorectal carcinoma cell lines by histone deacetylase inhibition

    International Nuclear Information System (INIS)

    Flatmark, Kjersti; Nome, Ragnhild V; Folkvord, Sigurd; Bratland, Åse; Rasmussen, Heidi; Ellefsen, Mali Strand; Fodstad, Øystein; Ree, Anne Hansen

    2006-01-01

    The tumor response to preoperative radiotherapy of locally advanced rectal cancer varies greatly, warranting the use of experimental models to assay the efficacy of molecular targeting agents in rectal cancer radiosensitization. Histone deacetylase (HDAC) inhibitors, agents that cause hyperacetylation of histone proteins and thereby remodeling of chromatin structure, may override cell cycle checkpoint responses to DNA damage and amplify radiation-induced tumor cell death. Human colorectal carcinoma cell lines were exposed to ionizing radiation and HDAC inhibitors, and cell cycle profiles and regulatory factors, as well as clonogenicity, were analyzed. In addition to G 2 /M phase arrest following irradiation, the cell lines displayed cell cycle responses typical for either intact or defective p53 function (the presence or absence, respectively, of radiation-induced expression of the cell cycle inhibitor p21 and subsequent accumulation of G 1 phase cells). In contrast, histone acetylation was associated with complete depletion of the G 1 population of cells with functional p53 but accumulation of both G 1 and G 2 /M populations of cells with defective p53. The cellular phenotypes upon HDAC inhibition were consistent with the observed repression of Polo-like kinase-1, a regulatory G 2 /M phase kinase. Following pre-treatment with HDAC inhibitors currently undergoing clinical investigation, the inhibitory effect of ionizing radiation on clonogenicity was significantly amplified. In these experimental models, HDAC inhibition sensitized the tumor cells to ionizing radiation, which is in accordance with the concept of increased probability of tumor cell death when chromatin structure is modified

  13. LET effects on normal and radiosensitive cell lines

    International Nuclear Information System (INIS)

    Geard, C.R.; Travisano, M.

    1986-01-01

    Charged particles in the track segment mode were produced by the RARAF Van de Graaff accelerator and used to irradiate two CHO cell lines, a radiosensitive hypermutable line EM9 and its normal parent AA8. Asynchronous cells were irradiated attached to 6 micrometer thick Mylar with protons, deuterons and helium-3 particles at LETs ranging from 10 to 150 keV per micrometer. A 50 kVp x-ray tube integrated into the track segment facility provided a low LET comparison. Following irradiation cells were monitored for clonogenicity, and in a separate series of experiments frequencies of sister chromatid exchanges. Up to 9 experiments were carried out at each LET, with a total of 8 radiations of different LETs being compared. The optimally effective LET for cell survival was between 80 and 120 keV per micrometer, with the 150 keV per micrometer particles indicating energy wastage. The differential between the normal and radiosensitive cell lines was maintained at all LETs

  14. ROS-induced toxicity: exposure of 3T3, RAW264.7, and MCF7 cells to superparamagnetic iron oxide nanoparticles results in cell death by mitochondria-dependent apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Hsieh, Hui-Chen, E-mail: d93548008@ntu.edu.tw; Chen, Chung-Ming, E-mail: chung@ntu.edu.tw [National Taiwan University, Institute of Biomedical Engineering (China); Hsieh, Wen-Yuan, E-mail: hsiehw@itri.org.tw [Industrial Technology Research Institute, Biomedical Technology and Device Research Labs (China); Chen, Ching-Yun, E-mail: chingyun523@gmail.com; Liu, Chia-Ching, E-mail: d95548005@ntu.edu.tw; Lin, Feng-Huei, E-mail: double@ntu.edu.tw [National Taiwan University, Institute of Biomedical Engineering (China)

    2015-02-15

    Superparamagnetic nanoparticles (Fe{sub 3}O{sub 4}, SPIO) have been used as magnetic resonance imaging enhancers for years. However, bio-safety issues concerning nanoparticles remain largely unexplored. Of particular concern is the possible cellular impact of nanoparticles during SPIO uptake and subsequent oxidative stress. SPIO causes cell death by apoptosis via a little understood mitochondrial pathway. To more closely examine this process, three kinds of cells—3T3, RAW264.7, and MCF7—were treated with SPIO coated with polyethylene glycol (SPIO-PEG) and monitored by transmission electron microscopy (TEM), using cytotoxicity evaluation, mitochondrial activity, reactive oxygen species (ROS) generation, and Annexin V assay. TEM revealed that SPIO-PEG nanoparticles surrounded the cellular endosome membrane, creating a bulge in the endosome. Compared to 3T3 cells, greater numbers of SPIO-PEG nanoparticles infiltrated the mitochondria of RAW264.7 and MCF7 cells. SPIO-PEG residency is associated with boosted ROS, with elevated levels of mitochondrial activity, and advancement of cell apoptosis. Furthermore, correlation analysis showed that a polynomial model demonstrates a better fit than a linear model in MCF7, implying that cytotoxicity may have alternative impacts on cell death at different concentrations. Thus, we believe that MCF7 cell death results from the apoptosis pathway triggered by mitochondria, and we find lower cytotoxicity in 3T3. We propose that optimal levels of SPIO-PEG nanoparticles lead to increased levels of ROS and a resulting oxidative stress environment which will kill only cancer cells while sparing normal cells. This finding has great potential for use in cancer therapies in the future.

  15. Isolation, Characterization, and Establishment of Spontaneously Immortalized Cell Line HRPE-2S With Stem Cell Properties.

    Science.gov (United States)

    Shams Najafabadi, Hoda; Soheili, Zahra-Soheila; Samiei, Shahram; Ahmadieh, Hamid; Ranaei Pirmardan, Ehsan; Masoumi, Maryam

    2017-10-01

    The retinal pigment epithelium is a monolayer of highly specialized pigmented cells located between the neural retina and the Bruch's membrane of the choroid. RPE cells play a crucial role in the maintenance and function of the underlying photoreceptors. This study introduces a spontaneously arising human retinal pigment epithelial cell line, HRPE-2S, which was isolated from primary RPE cell culture of 2 days old male donor. We characterized morphology and functional properties of the new cell line. The immortalized cell line was maintained in culture for more than 70 passages and 240 divisions. The average doubling time of the cells was approximately 22 h and got freezed at 26th passage. The cell line expressed RPE-specific markers RPE65 and cell junction protein ZO1 as an epithelial cell marker. It also expressed CHX10, PAX6, Nestin, SOX2 as stem and retinal progenitor cell markers. Ki67 as a marker of cell proliferation was expressed in all HRPE-2S cells. It represented typical epithelial cobblestone morphology and did not phenotypically change through several passages. Stem cell-like aggregations (neurospheres) were observed in SEM microscopy. The cells represented high mitotic index. They could be viable under hypoxic conditions and serum deprivation. According to functional studies, the cell line exhibited stem cell-like behaviors with particular emphasis on its self-renewal capacity. LDH isoenzymes expression pattern confirmed the same cellular source for both of the HRPE-2S cells and primary RPE cells. Characteristics of HRPE-2S cells promise it as an in vitro model for RPE stem cell-based researches. J. Cell. Physiol. 232: 2626-2640, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  16. Plasmids and packaging cell lines for use in phage display

    Science.gov (United States)

    Bradbury, Andrew M.

    2012-07-24

    The invention relates to a novel phagemid display system for packaging phagemid DNA into phagemid particles which completely avoids the use of helper phage. The system of the invention incorporates the use of bacterial packaging cell lines which have been transformed with helper plasmids containing all required phage proteins but not the packaging signals. The absence of packaging signals in these helper plasmids prevents their DNA from being packaged in the bacterial cell, which provides a number of significant advantages over the use of both standard and modified helper phage. Packaged phagemids expressing a protein or peptide of interest, in fusion with a phage coat protein such as g3p, are generated simply by transfecting phagemid into the packaging cell line.

  17. CD40 expression in Wehi-164 cell line.

    Science.gov (United States)

    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.

  18. The effect of anthralin (dithranol) on mitochondria.

    Science.gov (United States)

    Morlière, P; Dubertret, L; Sa e Melo, T; Salet, C; Fosse, M; Santus, R

    1985-05-01

    The short-term effect of topical application of anthralin (dithranol) on normal human skin was investigated by electron microscopy. Mitochondria appeared markedly damaged. By contrast other cellular structures, particularly the nuclear and cytoplasmic membranes were unchanged. In vitro experiments were therefore performed on isolated rat liver mitochondria and it was shown that anthralin acts as an uncoupler of oxidative phosphorylation. These results suggest that anthralin can inhibit the adenosine triphosphate supply in epidermal cells. This loss of energy supply in keratinocytes could explain, at least in part, the therapeutic efficiency of anthralin in psoriasis.

  19. Effects of irradiation on cytokine production in glioma cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Yamanaka, Ryuya; Tanaka, Ryuichi; Yoshida, Seiichi [Niigata Univ. (Japan). Brain Research Inst.

    1993-11-01

    The effects of irradiation on cytokine production in glioma cell lines, NP1, NP2 and NP3, were studied. Culture supernatants were collected after 6, 24, 48 or 72 hours and the concentrations of interleukin (IL)-6 and IL-8 measured by enzyme-linked immunosorbent assay. Spontaneous and IL-1[beta]-stimulated productions were analyzed. Some cells were given a single dose of Lineac irradiation (10 or 20 Gy). Production of IL-6 (with or without IL-1[beta] stimulation) increased gradually to a maximum after 72 hours, more in the 20 Gy-irradiated cells than 10 Gy cells (p<0.01). Production of IL-8 increased gradually to a maximum after 48 or 72 hours. Spontaneous production of IL-8 increased more in 20 Gy-irradiated cells than 10 Gy cells after 6 and 24 hours (p<0.01), but increased more in 10 Gy cells than 20 Gy cells after 48 and 72 hours (p<0.01). The production of IL-8 stimulated by IL-1[beta] increased more in 10 Gy cells than 20 Gy cells 24 hours later (p<0.01). IL-6 and IL-8 production differed in the response to irradiation. Our data suggest that bidirectional communication between the immune system and glioma cells changes after radiotherapy. (author).

  20. Catecholamine secretion by chemical hypoxia in guinea-pig, but not rat, adrenal medullary cells: differences in mitochondria.

