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Sample records for regulates apoptotic cell

  1. The regulation of apoptotic cell death

    Directory of Open Access Journals (Sweden)

    Amarante-Mendes G.P.

    1999-01-01

    Full Text Available Apoptosis is a fundamental biological phenomenon in which the death of a cell is genetically and biochemically regulated. Different molecules are involved in the regulation of the apoptotic process. Death receptors, coupled to distinct members of the caspases as well as other adapter molecules, are involved in the initiation of the stress signals (The Indictment. Members of the Bcl-2 family control at the mitochondrial level the decision between life and death (The Judgement. The effector caspases are responsible for all morphological and biochemical changes related to apoptosis including the "eat-me" signals perceived by phagocytes and neighboring cells (The Execution. Finally, apoptosis would have little biological significance without the recognition and removal of the dying cells (The Burial.

  2. Apoptotic Regulation

    National Research Council Canada - National Science Library

    Thress, Kenneth

    2000-01-01

    ... for implementation of the cell death program. In an extensive analysis of chromosomal deletion mutants in the fly, Drosophila Melanogaster, Steller and colleagues identified a chromosomal region containing a number of genes critical...

  3. Mitochondrial apoptotic pathways induced by Drosophila programmed cell death regulators

    International Nuclear Information System (INIS)

    Claveria, Cristina; Torres, Miguel

    2003-01-01

    Multicellular organisms eliminate unwanted or damaged cells by cell death, a process essential to the maintenance of tissue homeostasis. Cell death is a tightly regulated event, whose alteration by excess or defect is involved in the pathogenesis of many diseases such as cancer, autoimmune syndromes, and neurodegenerative processes. Studies in model organisms, especially in the nematode Caenorhabditis elegans, have been crucial in identifying the key molecules implicated in the regulation and execution of programmed cell death. In contrast, the study of cell death in Drosophila melanogaster, often an excellent model organism, has identified regulators and mechanisms not obviously conserved in other metazoans. Recent molecular and cellular analyses suggest, however, that the mechanisms of action of the main programmed cell death regulators in Drosophila include a canonical mitochondrial pathway

  4. Regulation of Intrinsic and Extrinsic Apoptotic Pathways in Osteosarcoma Cells Following Oleandrin Treatment.

    Science.gov (United States)

    Ma, Yunlong; Zhu, Bin; Yong, Lei; Song, Chunyu; Liu, Xiao; Yu, Huilei; Wang, Peng; Liu, Zhongjun; Liu, Xiaoguang

    2016-11-23

    Our previous study has reported the anti-tumor effect of oleandrin on osteosarcoma (OS) cells. In the current study, we mainly explored its potential regulation on intrinsic and extrinsic apoptotic pathway in OS cells. Cells apoptosis, reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were detected using fluorescence staining and flow cytometry. Caspase-3 activity was detected using a commercial kit. The levels of cytoplasmic cytochrome c, mitochondrial cytochrome c, bcl-2, bax, caspase-9, Fas, FasL, caspase-8 and caspase-3 were detected by Western blotting. z-VAD-fmk was applied to block both intrinsic and extrinsic apoptosis pathways, and cells apoptosis was also tested. Furthermore, we used z-LEHD-fmk and Fas blocking antibody to inhibit intrinsic and extrinsic pathways, separately, and the selectivity of oleandrin on these pathways was explored. Results showed that oleandrin induced the apoptosis of OS cells, which was accompanied by an increase in ROS and a decrease in MMP. Furthermore, cytochrome c level was reduced in mitochondria but elevated in the cytoplasm. Caspase-3 activity was enhanced by oleandrin in a concentration- and time-dependent manner. Oleandrin also down-regulated the expression of bcl-2, but up-regulated bax, caspase-9, Fas, FasL, caspase-8 and caspase-3. In addition, the suppression of both apoptotic pathways by z-VAD-fmk greatly reverted the oleandrin-induced apoptosis. Moreover, the suppression of one pathway by a corresponding inhibitor did not affect the regulation of oleandrin on another pathway. Taken together, we concluded that oleandrin induced apoptosis of OS cells via activating both intrinsic and extrinsic apoptotic pathways.

  5. Role and regulation of apoptotic cell death in the kidney. Y2K update.

    Science.gov (United States)

    Ortiz, A; Lorz, C; Catalan, M P; Justo, P; Egido, J

    2000-08-01

    Apoptosis is an active form of cell death that, in balance with mitosis, regulates cell number. Cell number abnormalities are a frequent feature of renal disease. We now review current concepts on the molecular regulation of apoptotic cell death, including the influence of survival and lethal factors from the extracellular microenvironment as well as the role of intracellular regulators of apoptosis, such as death receptors, proapoptotic and antiapoptotic bcl2-related proteins, the mitochondria and caspases. In addition the role of apoptosis in the genesis, persistence and progression and remodeling and resolution of renal injury is discussed. Information on the expression and function of apoptosis regulatory proteins in specific renal syndromes is summarized. Finally, future perspectives in research and clinical intervention are discussed.

  6. Poncirin Induces Apoptosis in AGS Human Gastric Cancer Cells through Extrinsic Apoptotic Pathway by up-Regulation of Fas Ligand.

    Science.gov (United States)

    Saralamma, Venu Venkatarame Gowda; Nagappan, Arulkumar; Hong, Gyeong Eun; Lee, Ho Jeong; Yumnam, Silvia; Raha, Suchismita; Heo, Jeong Doo; Lee, Sang Joon; Lee, Won Sup; Kim, Eun Hee; Kim, Gon Sup

    2015-09-18

    Poncirin, a natural bitter flavanone glycoside abundantly present in many species of citrus fruits, has various biological benefits such as anti-oxidant, anti-microbial, anti-inflammatory and anti-cancer activities. The anti-cancer mechanism of Poncirin remains elusive to date. In this study, we investigated the anti-cancer effects of Poncirin in AGS human gastric cancer cells (gastric adenocarcinoma). The results revealed that Poncirin could inhibit the proliferation of AGS cells in a dose-dependent manner. It was observed Poncirin induced accumulation of sub-G1 DNA content, apoptotic cell population, apoptotic bodies, chromatin condensation, and DNA fragmentation in a dose-dependent manner in AGS cells. The expression of Fas Ligand (FasL) protein was up-regulated dose dependently in Poncirin-treated AGS cells Moreover, Poncirin in AGS cells induced activation of Caspase-8 and -3, and subsequent cleavage of poly(ADP-ribose) polymerase (PARP). Inhibitor studies' results confirm that the induction of caspase-dependent apoptotic cell death in Poncirin-treated AGS cells was led by the Fas death receptor. Interestingly, Poncirin did not show any effect on mitochondrial membrane potential (ΔΨm), pro-apoptotic proteins (Bax and Bak) and anti-apoptotic protein (Bcl-xL) in AGS-treated cells followed by no activation in the mitochondrial apoptotic protein caspase-9. This result suggests that the mitochondrial-mediated pathway is not involved in Poncirin-induced cell death in gastric cancer. These findings suggest that Poncirin has a potential anti-cancer effect via extrinsic pathway-mediated apoptosis, possibly making it a strong therapeutic agent for human gastric cancer.

  7. Cross-talk between miR-471-5p and autophagy component proteins regulates LC3-associated phagocytosis (LAP) of apoptotic germ cells.

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    Panneerdoss, Subbarayalu; Viswanadhapalli, Suryavathi; Abdelfattah, Nourhan; Onyeagucha, Benjamin C; Timilsina, Santosh; Mohammad, Tabrez A; Chen, Yidong; Drake, Michael; Vuori, Kristiina; Kumar, T Rajendra; Rao, Manjeet K

    2017-09-19

    Phagocytic clearance of apoptotic germ cells by Sertoli cells is vital for germ cell development and differentiation. Here, using a tissue-specific miRNA transgenic mouse model, we show that interaction between miR-471-5p and autophagy member proteins regulates clearance of apoptotic germ cells via LC3-associated phagocytosis (LAP). Transgenic mice expressing miR-471-5p in Sertoli cells show increased germ cell apoptosis and compromised male fertility. Those effects are due to defective engulfment and impaired LAP-mediated clearance of apoptotic germ cells as miR-471-5p transgenic mice show lower levels of Dock180, LC3, Atg12, Becn1, Rab5 and Rubicon in Sertoli cells. Our results reveal that Dock180 interacts with autophagy member proteins to constitute a functional LC3-dependent phagocytic complex. We find that androgen regulates Sertoli cell phagocytosis by controlling expression of miR-471-5p and its target proteins. These findings suggest that recruitment of autophagy machinery is essential for efficient clearance of apoptotic germ cells by Sertoli cells using LAP.Although phagocytic clearance of apoptotic germ cells by Sertoli cells is essential for spermatogenesis, little of the mechanism is known. Here the authors show that Sertoli cells employ LC3-associated phagocytosis (LAP) by recruiting autophagy member proteins to clear apoptotic germ cells.

  8. Apoptotic Tumor Cell-Derived Extracellular Vesicles as Important Regulators of the Onco-Regenerative Niche

    Directory of Open Access Journals (Sweden)

    Christopher D. Gregory

    2018-05-01

    Full Text Available Cells undergoing apoptosis produce heterogeneous populations of membrane delimited extracellular vesicles (Apo-EVs which vary not only in size—from tens of nanometers to several microns—but also in molecular composition and cargo. Apo-EVs carry a variety of potentially biologically active components, including small molecules, proteins, and nucleic acids. Larger forms of Apo-EVs, commonly termed “apoptotic bodies,” can carry organelles, such as mitochondria and nuclear fragments. Molecules displayed on the surface of extracellular vesicles (EVs can contribute substantially to their size, as well as their functions. Thus far, relatively little is known of the functional significance of Apo-EVs apart from their roles in fragmentation of dying cells and indicated immunomodulatory activities. Here, we discuss EV production by dying tumor cells and consider the possible roles of Apo-EVs in a cell death-driven sector of the tumor microenvironment known as the onco-regenerative niche (ORN. We propose that tumor-derived Apo-EVs are significant vehicles of the ORN, functioning as critical intercellular communicators that activate oncogenic tissue repair and regeneration pathways. We highlight important outstanding questions and suggest that Apo-EVs may harbor novel therapeutic targets.

  9. Immunosuppressive effects of apoptotic cells

    Science.gov (United States)

    Voll, Reinhard E.; Herrmann, Martin; Roth, Edith A.; Stach, Christian; Kalden, Joachim R.; Girkontaite, Irute

    1997-11-01

    Apoptotic cell death is important in the development and homeostasis of multicellular organisms and is a highly controlled means of eliminating dangerous, damaged or unnecessary cells without causing an inflammatory response or tissue damage,. We now show that the presence of apoptotic cells during monocyte activation increases their secretion of the anti-inflammatory and immunoregulatory cytokine interleukin 10 (IL-10) and decreases secretion of the proinflammatory cytokines tumour necrosis factor-α (TNF-α), IL-1 and IL-12. This may inhibit inflammation and contribute to impaired cell-mediated immunity in conditions associated with increased apoptosis, such as viral infections, pregnancy, cancer and exposure to radiation.

  10. 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.

  11. Differential regulation of caspase-9 by ionizing radiation- and UV-induced apoptotic pathways in thymic cells

    Energy Technology Data Exchange (ETDEWEB)

    Okamoto, Mayumi; Koga, Satomi [Department of Life Sciences, Faculty of Life and Environmental Sciences, Prefectural University of Hiroshima, Hiroshima 727-0023 (Japan); Tatsuka, Masaaki, E-mail: tatsuka@pu-hiroshima.ac.jp [Department of Life Sciences, Faculty of Life and Environmental Sciences, Prefectural University of Hiroshima, Hiroshima 727-0023 (Japan)

    2010-06-01

    In mouse thymic lymphoma 3SB cells bearing wild type p53, ionizing radiation (IR) and UV light are potent triggers of caspase-3-dependent apoptosis. Although cytochrome c was released from mitochondria as expected, caspase-9 activation was not observed in UV-exposed cells. Laser scanning confocal microscopy analysis showed that caspase-9 is localized in an unusual punctuated pattern in UV-induced apoptotic cells. In agreement with differences in the status of caspase-9 activation between IR and UV, subcellular protein fractionation experiments showed that pro-apoptotic apoptosis protease-activating factor 1 (Apaf-1), normally a part of the apoptosome assembled in response to the release of cytochrome c from mitochondria, and B-cell lymphoma extra long (Bcl-xL), an inhibitor of the change in mitochondrial membrane permeability, were redistributed by the IR-exposure but not by the UV-exposure. Instead of the sequestration of the capase-9/apoptosome activation in UV-induced apoptotic cells, the extrinsic apoptotic signaling generated by caspase-8 activation and consequent activation of B-cell lymphoma extra long (Bid) to release cytochrome c from mitochondria was observed. Thus, the post-mitochondrial apoptotic pathway downstream of cytochrome c release cannot operate the apoptosome function in UV-induced apoptosis in thymic 3SB cells. The intracellular redistribution and sequestration of apoptosis-related proteins upon mitochondrion-based apoptotic signaling was identified as a novel cellular mechanism to respond to DNA damage in an agent type-specific manner. This finding suggests that the kind of the critical ultimate apoptosis-inducing DNA lesion complex form resulting from the agent-specific DNA damage responses is important to determine which of apoptosis signals would be activated.

  12. Induction of Non-Apoptotic Cell Death by Activated Ras Requires Inverse Regulation of Rac1 and Arf6

    OpenAIRE

    Bhanot, Haymanti; Young, Ashley M.; Overmeyer, Jean H.; Maltese, William A.

    2010-01-01

    Methuosis is a unique form of non-apoptotic cell death triggered by alterations in the trafficking of clathrin-independent endosomes, ultimately leading to extreme vacuolization and rupture of the cell. Methuosis can be induced in glioblastoma cells by expression of constitutively active Ras. This study identifies the small GTPases, Rac1 and Arf6, and the Arf6 GTPase-activating-protein, GIT1, as key downstream components of the signaling pathway underlying Ras-induced methuosis. The extent to...

  13. Pro-apoptotic protein Noxa regulates memory T cell population size and protects against lethal immunopathology

    NARCIS (Netherlands)

    Wensveen, Felix M.; Klarenbeek, Paul L.; van Gisbergen, Klaas P. J. M.; Pascutti, Maria F.; Derks, Ingrid A. M.; van Schaik, Barbera D. C.; ten Brinke, Anja; de Vries, Niek; Cekinovic, Durdica; Jonjic, Stipan; van Lier, René A. W.; Eldering, Eric

    2013-01-01

    Memory T cells form a highly specific defense layer against reinfection with previously encountered pathogens. In addition, memory T cells provide protection against pathogens that are similar, but not identical to the original infectious agent. This is because each T cell response harbors multiple

  14. Nogo-B (Reticulon-4B) functions as a negative regulator of the apoptotic pathway through the interaction with c-FLIP in colorectal cancer cells.

    Science.gov (United States)

    Kawaguchi, Nao; Tashiro, Keitaro; Taniguchi, Kohei; Kawai, Masaru; Tanaka, Keitaro; Okuda, Junji; Hayashi, Michihiro; Uchiyama, Kazuhisa

    2018-08-01

    Nogo-B is a member of the Nogo/Reticulon-4 family and has been reported to be an inducer of apoptosis in certain types of cancer cells. However, the role of Nogo-B in human cancer remains less understood. Here, we demonstrated the functions of Nogo-B in colorectal cancer cells. In clinical colorectal cancer specimens, Nogo-B was obviously overexpressed, as determined by immunohistochemistry; and Western blot analysis showed its expression level to be significantly up-regulated. Furthermore, knockdown of Nogo-B in two colorectal cancer cell lines, SW480 and DLD-1, by transfection with si-RNA (siR) resulted in significantly reduced cell viability and a dramatic increase in apoptosis with insistent overexpression of cleaved caspase-8 and cleaved PARP. The transfection with Nogo-B plasmid cancelled that apoptosis induced by siRNogoB in SW480 cells. Besides, combinatory treatment with siR-Nogo-B/staurosporine (STS) or siR-Nogo-B/Fas ligand (FasL) synergistically reduced cell viability and increased the expression of apoptotic signaling proteins in colorectal cancer cells. These results strongly support our contention that Nogo-B most likely played an oncogenic role in colorectal cancer cells, mainly by negatively regulating the extrinsic apoptotic pathway in them. Finally, we revealed that suppression of Nogo-B caused down-regulation of c-FLIP, known as a major anti-apoptotic protein, and activation of caspase-8 in the death receptor pathway. Interaction between Nogo-B and c-FLIP was shown by immunoprecipitation and immunofluorescence studies. In conclusion, Nogo-B was shown to play an important negative role in apoptotic signaling through its interaction with c-FLIP in colorectal cancer cells, and may thus become a novel therapeutic target for colorectal cancer. Copyright © 2018 Elsevier B.V. All rights reserved.

  15. Cell Cycle Regulation and Apoptotic Responses of the Embryonic Chick Retina by Ionizing Radiation.

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    Margot Mayer

    Full Text Available Ionizing radiation (IR exerts deleterious effects on the developing brain, since proliferative neuronal progenitor cells are highly sensitive to IR-induced DNA damage. Assuming a radiation response that is comparable to mammals, the chick embryo would represent a lower vertebrate model system that allows analysis of the mechanisms underlying this sensitivity, thereby contributing to the reduction, refinement and replacement of animal experiments. Thus, this study aimed to elucidate the radiation response of the embryonic chick retina in three selected embryonic stages. Our studies reveal a lack in the radiation-induced activation of a G1/S checkpoint, but rapid abrogation of G2/M progression after IR in retinal progenitors throughout development. Unlike cell cycle control, radiation-induced apoptosis (RIA showed strong variations between its extent, dose dependency and temporal occurrence. Whereas the general sensitivity towards RIA declined with ongoing differentiation, its dose dependency constantly increased with age. For all embryonic stages RIA occurred during comparable periods after irradiation, but in older animals its maximum shifted towards earlier post-irradiation time points. In summary, our results are in good agreement with data from the developing rodent retina, strengthening the suitability of the chick embryo for the analysis of the radiation response in the developing central nervous system.

  16. Lactobacillus casei Exerts Anti-Proliferative Effects Accompanied by Apoptotic Cell Death and Up-Regulation of TRAIL in Colon Carcinoma Cells

    Science.gov (United States)

    Santarmaki, Valentina; Aindelis, Georgios; Tompoulidou, Evgenia; Lamprianidou, Eleftheria E.; Saxami, Georgia; Ypsilantis, Petros; Lampri, Evangeli S.; Simopoulos, Constantinos; Kotsianidis, Ioannis; Galanis, Alex; Kourkoutas, Yiannis; Dimitrellou, Dimitra; Chlichlia, Katerina

    2016-01-01

    Probiotic microorganisms such as lactic acid bacteria (LAB) exert a number of strain-specific health-promoting activities attributed to their immunomodulatory, anti-inflammatory and anti-carcinogenic properties. Despite recent attention, our understanding of the biological processes involved in the beneficial effects of LAB strains is still limited. To this end, the present study investigated the growth-inhibitory effects of Lactobacillus casei ATCC 393 against experimental colon cancer. Administration of live Lactobacillus casei (as well as bacterial components thereof) on murine (CT26) and human (HT29) colon carcinoma cell lines raised a significant concentration- and time-dependent anti-proliferative effect, determined by cell viability assays. Specifically, a dramatic decrease in viability of colon cancer cells co-incubated with 109 CFU/mL L. casei for 24 hours was detected (78% for HT29 and 52% for CT26 cells). In addition, live L. casei induced apoptotic cell death in both cell lines as revealed by annexin V and propidium iodide staining. The significance of the in vitro anti-proliferative effects was further confirmed in an experimental tumor model. Oral daily administration of 109 CFU live L. casei for 13 days significantly inhibited in vivo growth of colon carcinoma cells, resulting in approximately 80% reduction in tumor volume of treated mice. Tumor growth inhibition was accompanied by L. casei-driven up-regulation of the TNF-related apoptosis-inducing ligand TRAIL and down-regulation of Survivin. Taken together, these findings provide evidence for beneficial tumor-inhibitory, anti-proliferative and pro-apoptotic effects driven by this probiotic LAB strain. PMID:26849051

  17. Lactobacillus casei Exerts Anti-Proliferative Effects Accompanied by Apoptotic Cell Death and Up-Regulation of TRAIL in Colon Carcinoma Cells.