    Science.gov (United States)

    Harada, K; Endo, Y; Warashina, A; Inoue, M

    2015-08-20

    The effects of mitochondrial inhibitors (CN(-), a complex IV inhibitor and CCCP, protonophore) on catecholamine (CA) secretion and mitochondrial function were explored functionally and biochemically in rat and guinea-pig adrenal chromaffin cells. Guinea-pig chromaffin cells conspicuously secreted CA in response to CN(-) or CCCP, but rat cells showed a little, if any, secretory response to either of them. The resting metabolic rates in rat adrenal medullae did not differ from those in guinea-pig adrenal medullae. On the other hand, the time course of depolarization of the mitochondrial membrane potential (ΔΨm) in guinea-pig chromaffin cells in response to CN(-) was slower than that in rat chromaffin cells, and this difference was abolished by oligomycin, an F1F0-ATPase inhibitor. The extent of CCCP-induced decrease in cellular ATP in guinea-pig chromaffin cells, which was indirectly measured using a Mg(2+) indicator, was smaller than that in rat chromaffin cells. Relative expression levels of F1F0-ATPase inhibitor factor in guinea-pig adrenal medullae were smaller than in rat adrenal medullae, and the opposite was true for F1F0-ATPase α subunit. The present results indicate that guinea-pig chromaffin cells secrete more CA in response to a mitochondrial inhibitor than rat chromaffin cells and this higher susceptibility in the former is accounted for by a larger extent of reversed operation of F1F0-ATPase with the consequent decrease in ATP under conditions where ΔΨm is depolarized. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

  1. Survey of Differentially Methylated Promoters in Prostate Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Yipeng Wang

    2005-08-01

    Full Text Available DNA methylation, copy number in the genomes of three immortalized prostate epithelial, five cancer cell lines (LNCaP, PC3, PC3M, PC3M-Pro4, PC3MLN4 were compared using a microarray-based technique. Genomic DNA is cut with a methylation-sensitive enzyme Hpall, followed by linker ligation, polymerase chain reaction (PCR amplification, labeling, hybridization to an array of promoter sequences. Only those parts of the genomic DNA that have unmethylated restriction sites within a few hundred base pairs generate PCR products detectable on an array. Of 2732 promoter sequences on a test array, 504 (18.5% showed differential hybridization between immortalized prostate epithelial, cancer cell lines. Among candidate hypermethylated genes in cancer-derived lines, there were eight (CD44, CDKN1A, ESR1, PLAU, RARB, SFN, TNFRSF6, TSPY previously observed in prostate cancer, 13 previously known methylation targets in other cancers (ARHI, bcl-2, BRCA1, CDKN2C, GADD45A, MTAP, PGR, SLC26A4, SPARC, SYK, TJP2, UCHL1, WIT-1. The majority of genes that appear to be both differentially methylated, differentially regulated between prostate epithelial, cancer cell lines are novel methylation targets, including PAK6, RAD50, TLX3, PIR51, MAP2K5, INSR, FBN1, GG2-1, representing a rich new source of candidate genes used to study the role of DNA methylation in prostate tumors.

  2. The cytoskeleton of Drosophila-derived Schneider line-1 and Kc23 cells undergoes significant changes during long-term culture

    Science.gov (United States)

    Schatten, H.; Hedrick, J.; Chakrabarti, A.

    1998-01-01

    Insect cell cultures derived from Drosophila melanogaster are increasingly being used as an alternative system to mammalian cell cultures, as they are amenable to genetic manipulation. Although Drosophila cells are an excellent tool for the study of genes and expression of proteins, culture conditions have to be considered in the interpretation of biochemical results. Our studies indicate that significant differences occur in cytoskeletal structure during the long-term culture of the Drosophila-derived cell lines Schneider Line-1 (S1) and Kc23. Scanning, transmission-electron, and immunofluorescence microscopy studies reveal that microfilaments, microtubules, and centrosomes become increasingly different during the culture of these cells from 24 h to 7-14 days. Significant cytoskeletal changes are observed at the cell surface where actin polymerizes into microfilaments, during the elongation of long microvilli. Additionally, long protrusions develop from the cell surface; these protrusions are microtubule-based and establish contact with neighboring cells. In contrast, the microtubule network in the interior of the cells becomes disrupted after four days of culture, resulting in altered transport of mitochondria. Microtubules and centrosomes are also affected in a small percent of cells during cell division, indicating an instability of centrosomes. Thus, the cytoskeletal network of microfilaments, microtubules, and centrosomes is affected in Drosophila cells during long-term culture. This implies that gene regulation and post-translational modifications are probably different under different culture conditions.

  3. Pathology of mitochondria in MELAS syndrome: an ultrastructural study.

    Science.gov (United States)

    Felczak, Paulina; Lewandowska, Eliza; Stępniak, Iwona; Ołdak, Monika; Pollak, Agnieszka; Lechowicz, Urszula; Pasennik, Elżbieta; Stępień, Tomasz; Wierzba-Bobrowicz, Teresa

    Ultrastructural changes in skeletal muscle biopsy in a 24-year-old female patient with clinically suspected mitochondrial encephalomyopathy lactic acidosis and stroke-like episodes (MELAS) syndrome are presented. We observed proliferation and/or pleomorphism of mitochondria in skeletal muscle and smooth muscle cells of arterioles, as well as in pericytes of capillaries. Paracrystalline inclusions were found only in damaged mitochondria of skeletal muscle. Genetic testing revealed a point mutation in A3243G tRNALeu(UUR) typical for MELAS syndrome. We conclude that differentiated pathological changes of mitochondria in the studied types of cells may be associated with the different energy requirements of these cells.

  4. Short-chain fatty acids induced autophagy serves as an adaptive strategy for retarding mitochondria-mediated apoptotic cell death

    OpenAIRE

    Tang, Y; Chen, Y; Jiang, H; Nie, D

    2010-01-01

    Short-chain fatty acids (SCFAs) are the major by-products of bacterial fermentation of undigested dietary fibers in the large intestine. SCFAs, mostly propionate and butyrate, inhibit proliferation and induce apoptosis in colon cancer cells, but clinical trials had mixed results regarding the anti-tumor activities of SCFAs. Herein we demonstrate that propionate and butyrate induced autophagy in human colon cancer cells to dampen apoptosis whereas inhibition of autophagy potentiated SCFA induc...

  5. Change of cell cycle arrest of tumor cell lines after 60Co γ-irradiation

    International Nuclear Information System (INIS)

    Tang Yi; Liu Wenli; Zhou Jianfeng; Gao Qinglei; Wu Jianhong

    2003-01-01

    Objective: To observe the cell cycle arrest changes in peripheral blood mononuclear cells (PBMNCs) of normal persons and several kinds of tumor cell lines after 60 Co γ-irradiation. Methods: PBMNCs of normal persons, HL-60, K562, SiHA and 113 tumor cell lines were irradiated with 60 Co γ-rays at the absorbed doses of 6, 10,15 Gy. Cell cycles changes were checked 6, 12, 24, 48 and 60 h after the irradiation. Results: A stasis state was observed in normal person PBMNCs, 95 percents of which were in G 1 phase, and they still remained stasis after the irradiation. Except the 113 cell line manifesting G 1 phase arrest, all other tumor cell lines showed G 2 /M phase arrest after irradiation. The radiation sensitivity of HL-60 was higher than that of SiHA cell line. Conclusion: Different cell lines have different cell cycle arrest reaction to radiation and their radiation sensitivity are also different

  6. Naja nigricollis CMS-9 enhances the mitochondria-mediated death pathway in adaphostin-treated human leukaemia U937 cells.

    Science.gov (United States)

    Chen, Ying-Jung; Wang, Jeh-Jeng; Chang, Long-Sen

    2011-11-01

    1. The aim of the present study was to explore the effect of the Naja nigricollis phospholipase A(2) CMS-9 on adaphostin-induced death of human leukaemia U937 cells. 2. Leukaemia U937 cells (Bcr/Abl-negative cells) were treated with adaphostin (0-10 μmol/L) and CMS-9 (0-1 μmol/L). The effects of CMS-9, adaphostin and their combination on cell viability, the generation reactive oxygen species (ROS), [Ca(2+) ](i) , p38 mitogen-activated protein kinase (MAPK) activation, Akt and extracellular signal-regulated kinase (ERK) inactivation, mitochondrial membrane potential (ΔΨ(m) ) and Bcl-2 family proteins were analysed. 3. Both adaphostin and CMS-9 induced U937 cell apoptosis, characterized by dissipation of ΔΨ(m) and ROS generation. Combined treatment further increased ΔΨ(m) loss and reduced the viability of adaphostin-treated cells. Unlike in CMS-9-treated cells, in adaphostin-treated cells ROS-induced increases in [Ca(2+) ](i) were observed. CMS-9-induced ROS generation resulted in p38 MAPK activation, whereas adaphostin treatment elicited ROS/Ca(2+) -mediated inactivation of Akt and ERK. Moreover, Akt was found to be involved in ERK phosphorylation. Suppression of p38 MAPK activation blocked CMS-9-induced ΔΨ(m) loss and Bcl-xL downregulation. Overexpression of constitutively active Akt and mitogen-activated protein kinase kinase (MEK) 1 rescued adaphostin-induced ΔΨ(m) loss and Bcl-2 downregulation. Similarly, CMS-9 augmented adaphostin toxicity in human leukaemia K562 cells via increased mitochondrial alterations. 4. The results suggest that two distinct pathways mediate adaphostin- and CMS-9-induced mitochondrial damage (i.e. the ROS-Ca(2+) -Akt-ERK and ROS-p38 MAPK pathways, respectively). These distinct pathway explain the augmentation by CMS-9 of ΔΨ(m) loss and apoptosis in adaphostin-treated U937 cells. © 2011 The Authors. Clinical and Experimental Pharmacology and Physiology © 2011 Blackwell Publishing Asia Pty Ltd.

  7. Redox interplay between mitochondria and peroxisomes

    Directory of Open Access Journals (Sweden)

    Celien eLismont

    2015-05-01

    Full Text Available Reduction-oxidation or ‘redox’ reactions are an integral part of a broad range of cellular processes such as gene expression, energy metabolism, protein import and folding, and autophagy. As many of these processes are intimately linked with cell fate decisions, transient or chronic changes in cellular redox equilibrium are likely to contribute to the initiation and progression of a plethora of human diseases. Since a long time, it is known that mitochondria are major players in redox regulation and signaling. More recently, it has become clear that also peroxisomes have the capacity to impact redox-linked physiological processes. To serve this function, peroxisomes cooperate with other organelles, including mitochondria. This review provides a comprehensive picture of what is currently known about the redox interplay between mitochondria and peroxisomes in mammals. We first outline the pro- and antioxidant systems of both organelles and how they may function as redox signaling nodes. Next, we critically review and discuss emerging evidence that peroxisomes and mitochondria share an intricate redox-sensitive relationship and cooperate in cell fate decisions. Key issues include possible physiological roles, messengers, and mechanisms. We also provide examples of how data mining of publicly-available datasets from ‘omics’ technologies can be a powerful means to gain additional insights into potential redox signaling pathways between peroxisomes and mitochondria. Finally, we highlight the need for more studies that seek to clarify the mechanisms of how mitochondria may act as dynamic receivers, integrators, and transmitters of peroxisome-derived mediators of oxidative stress. The outcome of such studies may open up exciting new avenues for the community of researchers working on cellular responses to organelle-derived oxidative stress, a research field in which the role of peroxisomes is currently highly underestimated and an issue of

  8. THP-1 cell line: an in vitro cell model for immune modulation approach.

    Science.gov (United States)

    Chanput, Wasaporn; Mes, Jurriaan J; Wichers, Harry J

    2014-11-01

    THP-1 is a human leukemia monocytic cell line, which has been extensively used to study monocyte/macrophage functions, mechanisms, signaling pathways, and nutrient and drug transport. This cell line has become a common model to estimate modulation of monocyte and macrophage activities. This review attempts to summarize and discuss recent publications related to the THP-1 cell model. An overview on the biological similarities and dissimilarities between the THP-1 cell line and human peripheral blood mononuclear cell (PBMC) derived-monocytes and macrophages, as well as the advantages and disadvantages of the use of THP-1 cell line, is included. The review summarizes different published co-cultivation studies of THP-1 cells with other cell types, for instance, intestinal cells, adipocytes, T-lymphocytes, platelets, and vascular smooth muscle cells, which can be an option to study cell-cell interaction in vitro and can be an approach to better mimic in vivo conditions. Macrophage polarization is a relatively new topic which gains interest for which the THP-1 cell line also may be relevant. Besides that an overview of newly released commercial THP-1 engineered-reporter cells and THP-1 inflammasome test-cells is also given. Evaluation of recent papers leads to the conclusion that the THP-1 cell line has unique characteristics as a model to investigate/estimate immune-modulating effects of compounds in both activated and resting conditions of the cells. Although the THP-1 response can hint to potential responses that might occur ex vivo or in vivo, these should be, however, validated by in vivo studies to draw more definite conclusions. Copyright © 2013. Published by Elsevier B.V.

  9. Fibronectin synthesized by a human hepatoma cell line

    International Nuclear Information System (INIS)

    Glasgow, J.E.; Colman, R.W.

    1984-01-01

    Fibronectin is a family of immunologically similar glycoproteins which mediate a variety of cell-cell and cell-substratum interactions. It is a constituent of the extracellular matrix of connective tissue and circulates in plasma. When suspension and adherent cultures of a human hepatoma cell line (SK-HEP-1) were incubated in serum-free medium, the resulting conditioned medium contained material which was specifically immunoprecipitated by antisera to human plasma fibronectin. By double immunodiffusion, a component in the conditioned culture medium was shown to form a line of identity with fibronectin in human plasma and to migrate as an alpha 2- to beta-globulin during immunoelectrophoresis. Human fibronectin was quantified in conditioned medium by electroimmunodiffusion, and was found to increase for at least three days at about 0.1 micrograms/10(6) cells/day. Adherent cultures of SK-HEP-1 cells were incubated with L-[ 35 S]methionine to label newly synthesized proteins. Labeled fibronectin in conditioned medium or in cell extracts comigrated with fibronectin in human plasma as shown by autoradiography following crossed-immunoelectrophoresis. Fibronectin was demonstrated in the extra-cellular matrix of adherent SK-HEP-1 cultures by immunofluorescence. It was shown previously that SK-HEP-1 cells synthesize alpha 1-protease inhibitor, one of the products of normal hepatocytes. The finding that these hepatoma cells also synthesize fibronectin supports the concept that the hepatocyte may be one source of circulating fibronectin, a possibility consistent with the established role of this cell type in blood plasma protein synthesis

  10. Sensitivity of breast cancer cell lines to recombinant thiaminase I.

    Science.gov (United States)

    Liu, Shuqian; Monks, Noel R; Hanes, Jeremiah W; Begley, Tadhg P; Yu, Hui; Moscow, Jeffrey A

    2010-05-01

    We have previously shown that the expression of the thiamine transporter THTR2 is decreased sevenfold in breast cancer, which may leave breast cancer cells vulnerable to acute thiamine starvation. This concept was supported by the observation that MDA231 breast cancer xenografts demonstrated growth inhibition in mice fed a thiamine-free diet. We purified recombinant Bacillus thiaminolyticus thiaminase I enzyme, which digests thiamine, to study acute thiamine starvation in breast cancer. Thiaminase I enzyme was cytotoxic in six breast cancer cell lines with IC(50)s ranging from 0.012 to 0.022 U/ml. The growth inhibitory effects of the combination of thiaminase I with either doxorubicin or paclitaxel were also examined. Over a wide range of drug concentrations, thiaminase 1 was consistently synergistic or additive with doxorubicin and paclitaxel in MCF-7, ZR75, HS578T and T47D cell lines, with most combinations having a calculated combination index (CI) of less than 0.8, indicating synergy. Although thiaminase I exposure did not stimulate the energy-sensing signaling kinases AKT, AMPK and GSK-3beta in MCF-7, ZR75, HS578T and T47D cell lines, thiaminase I exposure did stimulate expression of the ER stress response protein GRP78. In summary, thiaminase I is cytotoxic in breast cancer cell lines and triggers the unfolded protein response. These findings suggest that THTR2 down-regulation in breast tumors may present a nutritional vulnerability that could be exploited by thiaminase I enzyme therapy.

  11. Regulation of apoptosis by resveratrol through JAK/STAT and mitochondria mediated pathway in human epidermoid carcinoma A431 cells

    International Nuclear Information System (INIS)

    Madan, Esha; Prasad, Sahdeo; Roy, Preeti; George, Jasmine; Shukla, Yogeshwer

    2008-01-01

    Resveratrol (trans-3,4',5-trihydroxystilbene), a polyphenolic phytoalexin present mainly in grapes, red wine and berries, is known to possess strong chemopreventive and anticancer properties. Here, we demonstrated the anti-proliferative and apoptosis-inducing activities of resveratrol in human epidermoid carcinoma A431 cells. Resveratrol has cytotoxic effects through inhibiting cellular proliferation of A431 cells, which leads to the induction of apoptosis, as evident by an increase in the fraction of cells in the sub-G 1 phase of the cell cycle and Annexin-V binding of externalized phosphatidylserine. Results revealed that inhibition of proliferation is associated with regulation of the JAK/STAT pathway, where resveratrol prevents phosphorylation of JAK, thereby inhibiting STAT1 phosphorylation. Furthermore, resveratrol treatment actively stimulated reactive oxygen species (ROS) and mitochondrial membrane depolarization. Consequently, an imbalance in the Bax/Bcl-2 ratio triggered the caspase cascade and subsequent cleavage of PARP, thereby shifting the balance in favor of apoptosis. These observations indicate that resveratrol treatment inhibits JAK/STAT-mediated gene transcription and induce the mitochondrial cell death pathway.

  12. Caffeine markedly sensitizes human mesothelioma cell lines to pemetrexed

    Science.gov (United States)

    Min, Sang Hee; Goldman, I. David; Zhao, Rongbao

    2013-01-01

    Pemetrexed is a new generation antifolate approved for the treatment of mesothelioma and non-small cell lung cancer. Caffeine is known to augment radiation or chemotherapeutic drug-induced cell killing. The current study addresses the impact of caffeine on the activity of pemetrexed in mesothelioma cell lines. Caffeine enhanced pemetrexed activity in all four mesothelioma cell lines tested (H2052, H2373, H28 and MSTO-211H). Caffeine sensitized H2052 cells in a dose- and schedule-dependent manner, and was associated with a markedly decreased clonogenic survival. Caffeine sensitization occurred only in cells subjected to pulse, but not continuous, exposure to pemetrexed. Similar pemetrexed sensitization was also observed with the clinically better tolerated caffeine analog, theobromine. Pemetrexed sensitization by caffeine was associated with an increase in pemetrexed-induced phosphorylation of ataxia-telangiectasia-mutated (ATM) and Chk1. These data indicate that caffeine and its analog, theobromine, may be a useful approach to enhance pemetrexed-based chemotherapy. PMID:17594092

  13. Melatonin, mitochondria, and the skin.

    Science.gov (United States)

    Slominski, Andrzej T; Zmijewski, Michal A; Semak, Igor; Kim, Tae-Kang; Janjetovic, Zorica; Slominski, Radomir M; Zmijewski, Jaroslaw W

    2017-11-01

    The skin being a protective barrier between external and internal (body) environments has the sensory and adaptive capacity to maintain local and global body homeostasis in response to noxious factors. An important part of the skin response to stress is its ability for melatonin synthesis and subsequent metabolism through the indolic and kynuric pathways. Indeed, melatonin and its metabolites have emerged as indispensable for physiological skin functions and for effective protection of a cutaneous homeostasis from hostile environmental factors. Moreover, they attenuate the pathological processes including carcinogenesis and other hyperproliferative/inflammatory conditions. Interestingly, mitochondria appear to be a central hub of melatonin metabolism in the skin cells. Furthermore, substantial evidence has accumulated on the protective role of the melatonin against ultraviolet radiation and the attendant mitochondrial dysfunction. Melatonin and its metabolites appear to have a modulatory impact on mitochondrion redox and bioenergetic homeostasis, as well as the anti-apoptotic effects. Of note, some metabolites exhibit even greater impact than melatonin alone. Herein, we emphasize that melatonin-mitochondria axis would control integumental functions designed to protect local and perhaps global homeostasis. Given the phylogenetic origin and primordial actions of melatonin, we propose that the melatonin-related mitochondrial functions represent an evolutionary conserved mechanism involved in cellular adaptive response to skin injury and repair.

  14. Study of radiosensitization of chloroquine on esophageal cancer cell line

    International Nuclear Information System (INIS)

    Yuan Xiaoli; Li Tao; Huang Jianming; Zha Xiao; Deng Bifang; Lang Jinyi

    2014-01-01

    Objective: To investigate the possibility of chloroquine radiosensitization of esophageal cancer cell line TE-1 and its further mechanism. Methods: Effect of chloroquine on cell viability of TE-1 cells was determined by MTT method. Expression of LC3, Beclin-1 and formation of acidic vesicular organelles (AVOs) were determined by Western blot, and fluorescence staining with Lyso-Tracker Red DND-99, respectively. Clonogenic survival of TE-1 cells was examined by clonogenic forming assay. Results: Chloroquine showed dose-dependent inhibition of TE-1 cell growth, and its values of IC_5_0 and IC_1_0 were (72.33±5.28) and (15.42±3.33) μmol/L, respectively. The expression of Beclin-1 and LC3-II/I markedly increased in irradiated TE-1 cells. The addition of chloroquine with IC_1_0 concentration significantly reduced the fluorescence and intensity of AVOs accumulation in the cytoplasm of TE-1 cells. Clonogenic survival fraction decreased obviously in TE-1 cells with addition of chloroquine after radiation and the value of SERD0 was 1.439. Conclusions: Chloroquine could radiosensitize esophageal cancer cells by blocking autophagy-lysosomal pathway and be used as a potential radiosensitizing strategy. (authors)

  15. Derivation of novel genetically diverse human embryonic stem cell lines.

    Science.gov (United States)

    Stefanova, Valentina T; Grifo, James A; Hansis, Christoph

    2012-06-10

    Human embryonic stem cells (hESCs) have the potential to revolutionize many biomedical fields ranging from basic research to disease modeling, regenerative medicine, drug discovery, and toxicity testing. A multitude of hESC lines have been derived worldwide since the first 5 lines by Thomson et al. 13 years ago, but many of these are poorly characterized, unavailable, or do not represent desired traits, thus making them unsuitable for application purposes. In order to provide the scientific community with better options, we have derived 12 new hESC lines at New York University from discarded genetically normal and abnormal embryos using the latest techniques. We examined the genetic status of the NYUES lines in detail as well as their molecular and cellular features and DNA fingerprinting profile. Furthermore, we differentiated our hESCs into the tissues most affected by a specific condition or into clinically desired cell types. To our knowledge, a number of characteristics of our hESCs have not been previously reported, for example, mutation for alpha thalassemia X-linked mental retardation syndrome, linkage to conditions with a genetic component such as asthma or poor sperm morphology, and novel combinations of ethnic backgrounds. Importantly, all of our undifferentiated euploid female lines tested to date did not show X chromosome inactivation, believed to result in superior potency. We continue to derive new hESC lines and add them to the NIH registry and other registries. This should facilitate the use of our hESCs and lead to advancements for patient-benefitting applications.