    Directory of Open Access Journals (Sweden)

    Angeliki Tiptiri-Kourpeti

    Full Text Available Probiotic microorganisms such as lactic acid bacteria (LAB exert a number of strain-specific health-promoting activities attributed to their immunomodulatory, anti-inflammatory and anti-carcinogenic properties. Despite recent attention, our understanding of the biological processes involved in the beneficial effects of LAB strains is still limited. To this end, the present study investigated the growth-inhibitory effects of Lactobacillus casei ATCC 393 against experimental colon cancer. Administration of live Lactobacillus casei (as well as bacterial components thereof on murine (CT26 and human (HT29 colon carcinoma cell lines raised a significant concentration- and time-dependent anti-proliferative effect, determined by cell viability assays. Specifically, a dramatic decrease in viability of colon cancer cells co-incubated with 10(9 CFU/mL L. casei for 24 hours was detected (78% for HT29 and 52% for CT26 cells. In addition, live L. casei induced apoptotic cell death in both cell lines as revealed by annexin V and propidium iodide staining. The significance of the in vitro anti-proliferative effects was further confirmed in an experimental tumor model. Oral daily administration of 10(9 CFU live L. casei for 13 days significantly inhibited in vivo growth of colon carcinoma cells, resulting in approximately 80% reduction in tumor volume of treated mice. Tumor growth inhibition was accompanied by L. casei-driven up-regulation of the TNF-related apoptosis-inducing ligand TRAIL and down-regulation of Survivin. Taken together, these findings provide evidence for beneficial tumor-inhibitory, anti-proliferative and pro-apoptotic effects driven by this probiotic LAB strain.

  18. Lactobacillus casei Exerts Anti-Proliferative Effects Accompanied by Apoptotic Cell Death and Up-Regulation of TRAIL in Colon Carcinoma Cells.

    Science.gov (United States)

    Tiptiri-Kourpeti, Angeliki; Spyridopoulou, Katerina; Santarmaki, Valentina; Aindelis, Georgios; Tompoulidou, Evgenia; Lamprianidou, Eleftheria E; Saxami, Georgia; Ypsilantis, Petros; Lampri, Evangeli S; Simopoulos, Constantinos; Kotsianidis, Ioannis; Galanis, Alex; Kourkoutas, Yiannis; Dimitrellou, Dimitra; Chlichlia, Katerina

    2016-01-01

    Probiotic microorganisms such as lactic acid bacteria (LAB) exert a number of strain-specific health-promoting activities attributed to their immunomodulatory, anti-inflammatory and anti-carcinogenic properties. Despite recent attention, our understanding of the biological processes involved in the beneficial effects of LAB strains is still limited. To this end, the present study investigated the growth-inhibitory effects of Lactobacillus casei ATCC 393 against experimental colon cancer. Administration of live Lactobacillus casei (as well as bacterial components thereof) on murine (CT26) and human (HT29) colon carcinoma cell lines raised a significant concentration- and time-dependent anti-proliferative effect, determined by cell viability assays. Specifically, a dramatic decrease in viability of colon cancer cells co-incubated with 10(9) CFU/mL L. casei for 24 hours was detected (78% for HT29 and 52% for CT26 cells). In addition, live L. casei induced apoptotic cell death in both cell lines as revealed by annexin V and propidium iodide staining. The significance of the in vitro anti-proliferative effects was further confirmed in an experimental tumor model. Oral daily administration of 10(9) CFU live L. casei for 13 days significantly inhibited in vivo growth of colon carcinoma cells, resulting in approximately 80% reduction in tumor volume of treated mice. Tumor growth inhibition was accompanied by L. casei-driven up-regulation of the TNF-related apoptosis-inducing ligand TRAIL and down-regulation of Survivin. Taken together, these findings provide evidence for beneficial tumor-inhibitory, anti-proliferative and pro-apoptotic effects driven by this probiotic LAB strain.

  19. Induction of Non-Apoptotic Cell Death by Activated Ras Requires Inverse Regulation of Rac1 and Arf6

    Science.gov (United States)

    Bhanot, Haymanti; Young, Ashley M.; Overmeyer, Jean H.; Maltese, William A.

    2010-01-01

    Methuosis is a unique form of non-apoptotic cell death triggered by alterations in the trafficking of clathrin-independent endosomes, ultimately leading to extreme vacuolization and rupture of the cell. Methuosis can be induced in glioblastoma cells by expression of constitutively active Ras. This study identifies the small GTPases, Rac1 and Arf6, and the Arf6 GTPase-activating-protein, GIT1, as key downstream components of the signaling pathway underlying Ras-induced methuosis. The extent to which graded expression of active H-Ras(G12V) triggers cytoplasmic vacuolization correlates with the amount of endogenous Rac1 in the active GTP state. Blocking Rac1 activation with the specific Rac inhibitor, EHT 1864, or co-expression of dominant-negative Rac1(T17N), prevents the accumulation of vacuoles induced by H-Ras(G12V). Coincident with Rac1 activation, H-Ras(G12V) causes a decrease in the amount of active Arf6, a GTPase that functions in recycling of clathrin-independent endosomes. The effect of H-Ras(G12V) on Arf6 is blocked by EHT 1864, indicating that the decrease in Arf6-GTP is directly linked to activation of Rac1. Constitutively active Rac1(G12V) interacts with GIT1 in immunoprecipitation assays. Ablation of GIT1 by shRNA prevents the decrease in active Arf6, inhibits vacuolization, and prevents loss of cell viability in cells expressing Rac1(G12V). Together the results suggest that perturbations of endosome morphology associated with Ras-induced methuosis are due to downstream activation of Rac1, combined with reciprocal inactivation of Arf6. The latter appears to be mediated through Rac1 stimulation of GIT1. Further insights into this pathway could suggest opportunities for induction of methuosis in cancers that are resistant to apoptotic cell death. PMID:20713492

  20. Studying apoptotic cell death by flow cytometry

    International Nuclear Information System (INIS)

    Ormerod, Michael G.

    1998-01-01

    Full text: Programmed cell death (PCD) is of fundamental importance in the normal development of an animal and also in tumour biology and radiation biology. During PCD a sequence of changes occurs in cells giving rise to an apoptotic cascade of events. The main elements of this cascade are rapidly being elucidated. Flow cytometry has been used to follow many of these changes. It also has been used to quantify the number of apoptotic cells in a culture and, more recently, in clinical samples. In this review, the properties of apoptotic cells and the main feature of apoptotic cascade will be described. How flow cytometry can be used to follow changes during the apoptotic cascade will be discussed

  1. De-novo NAD+ synthesis regulates SIRT1-FOXO1 apoptotic pathway in response to NQO1 substrates in lung cancer cells.

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    Liu, Huiying; Xing, Rong; Cheng, Xuefang; Li, Qingran; Liu, Fang; Ye, Hui; Zhao, Min; Wang, Hong; Wang, Guangji; Hao, Haiping

    2016-09-20

    Tryptophan metabolism is essential in diverse kinds of tumors via regulating tumor immunology. However, the direct role of tryptophan metabolism and its signaling pathway in cancer cells remain largely elusive. Here, we establish a mechanistic link from L-type amino acid transporter 1 (LAT1) mediated transport of tryptophan and the subsequent de-novo NAD+ synthesis to SIRT1-FOXO1 regulated apoptotic signaling in A549 cells in response to NQO1 activation. In response to NQO1 activation, SIRT1 is repressed leading to the increased cellular accumulation of acetylated FOXO1 that transcriptionally activates apoptotic signaling. Decreased uptake of tryptophan due to the downregulation of LAT1 coordinates with PARP-1 hyperactivation to induce rapid depletion of NAD+ pool. Particularly, the LAT1-NAD+-SIRT1 signaling is activated in tumor tissues of patients with non-small cell lung cancer. Because NQO1 activation is characterized with oxidative challenge induced DNA damage, these results suggest that LAT1 and de-novo NAD+ synthesis in NSCLC cells may play essential roles in sensing excessive oxidative stress.

  2. Stabilization Of Apoptotic Cells: Generation Of Zombie Cells

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    José A. Sánchez Alcázar

    2015-08-01

    Stabilization of apoptotic cells can be used for reliable detection and quantification of apoptosis in cultured cells and may allow a safer administration of apoptotic cells in clinical applications. Furthermore, it opens new avenues in the functional reconstruction of apoptotic cells for longer preservation.

  3. Apoptotic pathways as regulators of recombination

    International Nuclear Information System (INIS)

    Gauny, S.S.; Kronenberg, A.; Liu, W.-C.

    2003-01-01

    Apoptosis, or programmed cell death (PCD), is a fundamental process that protects organismal integrity. In earlier work, we demonstrated that over-expression of either of two anti-apoptotic members of the BCL-2 family (BCL-2 or BCL-X L could elevate the frequency of radiation-induced mutations at the autosomal TK1 locus in human TK6 lymphoblasts that express wild-type TP53. Ectopic expression of BCL-X L also elevated the frequencies of double-strand break-induced gene conversion. The purpose of this study is to determine if BCL-2 family proteins promote radiation mutagenesis indirectly through their suppression of PCD, or whether the 'pro-mutagenic' function of these proteins can be separated from their anti-apoptotic function. We developed stable transfectants of TK6 cells that express a mutated form of BCL-X L with a single amino acid substitution in the BH1 domain that is known to interfere with the ability to suppress PCD (BCL-X L gly159ala). We also developed stable transfectants of TK6 cells that express a dominant negative caspase-9 that suppresses PCD. The results to date indicate that the mutated form of BCL-X L (gly159ala) does not suppress x-ray-induced PCD in TK6 cells, but it elevates radiation-induced TK1 mutant frequencies to the same extent as high level expression of wild-type BCL-X L . These data suggest that the anti-apoptotic function of BCL-2 family proteins is not required to elevate radiation mutagenesis. Separate experiments using TK6 cells that express a dominant negative caspase-9 indicate that this protein inhibits x-ray-induced PCD but TK1 mutant frequencies are not elevated. Taken together, the results suggest there is a separate function of BCL-2 family proteins that elevates radiation-induced mutagenesis independent of the well-known anti-apoptotic effect of these proteins of importance in human carcinogenesis

  4. FLASH knockdown sensitizes cells to Fas-mediated apoptosis via down-regulation of the anti-apoptotic proteins, MCL-1 and Cflip short.

    Directory of Open Access Journals (Sweden)

    Song Chen

    Full Text Available FLASH (FLICE-associated huge protein or CASP8AP2 is a large multifunctional protein that is involved in many cellular processes associated with cell death and survival. It has been reported to promote apoptosis, but we show here that depletion of FLASH in HT1080 cells by siRNA interference can also accelerate the process. As shown previously, depletion of FLASH halts growth by down-regulating histone biosynthesis and arrests the cell cycle in S-phase. FLASH knockdown followed by stimulating the cells with Fas ligand or anti-Fas antibodies was found to be associated with a more rapid cleavage of PARP, accelerated activation of caspase-8 and the executioner caspase-3 and rapid progression to cellular disintegration. As is the case for most anti-apoptotic proteins, FLASH was degraded soon after the onset of apoptosis. Depletion of FLASH also resulted in the reduced intracellular levels of the anti-apoptotic proteins, MCL-1 and the short isoform of cFLIP. FLASH knockdown in HT1080 mutant cells defective in p53 did not significantly accelerate Fas mediated apoptosis indicating that the effect was dependent on functional p53. Collectively, these results suggest that under some circumstances, FLASH suppresses apoptosis.

  5. The anti-apoptotic BAG3 protein is expressed in lung carcinomas and regulates small cell lung carcinoma (SCLC) tumor growth.

    Science.gov (United States)

    Chiappetta, Gennaro; Basile, Anna; Barbieri, Antonio; Falco, Antonia; Rosati, Alessandra; Festa, Michelina; Pasquinelli, Rosa; Califano, Daniela; Palma, Giuseppe; Costanzo, Raffaele; Barcaroli, Daniela; Capunzo, Mario; Franco, Renato; Rocco, Gaetano; Pascale, Maria; Turco, Maria Caterina; De Laurenzi, Vincenzo; Arra, Claudio

    2014-08-30

    BAG3, member the HSP70 co-chaperones family, has been shown to play a relevant role in the survival, growth and invasiveness of different tumor types. In this study, we investigate the expression of BAG3 in 66 specimens from different lung tumors and the role of this protein in small cell lung cancer (SCLC) tumor growth. Normal lung tissue did not express BAG3 while we detected the expression of BAG3 by immunohistochemistry in all the 13 squamous cell carcinomas, 13 adenocarcinomas and 4 large cell carcinomas. Furthermore, we detected BAG3 expression in 22 of the 36 SCLCs analyzed. The role on SCLC cell survival was determined by down-regulating BAG3 levels in two human SCLC cell lines, i.e. H69 and H446, in vitro and measuring cisplatin induced apoptosis. Indeed down-regulation of BAG3 determines increased cell death and sensitizes cells to cisplatin treatment. The effect of BAG3 down-regulation on tumor growth was also investigated in an in vivo xenograft model by treating mice with an adenovirus expressing a specific bag3 siRNA. Treatment with bag3 siRNA-Ad significantly reduced tumor growth and improved animal survival. In conclusion we show that a subset of SCLCs over express BAG3 that exerts an anti-apoptotic effect resulting in resistance to chemotherapy.

  6. Detection of apoptotic cells using immunohistochemistry

    NARCIS (Netherlands)

    Newbold, Andrea; Martin, Ben P.; Cullinane, Carleen; Bots, Michael

    2014-01-01

    Immunohistochemistry is commonly used to show the presence of apoptotic cells in situ. In this protocol, B-cell lymphoma cells are injected into recipient mice and, on tumor formation, the mice are treated with the apoptosis inducer vorinostat (a histone deacetylase inhibitor). Tumor samples are

  7. SYTO probes: markers of apoptotic cell demise.

    Science.gov (United States)

    Wlodkowic, Donald; Skommer, Joanna

    2007-10-01

    As mechanistic studies on tumor cell death advance towards their ultimate translational goal, there is a need for specific, rapid, and high-throughput analytical tools to detect diverse cell demise modes. Patented DNA-binding SYTO probes, for example, are gaining increasing interest as easy-to-use markers of caspase-dependent apoptotic cell death. They are proving convenient for tracking apoptosis in diverse hematopoietic cell lines and primary tumor samples, and, due to their spectral characteristics, appear to be useful for the development of multiparameter flow cytometry assays. Herein, several protocols for multiparametric assessment of apoptotic events using SYTO probes are provided. There are protocols describing the use of green fluorescent SYTO 16 and red fluorescent SYTO 17 dyes in combination with plasma membrane permeability markers. Another protocol highlights the multiparametric use of SYTO 16 dye in conjunction with the mitochondrial membrane potential sensitive probe, tetramethylrhodamine methyl ester (TMRM), and the plasma membrane permeability marker, 7-aminoactinomycin D (7-AAD).

  8. Detection of apoptotic cells in tumour paraffin sections

    International Nuclear Information System (INIS)

    Pizem, J.; Coer, A.

    2003-01-01

    Apoptosis is a distinct form of cell death characterised by specific morphological features and regulated by complex molecular mechanisms. Its deregulation is fundamental for tumour growth and progression and, moreover, anticancer therapies suppress tumour growth mainly by induction of apoptosis. Since the extent of apoptosis in a tumour may have prognostic as well as therapeutic implications, much effort has been invested in developing specific methods that can be routinely used to detect apoptotic cells in archival formalin- fixed paraffin-embedded tissue. Complex molecular pathways are involved in the regulation of apoptosis. Pro-apoptotic signals trigger activation of caspases that specifically cleave target proteins. Cleavage of proteins (caspase substrates) is responsible for morphological changes of apoptotic cells and DNA fragmentation. In the last decade, detection of apoptotic cells in formalin-fixed tumour tissue sections has been based mainly on morphology and characteristic DNA fragmentation. Recently, specific antibodies to activated caspases and cleaved target proteins (including cytokeratin 18, actin and PARP) have been produced that enable accurate detection of apoptosis in paraffin sections. (author)

  9. Induction of apoptotic cell death by putrescine

    DEFF Research Database (Denmark)

    Takao, Koichi; Rickhag, Karl Mattias; Hegardt, Cecilia

    2006-01-01

    that overexpression of a metabolically stable ODC in CHO cells induced a massive cell death unless the cells were grown in the presence of the ODC inhibitor alpha-difluoromethylornithine (DFMO). Cells overexpressing wild-type (unstable) ODC, on the other hand, were not dependent on the presence of DFMO...... for their growth. The induction of cell death was correlated with a dramatic increase in cellular putrescine levels. Analysis using flow cytometry revealed perturbed cell cycle kinetics, with a large accumulation of cells with sub-G1 amounts of DNA, which is a typical sign of apoptosis. Another strong indication...... of apoptosis was the finding that one of the key enzymes in the apoptotic process, caspase-3, was induced when DFMO was omitted from the growth medium. Furthermore, inhibition of the caspase activity significantly reduced the recruitment of cells to the sub-G1 fraction. In conclusion, deregulation of polyamine...

  10. Cobalt iron oxide nanoparticles induce cytotoxicity and regulate the apoptotic genes through ROS in human liver cells (HepG2).

    Science.gov (United States)

    Ahamed, Maqusood; Akhtar, Mohd Javed; Khan, M A Majeed; Alhadlaq, Hisham A; Alshamsan, Aws

    2016-12-01

    Cobalt iron oxide (CoFe 2 O 4 ) nanoparticles (CIO NPs) have been one of the most widely explored magnetic NPs because of their excellent chemical stability, mechanical hardness and heat generating potential. However, there is limited information concerning the interaction of CIO NPs with biological systems. In this study, we investigated the reactive oxygen species (ROS) mediated cytotoxicity and apoptotic response of CIO NPs in human liver cells (HepG2). Diameter of crystalline CIO NPs was found to be 23nm with a band gap of 1.97eV. CIO NPs induced cell viability reduction and membrane damage, and degree of induction was dose- and time-dependent. CIO NPs were also found to induce oxidative stress revealed by induction of ROS, depletion of glutathione and lower activity of superoxide dismutase enzyme. Real-time PCR data has shown that mRNA level of tumor suppressor gene p53 and apoptotic genes (bax, CASP3 and CASP9) were higher, while the expression level of anti-apoptotic gene bcl-2 was lower in cells following exposure to CIO NPs. Activity of caspase-3 and caspase-9 enzymes was also higher in CIO NPs exposed cells. Furthermore, co-exposure of N-acetyl-cysteine (ROS scavenger) efficiently abrogated the modulation of apoptotic genes along with the prevention of cytotoxicity caused by CIO NPs. Overall, we observed that CIO NPs induced cytotoxicity and apoptosis in HepG2 cells through ROS via p53 pathway. This study suggests that toxicity mechanisms of CIO NPs should be further investigated in animal models. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. The phosphatidylserine receptor has essential functions during embryogenesis but not in apoptotic cell removal

    Directory of Open Access Journals (Sweden)

    Hafner Martin

    2004-08-01

    Full Text Available Abstract Background Phagocytosis of apoptotic cells is fundamental to animal development, immune function and cellular homeostasis. The phosphatidylserine receptor (Ptdsr on phagocytes has been implicated in the recognition and engulfment of apoptotic cells and in anti-inflammatory signaling. To determine the biological function of the phosphatidylserine receptor in vivo, we inactivated the Ptdsr gene in the mouse. Results Ablation of Ptdsr function in mice causes perinatal lethality, growth retardation and a delay in terminal differentiation of the kidney, intestine, liver and lungs during embryogenesis. Moreover, eye development can be severely disturbed, ranging from defects in retinal differentiation to complete unilateral or bilateral absence of eyes. Ptdsr -/- mice with anophthalmia develop novel lesions, with induction of ectopic retinal-pigmented epithelium in nasal cavities. A comprehensive investigation of apoptotic cell clearance in vivo and in vitro demonstrated that engulfment of apoptotic cells was normal in Ptdsr knockout mice, but Ptdsr-deficient macrophages were impaired in pro- and anti-inflammatory cytokine signaling after stimulation with apoptotic cells or with lipopolysaccharide. Conclusion Ptdsr is essential for the development and differentiation of multiple organs during embryogenesis but not for apoptotic cell removal. Ptdsr may thus have a novel, unexpected developmental function as an important differentiation-promoting gene. Moreover, Ptdsr is not required for apoptotic cell clearance by macrophages but seems to be necessary for the regulation of macrophage cytokine responses. These results clearly contradict the current view that the phosphatidylserine receptor primarily functions in apoptotic cell clearance.