  16. Direct electric current treatment modifies mitochondrial function and lipid body content in the A549 cancer cell line.

    Science.gov (United States)

    Holandino, Carla; Teixeira, Cesar Augusto Antunes; de Oliveira, Felipe Alves Gomes; Barbosa, Gleyce Moreno; Siqueira, Camila Monteiro; Messeder, Douglas Jardim; de Aguiar, Fernanda Silva; da Veiga, Venicio Feo; Girard-Dias, Wendell; Miranda, Kildare; Galina, Antonio; Capella, Marcia Alves Marques; Morales, Marcelo Marcos

    2016-10-01

    Electrochemical therapy (EChT) entails treatment of solid tumors with direct electric current (DC). This work evaluated the specific effects of anodic flow generated by DC on biochemical and metabolic features of the A549 human lung cancer cell line. Apoptosis was evaluated on the basis of caspase-3 activity and mitochondrial transmembrane potential dissipation. Cell morphology was analyzed using transmission electron microscopy, and lipid droplets were studied through morphometric analysis and X-ray qualitative elemental microanalysis. High-resolution respirometry was used to assess mitochondrial respiratory parameters. Results indicated A549 viability decreased in a dose-dependent manner with a prominent drop between 18 and 24h after treatment (ppotential. Furthermore, treated cells demonstrated important ultrastructural mitochondria damage and a three-fold increase in the cytoplasmic lipid bodies' number, quantified by morphometrical analyses. Conversely, 24h after treatment, the cells presented a two-fold increase of residual oxygen consumption, accounting for 45.3% of basal oxygen consumption. These results show remarkable alterations promoted by anodic flow on human lung cancer cells which are possibly involved with the antitumoral effects of EChT. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Ultrastructural characteristics of three undifferentiated mouse embryonic stem cell lines and their differentiated three-dimensional derivatives: a comparative study.

    Science.gov (United States)

    Alharbi, Suzan; Elsafadi, Mona; Mobarak, Mohammed; Alrwili, Ali; Vishnubalaji, Radhakrishnan; Manikandan, Muthurangan; Al-Qudsi, Fatma; Karim, Saleh; Al-Nabaheen, May; Aldahmash, Abdullah; Mahmood, Amer

    2014-04-01

    The fine structures of mouse embryonic stem cells (mESCs) grown as colonies and differentiated in three-dimensional (3D) culture as embryoid bodies (EBs) were analyzed by transmission electron microscopy. Undifferentiated mESCs expressed markers that proved their pluripotency. Differentiated EBs expressed different differentiation marker proteins from the three germ layers. The ultrastructure of mESCs revealed the presence of microvilli on the cell surfaces, large and deep infolded nuclei, low cytoplasm-to-nuclear ratios, frequent lipid droplets, nonprominent Golgi apparatus, and smooth endoplasmic reticulum. In addition, we found prominent juvenile mitochondria and free ribosomes-rich cytoplasm in mESCs. Ultrastructure of the differentiated mESCs as EBs showed different cell arrangements, which indicate the different stages of EB development and differentiation. The morphologies of BALB/c and 129 W9.5 EBs were very similar at day 4, whereas C57BL/6 EBs were distinct from the others at day 4. This finding suggested that differentiation of EBs from different cell lines occurs in the same pattern but not at the same rate. Conversely, the ultrastructure results of BALB/c and 129 W9.5 ESCs revealed differentiating features, such as the dilated profile of a rough endoplasmic reticulum. In addition, we found low expression levels of undifferentiated markers on the outer cells of BALB/c and 129 W9.5 mESC colonies, which suggests a faster differentiation potential.

  18. Mitochondria-dependent apoptogenic activity of the aqueous root extract of Croton membranaceus against human BPH-1 cells.

    Science.gov (United States)

    Afriyie, D K; Asare, G A; Bugyei, K; Lin, J; Peng, J; Hong, Z

    2015-01-15

    Croton membranaceus aqueous root extract (CMARE) is among the widely used phytotherapeutics in Ghana for the management of benign prostatic hyperplasia (BPH) and prostate cancer. However, the mechanism of action of CMARE remains to be elucidated. This study aimed to establish whether apoptosis is involved in the antiproliferative effect of CMARE on human BPH-1 cells. We determined the effect of treatment with 0, 1, 3, and 5 mg/mL CMARE for 24, 48, and 72 h on the viability and morphology of BPH-1 cells using the MMT assay and phase-contrast microscopy, respectively. We examined the apoptosis-inducing effects of CMARE after 48 h at the cellular level using Hoescht 33258 and JC-1 dye staining and flow cytometry analysis. We performed reverse transcription polymerase chain reaction and Western blotting to confirm the apoptotic effects of CMARE at the molecular level. CMARE induced a significant dose-dependent inhibition in the proliferation of BPH-1 cells (P BPH-1 cells may be a possible mechanism of action of CMARE.

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

    Science.gov (United States)

    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.

  20. Mitochondria and the evolutionary roots of cancer

    International Nuclear Information System (INIS)

    Davila, Alfonso F; Zamorano, Pedro

    2013-01-01

    Cancer disease is inherent to, and widespread among, metazoans. Yet, some of the hallmarks of cancer such as uncontrolled cell proliferation, lack of apoptosis, hypoxia, fermentative metabolism and free cell motility (metastasis) are akin to a prokaryotic lifestyle, suggesting a link between cancer disease and evolution. In this hypothesis paper, we propose that cancer cells represent a phenotypic reversion to the earliest stage of eukaryotic evolution. This reversion is triggered by the dysregulation of the mitochondria due to cumulative oxidative damage to mitochondrial and nuclear DNA. As a result, the phenotype of normal, differentiated cells gradually reverts to the phenotype of a facultative anaerobic, heterotrophic cell optimized for survival and proliferation in hypoxic environments. This phenotype matches the phenotype of the last eukaryotic common ancestor (LECA) that resulted from the endosymbiosis between an α-proteobacteria (which later became the mitochondria) and an archaebacteria. As such, the evolution of cancer within one individual can be viewed as a recapitulation of the evolution of the eukaryotic cell from fully differentiated cells to LECA. This evolutionary model of cancer is compatible with the current understanding of the disease, and explains the evolutionary basis for most of the hallmarks of cancer, as well as the link between the disease and aging. It could also open new avenues for treatment directed at reestablishing the synergy between the mitochondria and the cancerous cell. (paper)

  1. ROS-dependent mitochondria molecular mechanisms underlying antitumor activity of Pleurotus abalonus acidic polysaccharides in human breast cancer MCF-7 cells.

    Directory of Open Access Journals (Sweden)

    Xiaolong Shi

    Full Text Available BACKGROUND: A greater reduction in cancer risk associated with mushroom diet rich in fungus polysaccharides is generally accepted. Meanwhile, edible Pleurotus abalonus as a member of Abalone mushroom family is a popular nutritional supplement that purportedly prevents cancer occurrence. However, these anecdotal claims are supported by limited studies describing tumor-inhibitory responses to the promising polysaccharides, and the molecular mechanisms underlying these properties have not yet been elucidated. METHODOLOGY/PRINCIPAL FINDINGS: We here fractionated the crude polysaccharide preparation from the fruiting bodies of P. abalonus into three fractions, namely PAP-1, PAP-2 and PAP-3, and tested these fractions for antiproliferative activity in human breast cancer MCF-7 cells. The largest PAP-3, an acidic polysaccharide fraction with a molecular mass of 3.68×10(5 Da, was the most active in inhibiting MCF-7 cancer cells with an IC50 of 193 µg/mL. The changes in cell normal morphology were observed by DAPI staining and the PAP-3-induced apoptosis was confirmed by annexin V/propidium iodide staining. The apoptosis was involved in mitochondria-mediated pathway including the loss of mitochondrial membrane potential (Δψm, the increase of Bax/Bcl-2 ratio, caspase-9/3 activation, and poly(ADP-ribose polymerase (PARP degradation, as well as intracellular ROS production. PAP-3 also induced up-regulation of p53, and cell cycle arrest at the S phase. The incubation of MCF-7 cells with antioxidant superoxide dismutase (SOD and N-acetylcysteine (NAC significantly attenuated the ROS generation and apoptosis caused by PAP-3, indicating that intracellular ROS plays a pivotal role in cell death. CONCLUSIONS/SIGNIFICANCE: These findings suggest that the polysaccharides, especially acidic PAP-3, are very important nutritional ingredients responsible for, at least in part, the anticancer health benefits of P. abalonus via ROS-mediated mitochondrial apoptotic

  2. Generation, isolation, and maintenance of rodent mast cells and mast cell lines

    DEFF Research Database (Denmark)

    Jensen, Bettina M; Swindle, Emily J; Iwaki, Shoko

    2006-01-01

    Antigen-mediated mast cell activation, with subsequent mediator release, is a major initiator of the inflammatory allergic response associated with such conditions as asthma. A comprehensive understanding of the principles involved in this process therefore is key to the development of novel...... therapies for the treatment of these disease states. In vitro models of mast cell function have allowed significant progress to be made in the recognition of the fundamental principles of mast cell activation via the high-affinity IgE receptor (FcvarepsilonRI) and, more recently, other receptors expressed...... on mast cells. In addition to human mast cells, the major cell culture systems employed to investigate these responses are rat and mouse peritoneal mast cells, mouse bone-marrow-derived mast cells, the rat basophilic leukemia cell line RBL-2H3, and the mouse MC/9 mast cell line. In this unit, we describe...

  3. CD40 expression in Wehi-164 cell line

    OpenAIRE

    Karimi, Mohammad Hossein; 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 ex...

  4. Destabilization of Akt Promotes the Death of Myeloma Cell Lines

    Directory of Open Access Journals (Sweden)

    Yanan Zhang

    2014-01-01

    Full Text Available Constitutive activation of Akt is believed to be an oncogenic signal in multiple myeloma and is associated with poor patient prognosis and resistance to available treatment. The stability of Akt proteins is regulated by phosphorylating the highly conserved turn motif (TM of these proteins and the chaperone protein HSP90. In this study we investigate the antitumor effects of inhibiting mTORC2 plus HSP90 in myeloma cell lines. We show that chronic exposure of cells to rapamycin can inhibit mTORC2 pathway, and AKT will be destabilized by administration of the HSP90 inhibitor 17-allylamino-geldanamycin (17-AAG. Finally, we show that the rapamycin synergizes with 17-AAG and inhibits myeloma cells growth and promotes cell death to a greater extent than either drug alone. Our studies provide a clinical rationale of use mTOR inhibitors and chaperone protein inhibitors in combination regimens for the treatment of human blood cancers.

  5. RBE of neutrons for induction of cell reproductive death and chromosome aberrations in three cell lines

    International Nuclear Information System (INIS)

    Zoetelief, J.; Kuijpers, W.C.; Baten-Wittwer, A.; Barendsen, G.W.

    1983-01-01

    The authors have compared the RBE values for induction of dicentrics and centric rings with those for cell inactivation and with the mean or effective quality factors (Q) recommended for radiation protection. The induction of cell reproductive death and chromosome aberrations has been investigated in plateau phase cultures of established lines of a rat rhabdomyosarcoma, a rat ureter carcinoma and Chinese hamster cells for single doses of 300 kV X-rays and 0.5, 4.2 and 15 MeV neutrons. The different cell lines show considerable variations in sensitivity and the RBE values obtained are presented in tabular form. The mean RBE values for the rat rhabdomyosarcoma cells are lower than those for the other two relatively resistant cell lines. Those for the Chinese hamster cells extrapolated to levels according to low doses of X-rays are in good agreement with the quoted Q values. (Auth./C.F.)

  6. DNA polymerase gamma inhibition by vitamin K3 induces mitochondria-mediated cytotoxicity in human cancer cells.

    Science.gov (United States)

    Sasaki, Ryohei; Suzuki, Yoko; Yonezawa, Yuko; Ota, Yosuke; Okamoto, Yoshiaki; Demizu, Yusuke; Huang, Peng; Yoshida, Hiromi; Sugimura, Kazuro; Mizushina, Yoshiyuki

    2008-05-01

    Among the vitamin K (VK) compounds, VK3 exhibits distinct cytotoxic activity in cancer cells and is thought to affect redox cycling; however, the underlying mechanisms remain unclear. Here we demonstrate that VK3 selectively inhibits DNA polymerase (pol) gamma, the key enzyme responsible for mitochondrial DNA replication and repair. VK3 at 30 microM inhibited pol gamma by more than 80%, caused impairment of mitochondrial DNA replication and repair, and induced a significant increase in reactive oxygen species (ROS), leading to apoptosis. At a lower concentration (3 microM), VK3 did not cause a significant increase in ROS, but was able to effectively inhibit cell proliferation, which could be reversed by supplementing glycolytic substrates. The cytotoxic action of VK3 was independent of p53 tumor suppressor gene status. Interestingly, VK3 only inhibited pol gamma but did not affect other pol including human pol alpha, pol beta, pol delta, and pol epsilon. VK1 and VK2 exhibited no inhibitory effect on any of the pol tested. These data together suggest that the inhibition of pol gamma by VK3 is relatively specific, and that this compound seems to exert its anticancer activity by two possible mechanisms in a concentration-dependent manner: (1) induction of ROS-mediated cell death at high concentrations; and (2) inhibition of cell proliferation at lower concentrations likely through the suppression of mitochondrial respiratory function. These findings may explain various cytotoxic actions induced by VK3, and may pave the way for the further use of VK3.

  7. Boronated protoporphyrin (BOPP): localization in lysosomes of the human glioma cell line SF-767 with uptake modulated by lipoprotein levels

    International Nuclear Information System (INIS)

    Callahan, Daniel E.; Forte, Trudy M.; Javed Afzal, S.M.; Deen, Dennis F.; Kahl, Stephen B.; Bjornstad, Kathleen A.; Bauer, William F.; Blakely, Eleanor A.

    1999-01-01

    Purpose: Boronated protoporphyrin (BOPP) is a candidate for use in both boron neutron capture therapy (BNCT) and photodynamic therapy (PDT) of glioblastoma multiforme (GBM). Our objectives are to identify factors that influence the uptake and retention of BOPP in vitro and to determine BOPP distribution in a human glioma cell line in vitro. This information will aid the development of compounds and treatment strategies that increase the effectiveness of BNCT therapy for GBM. Methods and Materials: The amount, distribution pattern, and site of internalization of BOPP were assessed using fluorescence microscopy. Living human glioma (SF-767) cells were imaged after a 24-h exposure to BOPP (20-135.6 μg/ml, normal serum). Dose-dependent uptake of BOPP was determined using both fluorescence microscopy of individual living cells and inductively-coupled plasma-atomic emission spectroscopy (ICP-AES) analysis of cell pellets. Lysosome- or mitochondria-specific fluorescent probes were used to identify the cellular compartment containing BOPP. Two human fibroblast cell lines, AG-1522 (LDL receptor-positive) and GM019-15C (LDL receptor-deficient), were used to investigate LDL receptor-dependent BOPP uptake. The dependence of BOPP uptake on lipoproteins in the media was determined by exposing each of the three cell types to BOPP in medium containing either normal (NS) or lipoprotein deficient serum (LPDS). Results: BOPP accumulated in the lysosomes of human glioma cells in vitro, and not in the mitochondria, as reported for C6 rat glioma cells in vitro. BOPP uptake was concentration-dependent and was also dependent on the amount of lipoproteins in the medium. Over the range of incubation concentrations studied and at the single exposure duration time point investigated (24 h), all cells retained a similar amount of BOPP. At the lowest incubation concentration (20 μg/ml, NS), the amount of boron retained was near 10 9 atoms per cell (15 μg B/g cells). Lysosomes containing high

  8. Modelling cell population growth with applications to cancer therapy in human tumour cell lines.

    Science.gov (United States)

    Basse, Britta; Baguley, Bruce C; Marshall, Elaine S; Wake, Graeme C; Wall, David J N

    2004-01-01

    In this paper we present an overview of the work undertaken to model a population of cells and the effects of cancer therapy. We began with a theoretical one compartment size structured cell population model and investigated its asymptotic steady size distributions (SSDs) (On a cell growth model for plankton, MMB JIMA 21 (2004) 49). However these size distributions are not similar to the DNA (size) distributions obtained experimentally via the flow cytometric analysis of human tumour cell lines (data obtained from the Auckland Cancer Society Research Centre, New Zealand). In our one compartment model, size was a generic term, but in order to obtain realistic steady size distributions we chose size to be DNA content and devised a multi-compartment mathematical model for the cell division cycle where each compartment corresponds to a distinct phase of the cell cycle (J. Math. Biol. 47 (2003) 295). We then incorporated another compartment describing the possible induction of apoptosis (cell death) from mitosis phase (Modelling cell death in human tumour cell lines exposed to anticancer drug paclitaxel, J. Math. Biol. 2004, in press). This enabled us to compare our model to flow cytometric data of a melanoma cell line where the anticancer drug, paclitaxel, had been added. The model gives a dynamic picture of the effects of paclitaxel on the cell cycle. We hope to use the model to describe the effects of other cancer therapies on a number of different cell lines. Copyright 2004 Elsevier Ltd.

  9. Snail regulates cell survival and inhibits cellular senescence in human metastatic prostate cancer cell lines.

    Science.gov (United States)

    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.

  10. Colony, hanging drop, and methylcellulose three dimensional hypoxic growth optimization of renal cell carcinoma cell lines.

    Science.gov (United States)

    Matak, Damian; Brodaczewska, Klaudia K; Lipiec, Monika; Szymanski, Łukasz; Szczylik, Cezary; Czarnecka, Anna M

    2017-08-01

    Renal cell carcinoma (RCC) is the most lethal of the common urologic malignancies, comprising 3% of all human neoplasms, and the incidence of kidney cancer is rising annually. We need new approaches to target tumor cells that are resistant to current therapies and that give rise to recurrence and treatment failure. In this study, we focused on low oxygen tension and three-dimensional (3D) cell culture incorporation to develop a new RCC growth model. We used the hanging drop and colony formation methods, which are common in 3D culture, as well as a unique methylcellulose (MC) method. For the experiments, we used human primary RCC cell lines, metastatic RCC cell lines, human kidney cancer stem cells, and human healthy epithelial cells. In the hanging drop assay, we verified the potential of various cell lines to create solid aggregates in hypoxic and normoxic conditions. With the semi-soft agar method, we also determined the ability of various cell lines to create colonies under different oxygen conditions. Different cell behavior observed in the MC method versus the hanging drop and colony formation assays suggests that these three assays may be useful to test various cell properties. However, MC seems to be a particularly valuable alternative for 3D cell culture, as its higher efficiency of aggregate formation and serum independency are of interest in different areas of cancer biology.

  11. Discovery of HeLa Cell Contamination in HES Cells: Call for Cell Line Authentication in Reproductive Biology Research.

    Science.gov (United States)

    Kniss, Douglas A; Summerfield, Taryn L

    2014-08-01

    Continuous cell lines are used frequently in reproductive biology research to study problems in early pregnancy events and parturition. It has been recognized for 50 years that many mammalian cell lines contain inter- or intraspecies contaminations with other cells. However, most investigators do not routinely test their culture systems for cross-contamination. The most frequent contributor to cross-contamination of cell lines is the HeLa cell isolated from an aggressive cervical adenocarcinoma. We report on the discovery of HeLa cell contamination of the human endometrial epithelial cell line HES isolated in our laboratory. Short tandem repeat analysis of 9 unique genetic loci demonstrated molecular identity between HES and HeLa cells. In addition, we verified that WISH cells, isolated originally from human amnion epithelium, were also contaminated with HeLa cells. Inasmuch as our laboratory did not culture HeLa cells at the time of HES cell derivations, the source of contamination was the WISH cell line. These data highlight the need for continued diligence in authenticating cell lines used in reproductive biology research. © The Author(s) 2014.