  12. Significance of apoptotic cell death after γ-irradiation

    International Nuclear Information System (INIS)

    Wu, H.G.; Kim, I.H.; Ha, S.W.; Park, C.I.

    2003-01-01

    Full text: The objectives of this study are to investigate the significance of apoptotic death compared to total cell death after γ-ray irradiation in human Hand N cancer cell lines and to find out correlation between apoptosis and radiation sensitivity. Materials and Method: Head and neck cancer cell lines (PCI-1, PCI-13, and SNU-1066), leukemia cell line (CCRF-CEM), and fibroblast cell line (LM217) as a normal control were used for this study. Cells were irradiated using Cs-137 animal experiment irradiator. Total cell death was measured by clonogenic assay. Annexin-V staining was used to detect the fraction of apoptotic death. The resulting data was analyzed with two parameters, apoptotic index (AI) and apoptotic fraction(AF). AI is ratio of apoptotic cells to total cells, and AF is ration of apoptotic cell death to mutant frequencytion ex(Number of apoptotic cells) / (Number of total cells counted) AF = {(AI) / (1-survival fraction)} x 100 (%) Results. Surviving fraction at 2 Gy (SF2) were 0.741, 0.544, 0.313, 0.302, and 0.100 for PCI-1, PCI-13, SNU-1066, CCRF-CEM, and LM217 cell lines, respectively. Apoptosis was detected in all cell lines. Apoptotic index reached peak value at 72 hours after irradiation in head and neck cancer cell lines, and that was at 24 hours in CCRF-CEM and LM217. Total cell death increased exponentially with increasing radiation dose from 0 Gy to 8 Gy, but the change was minimal in apoptotic index (Fig. 1.). Apoptotic fractions at 2 Gy were 46%, 48%, 46%, 24%, and 19% and at 6 Gy were 20%, 33%, 35%, 17%, and 20% for PCI-1, PCI-13, SNU-1066, CCRF-CEM, and LM217, respectively. The radioresistant cell lines showed more higher apoptotic fraction at 2 Gy (Table 1.), but there was not such correlation at 6 Gy. Conclusion: All cell lines used in this study showed apoptosis after irradiation, but time course of apoptosis was different from that of leukemia cell line and normal fibroblast cell line. Reproductive cell death was more important

  13. The Apoptotic Effect of Ursolic Acid on SK-Hep-1 Cells is Regulated by the PI3K/Akt, p38 and JNK MAPK Signaling Pathways

    Directory of Open Access Journals (Sweden)

    Wan-Ling Chuang

    2016-04-01

    Full Text Available Ursolic acid (UA is a pentacyclic triterpene acid that is present in a wide variety of medicinal herbs and edible plants. This study investigated the effect of UA on apoptosis and proliferation of hepatocellular carcinoma SK-Hep-1 cells. After treatment of SK-Hep-1 cells with different concentrations of UA, we observed that cell viability was reduced in a dose- and time-dependent manner. Furthermore, there was a dose-dependent increase in the percentage of cells in the sub-G1 and G2/M phases, with cells treated with 60 μM showing the highest percentages of cells in those phases. UA-induced chromatin condensation of nuclei was observed by using DAPI staining. The western blot results revealed that exposure to UA was associated with decreased expression of the anti-apoptotic proteins Mcl-1, Bcl-xL, Bcl-2, and TCTP and increased expression of apoptosis-related proteins TNF-α, Fas, FADD, Bax, cleaved caspase-3, caspase-8, caspase-9, and PARP. Immunocytochemistry staining showed that treatment with UA resulted in increased expression of caspase-3. Moreover, exposure to UA resulted in the inhibition of the PI3K/Akt and p38 MAPK signaling pathways. These findings suggest that UA inhibits the proliferation of SK-Hep-1 cells and induces apoptosis.

  14. Ethyl acetate extract of Chinese medicinal herb Sarcandra glabra induces growth inhibition on human leukemic HL-60 cells, associated with cell cycle arrest and up-regulation of pro-apoptotic Bax/Bcl-2 ratio.

    Science.gov (United States)

    Li, W Y; Chiu, Lawrence C M; Lam, W S; Wong, W Y; Chan, Y T; Ho, Y P; Wong, Elaine Y L; Wong, Y S; Ooi, Vincent E C

    2007-02-01

    Sarcandra glabra (Thunb.) Nakai, colloquially known as Caoshanhu, is a Chinese medicinal herb with reported anti-tumor, anti-inflammatory, anti-viral and non-specific immunoenhancing properties. Although the plant has been clinically used for treating a variety of diseases, its bioactive ingredients are largely unknown and its mode of action has never been investigated. In this study, the anti-tumor property of ethyl acetate (EA) extract of S. glabra was investigated by determining its in vitro growth-inhibitory effects on a panel of human cancer cell lines of different histotypes. Growth inhibition of the EA extract on the cancer cells seemed to be selective, and the leukemic HL-60 was found to be the most responsive after 48 h of treatment (IC50=58 microg/ml). Flow cytometric studies further illustrated that the extract might interfere with DNA replication and thus arrested the cell cycle at S phase in the leukemic cells, followed by DNA fragmentation and loss of phospholipid asymmetry in the plasma membrane after 72 h of treatment. Concurrently, the pro-apoptotic Bax/Bcl-2 ratio was also up-regulated by more than 178% of the control level. All these findings suggested that the extract had initiated apoptosis to kill the leukemic cells. Results from this pioneer study help to establish a scientific foundation for future research and development of the bioactive ingredients in EA extract of S. glabra as efficacious anti-cancer agents.

  15. TAM receptors in apoptotic cell clearance, autoimmunity, and cancer.

    Science.gov (United States)

    Nguyen, Khanh-Quynh; Tsou, Wen-I; Kotenko, Sergei; Birge, Raymond B

    2013-08-01

    Receptor tyrosine kinases, Tyro-3, Axl and Mer, collectively designated as TAM, are involved in the clearance of apoptotic cells. TAM ligands, Gas6 and Protein S, bind to the surfaces of apoptotic cells, and at the same time, interact directly with TAM expressed on phagocytes, impacting the engulfment and clearance of apoptotic cells and debris. The well-tuned and balanced actions of TAM may affect a variety of human pathologies including autoimmunity, retinal degeneration, and cancer. This article emphasizes some of the emerging findings and mechanistic insights into TAM functions that are clinically relevant and possibly therapeutically targeted.

  16. Harnessing Apoptotic Cell Clearance to Treat Autoimmune Arthritis

    Directory of Open Access Journals (Sweden)

    Philippe Saas

    2017-10-01

    Full Text Available Early-stage apoptotic cells possess immunomodulatory properties. Proper apoptotic cell clearance during homeostasis has been shown to limit subsequent immune responses. Based on these observations, early-stage apoptotic cell infusion has been used to prevent unwanted inflammatory responses in different experimental models of autoimmune diseases or transplantation. Moreover, this approach has been shown to be feasible without any toxicity in patients undergoing allogeneic hematopoietic cell transplantation to prevent graft-versus-host disease. However, whether early-stage apoptotic cell infusion can be used to treat ongoing inflammatory disorders has not been reported extensively. Recently, we have provided evidence that early-stage apoptotic cell infusion is able to control, at least transiently, ongoing collagen-induced arthritis. This beneficial therapeutic effect is associated with the modulation of antigen-presenting cell functions mainly of macrophages and plasmacytoid dendritic cells, as well as the induction of collagen-specific regulatory CD4+ T cells (Treg. Furthermore, the efficacy of this approach is not altered by the association with two standard treatments of rheumatoid arthritis (RA, methotrexate and tumor necrosis factor (TNF inhibition. Here, in the light of these observations and recent data of the literature, we discuss the mechanisms of early-stage apoptotic cell infusion and how this therapeutic approach can be transposed to patients with RA.

  17. Cell shape and organelle modification in apoptotic U937 cells

    Directory of Open Access Journals (Sweden)

    MR Montinari

    2009-12-01

    Full Text Available U937 cells induced to apoptosis, progressively and dramatically modified their cell shape by intense blebbing formation, leading to the production of apoptotic bodies. The blebs evolved with time; milder forms of blebbing involving only a region or just the cortical part of the cytoplasm were observed within the first hour of incubation with puromycin; blebbing involving the whole cell body with very deep constrictions is the most frequent event observed during late times of incubation. The ultrastructural analysis of apoptotic cells revealed characteristic features of nuclear fragmentation (budding and cleavage mode and cytoplasmatic modifications. The cytoplasm of blebs does not contain organelles, such as ribosomes or mitochondria. Scarce presence of endoplasmic reticulum can be observed at the site of bleb detachment. However, blebbing is a dispensable event as evaluated by using inhibitor of actin polymerization. In the present study, the progressive modifications of the nucleus, mitochondria, nuclear fragmentation, cytoplasmic blebs formation and production of apoptotic bodies in U937 monocytic cells induced to apoptosis by puromycin (an inhibitor of protein synthesis were simultaneously analyzed.

  18. NOX4-mediated ROS production induces apoptotic cell death via down-regulation of c-FLIP and Mcl-1 expression in combined treatment with thioridazine and curcumin.

    Science.gov (United States)

    Seo, Seung Un; Kim, Tae Hwan; Kim, Dong Eun; Min, Kyoung-Jin; Kwon, Taeg Kyu

    2017-10-01

    Thioridazine is known to have anti-tumor effects by inhibiting PI3K/Akt signaling, which is an important signaling pathway in cell survival. However, thioridazine alone does not induce apoptosis in head and neck squamous cell carcinoma (AMC-HN4), human breast carcinoma (MDA-MB231), and human glioma (U87MG) cells. Therefore, we investigated whether combined treatment with thioridazine and curcumin induces apoptosis. Combined treatment with thioridazine and curcumin markedly induced apoptosis in cancer cells without inducing apoptosis in human normal mesangial cells and human normal umbilical vein cells (EA.hy926). We found that combined treatment with thioridazine and curcumin had synergistic effects in AMC-HN4 cells. Among apoptosis-related proteins, thioridazine plus curcumin induced down-regulation of c-FLIP and Mcl-1 expression at the post-translational levels in a proteasome-dependent manner. Augmentation of proteasome activity was related to the up-regulation of proteasome subunit alpha 5 (PSMA5) expression in curcumin plus thioridazine-treated cells. Combined treatment with curcumin and thioridazine produced intracellular ROS in a NOX4-dependent manner, and ROS-mediated activation of Nrf2/ARE signaling played a critical role in the up-regulation of PSMA5 expression. Furthermore, ectopic expression of c-FLIP and Mcl-1 inhibited apoptosis in thioridazine and curcumin-treated cells. Therefore, we demonstrated that thioridazine plus curcumin induces proteasome activity by up-regulating PSMA5 expression via NOX4-mediated ROS production and that down-regulation of c-FLIP and Mcl-1 expression post-translationally is involved in apoptosis. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  19. NOX4-mediated ROS production induces apoptotic cell death via down-regulation of c-FLIP and Mcl-1 expression in combined treatment with thioridazine and curcumin

    Directory of Open Access Journals (Sweden)

    Seung Un Seo

    2017-10-01

    Full Text Available Thioridazine is known to have anti-tumor effects by inhibiting PI3K/Akt signaling, which is an important signaling pathway in cell survival. However, thioridazine alone does not induce apoptosis in head and neck squamous cell carcinoma (AMC-HN4, human breast carcinoma (MDA-MB231, and human glioma (U87MG cells. Therefore, we investigated whether combined treatment with thioridazine and curcumin induces apoptosis. Combined treatment with thioridazine and curcumin markedly induced apoptosis in cancer cells without inducing apoptosis in human normal mesangial cells and human normal umbilical vein cells (EA.hy926. We found that combined treatment with thioridazine and curcumin had synergistic effects in AMC-HN4 cells. Among apoptosis-related proteins, thioridazine plus curcumin induced down-regulation of c-FLIP and Mcl-1 expression at the post-translational levels in a proteasome-dependent manner. Augmentation of proteasome activity was related to the up-regulation of proteasome subunit alpha 5 (PSMA5 expression in curcumin plus thioridazine-treated cells. Combined treatment with curcumin and thioridazine produced intracellular ROS in a NOX4-dependent manner, and ROS-mediated activation of Nrf2/ARE signaling played a critical role in the up-regulation of PSMA5 expression. Furthermore, ectopic expression of c-FLIP and Mcl-1 inhibited apoptosis in thioridazine and curcumin-treated cells. Therefore, we demonstrated that thioridazine plus curcumin induces proteasome activity by up-regulating PSMA5 expression via NOX4-mediated ROS production and that down-regulation of c-FLIP and Mcl-1 expression post-translationally is involved in apoptosis.

  20. Effect of Apoptotic Cell Recognition on Macrophage Polarization and Mycobacterial Persistence

    Science.gov (United States)

    de Oliveira Fulco, Tatiana; Andrade, Priscila Ribeiro; de Mattos Barbosa, Mayara Garcia; Pinto, Thiago Gomes Toledo; Ferreira, Paula Fernandez; Ferreira, Helen; da Costa Nery, José Augusto; Real, Suzana Côrte; Borges, Valéria Matos; Moraes, Milton Ozório; Sarno, Euzenir Nunes; Sampaio, Elizabeth Pereira

    2014-01-01

    Intracellular Mycobacterium leprae infection modifies host macrophage programming, creating a protective niche for bacterial survival. The milieu regulating cellular apoptosis in the tissue plays an important role in defining susceptible and/or resistant phenotypes. A higher density of apoptotic cells has been demonstrated in paucibacillary leprosy lesions than in multibacillary ones. However, the effect of apoptotic cell removal on M. leprae-stimulated cells has yet to be fully elucidated. In this study, we investigated whether apoptotic cell removal (efferocytosis) induces different phenotypes in proinflammatory (Mϕ1) and anti-inflammatory (Mϕ2) macrophages in the presence of M. leprae. We stimulated Mϕ1 and Mϕ2 cells with M. leprae in the presence or absence of apoptotic cells and subsequently evaluated the M. leprae uptake, cell phenotype, and cytokine pattern in the supernatants. In the presence of M. leprae and apoptotic cells, Mϕ1 macrophages changed their phenotype to resemble the Mϕ2 phenotype, displaying increased CD163 and SRA-I expression as well as higher phagocytic capacity. Efferocytosis increased M. leprae survival in Mϕ1 cells, accompanied by reduced interleukin-15 (IL-15) and IL-6 levels and increased transforming growth factor beta (TGF-β) and IL-10 secretion. Mϕ1 cells primed with M. leprae in the presence of apoptotic cells induced the secretion of Th2 cytokines IL-4 and IL-13 in autologous T cells compared with cultures stimulated with M. leprae or apoptotic cells alone. Efferocytosis did not alter the Mϕ2 cell phenotype or cytokine secretion profile, except for TGF-β. Based on these data, we suggest that, in paucibacillary leprosy patients, efferocytosis contributes to mycobacterial persistence by increasing the Mϕ2 population and sustaining the infection. PMID:25024361

  1. Surface code—biophysical signals for apoptotic cell clearance

    International Nuclear Information System (INIS)

    Biermann, Mona; Maueröder, Christian; Brauner, Jan M; Chaurio, Ricardo; Herrmann, Martin; Muñoz, Luis E; Janko, Christina

    2013-01-01

    Apoptotic cell death and the clearance of dying cells play an important and physiological role in embryonic development and normal tissue turnover. In contrast to necrosis, apoptosis proceeds in an anti-inflammatory manner. It is orchestrated by the timed release and/or exposure of so-called ‘find-me’, ‘eat me’ and ‘tolerate me’ signals. Mononuclear phagocytes are attracted by various ‘find-me’ signals, including proteins, nucleotides, and phospholipids released by the dying cell, whereas the involvement of granulocytes is prevented via ‘stay away’ signals. The exposure of anionic phospholipids like phosphatidylserine (PS) by apoptotic cells on the outer leaflet of the plasma membrane is one of the main ‘eat me’ signals. PS is recognized by a number of innate receptors as well as by soluble bridging molecules on the surface of phagocytes. Importantly, phagocytes are able to discriminate between viable and apoptotic cells both exposing PS. Due to cytoskeleton remodeling PS has a higher lateral mobility on the surfaces of apoptotic cells thereby promoting receptor clustering on the phagocyte. PS not only plays an important role in the engulfment process, but also acts as ‘tolerate me’ signal inducing the release of anti-inflammatory cytokines by phagocytes. An efficient and fast clearance of apoptotic cells is required to prevent secondary necrosis and leakage of intracellular danger signals into the surrounding tissue. Failure or prolongation of the clearance process leads to the release of intracellular antigens into the periphery provoking inflammation and development of systemic inflammatory autoimmune disease like systemic lupus erythematosus. Here we review the current findings concerning apoptosis-inducing pathways, important players of apoptotic cell recognition and clearance as well as the role of membrane remodeling in the engulfment of apoptotic cells by phagocytes. (paper)

  2. Response rate of fibrosarcoma cells to cytotoxic drugs on the expression level correlates to the therapeutic response rate of fibrosarcomas and is mediated by regulation of apoptotic pathways

    International Nuclear Information System (INIS)

    Lehnhardt, Marcus; Mueller, Oliver; Klein-Hitpass, Ludger; Kuhnen, Cornelius; Homann, Heinz Herbert; Daigeler, Adrien; Steinau, Hans Ulrich; Roehrs, Sonja; Schnoor, Laura; Steinstraesser, Lars

    2005-01-01

    Because of the high resistance rate of fibrosarcomas against cytotoxic agents clinical chemotherapy of these tumors is not established. A better understanding of the diverse modes of tumor cell death following cytotoxic therapies will provide a molecular basis for new chemotherapeutic strategies. In this study we elucidated the response of a fibrosarcoma cell line to clinically used cytostatic agents on the level of gene expression. HT1080 fibrosarcoma cells were exposed to the chemotherapeutic agents doxorubicin, actinomycin D or vincristine. Total RNA was isolated and the gene expression patterns were analyzed by microarray analysis. Expression levels for 46 selected candidate genes were validated by quantitative real-time PCR. The analysis of the microarray data resulted in 3.309 (actinomycin D), 1.019 (doxorubicin) and 134 (vincristine) probesets that showed significant expression changes. For the RNA synthesis blocker actinomycin D, 99.4% of all differentially expressed probesets were under-represented. In comparison, probesets down-regulated by doxorubicin comprised only 37.4% of all genes effected by this agent. Closer analysis of the differentially regulated genes revealed that doxorubicin induced cell death of HT1080 fibrosarcoma cells mainly by regulating the abundance of factors mediating the mitochondrial (intrinsic) apoptosis pathway. Furthermore doxorubicin influences other pathways and crosstalk to other pathways (including to the death receptor pathway) at multiple levels. We found increased levels of cytochrome c, APAF-1 and members of the STAT-family (STAT1, STAT3), while Bcl-2 expression was decreased. Caspase-1, -3, -6, -8, and -9 were increased indicating that these proteases are key factors in the execution of doxorubicin mediated apoptosis. This study demonstrates that chemotherapy regulates the expression of apoptosis-related factors in fibrosarcoma cells. The number and the specific pattern of the genes depend on the used cytotoxic drug

  3. c-Met Overexpression Contributes to the Acquired Apoptotic Resistance of Nonadherent Ovarian Cancer Cells through a Cross Talk Mediated by Phosphatidylinositol 3-Kinase and Extracellular Signal-Regulated Kinase 1/2

    Directory of Open Access Journals (Sweden)

    Maggie K.S. Tang

    2010-02-01

    Full Text Available Ovarian cancer is the most lethal gynecologic cancer mainly because of widespread peritoneal dissemination and malignant ascites. Key to this is the capacity of tumor cells to escape suspension-induced apoptosis (anoikis, which also underlies their resistance to chemotherapy. Here, we used a nonadherent cell culture model to investigate the molecular mechanisms of apoptotic resistance of ovarian cancer cells that may mimic the chemoresistance found in solid tumors. We found that ovarian cancer cells acquired a remarkable resistance to anoikis and apoptosis induced by exposure to clinically relevant doses of two front-line chemotherapeutic drugs cisplatin and paclitaxel when grown in three-dimensional than monolayer cultures. Inhibition of the hepatocyte growth factor (HGF receptor c-Met, which is frequently overexpressed in ovarian cancer, by a specific inhibitor or small interfering RNA blocked the acquired anoikis resistance and restored chemosensitivity in three-dimensional not in two-dimensional cultures. These effects were found to be dependent on both phosphatidylinositol 3-kinase (PI3K/Akt and extracellular signal-regulated kinase (ERK 1/2 signaling pathways. Inhibitors of PI3K/Akt abrogated ERK1/2 activation and its associated anoikis resistance in response to HGF, suggesting a signaling relay between these two pathways. Furthermore, we identified a central role of Ras as a mechanism of this cross talk. Interestingly, Ras did not lie upstream of PI3K/Akt, whereas PI3K/Akt signaling to ERK1/2 involved Ras. These findings shed new light on the apoptotic resistance mechanism of nonadherent ovarian cancer ascites cells and may have important clinical implications.