  12. Potassium channels in brain mitochondria.

    Science.gov (United States)

    Bednarczyk, Piotr

    2009-01-01

    Potassium channels are the most widely distributed class of ion channels. These channels are transmembrane proteins known to play important roles in both normal and pathophysiological functions in all cell types. Various potassium channels are recognised as potential therapeutic targets in the treatment of Parkinson's disease, Alzheimer's disease, brain/spinal cord ischaemia and sepsis. In addition to their importance as therapeutic targets, certain potassium channels are known for their beneficial roles in anaesthesia, cardioprotection and neuroprotection. Some types of potassium channels present in the plasma membrane of various cells have been found in the inner mitochondrial membrane as well. Potassium channels have been proposed to regulate mitochondrial membrane potential, respiration, matrix volume and Ca(+) ion homeostasis. It has been proposed that mitochondrial potassium channels mediate ischaemic preconditioning in various tissues. However, the specificity of a pharmacological agents and the mechanisms underlying their effects on ischaemic preconditioning remain controversial. The following potassium channels from various tissues have been identified in the inner mitochondrial membrane: ATP-regulated (mitoK(ATP)) channel, large conductance Ca(2+)-regulated (mitoBK(Ca)) channel, intermediate conductance Ca(2+)-regulated (mitoIK(Ca)) channel, voltage-gated (mitoKv1.3 type) channel, and twin-pore domain (mitoTASK-3) channel. It has been shown that increased potassium flux into brain mitochondria induced by either the mitoK(ATP) channel or mitoBK(Ca) channel affects the beneficial effects on neuronal cell survival under pathological conditions. Recently, differential distribution of mitoBK(Ca) channels has been observed in neuronal mitochondria. These findings may suggest a neuroprotective role for the mitoBK(Ca) channel in specific brain structures. This minireview summarises current data on brain mitochondrial potassium channels and the efforts to identify

  13. Expression of cadherin and NCAM in human small cell lung cancer cell lines and xenografts

    DEFF Research Database (Denmark)

    Rygaard, K; Møller, C; Bock, E

    1992-01-01

    characterised, the cadherin family and the Ig superfamily member, neural cell adhesion molecule (NCAM). We investigated expression of these two adhesion molecule families in small cell lung cancer (SCLC) cell lines and xenografts by immunoblotting. Nineteen tumours established from 15 patients with SCLC were......Tumour cell adhesion, detachment and aggregation seem to play an important part in tumour invasion and metastasis, and numerous cell adhesion molecules are expressed by tumour cells. Several families of cell-cell adhesion molecules have been described, of which two groups are particularly well...... embryonic development, which may play a role in connection with tumour invasion and metastasis, was found in 14/18 NCAM expressing SCLC tumours. Individual tumours grown as cell lines and as nude mouse xenografts showed no qualitative differences in cadherin or NCAM expression....

  14. Glucose-Modulated Mitochondria Adaptation in Tumor Cells: A Focus on ATP Synthase and Inhibitor Factor 1

    Directory of Open Access Journals (Sweden)

    Irene Mavelli

    2012-02-01

    Full Text Available Warburg’s hypothesis has been challenged by a number of studies showing that oxidative phosphorylation is repressed in some tumors, rather than being inactive per se. Thus, treatments able to shift energy metabolism by activating mitochondrial pathways have been suggested as an intriguing basis for the optimization of antitumor strategies. In this study, HepG2 hepatocarcinoma cells were cultivated with different metabolic substrates under conditions mimicking “positive” (activation/biogenesis or “negative” (silencing mitochondrial adaptation. In addition to the expected up-regulation of mitochondrial biogenesis, glucose deprivation caused an increase in phosphorylating respiration and a rise in the expression levels of the ATP synthase β subunit and Inhibitor Factor 1 (IF1. Hyperglycemia, on the other hand, led to a markedly decreased level of the transcriptional coactivator PGC-α suggesting down-regulation of mitochondrial biogenesis, although no change in mitochondrial mass and no impairment of phosphorylating respiration were observed. Moreover, a reduction in mitochondrial networking and in ATP synthase dimer stability was produced. No effect on β-ATP synthase expression was elicited. Notably, hyperglycemia caused an increase in IF1 expression levels, but it did not alter the amount of IF1 associated with ATP synthase. These results point to a new role of IF1 in relation to high glucose utilization by tumor cells, in addition to its well known effect upon mitochondrial ATP synthase regulation.

  15. Assessment of citalopram and escitalopram on neuroblastoma cell lines: Cell toxicity and gene modulation

    Science.gov (United States)

    Sakka, Laurent; Delétage, Nathalie; Chalus, Maryse; Aissouni, Youssef; Sylvain-Vidal, Valérie; Gobron, Stéphane; Coll, Guillaume

    2017-01-01

    Selective serotonin reuptake inhibitors (SSRI) are common antidepressants which cytotoxicity has been assessed in cancers notably colorectal carcinomas and glioma cell lines. We assessed and compared the cytotoxicity of 2 SSRI, citalopram and escitalopram, on neuroblastoma cell lines. The study was performed on 2 non-MYCN amplified cell lines (rat B104 and human SH-SY5Y) and 2 human MYCN amplified cell lines (IMR32 and Kelly). Citalopram and escitalopram showed concentration-dependent cytotoxicity on all cell lines. Citalopram was more cytotoxic than escitalopram. IMR32 was the most sensitive cell line. The absence of toxicity on human primary Schwann cells demonstrated the safety of both molecules for myelin. The mechanisms of cytotoxicity were explored using gene-expression profiles and quantitative real-time PCR (qPCR). Citalopram modulated 1 502 genes and escitalopram 1 164 genes with a fold change ≥ 2. 1 021 genes were modulated by both citalopram and escitalopram; 481 genes were regulated only by citalopram while 143 genes were regulated only by escitalopram. Citalopram modulated 69 pathways (KEGG) and escitalopram 42. Ten pathways were differently modulated by citalopram and escitalopram. Citalopram drastically decreased the expression of MYBL2, BIRC5 and BARD1 poor prognosis factors of neuroblastoma with fold-changes of -107 (pescitalopram. PMID:28467792

  16. Assessment of citalopram and escitalopram on neuroblastoma cell lines. Cell toxicity and gene modulation.

    Science.gov (United States)

    Sakka, Laurent; Delétage, Nathalie; Chalus, Maryse; Aissouni, Youssef; Sylvain-Vidal, Valérie; Gobron, Stéphane; Coll, Guillaume

    2017-06-27

    Selective serotonin reuptake inhibitors (SSRI) are common antidepressants which cytotoxicity has been assessed in cancers notably colorectal carcinomas and glioma cell lines. We assessed and compared the cytotoxicity of 2 SSRI, citalopram and escitalopram, on neuroblastoma cell lines. The study was performed on 2 non-MYCN amplified cell lines (rat B104 and human SH-SY5Y) and 2 human MYCN amplified cell lines (IMR32 and Kelly). Citalopram and escitalopram showed concentration-dependent cytotoxicity on all cell lines. Citalopram was more cytotoxic than escitalopram. IMR32 was the most sensitive cell line. The absence of toxicity on human primary Schwann cells demonstrated the safety of both molecules for myelin. The mechanisms of cytotoxicity were explored using gene-expression profiles and quantitative real-time PCR (qPCR). Citalopram modulated 1 502 genes and escitalopram 1 164 genes with a fold change ≥ 2. 1 021 genes were modulated by both citalopram and escitalopram; 481 genes were regulated only by citalopram while 143 genes were regulated only by escitalopram. Citalopram modulated 69 pathways (KEGG) and escitalopram 42. Ten pathways were differently modulated by citalopram and escitalopram. Citalopram drastically decreased the expression of MYBL2, BIRC5 and BARD1 poor prognosis factors of neuroblastoma with fold-changes of -107 (pescitalopram.

  17. Establishment of a novel human medulloblastoma cell line characterized by highly aggressive stem-like cells.

    Science.gov (United States)

    Silva, Patrícia Benites Gonçalves da; Rodini, Carolina Oliveira; Kaid, Carolini; Nakahata, Adriana Miti; Pereira, Márcia Cristina Leite; Matushita, Hamilton; Costa, Silvia Souza da; Okamoto, Oswaldo Keith

    2016-08-01

    Medulloblastoma is a highly aggressive brain tumor and one of the leading causes of morbidity and mortality related to childhood cancer. These tumors display differential ability to metastasize and respond to treatment, which reflects their high degree of heterogeneity at the genetic and molecular levels. Such heterogeneity of medulloblastoma brings an additional challenge to the understanding of its physiopathology and impacts the development of new therapeutic strategies. This translational effort has been the focus of most pre-clinical studies which invariably employ experimental models using human tumor cell lines. Nonetheless, compared to other cancers, relatively few cell lines of human medulloblastoma are available in central repositories, partly due to the rarity of these tumors and to the intrinsic difficulties in establishing continuous cell lines from pediatric brain tumors. Here, we report the establishment of a new human medulloblastoma cell line which, in comparison with the commonly used and well-established cell line Daoy, is characterized by enhanced proliferation and invasion capabilities, stem cell properties, increased chemoresistance, tumorigenicity in an orthotopic metastatic model, replication of original medulloblastoma behavior in vivo, strong chromosome structural instability and deregulation of genes involved in neural development. These features are advantageous for designing biologically relevant experimental models in clinically oriented studies, making this novel cell line, named USP-13-Med, instrumental for the study of medulloblastoma biology and treatment.

  18. Characterization of cell lines stably transfected with rubella virus replicons

    International Nuclear Information System (INIS)

    Tzeng, Wen-Pin; Xu, Jie; Frey, Teryl K.

    2012-01-01

    Rubella virus (RUBV) replicons expressing a drug resistance gene and a gene of interest were used to select cell lines uniformly harboring the replicon. Replicons expressing GFP and a virus capsid protein GFP fusion (C-GFP) were compared. Vero or BHK cells transfected with either replicon survived drug selection and grew into a monolayer. However, survival was ∼9-fold greater following transfection with the C-GFP-replicon than with the GFP-expressing replicon and while the C-GFP-replicon cells grew similarly to non-transfected cells, the GFP-replicon cells grew slower. Neither was due to the ability of the CP to enhance RNA synthesis but survival during drug selection was correlated with the ability of CP to inhibit apoptosis. Additionally, C-GFP-replicon cells were not cured of the replicon in the absence of drug selection. Interferon-alpha suppressed replicon RNA and protein synthesis, but did not cure the cells, explaining in part the ability of RUBV to establish persistent infections.

  19. Characterization of cell lines stably transfected with rubella virus replicons

    Energy Technology Data Exchange (ETDEWEB)

    Tzeng, Wen-Pin; Xu, Jie [Department of Biology, Georgia State University, P.O. Box 4010, Atlanta GA 30302-4010 (United States); Frey, Teryl K., E-mail: tfrey@gsu.edu [Department of Biology, Georgia State University, P.O. Box 4010, Atlanta GA 30302-4010 (United States)

    2012-07-20

    Rubella virus (RUBV) replicons expressing a drug resistance gene and a gene of interest were used to select cell lines uniformly harboring the replicon. Replicons expressing GFP and a virus capsid protein GFP fusion (C-GFP) were compared. Vero or BHK cells transfected with either replicon survived drug selection and grew into a monolayer. However, survival was {approx}9-fold greater following transfection with the C-GFP-replicon than with the GFP-expressing replicon and while the C-GFP-replicon cells grew similarly to non-transfected cells, the GFP-replicon cells grew slower. Neither was due to the ability of the CP to enhance RNA synthesis but survival during drug selection was correlated with the ability of CP to inhibit apoptosis. Additionally, C-GFP-replicon cells were not cured of the replicon in the absence of drug selection. Interferon-alpha suppressed replicon RNA and protein synthesis, but did not cure the cells, explaining in part the ability of RUBV to establish persistent infections.