  4. Anti-inflammatory drugs suppress proliferation and induce apoptosis through altering expressions of cell cycle regulators and pro-apoptotic factors in cultured human osteoblasts

    International Nuclear Information System (INIS)

    Chang, J.-K.; Li, C.-J.; Liao, H.-J.; Wang, C.-K.; Wang, G.-J.; Ho, M.-L.

    2009-01-01

    It has been reported that anti-inflammatory drugs (AIDs) inhibited bone repair in animal studies, and suppressed proliferation and induced cell death in rat osteoblast cultures. In this study, we further investigated the molecular mechanisms of AID effects on proliferation and cell death in human osteoblasts (hOBs). We examined the effects of dexamethasone (10 -7 and 10 -6 M), non-selective non-steroidal anti-inflammatory drugs (NSAIDs): indomethacin, ketorolac, piroxicam and diclofenac (10 -5 and 10 -4 M), and COX-2 inhibitor: celecoxib (10 -6 and 10 -5 M) on proliferation, cytotoxicity, cell death, and mRNA and protein levels of cell cycle and apoptosis-related regulators in hOBs. All the tested AIDs significantly inhibited proliferation and arrested cell cycle at G0/G1 phase in hOBs. Celecoxib and dexamethasone, but not non-selective NSAIDs, were found to have cytotoxic effects on hOB, and further demonstrated to induce apoptosis and necrosis (at higher concentration) in hOBs. We further found that indomethacin, celecoxib and dexamethasone increased the mRNA and protein expressions of p27 kip1 and decreased those of cyclin D2 and p-cdk2 in hOBs. Bak expression was increased by celecoxib and dexamethasone, while Bcl-XL level was declined only by dexamethasone. Furthermore, the replenishment of PGE1, PGE2 or PGF2α did not reverse the effects of AIDs on proliferation and expressions of p27 kip1 and cyclin D2 in hOBs. We conclude that the changes in expressions of regulators of cell cycle (p27 kip1 and cyclin D2) and/or apoptosis (Bak and Bcl-XL) by AIDs may contribute to AIDs caused proliferation suppression and apoptosis in hOBs. This effect might not relate to the blockage of prostaglandin synthesis by AIDs

  5. Detection of apoptotic cells using propidium iodide staining

    NARCIS (Netherlands)

    Newbold, Andrea; Martin, Ben P.; Cullinane, Carleen; Bots, Michael

    2014-01-01

    Flow cytometry assays are often used to detect apoptotic cells in in vitro cultures. Depending on the experimental model, these assays can also be useful in evaluating apoptosis in vivo. In this protocol, we describe a propidium iodide (PI) flow cytometry assay to evaluate B-cell lymphomas that have

  6. Macrophages discriminate glycosylation patterns of apoptotic cell-derived microparticles.

    Science.gov (United States)

    Bilyy, Rostyslav O; Shkandina, Tanya; Tomin, Andriy; Muñoz, Luis E; Franz, Sandra; Antonyuk, Volodymyr; Kit, Yuriy Ya; Zirngibl, Matthias; Fürnrohr, Barbara G; Janko, Christina; Lauber, Kirsten; Schiller, Martin; Schett, Georg; Stoika, Rostyslav S; Herrmann, Martin

    2012-01-02

    Inappropriate clearance of apoptotic remnants is considered to be the primary cause of systemic autoimmune diseases, like systemic lupus erythematosus. Here we demonstrate that apoptotic cells release distinct types of subcellular membranous particles (scMP) derived from the endoplasmic reticulum (ER) or the plasma membrane. Both types of scMP exhibit desialylated glycotopes resulting from surface exposure of immature ER-derived glycoproteins or from surface-borne sialidase activity, respectively. Sialidase activity is activated by caspase-dependent mechanisms during apoptosis. Cleavage of sialidase Neu1 by caspase 3 was shown to be directly involved in apoptosis-related increase of surface sialidase activity. ER-derived blebs possess immature mannosidic glycoepitopes and are prioritized by macrophages during clearance. Plasma membrane-derived blebs contain nuclear chromatin (DNA and histones) but not components of the nuclear envelope. Existence of two immunologically distinct types of apoptotic blebs may provide new insights into clearance-related diseases.

  7. Macrophage Clearance of Apoptotic Cells: A Critical Assessment

    Directory of Open Access Journals (Sweden)

    Siamon Gordon

    2018-01-01

    Full Text Available As the body continues to grow and age, it becomes essential to maintain a balance between living and dying cells. Macrophages and dendritic cells play a central role in discriminating among viable, apoptotic, and necrotic cells, as selective and efficient phagocytes, without inducing inappropriate inflammation or immune responses. A great deal has been learnt concerning clearance receptors for modified and non-self-ligands on potential targets, mediating their eventual uptake, disposal, and replacement. In this essay, we assess current understanding of the phagocytic recognition of apoptotic cells within their tissue environment; we conclude that efferocytosis constitutes a more complex process than simply removal of corpses, with regulatory interactions between the target and effector cells, which determine the outcome of this homeostatic process.

  8. Protein C Inhibitor (PCI) Binds to Phosphatidylserine Exposing Cells with Implications in the Phagocytosis of Apoptotic Cells and Activated Platelets

    Science.gov (United States)

    Rieger, Daniela; Assinger, Alice; Einfinger, Katrin; Sokolikova, Barbora; Geiger, Margarethe

    2014-01-01

    Protein C Inhibitor (PCI) is a secreted serine protease inhibitor, belonging to the family of serpins. In addition to activated protein C PCI inactivates several other proteases of the coagulation and fibrinolytic systems, suggesting a regulatory role in hemostasis. Glycosaminoglycans and certain negatively charged phospholipids, like phosphatidylserine, bind to PCI and modulate its activity. Phosphatidylerine (PS) is exposed on the surface of apoptotic cells and known as a phagocytosis marker. We hypothesized that PCI might bind to PS exposed on apoptotic cells and thereby influence their removal by phagocytosis. Using Jurkat T-lymphocytes and U937 myeloid cells, we show here that PCI binds to apoptotic cells to a similar extent at the same sites as Annexin V, but in a different manner as compared to live cells (defined spots on ∼10–30% of cells). PCI dose dependently decreased phagocytosis of apoptotic Jurkat cells by U937 macrophages. Moreover, the phagocytosis of PS exposing, activated platelets by human blood derived monocytes declined in the presence of PCI. In U937 cells the expression of PCI as well as the surface binding of PCI increased with time of phorbol ester treatment/macrophage differentiation. The results of this study suggest a role of PCI not only for the function and/or maturation of macrophages, but also as a negative regulator of apoptotic cell and activated platelets removal. PMID:25000564

  9. Caloric restriction suppresses apoptotic cell death in the mammalian cochlea and leads to prevention of presbycusis.

    Science.gov (United States)

    Someya, Shinichi; Yamasoba, Tatsuya; Weindruch, Richard; Prolla, Tomas A; Tanokura, Masaru

    2007-10-01

    Presbycusis is characterized by an age-related progressive decline of auditory function, and arises mainly from the degeneration of hair cells or spiral ganglion (SG) cells in the cochlea. Here we show that caloric restriction suppresses apoptotic cell death in the mouse cochlea and prevents late onset of presbycusis. Calorie restricted (CR) mice, which maintained body weight at the same level as that of young control (YC) mice, retained normal hearing and showed no cochlear degeneration. CR mice also showed a significant reduction in the number of TUNEL-positive cells and cleaved caspase-3-positive cells relative to middle-age control (MC) mice. Microarray analysis revealed that CR down-regulated the expression of 24 apoptotic genes, including Bak and Bim. Taken together, our findings suggest that loss of critical cells through apoptosis is an important mechanism of presbycusis in mammals, and that CR can retard this process by suppressing apoptosis in the inner ear tissue.

  10. A Novel Non-Apoptotic Role of Procaspase-3 in the Regulation of Mitochondrial Biogenesis Activators.

    Science.gov (United States)

    Kim, Ji-Soo; Ha, Ji-Young; Yang, Sol-Ji; Son, Jin H

    2018-01-01

    The executioner caspase-3 has been proposed as a pharmacological intervention target to preserve degenerating dopaminergic (DA) neurons because apoptotic mechanisms involving caspase-3 contribute, at least in part, to the loss of DA neurons in patients and experimental models of Parkinson's disease (PD). Here, we determined that genetic intervention of caspase-3 was sufficient to prevent cell death against oxidative stress (OS), accompanied by unexpected severe mitochondrial dysfunction. Specifically, as we expected, caspase-3-deficient DA neuronal cells were very significantly resistant to OS-induced cell death, while the activation of the initiator caspase-9 by OS was preserved. Moreover, detailed phenotypic characterization of caspase-3-deficient DA cells revealed severe mitochondrial dysfunction, including an accumulation of damaged mitochondria with a characteristic swollen structure and broken cristae, reduced membrane potential, increased levels of reactive oxygen species (ROS), and deficits in mitochondrial oxidative phosphorylation (OXPHOS) enzymes. Of great interest, we found that mitochondrial biogenesis was dramatically decreased in caspase-3-deficient DA cells, whereas their capability of mitophagy was normal. In accordance with this observation, caspase-3 gene knock down (KD) resulted in dramatically decreased expression of the key transcriptional activators of mitochondrial biogenesis, such as Tfam and Nrf-1, implicating a non-apoptotic role of procaspase-3 in mitochondrial biogenesis. Therefore, a prolonged anti-apoptotic intervention targeting caspase-3 should be considered with caution due to the potential adverse effects in mitochondria dynamics resulting from a novel potential functional role of procaspase-3 in mitochondrial biogenesis via regulating the expression of mitochondrial biogenesis activators. J. Cell. Biochem. 119: 347-357, 2018. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  11. Ion transport in a human lens epithelial cell line exposed to hyposmotic and apoptotic stress.

    Science.gov (United States)

    Chimote, Ameet A; Adragna, Norma C; Lauf, Peter K

    2010-04-01

    Membrane transport changes in human lens epithelial (HLE-B3) cells under hyposmotic and apoptotic stress were compared. Cell potassium content, K(i), uptake of the K congener rubidium, Rb(i), and water content were measured after hyposmotic stress induced by hypotonicity, and apoptotic stress by the protein-kinase inhibitor staurosporine (STP). Cell water increased in hyposmotic (150 mOsm) as compared to isosmotic (300 mOsm) balanced salt solution (BSS) by >2-fold at 5 min and decreased within 15 min to baseline values accompanied by a 40% K(i) loss commensurate with cell swelling and subsequent cell shrinkage likely due to regulatory volume decrease (RVD). Loss of K(i), and accompanying water, and Rb(i) uptake in hyposmotic BSS were prevented by clotrimazole (CTZ) suggesting water shifts associated with K and Rb flux via intermediate conductance K (IK) channels, also detected at the mRNA and protein level. In contrast, 2 h after 2 microM STP exposure, the cells lost approximately 40% water and approximately 60% K(i), respectively, consistent with apoptotic volume decrease (AVD). Indeed, water and K(i) loss was at least fivefold greater after hyposmotic than after apoptotic stress. High extracellular K and 2 mM 4-aminopyridine (4-AP) but not CTZ significantly reduced apoptosis. Annexin labeling phosphatidylserine (PS) at 15 min suggested loss of lipid asymmetry. Quantitative PCR revealed significant IK channel expression during prolonged hyposmotic stress. Results suggest in HLE-B3 cells, IK channels likely partook in and were down regulated after RVD, whereas pro-apoptotic STP-activation of 4-AP-sensitive voltage-gated K channels preceded or accompanied PS externalization before subsequent apoptosis. J. Cell. Physiol. 223: 110-122, 2010. (c) 2009 Wiley-Liss, Inc.

  12. Apoptotic potential and cell sensitivity to fractionated radiotherapy

    International Nuclear Information System (INIS)

    Rupnow, Brent A.; Murtha, Albert D.; Alarcon, Rodolfo M.; Giaccia, Amato J.; Knox, Susan J.

    1997-01-01

    Purpose/Objective: At present, the relationship between sensitivity to radiation-induced apoptosis and overall cellular radiosensitivity remains unclear. In particular, the relationship of apoptotic sensitivity to the survival of cells following fractionated irradiation has not been well studied. The purpose of the present study was to determine if increasing cell sensitivity to radiation-induced apoptosis would result in decreased clonogenic survival following single dose and fractionated irradiation in vitro. Materials and Methods: To address this, we chose a cell line (Rat-1MycER) in which the sensitivity to radiation-induced apoptosis could be altered by switching on or off the activity of a conditional c-Myc allele (c-MycER). The c-MycER construct expresses a full length c-Myc protein fused to a modified hormone binding domain of the estrogen receptor. Only in the presence of the estrogen analog 4-hydroxytamoxifen (4HT), does the conditional c-MycER become active. Apoptosis following irradiation in these cells (with and without c-MycER activation) was analyzed by flow cytometry to determine the percentage of cells undergoing apoptosis following various radiation doses and at different times after irradiation. Additionally, clonogenic survival analysis was performed following single radiation doses from 0 to 10 Gy and following five fractions of 2 or 4 Gy each. Survival of cells with and without c-MycER activation was compared. Furthermore, the effect of overexpressing the anti-apoptotic Bcl-2 gene on apoptosis induction and clonogenic survival of these cells was examined. Results: Rat-1MycER cells were strongly sensitized to radiation-induced apoptosis in a dose and time dependent manner when MycER was activated relative to cells treated without c-MycER activation. This c-Myc-mediated sensitivity to radiation-induced apoptosis was suppressed by overexpression of the anti-apoptotic protein Bcl-2. In addition to increasing apoptosis, activating c-MycER prior to

  13. Targeting multiple pro-apoptotic signaling pathways with curcumin in prostate cancer cells

    Science.gov (United States)

    Rivera, Mariela; Ramos, Yanilda; Rodríguez-Valentín, Madeline; López-Acevedo, Sheila; Cubano, Luis A.; Zou, Jin; Zhang, Qiang; Wang, Guangdi

    2017-01-01

    Curcumin, an extract from the turmeric rhizome (Curcuma longa), is known to exhibit anti-inflammatory, antioxidant, chemopreventive and antitumoral activities against aggressive and recurrent cancers. Accumulative data indicate that curcumin may induce cancer cell death. However, the detailed mechanism underlying its pro-apoptotic and anti-cancer effects remains to be elucidated. In the present study, we examined the signaling pathways triggered by curcumin, specifically, the exact molecular mechanisms of curcumin-induced apoptosis in highly metastatic human prostate cancer cells. The effect of curcumin was evaluated using for the first time in prostate cancer, a gel-free shotgun quantitative proteomic analysis coupled with Tandem Mass Tag isobaric labeling-based-signaling networks. Results were confirmed at the gene expression level by qRT-PCR and at the protein expression level by western blot and flow cytometry. Our findings revealed that curcumin induced an Endoplasmic Reticulum stress-mediated apoptosis in PC3. The mechanisms by which curcumin promoted cell death in these cells were associated with cell cycle arrest, increased reactive oxygen species, autophagy and the Unfolded Protein Response. Furthermore, the upregulation of ER stress was measured using key indicators of ER stress: Glucose-Regulated Protein 78, Inositol-Requiring Enzyme 1 alpha, Protein Disulfide isomerase and Calreticulin. Chronic ER stress induction was concomitant with the upregulation of pro-apoptotic markers (caspases 3,9,12) and Poly (ADP-ribose) polymerase. The downregulated proteins include anti-apoptotic and anti-tumor markers, supporting their curcumin-induced pro-apoptotic role in prostate cancer cells. Taken together, these data suggest that curcumin may serve as a promising anticancer agent by inducing a chronic ER stress mediated cell death and activation of cell cycle arrest, UPR, autophagy and oxidative stress responses. PMID:28628644

  14. Targeting multiple pro-apoptotic signaling pathways with curcumin in prostate cancer cells.

    Directory of Open Access Journals (Sweden)

    Mariela Rivera

    Full Text Available Curcumin, an extract from the turmeric rhizome (Curcuma longa, is known to exhibit anti-inflammatory, antioxidant, chemopreventive and antitumoral activities against aggressive and recurrent cancers. Accumulative data indicate that curcumin may induce cancer cell death. However, the detailed mechanism underlying its pro-apoptotic and anti-cancer effects remains to be elucidated. In the present study, we examined the signaling pathways triggered by curcumin, specifically, the exact molecular mechanisms of curcumin-induced apoptosis in highly metastatic human prostate cancer cells. The effect of curcumin was evaluated using for the first time in prostate cancer, a gel-free shotgun quantitative proteomic analysis coupled with Tandem Mass Tag isobaric labeling-based-signaling networks. Results were confirmed at the gene expression level by qRT-PCR and at the protein expression level by western blot and flow cytometry. Our findings revealed that curcumin induced an Endoplasmic Reticulum stress-mediated apoptosis in PC3. The mechanisms by which curcumin promoted cell death in these cells were associated with cell cycle arrest, increased reactive oxygen species, autophagy and the Unfolded Protein Response. Furthermore, the upregulation of ER stress was measured using key indicators of ER stress: Glucose-Regulated Protein 78, Inositol-Requiring Enzyme 1 alpha, Protein Disulfide isomerase and Calreticulin. Chronic ER stress induction was concomitant with the upregulation of pro-apoptotic markers (caspases 3,9,12 and Poly (ADP-ribose polymerase. The downregulated proteins include anti-apoptotic and anti-tumor markers, supporting their curcumin-induced pro-apoptotic role in prostate cancer cells. Taken together, these data suggest that curcumin may serve as a promising anticancer agent by inducing a chronic ER stress mediated cell death and activation of cell cycle arrest, UPR, autophagy and oxidative stress responses.