  20. Ginsenoside Rb1 Attenuates Oxygen-Glucose Deprivation-Induced Apoptosis in SH-SY5Y Cells via Protection of Mitochondria and Inhibition of AIF and Cytochrome c Release

    Directory of Open Access Journals (Sweden)

    Pengfei Ge

    2013-10-01

    Full Text Available To investigate the role of mitochondria in the protective effects of ginsenoside Rb1 on cellular apoptosis caused by oxygen-glucose deprivation, in this study, MTT assay, TUNEL staining, flow cytometry, immunocytochemistry and western blotting were used to examine the cellular viability, apoptosis, ROS level, mitochondrial membrane potential, and the distribution of apoptosis inducing factor, cytochrome c, Bax and Bcl-2 in nucleus, mitochondria and cytoplasm. We found that pretreatment with GRb1 improved the cellular viability damaged by OGD. Moreover, GRb1 inhibited apoptosis in SH-SY5Y cells induced by OGD. Further studies showed that the elevation of cellular reactive oxygen species levels and the reduction of mitochondrial membrane potential caused by OGD were both counteracted by GRb1. Additionally, GRb1 not only suppressed the translocation of apoptosis inducing factor into nucleus and cytochrome c into cytoplasm, but also inhibited the increase of Bax within mitochondria and alleviated the decrease of mitochondrial Bcl-2. Our study indicates that the protection of GRb1 on OGD-induced apoptosis in SH-SY5Y cells is associated with its protection on mitochondrial function and inhibition of release of AIF and cytochrome c.

  1. Hypoxia induces adipogenic differentitation of myoblastic cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Itoigawa, Yoshiaki [Tohoku University School of Medicine, Sendai (Japan); Juntendo University School of Medicine, Tokyo (Japan); Kishimoto, Koshi N., E-mail: kishimoto@med.tohoku.ac.jp [Tohoku University School of Medicine, Sendai (Japan); Okuno, Hiroshi; Sano, Hirotaka [Tohoku University School of Medicine, Sendai (Japan); Kaneko, Kazuo [Juntendo University School of Medicine, Tokyo (Japan); Itoi, Eiji [Tohoku University School of Medicine, Sendai (Japan)

    2010-09-03

    Research highlights: {yields} C2C12 and G8 myogenic cell lines treated by hypoxia differentiate into adipocytes. {yields} The expression of C/EBP{beta}, {alpha} and PPAR{gamma} were increased under hypoxia. {yields} Myogenic differentiation of C2C12 was inhibited under hypoxia. -- Abstract: Muscle atrophy usually accompanies fat accumulation in the muscle. In such atrophic conditions as back muscles of kyphotic spine and the rotator cuff muscles with torn tendons, blood flow might be diminished. It is known that hypoxia causes trans-differentiation of mesenchymal stem cells derived from bone marrow into adipocytes. However, it has not been elucidated yet if hypoxia turned myoblasts into adipocytes. We investigated adipogenesis in C2C12 and G8 murine myogenic cell line treated by hypoxia. Cells were also treated with the cocktail of insulin, dexamethasone and IBMX (MDI), which has been known to inhibit Wnt signaling and promote adipogenesis. Adipogenic differentiation was seen in both hypoxia and MDI. Adipogenic marker gene expression was assessed in C2C12. CCAAT/enhancer-binding protein (C/EBP) {beta}, {alpha} and peroxisome proliferator activating receptor (PPAR) {gamma} were increased by both hypoxia and MDI. The expression profile of Wnt10b was different between hypoxia and MDI. The mechanism for adipogenesis of myoblasts in hypoxia might be regulated by different mechanism than the modification of Wnt signaling.

  2. Multidrug resistance and retroviral transduction potential in human small cell lung cancer cell lines

    DEFF Research Database (Denmark)

    Theilade, M D; Gram, G J; Jensen, P B

    1999-01-01

    Multidrug resistance (MDR) remains a major problem in the successful treatment of small cell lung cancer (SCLC). New treatment strategies are needed, such as gene therapy specifically targeting the MDR cells in the tumor. Retroviral LacZ gene-containing vectors that were either pseudotyped...... for the gibbon ape leukemia virus (GALV-1) receptor or had specificity for the amphotropic murine leukemia virus (MLV-A) receptor were used for transduction of five SCLC cell lines differing by a range of MDR mechanisms. Transduction efficiencies in these cell lines were compared by calculating the percentage...... of blue colonies after X-Gal staining of the cells grown in soft agar. All examined SCLC cell lines were transducible with either vector. Transduction efficiencies varied from 5.7% to 33.5% independent of the presence of MDR. These results indicate that MDR does not severely impair transduction of SCLC...

  3. Endurance training increases the efficiency of rat skeletal muscle mitochondria.

    Science.gov (United States)

    Zoladz, Jerzy A; Koziel, Agnieszka; Woyda-Ploszczyca, Andrzej; Celichowski, Jan; Jarmuszkiewicz, Wieslawa

    2016-10-01

    Endurance training enhances mitochondrial oxidative capacity, but its effect on mitochondria functioning is poorly understood. In the present study, the influence of an 8-week endurance training on the bioenergetic functioning of rat skeletal muscle mitochondria under different assay temperatures (25, 35, and 42 °C) was investigated. The study was performed on 24 adult 4-month-old male Wistar rats, which were randomly assigned to either a treadmill training group (n = 12) or a sedentary control group (n = 12). In skeletal muscles, endurance training stimulated mitochondrial biogenesis and oxidative capacity. In isolated mitochondria, endurance training increased the phosphorylation rate and elevated levels of coenzyme Q. Moreover, a decrease in mitochondrial uncoupling, including uncoupling protein-mediated proton leak, was observed after training, which could explain the increased reactive oxygen species production (in nonphosphorylating mitochondria) and enhanced oxidative phosphorylation efficiency. At all studied temperatures, endurance training significantly augmented H2O2 production (and coenzyme Q reduction level) in nonphosphorylating mitochondria and decreased H2O2 production (and coenzyme Q reduction level) in phosphorylating mitochondria. Endurance training magnified the hyperthermia-induced increase in oxidative capacity and attenuated the hyperthermia-induced decline in oxidative phosphorylation efficiency and reactive oxygen species formation of nonphosphorylating mitochondria via proton leak enhancement. Thus, endurance training induces both quantitative and qualitative changes in muscle mitochondria that are important for cell signaling as well as for maintaining muscle energy homeostasis, especially at high temperatures.

  4. Interaction of plant cell signaling molecules, salicylic acid and jasmonic acid, with the mitochondria of Helicoverpa armigera.

    Science.gov (United States)

    Akbar, S M D; Sharma, H C; Jayalakshmi, S K; Sreeramulu, K

    2012-02-01

    The cotton bollworm, Helicoverpa armigera is a polyphagous pest in Asia, Africa, and the Mediterranean Europe. Salicylic acid (SA) and jasmonic acid (JA) are the cell signaling molecules produced in response to insect attack in plants. The effect of these signaling molecules was investigated on the oxidative phosphorylation and oxidative stress of H. armigera. SA significantly inhibited the state III and state IV respiration, respiratory control index (RCI), respiratory complexes I and II, induced mitochondrial swelling, and cytochrome c release in vitro. Under in vivo conditions, SA induced state IV respiration as well as oxidative stress in time- and dose-dependent manner, and also inhibited the larval growth. In contrast, JA did not affect the mitochondrial respiration and oxidative stress. SA affected the growth and development of H. armigera, in addition to its function as signaling molecules involved in both local defense reactions at feeding sites and the induction of systemic acquired resistance in plants.

  5. Radiation-induced apoptosis and cell cycle checkpoints in human colorectal tumour cell lines

    International Nuclear Information System (INIS)

    Playle, L.C.

    2001-03-01

    The p53 tumour suppressor gene is mutated in 75% of colorectal carcinomas and is critical for DNA damage-induced G1 cell cycle arrest. Data presented in this thesis demonstrate that after treatment with Ionizing Radiation (IR), colorectal tumour cell lines with mutant p53 are unable to arrest at G1 and undergo cell cycle arrest at G2. The staurosporine derivative, UCN-01, was shown to abrogate the IR-induced G2 checkpoint in colorectal tumour cell lines. Furthermore, in some cell lines, abrogation of the G2 checkpoint was associated with radiosensitisation. Data presented in this study demonstrate that 2 out of 5 cell lines with mutant p53 were sensitised to IR by UCN-01. In order to determine whether radiosensitisation correlated with lack of functional p53, transfected derivatives of an adenoma-derived cell line were studied, in which endogenous wild type p53 was disrupted by expression of a dominant negative p53 mutant protein (and with a vector control). In both these cell lines UCN-01 abrogated the G2 arrest however this was not associated with radiosensitisation, indicating that radiosensitisation is a cell type-specific phenomenon. Although 2 colorectal carcinoma cell lines, with mutant p53, were sensitised to IR by UCN-01, the mechanisms of p53-independent IR-induced apoptosis in the colon are essentially unknown. The mitogen-activated protein kinase (MAPK) pathways (that is the JNK, p38 and ERK pathways) have been implicated in apoptosis in a range of cell systems and in IR-induced apoptosis in some cell types. Data presented in this study show that, although the MAPKs can be activated by the known activator anisomycin, there is no evidence of a role for MAPKs in IR-induced apoptosis in colorectal tumour cell lines, regardless of p53 status. In summary, some colorectal tumour cell lines with mutant p53 can be sensitised to IR-induced cell death by G2 checkpoint abrogation and this may be an important treatment strategy, however mechanisms of IR-induced p53

  6. Intrinsic radiosensitivity and PLD repair in osteosarcoma cell lines

    International Nuclear Information System (INIS)

    Sugimoto, M.; Toguchida, J.; Kotoura, Y.; Yamamuro, T.; Utsumi, H.

    1992-01-01

    The response to radiation of seven osteosarcoma cell lines was analysed by in vitro colony-forming assay and compared with that of eight human fibroblast strains. The values of D 0 , the surviving fraction after 2 Gy (S2Gy), and the mean inactivation dose (D-bar) of osteosarcoma cells in log-phase culture were significantly higher than those of fibroblast strains (p<0.01). PLD (potentially lethal damage) repair of osteosarcoma cells evaluated in the plateau phase of growth showed great variation for enhancement of survival, although all of the values were maximised within 12 h after irradiation. In the osteosarcoma, intrinsic radiosensitivity in vitro reflected the clinical response to radiation. However, the capacity for PLD repair might not be a good indicator for predicting the results of radiation therapy. (author)

  7. UV light blocks EGFR signalling in human cancer cell lines

    DEFF Research Database (Denmark)

    Olsen, BB; Neves-Petersen, M T; Klitgaard, S

    2007-01-01

    UV light excites aromatic residues, causing these to disrupt nearby disulphide bridges. The EGF receptor is rich in aromatic residues near the disulphide bridges. Herein we show that laser-pulsed UV illumination of two different skin-derived cancer cell lines i.e. Cal-39 and A431, which both...... antibodies. There was a threshold level, below which the receptor could not be blocked. In addition, illumination caused the cells to upregulate the cyclin-dependent kinase inhibitor p21WAF1, irrespective of the p53 status. Since the EGF receptor is often overexpressed in cancers and other proliferative skin...... disorders, it might be possible to significantly reduce the proliferative potential of these cells making them good targets for laser-pulsed UV light treatment....

  8. Isolation of rat adrenocortical mitochondria

    Energy Technology Data Exchange (ETDEWEB)

    Solinas, Paola [Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, OH 44106 (United States); Department of Medicine, Center for Mitochondrial Disease, School of Medicine, Case Western Reserve University, Cleveland, OH 44106 (United States); Fujioka, Hisashi [Electron Microscopy Facility, Department of Pharmacology, Center for Mitochondrial Disease, School of Medicine, Case Western Reserve University, Cleveland, OH 44106 (United States); Tandler, Bernard [Department of Biological Sciences, School of Dental Medicine, Center for Mitochondrial Disease, School of Medicine, Case Western Reserve University, Cleveland, OH 44106 (United States); Hoppel, Charles L., E-mail: charles.hoppel@case.edu [Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, OH 44106 (United States); Department of Medicine, Center for Mitochondrial Disease, School of Medicine, Case Western Reserve University, Cleveland, OH 44106 (United States)

    2012-10-12

    Highlights: Black-Right-Pointing-Pointer A method for isolation of adrenocortical mitochondria from the adrenal gland of rats is described. Black-Right-Pointing-Pointer The purified isolated mitochondria show excellent morphological integrity. Black-Right-Pointing-Pointer The properties of oxidative phosphorylation are excellent. Black-Right-Pointing-Pointer The method increases the opportunity of direct analysis of adrenal mitochondria from small animals. -- Abstract: This report describes a relatively simple and reliable method for isolating adrenocortical mitochondria from rats in good, reasonably pure yield. These organelles, which heretofore have been unobtainable in isolated form from small laboratory animals, are now readily accessible. A high degree of mitochondrial purity is shown by the electron micrographs, as well as the structural integrity of each mitochondrion. That these organelles have retained their functional integrity is shown by their high respiratory control ratios. In general, the biochemical performance of these adrenal cortical mitochondria closely mirrors that of typical hepatic or cardiac mitochondria.

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

    Science.gov (United States)

    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.

  10. Improving the efficiency of CHO cell line generation using glutamine synthetase gene knockout cells.

    Science.gov (United States)

    Fan, Lianchun; Kadura, Ibrahim; Krebs, Lara E; Hatfield, Christopher C; Shaw, Margaret M; Frye, Christopher C

    2012-04-01

    Although Chinese hamster ovary (CHO) cells, with their unique characteristics, have become a major workhorse for the manufacture of therapeutic recombinant proteins, one of the major challenges in CHO cell line generation (CLG) is how to efficiently identify those rare, high-producing clones among a large population of low- and non-productive clones. It is not unusual that several hundred individual clones need to be screened for the identification of a commercial clonal cell line with acceptable productivity and growth profile making the cell line appropriate for commercial application. This inefficiency makes the process of CLG both time consuming and laborious. Currently, there are two main CHO expression systems, dihydrofolate reductase (DHFR)-based methotrexate (MTX) selection and glutamine synthetase (GS)-based methionine sulfoximine (MSX) selection, that have been in wide industrial use. Since selection of recombinant cell lines in the GS-CHO system is based on the balance between the expression of the GS gene introduced by the expression plasmid and the addition of the GS inhibitor, L-MSX, the expression of GS from the endogenous GS gene in parental CHOK1SV cells will likely interfere with the selection process. To study endogenous GS expression's potential impact on selection efficiency, GS-knockout CHOK1SV cell lines were generated using the zinc finger nuclease (ZFN) technology designed to specifically target the endogenous CHO GS gene. The high efficiency (∼2%) of bi-allelic modification on the CHO GS gene supports the unique advantages of the ZFN technology, especially in CHO cells. GS enzyme function disruption was confirmed by the observation of glutamine-dependent growth of all GS-knockout cell lines. Full evaluation of the GS-knockout cell lines in a standard industrial cell culture process was performed. Bulk culture productivity improved two- to three-fold through the use of GS-knockout cells as parent cells. The selection stringency was

  11. Synergistic inhibitory effect of berberine and d-limonene on human gastric carcinoma cell line MGC803.

    Science.gov (United States)

    Zhang, Xiu-Zhen; Wang, Ling; Liu, Dong-Wu; Tang, Guang-Yan; Zhang, Hong-Yu

    2014-09-01

    This study aims at evaluating the anticancer effects of berberine hydrochloride (berberine) and d-limonene, alone and in combination, on human gastric carcinoma cell line MGC803 to determine whether berberine and d-limonene work synergistically and elucidate their mechanisms. MGC803 cells were treated with berberine and d-limonene, alone and in combination, for 24-48 h. The inhibitory effects of these drugs on growth were determined by MTT assay. The combination index and drug reduction index were calculated with the Chou-Talalay method based on the median-effect principle. Flow cytometry and laser scanning confocal microscopy were employed to evaluate the effects of both drugs on cell-cycle perturbation and apoptosis, generation of reactive oxygen species (ROS), mitochondrial membrane potential, and expression of Bcl-2 and caspase-3 in MGC803 cells. Berberine or d-limonene alone can inhibit the growth of MGC803 cells in a dose- and time-dependent manner. Berberine and d-limonene at a combination ratio of 1:4 exhibited a synergistic effect on anti-MGC803 cells. The two drugs distinctly induced intracellular ROS generation, reduced the mitochondrial transmembrane potential (ΔΨm), enhanced the expression of caspase-3, and decreased the expression of Bcl-2. The combination of berberine and d-limonene showed more remarkable effects compared with drugs used singly in MGC803 cells. The combination of berberine and d-limonene exerted synergistic anticancer effects on MGC803 cells by cell-cycle arrest, ROS production, and apoptosis induction through the mitochondria-mediated intrinsic pathway.

  12. Functional somatostatin receptors on a rat pancreatic acinar cell line

    International Nuclear Information System (INIS)

    Viguerie, N.; Tahiri-Jouti, N.; Esteve, J.P.; Clerc, P.; Logsdon, C.; Svoboda, M.; Susini, C.; Vaysse, N.; Ribet, A.

    1988-01-01

    Somatostatin receptors from a rat pancreatic acinar cell line, AR4-2J, were characterized biochemically, structurally, and functionally. Binding of 125 I-[Tyr 11 ]Somatostatin to AR4-2J cells was saturable, exhibiting a single class of high-affinity binding sites with a maximal binding capacity of 258 ± 20 fmol/10 6 cells. Somatostatin receptor structure was analyzed by covalently cross-linking 125 I-[Tyr 11 ]somatostatin to its plasma membrane receptors. Gel electrophoresis and autoradiography of cross-linked proteins revealed a peptide containing the somatostatin receptor. Somatostatin inhibited vasoactive intestinal peptide (VIP)-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) formation in a dose-dependent manner. The concentration of somatostatin that caused half-maximal inhibition of cAMP formation was close to the receptor affinity for somatostatin. Pertussis toxin pretreatment of AR4-2J cells prevented somatostatin inhibition of VIP-stimulated cAMP formation as well as somatostatin binding. The authors conclude that AR4-2J cells exhibit functional somatostatin receptors that retain both specificity and affinity of the pancreatic acinar cell somatostatin receptors and act via the pertussis toxin-sensitive guanine nucleotide-binding protein N i to inhibit adenylate cyclase

  13. New model for gastroenteropancreatic large-cell neuroendocrine carcinoma: establishment of two clinically relevant cell lines.

    Directory of Open Access Journals (Sweden)

    Andreas Krieg

    Full Text Available Recently, a novel WHO-classification has been introduced that divided gastroenteropancreatic neuroendocrine neoplasms (GEP-NEN according to their proliferation index into G1- or G2-neuroendocrine tumors (NET and poorly differentiated small-cell or large-cell G3-neuroendocrine carcinomas (NEC. Our knowledge on primary NECs of the GEP-system is limited due to the rarity of these tumors and chemotherapeutic concepts of highly aggressive NEC do not provide convincing results. The aim of this study was to establish a reliable cell line model for NEC that could be helpful in identifying novel druggable molecular targets. Cell lines were established from liver (NEC-DUE1 or lymph node metastases (NEC-DUE2 from large cell NECs of the gastroesophageal junction and the large intestine, respectively. Morphological characteristics and expression of neuroendocrine markers were extensively analyzed. Chromosomal aberrations were mapped by array comparative genomic hybridization and DNA profiling was analyzed by DNA fingerprinting. In vitro and in vivo tumorigenicity was evaluated and the sensitivity against chemotherapeutic agents assessed. Both cell lines exhibited typical morphological and molecular features of large cell NEC. In vitro and in vivo experiments demonstrated that both cell lines retained their malignant properties. Whereas NEC-DUE1 and -DUE2 were resistant to chemotherapeutic drugs such as cisplatin, etoposide and oxaliplatin, a high sensitivity to 5-fluorouracil was observed for the NEC-DUE1 cell line. Taken together, we established and characterized the first GEP large-cell NEC cell lines that might serve as a helpful tool not only to understand the biology of these tumors, but also to establish novel targeted therapies in a preclinical setup.

  14. Metabolic characterization of invaded cells of the pancreatic cancer cell line, PANC?1

    OpenAIRE

    Fujita, Mayumi; Imadome, Kaori; Imai, Takashi

    2017-01-01

    We previously reported that about 0.4% of cells in the cultured human pancreatic cancer cell line, PANC?1, can invade matrigel during the transwell invasion assay, suggesting that these invaded PANC?1 cells may have specific characteristics to keep their invasive potential. To identify the metabolic characterization specific in the invaded PANC?1 cells, metabolome analysis of the invaded PANC?1 compared with the whole cultured PANC?1 was performed using CE?TOFMS, and concentrations of 110 met...

  15. Recommendation of short tandem repeat profiling for authenticating human cell lines, stem cells, and tissues

    OpenAIRE

    Barallon, Rita; Bauer, Steven R.; Butler, John; Capes-Davis, Amanda; Dirks, Wilhelm G.; Elmore, Eugene; Furtado, Manohar; Kline, Margaret C.; Kohara, Arihiro; Los, Georgyi V.; MacLeod, Roderick A. F.; Masters, John R. W.; Nardone, Mark; Nardone, Roland M.; Nims, Raymond W.

    2010-01-01

    Cell misidentification and cross-contamination have plagued biomedical research for as long as cells have been employed as research tools. Examples of misidentified cell lines continue to surface to this day. Efforts to eradicate the problem by raising awareness of the issue and by asking scientists voluntarily to take appropriate actions have not been successful. Unambiguous cell authentication is an essential step in the scientific process and should be an inherent consideration during peer...

  16. Oxidative stress induces mitochondrial dysfunction in a subset of autistic lymphoblastoid cell lines

    Science.gov (United States)

    Rose, S; Frye, R E; Slattery, J; Wynne, R; Tippett, M; Melnyk, S; James, S J

    2014-01-01

    There is an increasing recognition that mitochondrial dysfunction is associated with autism spectrum disorders. However, little attention has been given to the etiology of mitochondrial dysfunction and how mitochondrial abnormalities might interact with other physiological disturbances such as oxidative stress. Reserve capacity is a measure of the ability of the mitochondria to respond to physiological stress. In this study, we demonstrate, for the first time, that lymphoblastoid cell lines (LCLs) derived from children with autistic disorder (AD) have an abnormal mitochondrial reserve capacity before and after