  15. Proposed Pharmacological Countermeasures Against Apoptotic Cell Death in Experimental Models Mimicking Space Environment Damage

    Science.gov (United States)

    Lulli, Matteo; Papucci, Laura; Witort, Ewa; Donnini, Martino; Lapucci, Andrea; Lazzarano, Stefano; Mazzoni, Tiziano; Simoncini, Madine; Falciani, Piergiuseppe; Capaccioli, Sergio

    2008-06-01

    Several damaging agents have been suggested to affect human vision during long term space travels. Recently, apoptosis induced by DNA-damaging agents has emerged as frequent pathogenetic mechanism of ophthalmologic pathologies. Here, we propose two countermeasures: coenzyme Q10 and bcl-2 downregulation preventing antisense oligoribonucleotides (ORNs), aimed to inhibit cellular apoptotic death. Our studies have been carried out on retina and neuronal cultured cells treated with the following apoptotic stimuli mimicking space environment: a several-day exposure to either 3H-labeled tymidine or to the genotoxic drug doxorubicin, UV irradiation, hypoxia and glucose/growth factor starvation (Locke medium). The preliminary results clearly indicate that CoQ10, as well as bcl-2 down-regulation preventing ORNs, significantly counteract apoptosis in response to different DNA damaging agents in cultured eye and in neuronal cells. This supports the possibility that both could be optimal countermeasures against ophthalmologic lesions during space explorations.

  16. Apoptotic effects of bovine apo-lactoferrin on HeLa tumor cells.

    Science.gov (United States)

    Luzi, Carla; Brisdelli, Fabrizia; Iorio, Roberto; Bozzi, Argante; Carnicelli, Veronica; Di Giulio, Antonio; Lizzi, Anna Rita

    2017-01-01

    Lactoferrin (Lf), a cationic iron-binding glycoprotein of 80 kDa present in body secretions, is known as a compound with marked antimicrobial activity. In the present study, the apoptotic effect of iron-free bovine lactoferrin (apo-bLf) on human epithelial cancer (HeLa) cells was examined in association with reactive oxygen species and glutathione (GSH) levels. Apoptotic effect of iron-free bovine lactoferrin inhibited the growth of HeLa cells after 48 hours of treatment while the diferric-bLf was ineffective in the concentration range tested (from 1 to 12.5 μM). Western blot analysis showed that key apoptotic regulators including Bax, Bcl-2, Sirt1, Mcl-1, and PARP-1 were modulated by 1.25 μM of apo-bLf. In the same cell line, apo-bLf induced apoptosis together with poly (ADP-ribose) polymerase cleavage, caspase activation, and a significant drop of NAD + . In addition, apo-bLf-treated HeLa cells showed a marked increase of reactive oxygen species level and a significant GSH depletion. On the whole, apo-bLf triggered apoptosis of HeLa cells upon oxygen radicals burst and GSH decrease. Copyright © 2017 John Wiley & Sons, Ltd.

  17. 5-Lipoxygenase contributes to PPARγ activation in macrophages in response to apoptotic cells.

    Science.gov (United States)

    von Knethen, Andreas; Sha, Lisa K; Kuchler, Laura; Heeg, Annika K; Fuhrmann, Dominik; Heide, Heinrich; Wittig, Ilka; Maier, Thorsten J; Steinhilber, Dieter; Brüne, Bernhard

    2013-12-01

    Macrophage polarization to an anti-inflammatory phenotype upon contact with apoptotic cells is a contributing hallmark to immune suppression during the late phase of sepsis. Although the peroxisome proliferator-activated receptor γ (PPARγ) supports this macrophage phenotype switch, it remains elusive how apoptotic cells activate PPARγ. Assuming that a molecule causing PPARγ activation in macrophages originates in the cell membrane of apoptotic cells we analyzed lipid rafts from apoptotic, necrotic, and living human Jurkat T cells which showed the presence of 5-lipoxygenase (5-LO) in lipid rafts of apoptotic cells only. Incubating macrophages with lipid rafts of apoptotic, but not necrotic or living cells, induced PPAR responsive element (PPRE)-driven mRuby reporter gene expression in RAW 264.7 macrophages stably transduced with a 4xPPRE containing vector. Experiments with lipid rafts of apoptotic murine EL4 T cells revealed similar results. To verify the involvement of 5-LO in activating PPARγ in macrophages, Jurkat T cells were incubated with the 5-LO inhibitor MK-866 prior to induction of apoptosis, which failed to induce mRuby expression. Similar results were obtained with lipid rafts of apoptotic EL4 T cells preexposed to the 5-LO inhibitors zileuton and CJ-13610. Interestingly, Jurkat T cells overexpressing 5-LO failed to activate PPARγ in macrophages, while their 5-LO overexpressing apoptotic counterparts did. Our results suggest that during apoptosis 5-LO gets associated with lipid rafts and synthesizes ligands that in turn stimulate PPARγ in macrophages. © 2013.

  18. Apoptotic induction of skin cancer cell death by plant extracts.

    Science.gov (United States)

    Thuncharoen, Walairat; Chulasiri, Malin; Nilwarangkoon, Sirinun; Nakamura, Yukio; Watanapokasin, Ramida

    2013-01-01

    The aim of the present study was to investigate the effects of plant extracts on cancer apoptotic induction. Human epidermoid carcinoma A431 cell line, obtained from the American Type Culture Collection (ATCC, Manassas, VA), was maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 degrees C, 5% carbon dioxide (CO2). Plant extract solutions were obtained from S & J international enterprises public company limited. These plant extracts include 50% hydroglycol extracts from Etlingera elatior (Jack) R.M.Smith (torch ginger; EE), Rosa damascene (damask rose; DR) and Rafflesia kerrii Meijer (bua phut; RM). The cell viability, time and dose dependency were determined by MTT (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay. A431 cells were treated with the plant extracts and stained with Hoechst 33342 fluorescent staining dye. Cell viability was demonstrated by the inhibitory concentration 50% (IC50). The anti-proliferative effects were shown to be dependent on time and dose. Typical characteristics of apoptosis which are cell morphological changes and chromatin condensation were clearly observed. The plant extracts was shown to be effective for anti-proliferation and induction of apoptosis cell death in skin cancer cells. Therefore, mechanisms underlying the cell death and its potential use for treatment of skin cancer will be further studied.

  19. Scrambled Eggs: Apoptotic Cell Clearance by Non-Professional Phagocytes in the Drosophila Ovary

    Directory of Open Access Journals (Sweden)

    Sandy B. Serizier

    2017-11-01

    Full Text Available For half of a century, it has been known that non-professional phagocytes, such as fibroblasts, endothelial, and epithelial cells, are capable of efferocytosis (engulfment of apoptotic cells. Non-professional phagocytes differ from professional phagocytes in the range and efficiency of engulfment. Much of the recognition and underlying signaling machinery between non-professional and professional phagocytes is the same, but it is not known how the engulfment capacity of non-professional phagocytes is controlled. Moreover, the signaling networks involved in cell corpse recognition, engulfment, and phagosome maturation are only partially understood. The Drosophila ovary provides an excellent system to investigate the regulation of phagocytic activity by epithelial cells, a major class of non-professional phagocytes. During Drosophila oogenesis, mid-stage egg chambers undergo apoptosis of the germline in response to nutrient deprivation. Epithelial follicle cells then undergo major cell shape changes and concomitantly engulf the germline material. Our previous work has established that Draper and the integrin α-PS3/β-PS heterodimer are required in follicle cells for germline cell clearance. In addition, we have characterized phagosome maturation pathways, and found that the JNK pathway amplifies the engulfment response. In this review, we discuss recent advances on the interplay between engulfment pathways in the follicular epithelium for cell clearance in the Drosophila ovary. We also provide a comparison to apoptotic cell clearance mechanisms in C. elegans and mammals, illustrating strong conservation of efferocytosis mechanisms by non-professional phagocytes.

  20. Scrambled Eggs: Apoptotic Cell Clearance by Non-Professional Phagocytes in the Drosophila Ovary.

    Science.gov (United States)

    Serizier, Sandy B; McCall, Kimberly

    2017-01-01

    For half of a century, it has been known that non-professional phagocytes, such as fibroblasts, endothelial, and epithelial cells, are capable of efferocytosis (engulfment of apoptotic cells). Non-professional phagocytes differ from professional phagocytes in the range and efficiency of engulfment. Much of the recognition and underlying signaling machinery between non-professional and professional phagocytes is the same, but it is not known how the engulfment capacity of non-professional phagocytes is controlled. Moreover, the signaling networks involved in cell corpse recognition, engulfment, and phagosome maturation are only partially understood. The Drosophila ovary provides an excellent system to investigate the regulation of phagocytic activity by epithelial cells, a major class of non-professional phagocytes. During Drosophila oogenesis, mid-stage egg chambers undergo apoptosis of the germline in response to nutrient deprivation. Epithelial follicle cells then undergo major cell shape changes and concomitantly engulf the germline material. Our previous work has established that Draper and the integrin α-PS3/β-PS heterodimer are required in follicle cells for germline cell clearance. In addition, we have characterized phagosome maturation pathways, and found that the JNK pathway amplifies the engulfment response. In this review, we discuss recent advances on the interplay between engulfment pathways in the follicular epithelium for cell clearance in the Drosophila ovary. We also provide a comparison to apoptotic cell clearance mechanisms in C. elegans and mammals, illustrating strong conservation of efferocytosis mechanisms by non-professional phagocytes.

  1. SATB2 participates in regulation of menadione-induced apoptotic insults to osteoblasts.

    Science.gov (United States)

    Wei, Jyh-Ding; Lin, Yi-Ling; Tsai, Cheng-Hsiu; Shieh, Hui-Shan; Lin, Pei-I; Ho, Wei-Pin; Chen, Ruei-Ming

    2012-07-01

    Special AT-rich sequence binding protein 2 (SATB2), a nuclear matrix attachment region-binding protein, can regulate embryonic development, cell differentiation, and cell survival. Previous studies showed that SATB2 is involved in osteoblast differentiation and skeletal development. In this study, we evaluated the role of SATB2 in oxidative stress-induced apoptotic insults to human osteoblast-like MG63 cells and mouse MC3T3-E1 cells. Exposure of MG63 cells to menadione increased intracellular reactive oxygen species levels in a concentration- and time-dependent manner. Simultaneously, menadione-induced oxidative stress triggered cell shrinkage and decreased cell viability. In addition, treatment of MG63 cells with menadione time-dependently decreased the mitochondrial membrane potential but enhanced caspase-3 activity. As a result, menadione-induced DNA fragmentation and cell apoptosis. As to the mechanism, exposure of MG63 cells to menadione amplified SATB2 messenger (m)RNA and protein expression in a time-dependent manner. Knockdown of translation of SATB2 mRNA using RNA interference led to chromatin disruption and nuclear damage. When MG63 cells and MC3T3-E1 cells were treated with SATB2 small interfering RNA, menadione-induced cell apoptosis was increased. We conclude that menadione causes oxidative stress in human osteoblasts and induces cellular apoptosis via a mitochondrion-caspase protease pathway. In addition, SATB2 may play a crucial role in protecting against oxidative stress-induced osteoblast apoptosis. Copyright © 2012 Orthopaedic Research Society.

  2. Non-apoptotic cell death associated with perturbations of macropinocytosis.

    Science.gov (United States)

    Maltese, William A; Overmeyer, Jean H

    2015-01-01

    Although macropinocytosis is widely recognized as a distinct form of fluid-phase endocytosis in antigen-presenting dendritic cells, it also occurs constitutively in many other normal and transformed cell types. Recent studies have established that various genetic or pharmacological manipulations can hyperstimulate macropinocytosis or disrupt normal macropinosome trafficking pathways, leading to accumulation of greatly enlarged cytoplasmic vacuoles. In some cases, this extreme vacuolization is associated with a unique form of non-apoptotic cell death termed "methuosis," from the Greek methuo (to drink to intoxication). It remains unclear whether cell death related to dysfunctional macropinocytosis occurs in normal physiological contexts. However, the finding that some types of cancer cells are particularly vulnerable to this unusual form of cell death has raised the possibility that small molecules capable of altering macropinosome trafficking or function might be useful as therapeutic agents against cancers that are resistant to drugs that work by inducing apoptosis. Herein we review examples of cell death associated with dysfunctional macropinocytosis and summarize what is known about the underlying mechanisms.

  3. Non-apoptotic cell death associated with perturbations of macropinocytosis

    Directory of Open Access Journals (Sweden)

    William A. Maltese

    2015-02-01

    Full Text Available Although macropinocytosis is widely recognized as a distinct form of fluid-phase endocytosis in antigen-presenting dendritic cells, it also occurs constitutively in many other normal and transformed cell types. Recent studies have established that various genetic or pharmacological manipulations can hyperstimulate macropinocytosis or disrupt normal macropinosome trafficking pathways, leading to accumulation of greatly enlarged cytoplasmic vacuoles. In some cases, this extreme vacuolization is associated with a unique form of non-apoptotic cell death termed ‘methuosis’, from the Greek methuo (to drink to intoxication. It remains unclear whether cell death related to dysfunctional macropinocytosis occurs in normal physiological contexts. However, the finding that some types of cancer cells are particularly vulnerable to this unusual form of cell death has raised the possibility that small molecules capable of altering macropinosome trafficking or function might be useful as therapeutic agents against cancers that are resistant to drugs that work by inducing apoptosis. Herein we review examples of cell death associated with dysfunctional macropinocytosis and summarize what is known about the underlying mechanisms.

  4. Free cholesterol accumulation impairs antioxidant activities and aggravates apoptotic cell death in menadione-induced oxidative injury.

    Science.gov (United States)

    Lee, Waisin; Xu, Mingjing; Li, Yue; Gu, Yong; Chen, Jianping; Wong, Derek; Fung, Peter C W; Shen, Jiangang

    2011-10-01

    Although the relationship between hypercholesterolemia and oxidative stress has been extensively investigated, direct evidence regarding to the roles of cholesterol accumulation in the generations of reactive oxygen species (ROS) and apoptotic cell death under oxidative stress is lack. In this study, we investigated productions of superoxide anions (O(2)(-)) and nitric oxide (NO), and apoptotic cell death in wild type Chinese hamster ovary (CHO) cells and cholesterol accumulated CHO cells genetically and chemically. Oxidative stress was induced by menadione challenge. The results revealed that abundance of free cholesterol (FC) promoted menadione-induced O(2)(-) and NO productions. FC accumulation down-regulated eNOS expression but up-regulated NADPH oxidases, and inhibited the activities of superoxide dismutase (SOD) and catalase. Treatment of menadione increased the expressions of iNOS and qp91 phox, enhanced the activities of SOD and catalase in the wild-type CHO cells but inhibited the activity of glutathione peroxidase in the cholesterol accumulated CHO cells. Moreover, FC abundance promoted apoptotic cell death in these cells. Taken together, those results suggest that free cholesterol accumulation aggravates menadione-induced oxidative stress and exacerbates apoptotic cell death. Copyright © 2011 Elsevier Inc. All rights reserved.

  5. Anti-apoptotic effect of hyperglycemia can allow survival of potentially autoreactive T cells.

    Science.gov (United States)

    Ramakrishnan, P; Kahn, D A; Baltimore, D

    2011-04-01

    Thymocyte development is a tightly controlled multi-step process involving selective elimination of self-reactive and non-functional T cells by apoptosis. This developmental process depends on signaling by Notch, IL-7 and active glucose metabolism. In this study, we explored the requirement of glucose for thymocyte survival and found that in addition to metabolic regulation, glucose leads to the expression of anti-apoptotic genes. Under hyperglycemic conditions, both mouse and human thymocytes demonstrate enhanced survival. We show that glucose-induced anti-apoptotic genes are dependent on NF-κB p65 because high glucose is unable to attenuate normal ongoing apoptosis of thymocytes isolated from p65 knockout mice. Furthermore, we demonstrate that in vivo hyperglycemia decreases apoptosis of thymocytes allowing for survival of potentially self-reactive thymocytes. These results imply that hyperglycemic conditions could contribute to the development of autoimmunity through dysregulated thymic selection. © 2011 Macmillan Publishers Limited

  6. Andrographolide induces apoptotic and non-apoptotic death and enhances tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis in gastric cancer cells.

    Science.gov (United States)

    Lim, Sung-Chul; Jeon, Ho Jong; Kee, Keun Hong; Lee, Mi Ja; Hong, Ran; Han, Song Iy

    2017-05-01

    Andrographolide, a natural compound isolated from Andrographis paniculata , has been reported to possess antitumor activity. In the present study, the effect of andrographolide in human gastric cancer (GC) cells was investigated. Andrographolide induced cell death with apoptotic and non-apoptotic features. At a low concentration, andrographolide potentiated apoptosis and reduction of clonogenicity triggered by recombinant human tumor necrosis factor-related apoptosis-inducing ligand (rhTRAIL). Exposure of GC cells to andrographolide altered the expression level of several growth-inhibiting and apoptosis-regulating proteins, including death receptors. It was demonstrated that activity of the TRAIL-R2 (DR5) pathway was critical in the development of andrographolide-mediated rhTRAIL sensitization, since its inhibition significantly reduced the extent of apoptosis induced by the combination of rhTRAIL and andrographolide. In addition, andrographolide increased reactive oxygen species (ROS) generation in a dose-dependent manner. N-acetyl cysteine prevented andrographolide-mediated DR5 induction and the apoptotic effect induced by the combination of rhTRAIL and andrographolide. Collectively, the present study demonstrated that andrographolide enhances TRAIL-induced apoptosis through induction of DR5 expression. This effect appears to involve ROS generation in GCs.

  7. Expression of caspase-3 gene in apoptotic HL-60 cell and different human tumor cell lines

    International Nuclear Information System (INIS)

    Li Xiaoming; Song Tianbao

    1999-01-01

    Objective: To research the expression of caspase-3 gene in the apoptotic and the control HL-60 cells and in the different human tumor cell lines. Methods: Caspase-3 mRNA in the control and γ-radiation-induced apoptotic HL-60 cells, and in the 6 types of human tumor cell lines, was analysed by Northern blot. Results: The caspase-3 gene transcript was more highly expressed in leukemia cells HL-60, CEM, K562 and neuroblastoma SH-SY5Y than in cervical adenocarcinoma HeLa and breast carcinoma MCF7, and more highly in the radiation-induced apoptotic HL-60 than in the control HL-60 cells. Conclusion: The high level of expression of caspase-3 may aid the efforts to understand the tumor cell sensitivity to radiation, apoptosis and its inherent ability to survive

  8. Induction of discrete apoptotic pathways by bromo-substituted indirubin derivatives in invasive breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Nicolaou, Katerina A. [Department of Biological Sciences, University of Cyprus, Nicosia (Cyprus); Liapis, Vasilis; Evdokiou, Andreas [Department of Surgery, Basil Hetzel Institute, Adelaide University, Adelaide (Australia); Constantinou, Constantina [St. George' s University of London Medical School at the University of Nicosia, Nicosia (Cyprus); Magiatis, Prokopios; Skaltsounis, Alex L. [Faculty of Pharmacy, University of Athens, Athens (Greece); Koumas, Laura; Costeas, Paul A. [Center for Study of Hematological Malignancies, Nicosia (Cyprus); Constantinou, Andreas I., E-mail: andreasc@ucy.ac.cy [Department of Biological Sciences, University of Cyprus, Nicosia (Cyprus)

    2012-08-17

    Highlights: Black-Right-Pointing-Pointer The effects of 6BIO and 7BIO are evaluated against five breast cancer cell lines. Black-Right-Pointing-Pointer 6BIO induces a caspase dependent apoptotic effect via the intrinsic pathway. Black-Right-Pointing-Pointer 7BIO promotes G{sub 2}/M cells cycle arrest. Black-Right-Pointing-Pointer 7BIO triggers a caspase-8 mediated apoptotic pathway. Black-Right-Pointing-Pointer 7BIO triggers and a caspase independent pathway. -- Abstract: Indirubin derivatives gained interest in recent years for their anticancer and antimetastatic properties. The objective of the present study was to evaluate and compare the anticancer properties of the two novel bromo-substituted derivatives 6-bromoindirubin-3 Prime -oxime (6BIO) and 7-bromoindirubin-3 Prime -oxime (7BIO) in five different breast cancer cell lines. Cell viability assays identified that 6BIO and 7BIO are most effective in preventing the proliferation of the MDA-MB-231-TXSA breast cancer cell line from a total of five breast cancer cell lined examined. In addition it was found that the two compounds induce apoptosis via different mechanisms. 6BIO induces caspase-dependent programmed cell death through the intrinsic (mitochondrial) caspase-9 pathway. 7BIO up-regulates p21 and promotes G{sub 2}/M cell cycle arrest which is subsequently followed by the activation of two different apoptotic pathways: (a) a pathway that involves the upregulation of DR4/DR5 and activation of caspase-8 and (b) a caspase independent pathway. In conclusion, this study provides important insights regarding the molecular pathways leading to cell cycle arrest and apoptosis by two indirubin derivatives that can find clinical applications in targeted cancer therapeutics.

  9. Eclalbasaponin II induces autophagic and apoptotic cell death in human ovarian cancer cells

    Directory of Open Access Journals (Sweden)

    Yoon Jin Cho

    2016-09-01

    Full Text Available Triterpenoids echinocystic acid and its glycosides, isolated from several Eclipta prostrata, have been reported to possess various biological activities such as anti-inflammatory, anti-bacterial, and anti-diabetic activity. However, the cytotoxicity of the triterpenoids in human cancer cells and their molecular mechanism of action are poorly understood. In the present study, we found that eclalbasaponin II with one glucose moiety has potent cytotoxicity in three ovarian cancer cells and two endometrial cancer cells compared to an aglycone echinocystic acid and eclalbasaponin I with two glucose moiety. Eclalbasaponin II treatment dose-dependently increased sub G1 population. Annexin V staining revealed that eclalbasaponin II induced apoptosis in SKOV3 and A2780 ovarian cancer cells. In addition, eclalbasaponin II-induced cell death was associated with characteristics of autophagy; an increase in acidic vesicular organelle content and elevation of the levels of LC3-II. Interestingly, autophagy inhibitor BaF1 suppressed the eclalbasaponin II-induced apoptosis. Moreover, eclalbasaponin II activated JNK and p38 signaling and inhibited the mTOR signaling. We further demonstrated that pre-treatment with a JNK and p38 inhibitor and mTOR activator attenuated the eclalbasaponin II-induced autophagy. This suggests that eclalbasaponin II induces apoptotic and autophagic cell death through the regulation of JNK, p38, and mTOR signaling in human ovarian cancer cells.

  10. Barium inhibits arsenic-mediated apoptotic cell death in human squamous cell carcinoma cells.

    Science.gov (United States)

    Yajima, Ichiro; Uemura, Noriyuki; Nizam, Saika; Khalequzzaman, Md; Thang, Nguyen D; Kumasaka, Mayuko Y; Akhand, Anwarul A; Shekhar, Hossain U; Nakajima, Tamie; Kato, Masashi

    2012-06-01

    Our fieldwork showed more than 1 μM (145.1 μg/L) barium in about 3 μM (210.7 μg/L) arsenic-polluted drinking well water (n = 72) in cancer-prone areas in Bangladesh, while the mean concentrations of nine other elements in the water were less than 3 μg/L. The types of cancer include squamous cell carcinomas (SCC). We hypothesized that barium modulates arsenic-mediated biological effects, and we examined the effect of barium (1 μM) on arsenic (3 μM)-mediated apoptotic cell death of human HSC-5 and A431 SCC cells in vitro. Arsenic promoted SCC apoptosis with increased reactive oxygen species (ROS) production and JNK1/2 and caspase-3 activation (apoptotic pathway). In contrast, arsenic also inhibited SCC apoptosis with increased NF-κB activity and X-linked inhibitor of apoptosis protein (XIAP) expression level and decreased JNK activity (antiapoptotic pathway). These results suggest that arsenic bidirectionally promotes apoptotic and antiapoptotic pathways in SCC cells. Interestingly, barium in the presence of arsenic increased NF-κB activity and XIAP expression and decreased JNK activity without affecting ROS production, resulting in the inhibition of the arsenic-mediated apoptotic pathway. Since the anticancer effect of arsenic is mainly dependent on cancer apoptosis, barium-mediated inhibition of arsenic-induced apoptosis may promote progression of SCC in patients in Bangladesh who keep drinking barium and arsenic-polluted water after the development of cancer. Thus, we newly showed that barium in the presence of arsenic might inhibit arsenic-mediated cancer apoptosis with the modulation of the balance between arsenic-mediated promotive and suppressive apoptotic pathways.

  11. The antiproliferative and apoptotic effects of apigenin on glioblastoma cells.

    Science.gov (United States)

    Stump, Trevor A; Santee, Brittany N; Williams, Lauren P; Kunze, Rachel A; Heinze, Chelsae E; Huseman, Eric D; Gryka, Rebecca J; Simpson, Denise S; Amos, Samson

    2017-07-01

    Glioblastoma (GBM) is highly proliferative, infiltrative, malignant and the most deadly form of brain tumour. The epidermal growth factor receptor (EGFR) is overexpressed, amplified and mutated in GBM and has been shown to play key and important roles in the proliferation, growth and survival of this tumour. The goal of our study was to investigate the antiproliferative, apoptotic and molecular effects of apigenin in GBM. Proliferation and viability tests were carried out using the trypan blue exclusion, MTT and lactate dehydrogenase (LDH) assays. Flow cytometry was used to examine the effects of apigenin on the cell cycle check-points. In addition, we determined the effects of apigenin on EGFR-mediated signalling pathways by Western blot analyses. Our results showed that apigenin reduced cell viability and proliferation in a dose- and time-dependent manner while increasing cytotoxicity in GBM cells. Treatment with apigenin-induced is poly ADP-ribose polymerase (PARP) cleavage and caused cell cycle arrest at the G2M checkpoint. Furthermore, our data revealed that apigenin inhibited EGFR-mediated phosphorylation of mitogen-activated protein kinase (MAPK), AKT and mammalian target of rapamycin (mTOR) signalling pathways and attenuated the expression of Bcl-xL. Our results demonstrated that apigenin has potent inhibitory effects on pathways involved in GBM proliferation and survival and could potentially be used as a therapeutic agent for GBM. © 2017 Royal Pharmaceutical Society.

  12. Regulation of ROS in transmissible gastroenteritis virus-activated apoptotic signaling

    Energy Technology Data Exchange (ETDEWEB)

    Ding, Li [College of Veterinary Medicine, Northwest A and F University, Yangling, Shaanxi 712100 (China); College of Life Sciences, Hainan Normal University, Haikou, Hainan 571158 (China); Zhao, Xiaomin; Huang, Yong; Du, Qian; Dong, Feng; Zhang, Hongling; Song, Xiangjun; Zhang, Wenlong [College of Veterinary Medicine, Northwest A and F University, Yangling, Shaanxi 712100 (China); Tong, Dewen, E-mail: dwtong@nwsuaf.edu.cn [College of Veterinary Medicine, Northwest A and F University, Yangling, Shaanxi 712100 (China)

    2013-12-06

    Highlights: •TGEV infection induced ROS accumulation. •ROS accumulation is involved in TGEV-induced mitochondrial integrity impairment. •ROS is associated with p53 activation and apoptosis occurrence in TGEV-infected cells. -- Abstract: Transmissible gastroenteritis virus (TGEV), an enteropathogenic coronavirus, causes severe lethal watery diarrhea and dehydration in piglets. Previous studies indicate that TGEV infection induces cell apoptosis in host cells. In this study, we investigated the roles and regulation of reactive oxygen species (ROS) in TGEV-activated apoptotic signaling. The results showed that TGEV infection induced ROS accumulation, whereas UV-irradiated TGEV did not promote ROS accumulation. In addition, TGEV infection lowered mitochondrial transmembrane potential in PK-15 cell line, which could be inhibited by ROS scavengers, pyrrolidinedithiocarbamic (PDTC) and N-acetyl-L-cysteine (NAC). Furthermore, the two scavengers significantly inhibited the activation of p38 MAPK and p53 and further blocked apoptosis occurrence through suppressing the TGEV-induced Bcl-2 reduction, Bax redistribution, cytochrome c release and caspase-3 activation. These results suggest that oxidative stress pathway might be a key element in TGEV-induced apoptosis and TGEV pathogenesis.

  13. Apoptotic Cells Induced Signaling for Immune Homeostasis in Macrophages and Dendritic Cells

    Directory of Open Access Journals (Sweden)

    Uriel Trahtemberg

    2017-10-01

    Full Text Available Inefficient and abnormal clearance of apoptotic cells (efferocytosis contributes to systemic autoimmune disease in humans and mice, and inefficient chromosomal DNA degradation by DNAse II leads to systemic polyarthritis and a cytokine storm. By contrast, efficient clearance allows immune homeostasis, generally leads to a non-inflammatory state for both macrophages and dendritic cells (DCs, and contributes to maintenance of peripheral tolerance. As many as 3 × 108 cells undergo apoptosis every hour in our bodies, and one of the primary “eat me” signals expressed by apoptotic cells is phosphatidylserine (PtdSer. Apoptotic cells themselves are major contributors to the “anti-inflammatory” nature of the engulfment process, some by secreting thrombospondin-1 (TSP-1 or adenosine monophosphate and possibly other immune modulating “calm-down” signals that interact with macrophages and DCs. Apoptotic cells also produce “find me” and “tolerate me” signals to attract and immune modulate macrophages and DCs that express specific receptors for some of these signals. Neither macrophages nor DCs are uniform, and each cell type may variably express membrane proteins that function as receptors for PtdSer or for opsonins like complement or opsonins that bind to PtdSer, such as protein S and growth arrest-specific 6. Macrophages and DCs also express scavenger receptors, CD36, and integrins that function via bridging molecules such as TSP-1 or milk fat globule-EGF factor 8 protein and that differentially engage in various multi-ligand interactions between apoptotic cells and phagocytes. In this review, we describe the anti-inflammatory and pro-homeostatic nature of apoptotic cell interaction with the immune system. We do not review some forms of immunogenic cell death. We summarize the known apoptotic cell signaling events in macrophages and DCs that are related to toll-like receptors, nuclear factor kappa B, inflammasome, the lipid

  14. The calcimimetic R-568 induces apoptotic cell death in prostate cancer cells

    Directory of Open Access Journals (Sweden)

    Cheng Guangming

    2009-07-01

    Full Text Available Abstract Background Increased serum level of parathyroid hormone (PTH was found in metastatic prostate cancers. Calcimimetic R-568 was reported to reduce PTH expression, to suppress cell proliferation and to induce apoptosis in parathyroid cells. In this study, we investigated the effect of R-568 on cellular survival of prostate cancer cells. Methods Prostate cancer cell lines LNCaP and PC-3 were used in this study. Cellular survival was determined with MTT, trypan blue exclusion and fluorescent Live/Death assays. Western blot assay was utilized to assess apoptotic events induced by R-568 treatment. JC-1 staining was used to evaluate mitochondrial membrane potential. Results In cultured prostate cancer LNCaP and PC-3 cells, R-568 treatment significantly reduced cellular survival in a dose- and time-dependent manner. R-568-induced cell death was an apoptotic event, as evidenced by caspase-3 processing and PARP cleavage, as well as JC-1 color change in mitochondria. Knocking down calcium sensing receptor (CaSR significantly reduced R-568-induced cytotoxicity. Enforced expression of Bcl-xL gene abolished R-568-induced cell death, while loss of Bcl-xL expression led to increased cell death in R-568-treated LNCaP cells,. Conclusion Taken together, our data demonstrated that calcimimetic R-568 triggers an intrinsic mitochondria-related apoptotic pathway, which is dependent on the CaSR and is modulated by Bcl-xL anti-apoptotic pathway.

  15. Find-me and eat-me signals in apoptotic cell clearance: progress and conundrums

    Science.gov (United States)

    2010-01-01

    Everyday we turnover billions of cells. The quick, efficient, and immunologically silent disposal of the dying cells requires a coordinated orchestration of multiple steps, through which phagocytes selectively recognize and engulf apoptotic cells. Recent studies have suggested an important role for soluble mediators released by apoptotic cells that attract phagocytes (“find-me” signals). New information has also emerged on multiple receptors that can recognize phosphatidylserine, the key “eat-me” signal exposed on the surface of apoptotic cells. This perspective discusses recent exciting progress, gaps in our understanding, and the conflicting issues that arise from the newly acquired knowledge. PMID:20805564

  16. Aged garlic extract and S-allylcysteine prevent apoptotic cell death in a chemical hypoxia model

    Directory of Open Access Journals (Sweden)

    Marisol Orozco-Ibarra

    Full Text Available BACKGROUND: Aged garlic extract (AGE and its main constituent S-allylcysteine (SAC are natural antioxidants with protective effects against cerebral ischemia or cancer, events that involve hypoxia stress. Cobalt chloride (CoCl2 has been used to mimic hypoxic conditions through the stabilization of the α subunit of hypoxia inducible factor (HIF-Ια and up-regulation of HIF-1a-dependent genes as well as activation of hypoxic conditions such as reactive oxygen species (ROS generation, loss of mitochondrial membrane potential and apoptosis. The present study was designed to assess the effect of AGE and SAC on the CoCl2-chemical hypoxia model in PC12 cells RESULTS: We found that CoCl2 induced the stabilization of HIF-1a and its nuclear localization. CoCl2 produced ROS and apoptotic cell death that depended on hypoxia extent. The treatment with AGE and SAC decreased ROS and protected against CoCl2-induced apoptotic cell death which depended on the CoCl2 concentration and incubation time. SAC or AGE decreased the number of cells in the early and late stages of apoptosis. Interestingly, this protective effect was associated with attenuation in HIF-1a stabilization, activity not previously reported for AGE and SAC CONCLUSIONS: Obtained results show that AGE and SAC decreased apoptotic CoCl2-induced cell death. This protection occurs by affecting the activity of HIF-1a and supports the use of these natural compounds as a therapeutic alternative for hypoxic conditions

  17. Gingerol sensitizes TRAIL-induced apoptotic cell death of glioblastoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Dae-Hee, E-mail: leedneo@gmail.com [Departments of Surgery and Pharmacology and Cell Biology, School of Medicine, University of Pittsburgh, Pittsburgh, PA (United States); Kim, Dong-Wook [Department of Microbiology, Immunology, and Cancer Biology, University of VA (United States); Jung, Chang-Hwa [Division of Metabolism and Functionality Research, Korea Food Research Institute (Korea, Republic of); Lee, Yong J. [Departments of Surgery and Pharmacology and Cell Biology, School of Medicine, University of Pittsburgh, Pittsburgh, PA (United States); Park, Daeho, E-mail: daehopark@gist.ac.kr [School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju 500-712 (Korea, Republic of)

    2014-09-15

    Glioblastoma multiforme (GBM) is the most lethal and aggressive astrocytoma of primary brain tumors in adults. Although there are many clinical trials to induce the cell death of glioblastoma cells, most glioblastoma cells have been reported to be resistant to TRAIL-induced apoptosis. Here, we showed that gingerol as a major component of ginger can induce TRAIL-mediated apoptosis of glioblastoma. Gingerol increased death receptor (DR) 5 levels in a p53-dependent manner. Furthermore, gingerol decreased the expression level of anti-apoptotic proteins (survivin, c-FLIP, Bcl-2, and XIAP) and increased pro-apoptotic protein, Bax and truncate Bid, by generating reactive oxygen species (ROS). We also found that the sensitizing effects of gingerol in TRAIL-induced cell death were blocked by scavenging ROS or overexpressing anti-apoptotic protein (Bcl-2). Therefore, we showed the functions of gingerol as a sensitizing agent to induce cell death of TRAIL-resistant glioblastoma cells. This study gives rise to the possibility of applying gingerol as an anti-tumor agent that can be used for the purpose of combination treatment with TRAIL in TRAIL-resistant glioblastoma tumor therapy. - Highlights: • Most GBM cells have been reported to be resistant to TRAIL-induced apoptosis. • Gingerol enhances the expression level of anti-apoptotic proteins by ROS. • Gingerol enhances TRAIL-induced apoptosis through actions on the ROS–Bcl2 pathway.

  18. Gingerol sensitizes TRAIL-induced apoptotic cell death of glioblastoma cells

    International Nuclear Information System (INIS)

    Lee, Dae-Hee; Kim, Dong-Wook; Jung, Chang-Hwa; Lee, Yong J.; Park, Daeho

    2014-01-01

    Glioblastoma multiforme (GBM) is the most lethal and aggressive astrocytoma of primary brain tumors in adults. Although there are many clinical trials to induce the cell death of glioblastoma cells, most glioblastoma cells have been reported to be resistant to TRAIL-induced apoptosis. Here, we showed that gingerol as a major component of ginger can induce TRAIL-mediated apoptosis of glioblastoma. Gingerol increased death receptor (DR) 5 levels in a p53-dependent manner. Furthermore, gingerol decreased the expression level of anti-apoptotic proteins (survivin, c-FLIP, Bcl-2, and XIAP) and increased pro-apoptotic protein, Bax and truncate Bid, by generating reactive oxygen species (ROS). We also found that the sensitizing effects of gingerol in TRAIL-induced cell death were blocked by scavenging ROS or overexpressing anti-apoptotic protein (Bcl-2). Therefore, we showed the functions of gingerol as a sensitizing agent to induce cell death of TRAIL-resistant glioblastoma cells. This study gives rise to the possibility of applying gingerol as an anti-tumor agent that can be used for the purpose of combination treatment with TRAIL in TRAIL-resistant glioblastoma tumor therapy. - Highlights: • Most GBM cells have been reported to be resistant to TRAIL-induced apoptosis. • Gingerol enhances the expression level of anti-apoptotic proteins by ROS. • Gingerol enhances TRAIL-induced apoptosis through actions on the ROS–Bcl2 pathway

  19. 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...

  20. Regulation of apoptotic mediators reveals dynamic responses to thermal stress in the reef building coral Acropora millepora.

    Science.gov (United States)

    Pernice, Mathieu; Dunn, Simon R; Miard, Thomas; Dufour, Sylvie; Dove, Sophie; Hoegh-Guldberg, Ove

    2011-01-24

    Mass coral bleaching is increasing in scale and frequency across the world's coral reefs and is being driven primarily by increased levels of thermal stress arising from global warming. In order to understand the impacts of projected climate change upon corals reefs, it is important to elucidate the underlying cellular mechanisms that operate during coral bleaching and subsequent mortality. In this respect, increased apoptotic cell death activity is an important cellular process that is associated with the breakdown of the mutualistic symbiosis between the cnidarian host and their dinoflagellate symbionts. The PRESENT study reports the impacts of different stressors (colchicine and heat stress) on three phases of apoptosis: (i) the potential initiation by differential expression of Bcl-2 members, (ii) the execution of apoptotic events by activation of caspase 3-like proteases and (iii) and finally, the cell disposal indicated by DNA fragmentation in the reef building coral Acropora millepora. In corals incubated with colchicine, an increase in caspase 3-like activity and DNA fragmentation was associated with a relative down-regulation of Bcl-2, suggesting that the initiation of apoptosis may be mediated by the suppression of an anti-apoptotic mechanism. In contrast, in the early steps of heat stress, the induction of caspase-dependent apoptosis was related to a relative up-regulation of Bcl-2 consecutively followed by a delayed decrease in apoptosis activity. In the light of these results, we propose a model of heat stress in coral hosts whereby increasing temperatures engage activation of caspase 3-dependent apoptosis in cells designated for termination, but also the onset of a delayed protective response involving overexpression of Bcl-2 in surviving cells. This mitigating response to thermal stress could conceivably be an important regulatory mechanism for cell survival in corals exposed to sudden environmental changes.

  1. Regulation of apoptotic mediators reveals dynamic responses to thermal stress in the reef building coral Acropora millepora.

    Directory of Open Access Journals (Sweden)

    Mathieu Pernice

    2011-01-01

    Full Text Available Mass coral bleaching is increasing in scale and frequency across the world's coral reefs and is being driven primarily by increased levels of thermal stress arising from global warming. In order to understand the impacts of projected climate change upon corals reefs, it is important to elucidate the underlying cellular mechanisms that operate during coral bleaching and subsequent mortality. In this respect, increased apoptotic cell death activity is an important cellular process that is associated with the breakdown of the mutualistic symbiosis between the cnidarian host and their dinoflagellate symbionts.The PRESENT study reports the impacts of different stressors (colchicine and heat stress on three phases of apoptosis: (i the potential initiation by differential expression of Bcl-2 members, (ii the execution of apoptotic events by activation of caspase 3-like proteases and (iii and finally, the cell disposal indicated by DNA fragmentation in the reef building coral Acropora millepora. In corals incubated with colchicine, an increase in caspase 3-like activity and DNA fragmentation was associated with a relative down-regulation of Bcl-2, suggesting that the initiation of apoptosis may be mediated by the suppression of an anti-apoptotic mechanism. In contrast, in the early steps of heat stress, the induction of caspase-dependent apoptosis was related to a relative up-regulation of Bcl-2 consecutively followed by a delayed decrease in apoptosis activity.In the light of these results, we propose a model of heat stress in coral hosts whereby increasing temperatures engage activation of caspase 3-dependent apoptosis in cells designated for termination, but also the onset of a delayed protective response involving overexpression of Bcl-2 in surviving cells. This mitigating response to thermal stress could conceivably be an important regulatory mechanism for cell survival in corals exposed to sudden environmental changes.

  2. Bioactive compounds from crocodile (Crocodylus siamensis) white blood cells induced apoptotic cell death in hela cells.

    Science.gov (United States)

    Patathananone, Supawadee; Thammasirirak, Sompong; Daduang, Jureerut; Chung, Jing Gung; Temsiripong, Yosapong; Daduang, Sakda

    2016-08-01

    Crocodile (Crocodylus siamensis) white blood cell extracts (WBCex) were examined for anticancer activity in HeLa cell lines using the MTT assay. The percentage viability of HeLa cells significantly deceased after treatment with WBCex in a dose- and time-dependent manner. The IC50 dose was suggested to be approximately 225 μg/mL protein. Apoptotic cell death occurred in a time-dependent manner based on investigation by flow cytometry using annexin V-FITC and PI staining. DAPI nucleic acid staining indicated increased chromatin condensation. Caspase-3, -8 and -9 activities also increased, suggesting the induction of the caspase-dependent apoptotic pathway. Furthermore, the mitochondrial membrane potential (ΔΨm ) of HeLa cells was lost as a result of increasing levels of Bax and reduced levels of Bcl-2, Bcl-XL, Bcl-Xs, and XIAP. The decreased ΔΨm led to the release of cytochrome c and the activation of caspase-9 and -3. Apoptosis-inducing factor translocated into the nuclei, and endonuclease G (Endo G) was released from the mitochondria. These results suggest that anticancer agents in WBCex can induce apoptosis in HeLa cells via both caspase-dependent and -independent pathways. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 986-997, 2016. © 2015 Wiley Periodicals, Inc.

  3. Histochemical detection and comparison of apoptotic cells in the gingival epithelium using hematoxylin and eosin and methyl green-pyronin: A pilot study.

    Science.gov (United States)

    Nayak, Aarati; Raikar, Anita; Kotrashetti, Vijaylaxmi; Nayak, Ramakant; Shree, Sumedha; Kambali, Soumya

    2016-01-01

    Apoptosis plays a critical role in the regulation of inflammation and host immune response. It helps in tissue homeostasis and a disturbance in this is often associated with disease. The use of histochemical stains like hematoxylin and eosin (H and E) and methyl green-pyronin (MGP) can provide a simple and cost-effective method for the detection of apoptotic cells. Study intended to analyze the expression of apoptosis in the gingival epithelium of healthy subjects and in patients with chronic periodontitis, using H and E and MGP. It is also proposed to correlate the apoptotic index (AI) of healthy individuals and those with chronic periodontitis. Twenty gingival biopsies were harvested from which ten samples were of healthy subjects and ten subjects who suffered from chronic periodontitis. Apoptotic cells were analyzed using MGP and H and E under light microscopy. Apoptotic cells were identified at ×100 magnification and AI was calculated. Apoptotic cells were easily distinguishable in MGP stained sections when compared to those stained using H and E. Moreover, apoptotic cell count was higher in chronic periodontitis. Statistical analyses were done by Tukey's multiple post hoc procedure. The study reveals that MGP staining can be used in a routine basic laboratory set up as one of the cost-effective methods for the detection of apoptotic cells.

  4. Histochemical detection and comparison of apoptotic cells in the gingival epithelium using hematoxylin and eosin and methyl green-pyronin: A pilot study

    Directory of Open Access Journals (Sweden)

    Aarati Nayak

    2016-01-01

    Full Text Available Background: Apoptosis plays a critical role in the regulation of inflammation and host immune response. It helps in tissue homeostasis and a disturbance in this is often associated with disease. The use of histochemical stains like hematoxylin and eosin (H and E and methyl green-pyronin (MGP can provide a simple and cost-effective method for the detection of apoptotic cells. Aim: Study intended to analyze the expression of apoptosis in the gingival epithelium of healthy subjects and in patients with chronic periodontitis, using H and E and MGP. It is also proposed to correlate the apoptotic index (AI of healthy individuals and those with chronic periodontitis. Materials and Methods: Twenty gingival biopsies were harvested from which ten samples were of healthy subjects and ten subjects who suffered from chronic periodontitis. Apoptotic cells were analyzed using MGP and H and E under light microscopy. Results: Apoptotic cells were identified at ×100 magnification and AI was calculated. Apoptotic cells were easily distinguishable in MGP stained sections when compared to those stained using H and E. Moreover, apoptotic cell count was higher in chronic periodontitis. Statistical analyses were done by Tukey's multiple post hoc procedure. Conclusion: The study reveals that MGP staining can be used in a routine basic laboratory set up as one of the cost-effective methods for the detection of apoptotic cells.

  5. Nickel oxide nanoparticles exert cytotoxicity via oxidative stress and induce apoptotic response in human liver cells (HepG2).

    Science.gov (United States)

    Ahamed, Maqusood; Ali, Daoud; Alhadlaq, Hisham A; Akhtar, Mohd Javed

    2013-11-01

    Increasing use of nickel oxide nanoparticles (NiO NPs) necessitates an improved understanding of their potential impact on human health. Previously, toxic effects of NiO NPs have been investigated, mainly on airway cells. However, information on effect of NiO NPs on human liver cells is largely lacking. In this study, we investigated the reactive oxygen species (ROS) mediated cytotoxicity and induction of apoptotic response in human liver cells (HepG2) due to NiO NPs exposure. Prepared NiO NPs were crystalline and spherical shaped with an average diameter of 44 nm. NiO NPs induced cytotoxicity (cell death) and ROS generation in HepG2 cells in dose-dependent manner. Further, ROS scavenger vitamin C reduced cell death drastically caused by NiO NPs exposure indicating that oxidative stress plays an important role in NiO NPs toxicity. Micronuclei induction, chromatin condensation and DNA damage in HepG2 cells treated with NiO NPs suggest that NiO NPs induced cell death via apoptotic pathway. Quantitative real-time PCR analysis showed that following the exposure of HepG2 cells to NiO NPs, the expression level of mRNA of apoptotic genes (bax and caspase-3) were up-regulated whereas the expression level of anti-apoptotic gene bcl-2 was down-regulated. Moreover, activity of caspase-3 enzyme was also higher in NiO NPs treated cells. To the best of our knowledge this is the first report demonstrating that NiO NPs caused cytotoxicity via ROS and induced apoptosis in HepG2 cells, which is likely to be mediated through bax/bcl-2 pathway. This work warrants careful assessment of Ni NPs before their commercial and industrial applications. Copyright © 2013 Elsevier Ltd. All rights reserved.

  6. An apoptotic cell cycle mutant in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Villadsen, Ingrid

    1996-01-01

    The simple eukaryote Saccharomyces cerevisiae has proved to be a useful organism for elucidating the mechanisms that govern cell cycle progression in eukaryotic cells. The excellent in vivo system permits a cell cycle study using temperature sensitive mutants. In addition, it is possible to study...... many genes and gene products from higher eukaryotes in Saccharomyces cerevisiae because many genes and biological processes are homologous or similar in lower and in higher eukaryotes. The highly developed methods of genetics and molecular biology greatly facilitates studies of higher eukaryotic...... processes.Programmmed cell death with apoptosis plays a major role in development and homeostatis in most, if not all, animal cells. Apoptosis is a morphologically distinct form of death, that requires the activation of a highly regulated suicide program. Saccharomyces cerevisiae provides a new system...

  7. Apoptotic Effect of Nigella sativa on Human Lymphoma U937 Cells.

    Science.gov (United States)

    Arslan, Belkis Atasever; Isik, Fatma Busra; Gur, Hazal; Ozen, Fatih; Catal, Tunc

    2017-10-01

    Nigella sativa is from botanical Ranunculaceae family and commonly known as black seed. Apoptotic effect of N. sativa and its apoptotic signaling pathways on U937 lymphoma cells are unknown. In this study, we investigated selective cytotoxic and apoptotic effects of N. sativa extract and its apoptotic mechanisms on U937 cells. In addition, we also studied selective cytotoxic activity of thymoquinone that is the most active essential oil of N. sativa . Our results showed that N. sativa extract has selective cytotoxicity and apoptotic effects on U937 cells but not ECV304 control cells. However, thymoquinone had no significant cytotoxicity against on both cells. N. sativa extract increased significantly caspase-3, BAD, and p53 gene expressions in U937 cells. N. sativa may have anticancer drug potential and trigger p53-induced apoptosis in U937 lymphoma cells. This is the first study showing the apoptotic effect of Nigella sativa extract on U937 cells. Abbreviations used: CI: Cytotoxicity index, DMEM: Dulbecco's Modified Eagle Medium, HL: Hodgkin's lymphoma, MTT: 3-(4,5-dimethy lthiazol-2yl)-2,5-diphenyl tetrazolium bromide, RPMI: Roswell Park Memorial Institute medium.

  8. Anti-apoptotic effects of Z alpha1-antitrypsin in human bronchial epithelial cells.

    LENUS (Irish Health Repository)

    Greene, C M

    2010-05-01

    alpha(1)-antitrypsin (alpha(1)-AT) deficiency is a genetic disease which manifests as early-onset emphysema or liver disease. Although the majority of alpha(1)-AT is produced by the liver, it is also produced by bronchial epithelial cells, amongst others, in the lung. Herein, we investigate the effects of mutant Z alpha(1)-AT (ZAAT) expression on apoptosis in a human bronchial epithelial cell line (16HBE14o-) and delineate the mechanisms involved. Control, M variant alpha(1)-AT (MAAT)- or ZAAT-expressing cells were assessed for apoptosis, caspase-3 activity, cell viability, phosphorylation of Bad, nuclear factor (NF)-kappaB activation and induced expression of a selection of pro- and anti-apoptotic genes. Expression of ZAAT in 16HBE14o- cells, like MAAT, inhibited basal and agonist-induced apoptosis. ZAAT expression also inhibited caspase-3 activity by 57% compared with control cells (p = 0.05) and was a more potent inhibitor than MAAT. Whilst ZAAT had no effect on the activity of Bad, its expression activated NF-kappaB-dependent gene expression above control or MAAT-expressing cells. In 16HBE14o- cells but not HEK293 cells, ZAAT upregulated expression of cIAP-1, an upstream regulator of NF-kappaB. cIAP1 expression was increased in ZAAT versus MAAT bronchial biopsies. The data suggest a novel mechanism by which ZAAT may promote human bronchial epithelial cell survival.

  9. Cell-Centric View of Apoptosis and Apoptotic Cell Death-Inducing Antitumoral Strategies

    Directory of Open Access Journals (Sweden)

    Maria Dolores Boyano

    2011-03-01

    Full Text Available Programmed cell death and especially apoptotic cell death, occurs under physiological conditions and is also desirable under pathological circumstances. However, the more we learn about cellular signaling cascades, the less plausible it becomes to find restricted and well-limited signaling pathways. In this context, an extensive description of pathway-connections is necessary in order to point out the main regulatory molecules as well as to select the most appropriate therapeutic targets. On the other hand, irregularities in programmed cell death pathways often lead to tumor development and cancer-related mortality is projected to continue increasing despite the effort to develop more active and selective antitumoral compounds. In fact, tumor cell plasticity represents a major challenge in chemotherapy and improvement on anticancer therapies seems to rely on appropriate drug combinations. An overview of the current status regarding apoptotic pathways as well as available chemotherapeutic compounds provides a new perspective of possible future anticancer strategies.

  10. Andrographolide induces apoptotic and non-apoptotic death and enhances tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis in gastric cancer cells

    OpenAIRE

    Lim, Sung-Chul; Jeon, Ho Jong; Kee, Keun Hong; Lee, Mi Ja; Hong, Ran; Han, Song Iy

    2017-01-01

    Andrographolide, a natural compound isolated from Andrographis paniculata, has been reported to possess antitumor activity. In the present study, the effect of andrographolide in human gastric cancer (GC) cells was investigated. Andrographolide induced cell death with apoptotic and non-apoptotic features. At a low concentration, andrographolide potentiated apoptosis and reduction of clonogenicity triggered by recombinant human tumor necrosis factor-related apoptosis-inducing ligand (rhTRAIL)....

  11. Apoptotic cells can induce non-autonomous apoptosis through the TNF pathway

    Science.gov (United States)

    Pérez-Garijo, Ainhoa; Fuchs, Yaron; Steller, Hermann

    2013-01-01

    Apoptotic cells can produce signals to instruct cells in their local environment, including ones that stimulate engulfment and proliferation. We identified a novel mode of communication by which apoptotic cells induce additional apoptosis in the same tissue. Strong induction of apoptosis in one compartment of the Drosophila wing disc causes apoptosis of cells in the other compartment, indicating that dying cells can release long-range death factors. We identified Eiger, the Drosophila tumor necrosis factor (TNF) homolog, as the signal responsible for apoptosis-induced apoptosis (AiA). Eiger is produced in apoptotic cells and, through activation of the c-Jun N-terminal kinase (JNK) pathway, is able to propagate the initial apoptotic stimulus. We also show that during coordinated cell death of hair follicle cells in mice, TNF-α is expressed in apoptotic cells and is required for normal cell death. AiA provides a mechanism to explain cohort behavior of dying cells that is seen both in normal development and under pathological conditions. DOI: http://dx.doi.org/10.7554/eLife.01004.001 PMID:24066226

  12. Expression Profile of Apoptotic Mediators and Proliferative Markers in Oral Squamous Cell Carcinoma

    International Nuclear Information System (INIS)

    Ahmed, M.M.

    2009-01-01

    Oral squamous cell carcinoma (OSCC) represents a major health problem worldwide. It is therefore essential to develop a deeper understanding of its biology. Beside the recent hypothesis of cancer stem cells, the consideration of its cell death and cell proliferation has emerged as important diagnostic and prognostic tools. Purpose of the Study: Detection of the proportion of cell loss monitored by apoptosis-related genes, p53, p21 and Bcl2, and their relationship to the pathological proliferation parameter, PCNA in OSCC. Furthermore, discussion of the hypothesis of cancer stem cell biology in OSCC would be anticipated. Material and Methods: Archival 35 tissue embedded paraffin blocks, that were previously diagnosed as well to moderately differentiated OSCC, were immunohistochemically stained using a panel of antibodies including apoptotic mediators, p53, p21, Bcl2, and proliferation marker, PCNA. Immuno expression was scored using a semiquantitative scale and statistically analyzed. Results: The clinico-pathological data revealed that mean age was 46.9±8.2 and the tongue was the most affected site, followed by the palate then the floor of the mouth. There was no significant difference between metastasizing and non-metastasizing patients regarding age or gender (p=0.174, 0.404, respectively). On the other hand, variable profile patterns of the investigated indicators existed, where PCNA positively immunostaining cells was 100% while P21 recorded the higher percentage of negatively immunoreactive cells (42.9%). A common trait for the studied cell cycle indicators was that the basal and supra basal epithelial cells as well as the peripheral cells of the invading nests were the harbor of immunoreactivity. Meanwhile, Pca immuno positivity was revealed in all epithelial layers plus stromal cells. Conclusions: Assessment of the studied cell cycle regulators may be valuable to judge tumorigenesis of Osac. Furthermore, deregulation of cell cycle control might aid in the

  13. The TAM-family receptor Mer mediates production of HGF through the RhoA-dependent pathway in response to apoptotic cells.

    Science.gov (United States)

    Park, Hyun-Jung; Baen, Ji-Yeon; Lee, Ye-Ji; Choi, Youn-Hee; Kang, Jihee Lee

    2012-08-01

    The TAM receptor protein tyrosine kinases Tyro3, Axl, and Mer play important roles in macrophage function. We investigated the roles of the TAM receptors in mediating the induction of hepatocyte growth factor (HGF) during the interaction of macrophages with apoptotic cells. Mer-specific neutralizing antibody, small interfering RNA (siRNA), and a recombinant Mer protein (Mer/Fc) inhibited HGF mRNA and protein expression, as well as activation of RhoA, Akt, and specific mitogen-activated protein (MAP) kinases in response to apoptotic cells. Inhibition of Axl or Tyro3 with specific antibodies, siRNA, or Fc-fusion proteins did not prevent apoptotic cell-induced HGF mRNA and protein expression and did not inhibit activation of the postreceptor signaling molecules RhoA and certain MAP kinases, including extracellular signal-regulated protein kinase and c-Jun NH(2)-terminal kinase. However, Axl- and Tyro3-specific blockers did inhibit the activation of Akt and p38 MAP kinase in response to apoptotic cells. In addition, none of the TAM receptors mediated the effects of apoptotic cells on transforming growth factor-β or epidermal growth factor mRNA expression. However, they were involved in the induction of vascular endothelial growth factor mRNA expression. Our data provide evidence that when macrophages interact with apoptotic cells, only Mer of the TAM-family receptors is responsible for mediating transcriptional HGF production through a RhoA-dependent pathway.

  14. Intrinsic, pro-apoptotic effects of IGFBP-3 on breast cancer cells are reversible: Involvement of PKA, Rho and ceramide.

    Directory of Open Access Journals (Sweden)

    Claire M Perks

    2011-05-01

    Full Text Available We established previously that IGFBP-3 could exert positive or negative effects on cell function depending upon the extracellular matrix composition and by interacting with integrin signalling. To elicit its pro-apoptotic effects IGFBP-3 bound to caveolin-1 and the beta 1 integrin receptor and increased their association culminating in MAPK activation. Disruption of these complexes or blocking the beta 1 integrin receptor reversed these intrinsic actions of IGFBP-3. In this study we have examined the signalling pathway between integrin receptor binding and MAPK activation that mediates the intrinsic, pro-apoptotic actions of IGFBP-3. We found on inhibiting protein kinase A(PKA, Rho associated kinase (ROCK and ceramide, the accentuating effects of IGFBP-3 on apoptotic triggers were reversed, such that IGFBP-3 then conferred cell survival. We established that IGFBP-3 activated Rho, the upstream regulator of ROCK and that beta1 integrin and PKA were upstream of Rho activation, whereas the involvement of ceramide was downstream. The beta 1 integrin, PKA, Rho and ceramide were all upstream of MAPK activation. These data highlight key components involved in the pro-apoptotic effects of IGFBP-3 and that inhibiting them leads to a reversal in the action of IGFBP-3.

  15. Methadone as an inducer of apoptotic process in cheek mucosae cells in rats

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    Małgorzata Stępień

    2017-11-01

    Full Text Available Methadone is an opioid medication which can reduce withdrawal symptoms in people addicted to heroin and other drugs. Methadone is used also as a pain reliever and as part of drug addiction detoxification program. Apoptosis is the physiological process that plays a critical role in development and tissue homeostasis. The progress of apoptosis is regulated by signal cascades. The aim of this study was to asses how methadone induces apoptotic process in cheek mucosae cells in rats. Forty albino rats wares divided into two parts and five subgroups each. The biggest histological changes of cheek mucosae was observed in the groups with methadone. There is no indication of ability to regeneration in short time after treatment.

  16. Tuberin and PRAS40 are anti-apoptotic gatekeepers during early human amniotic fluid stem-cell differentiation.

    Science.gov (United States)

    Fuchs, Christiane; Rosner, Margit; Dolznig, Helmut; Mikula, Mario; Kramer, Nina; Hengstschläger, Markus

    2012-03-01

    Embryoid bodies (EBs) are three-dimensional multicellular aggregates allowing the in vitro investigation of stem-cell differentiation processes mimicking early embryogenesis. Human amniotic fluid stem (AFS) cells harbor high proliferation potential, do not raise the ethical issues of embryonic stem cells, have a lower risk for tumor development, do not need exogenic induction of pluripotency and are chromosomal stable. Starting from a single human AFS cell, EBs can be formed accompanied by the differentiation into cells of all three embryonic germ layers. Here, we report that siRNA-mediated knockdown of the endogenous tuberous sclerosis complex-2 (TSC2) gene product tuberin or of proline-rich Akt substrate of 40 kDa (PRAS40), the two major negative regulators of mammalian target of rapamycin (mTOR), leads to massive apoptotic cell death during EB development of human AFS cells without affecting the endodermal, mesodermal and ectodermal cell differentiation spectrum. Co-knockdown of endogenous mTOR demonstrated these effects to be mTOR-dependent. Our findings prove this enzyme cascade to be an essential anti-apoptotic gatekeeper of stem-cell differentiation during EB formation. These data allow new insights into the regulation of early stem-cell maintenance and differentiation and identify a new role of the tumor suppressor tuberin and the oncogenic protein PRAS40 with the relevance for a more detailed understanding of the pathogenesis of diseases associated with altered activities of these gene products.

  17. Molecular analysis of the apoptotic effects of BPA in acute myeloid leukemia cells

    Directory of Open Access Journals (Sweden)

    Del Pozzo Giovanna

    2009-06-01

    Full Text Available Abstract Background: BPA (bisphenol A or 2,2-bis(4-hydroxy-phenolpropane is present in the manufacture of polycarbonate plastic and epoxy resins, which can be used in impact-resistant safety equipment and baby bottles, as protective coatings inside metal food containers, and as composites and sealants in dentistry. Recently, attention has focused on the estrogen-like and carcinogenic adverse effects of BPA. Thus, it is necessary to investigate the cytotoxicity and apoptosis-inducing activity of this compound. Methods: Cell cycle, apoptosis and differentiation analyses; western blots. Results: BPA is able to induce cell cycle arrest and apoptosis in three different acute myeloid leukemias. Although some granulocytic differentiation concomitantly occurred in NB4 cells upon BPA treatment, the major action was the induction of apoptosis. BPA mediated apoptosis was caspase dependent and occurred by activation of extrinsic and intrinsic cell death pathways modulating both FAS and TRAIL and by inducing BAD phosphorylation in NB4 cells. Finally, also non genomic actions such as the early decrease of both ERK and AKT phosphorylation were induced by BPA thus indicating that a complex intersection of regulations occur for the apoptotic action of BPA. Conclusion: BPA is able to induce apoptosis in leukemia cells via caspase activation and involvement of both intrinsic and extrinsic pathways of apoptosis.

  18. The effect of bystander medium on the apoptotic process in HPV-G cells

    International Nuclear Information System (INIS)

    Maguire, P.; Lyng, F.; Seymour, C.; Mothersill, C.

    2003-01-01

    Full text: It has been shown in recent years that in both in vivo and in vitro situations irradiated cells cause what is known as the bystander effect. This presently unknown factor causes chromosomal aberrations, initiation of apoptosis and reduced clonogenic survival. Using the medium transfer method to study the bystander effect, this study investigated early events in the apoptotic cascade, which leads to cell death in cells receiving medium from irradiated cells but which were not themselves irradiated. Medium from irradiated ( 0.005Gy to 5Gy) human HPV G keratinocytes was harvested one hour after irradiation, sterile filtered and transferred on to unirradiated HPV-G cells. The appearance of apoptotic markers in the apoptotic cascade was monitored over a period of 48 hours following medium transfer. These apoptotic markers include loss of mitochondrial membrane potential, cytochrome c release and the activity of the death inducing caspase 3. Clonogenic survival of HPV-G cells over a nine day period was also monitored to assess the final survival of the cells. A TUNEL assay, which indicated the level of apoptosis over a 72 hour period after exposure to bystander medium was also performed. Data collected in this study indicates that for very low doses (0.005Gy) the appearance of well-characterised early 'apoptotic' markers such as changes in mitochondrial membrane potential doesn't mean the cell has committed to the apoptotic cascade leading to cell death. This has been illustrated for bystander medium from 0.005Gy irradiated cells, which causes mitochondrial membrane potential depolarisation after six-hour exposure but little difference has been noted for clonogenic survival for exposure to 0.005Gy bystander medium from that of the control. The results may help clarify how cells sector to death or survival following receipt of a signal from a radiation event

  19. Opening up of plasmalemma type-1 VDAC to form apoptotic "find me signal" pathways is essential in early apoptosis - evidence from the pathogenesis of cystic fibrosis resulting from failure of apoptotic cell clearance followed by sterile inflammation.

    Science.gov (United States)

    Thinnes, Friedrich P

    2014-04-01

    Cell membrane-standing type-1 VDAC is involved in cell volume regulation and thus apoptosis. The channel has been shown to figure as a pathway for osmolytes of varying classes, ATP included. An early event in apoptotic cell death is the release of "find me signals" by cells that enter the apoptotic process. ATP is one of those signals. Apoptotic cells this way attract phagocytes for an immunologically silent cell clearance. Thus, whenever apoptosis fails by a blockade of plasmalemma type-1 VDAC processes of sterile inflammation must be assumed for cell elimination. This is evident from a close look on the pathogenetic process of cystic fibrosis (CF). However, in normal airway epithelia two different anion channels cooperate to guarantee an appropriate volume of airway surface liquid (ASL) necessary for surface clearing: the cystic fibrosis conductance regulator (CFTR) and the outwardly rectifying chloride channel (ORCC) complex also called "alternate chloride channel" and under the control of the CFTR. There are arguments, that type-1 VDAC forms the channel part of the ORCC complex, and it has been shown that CFTR and type-1 VDAC co-localize in the apical membranes of human surface respiratory epithelium. In cystic fibrosis, the central cAMP-dependent regulation of ion and water transport via functional CFTR is lost. Here, CFTR molecules do not reach the apical membranes of airway epithelia anymore or work in an insufficient way, respectively. In addition, type-1 VDAC is no longer available to work as a "find me signal" pathway. In consequence, clearing away of apoptotic cells is blocked. There are experimental data on the channel characteristics of type-1 VDAC under the anion channel blocker DIDS (4,4-diisothiocyanato-stilbenedisulphonic acid) that argue in favor of this hypothesis. Together, type-1 VDAC should be kept as a "find me signal" pathway, which may give way to several classes of such signals. Copyright © 2014 Elsevier Inc. All rights reserved.

  20. Reactive oxygen species induced by Streptococcus pyogenes invasion trigger apoptotic cell death in infected epithelial cells.

    Science.gov (United States)

    Aikawa, Chihiro; Nozawa, Takashi; Maruyama, Fumito; Tsumoto, Kohei; Hamada, Shigeyuki; Nakagawa, Ichiro

    2010-06-01

    Streptococcus pyogenes (group A streptococcus, GAS), one of the most common pathogens of humans, attaches and invades into human pharyngeal or skin epithelial cells. We have previously reported that induction of apoptosis is associated with GAS invasion, which induces mitochondrial dysfunction and apoptotic cell death. We demonstrate here that GAS-induced apoptosis is mediated by reactive oxygen species (ROS) production. Both the induction of apoptosis and ROS production markedly increased upon invasion of wild-type GAS strain JRS4 into HeLa cells; however, the apoptotic response was not observed in fibronectin-binding protein F1-disrupted mutant SAM1-infected cells. In Bcl-2-overexpressing HeLa cells (HBD98-2-4), the induction of apoptosis, ROS production and mitochondrial dysfunction were significantly suppressed, whereas the numbers of invaded GAS was not different between HeLa (mock cells) and the HeLa HBD98-2-4 cells. Whereas Rac1 activation occurred during GAS invasion, ROS production in GAS-infected cells was clearly inhibited by transfection with the Rac1 mutants (L37 or V12L37), but not by the dominant active mutant (V12L61) or by the dominant negative mutant (N17). These observations indicate that GAS invasion triggers ROS production through Rac1 activation and generated ROS induced mitochondrial dysfunction leading to cellular apoptosis.

  1. T Cell Zone Resident Macrophages Silently Dispose of Apoptotic Cells in the Lymph Node.

    Science.gov (United States)

    Baratin, Myriam; Simon, Léa; Jorquera, Audrey; Ghigo, Clément; Dembele, Doulaye; Nowak, Jonathan; Gentek, Rebecca; Wienert, Stephan; Klauschen, Frederick; Malissen, Bernard; Dalod, Marc; Bajénoff, Marc

    2017-08-15

    In lymph nodes (LNs), dendritic cells (DCs) are thought to dispose of apoptotic cells, a function pertaining to macrophages in other tissues. We found that a population of CX3CR1 + MERTK + cells located in the T cell zone of LNs, previously identified as DCs, are efferocytic macrophages. Lineage-tracing experiments and shield chimeras indicated that these T zone macrophages (TZM) are long-lived macrophages seeded in utero and slowly replaced by blood monocytes after birth. Imaging the LNs of mice in which TZM and DCs express different fluorescent proteins revealed that TZM-and not DCs-act as the only professional scavengers, clearing apoptotic cells in the LN T cell zone in a CX3CR1-dependent manner. Furthermore, similar to other macrophages, TZM appear inefficient in priming CD4 T cells. Thus, efferocytosis and T cell activation in the LN are uncoupled processes designated to macrophages and DCs, respectively, with implications to the maintenance of immune homeostasis. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Effects of Malnutrition on Neutrophil/Mononuclear Cell Apoptotic Functions in Children with Acute Lymphoblastic Leukemia.

    Science.gov (United States)

    Cakir, Fatma Betul; Berrak, Su Gülsün; Aydogan, Gonul; Tulunay, Aysin; Timur, Cetin; Canpolat, Cengiz; Eksioglu Demiralp, Emel

    2017-04-01

    Recent studies claim that apoptosis may explain immune dysfunction observed in malnutrition. The objective of this study was to determine the effect of malnutrition on apoptotic functions of phagocytic cells in acute lymphoblastic leukemia (ALL). Twenty-eight ALL patients (13 with malnutrition) and thirty controls were enrolled. Neutrophil and mononuclear cell apoptosis of ALL patients and the control group were studied on admission before chemotherapy and repeated at a minimum of three months after induction of chemotherapy or when the nutritional status of leukemic children improved. The apoptotic functions of both ALL groups on admission were significantly lower than those of the control group. The apoptotic functions were lower in ALL patients with malnutrition than those in ALL patients without malnutrition, but this was not statistically significant. The repeated apoptotic functions of both ALL groups were increased to similar values with the control group. This increase was found to be statistically significant. The apoptotic functions in ALL patients were not found to be affected by malnutrition. However, after dietary intervention, increased apoptotic functions in both ALL patient groups deserve mentioning. Dietary intervention should always be recommended as malnutrition or cachexia leads to multiple complications. Enhanced apoptosis might originate also from remission state of cancer.

  3. Involvement of caspase-12-dependent apoptotic pathway in ionic radiocontrast urografin-induced renal tubular cell injury

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Cheng Tien [Institute of Toxicology, College of Medicine, National Taiwan University, Taipei, Taiwan (China); Weng, Te I. [Department of Forensic Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan (China); Chen, Li Ping [Department of Dentistry, Chang Gang Memorial Hospital, Chang Gang University, Taoyuan, Taiwan (China); Chiang, Chih Kang [Department of Integrated Diagnostics and Therapeutics, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan (China); Department of Internal Medicine, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan (China); Liu, Shing Hwa, E-mail: shinghwaliu@ntu.edu.tw [Institute of Toxicology, College of Medicine, National Taiwan University, Taipei, Taiwan (China); Department of Urology, National Taiwan University Hospital, Taipei, Taiwan (China)

    2013-01-01

    Contrast medium (CM) induces a direct toxic effect on renal tubular cells. This toxic effect subjects in the disorder of CM-induced nephropathy. Our previous work has demonstrated that CM shows to activate the endoplasmic reticulum (ER)-related adaptive unfolding protein response (UPR) activators. Glucose-regulated protein 78 (GRP78)/eukaryotic initiation factor 2α (eIF2α)-related pathways play a protective role during the urografin (an ionic CM)-induced renal tubular injury. However, the involvement of ER stress-related apoptotic signals in the urografin-induced renal tubular cell injury remains unclear. Here, we examined by the in vivo and in vitro experiments to explore whether ER stress-regulated pro-apoptotic activators participate in urografin-induced renal injury. Urografin induced renal tubular dilation, tubular cells detachment, and necrosis in the kidneys of rats. The tubular apoptosis, ER stress-related pro-apoptotic transcriptional factors, and kidney injury marker-1 (kim-1) were also conspicuously up-regulated in urografin-treated rats. Furthermore, treatment of normal rat kidney (NRK)-52E tubular cells with urografin augmented the expressions of activating transcription factor-6 (ATF-6), C/EBP homologous protein (CHOP), Bax, caspase-12, JNK, and inositol-requiring enzyme (IRE) 1 signals. Urografin-induced renal tubular cell apoptosis was not reversed by the inhibitors of ATF-6, JNK signals or CHOP siRNA transfection, but it could be partially reversed by the inhibitor of caspase-12. Taken together, the present results and our previous findings suggest that exposure of CM/urografin activates the ER stress-regulated survival- and apoptosis-related signaling pathways in renal tubular cells. Caspase-12-dependent apoptotic pathway may be partially involved in the urografin-induced nephropathy. -- Highlights: ► Ionic contrast medium-urografin induces renal tubular cell apoptosis. ► Urografin induces the ER stress-regulated survival and apoptosis

  4. RACK1 downregulates levels of the pro-apoptotic protein Fem1b in apoptosis-resistant colon cancer cells.

    Science.gov (United States)

    Subauste, M Cecilia; Ventura-Holman, Tereza; Du, Liqin; Subauste, Jose S; Chan, Shing-Leng; Yu, Victor C; Maher, Joseph F

    2009-12-01

    Evasion of apoptosis plays an important role in colon cancer progression. Following loss of the Apc tumor suppressor gene in mice, the gene encoding Fem1b is upregulated early in neoplastic intestinal epithelium. Fem1b is a pro-apoptotic protein that interacts with Fas, TNFR1 and Apaf-1, and increased expression of Fem1b induces apoptosis of cancer cells. Fem1b is a homolog of FEM-1, a protein in Caenorhabditis elegans that is negatively regulated by ubiquitination and proteasomal degradation. To study Fem1b regulation in colon cancer progression, we used apoptotis-sensitive SW480 cells, derived from a primary colon cancer, and their isogenic, apoptosis-resistant counterparts SW620 cells, derived from a subsequent metastatic lesion in the same patient. Treatment with proteasome inhibitor increased Fem1b protein levels in SW620 cells, but not in SW480 cells. In SW620 cells we found that endogenous Fem1b co-immunoprecipitates in complexes with RACK1, a protein known to mediate ubiquitination and proteasomal degradation of other pro-apoptotic proteins and to be upregulated in colon cancer. Full-length Fem1b, or the N-terminal region of Fem1b, associated with RACK1 when co-expressed in HEK293T cells, and RACK1 stimulated ubiquitination of Fem1b. RACK1 overexpression in SW620 cells led to downregulation of Fem1b protein levels. Conversely, downregulation of RACK1 led to upregulation of Fem1b protein levels, associated with induction of apoptosis, and this apoptosis was inhibited by blocking Fem1b protein upregulation. In conclusion, RACK1 downregulates levels of the pro-apoptotic protein Fem1b in metastatic, apoptosis-resistant colon cancer cells, which may promote apoptosis-resistance during progression of colon cancer.

  5. Autophagy postpones apoptotic cell death in PRRSV infection through Bad-Beclin1 interaction.

    Science.gov (United States)

    Zhou, Ao; Li, Shuaifeng; Khan, Faheem Ahmed; Zhang, Shujun

    2016-01-01

    Autophagy and apoptosis play significant roles in PRRSV infection and replication. However, the interaction between these 2 processes in PRRSV replication is still far from been completely understood. In our studies, the exposure of MARC-145 cells to PRRSV confirmed the activation of autophagy and subsequent induction of apoptosis. The inhibition of autophagy by 3-methyladenine (3-MA) caused a significant increase in PRRSV-induced apoptosis, showing a potential connection between both mechanisms. Moreover, we observed an increase in Bad expression (a pro-apoptotic protein) and Beclin1 (an autophagy regulator) in virus-infected cells up to 36h. Co-immunoprecipitation assays showed the formation of Bad and Beclin1 complex in PRRSV infected cells. Accordingly, Bad co-localized with Beclin1 in MARC-145 infected cells. Knockdown of Beclin1 significantly decreased PRRSV replication and PRRSV-induced autophagy, while Bad silencing resulted in increased autophagy and enhanced viral replication. Furthermore, PRRSV infection phosphorylated Bad (Ser112) to promote cellular survival. These results demonstrate that autophagy can favor PRRSV replication by postponing apoptosis through the formation of a Bad-Beclin1 complex.

  6. Lithium delays the radiation-induced apoptotic process in external granule cells of mouse cerebellum

    International Nuclear Information System (INIS)

    Inouye, Minoru; Yamamura, Hideki; Nakano, Atsuhiro.

    1995-01-01

    Proliferating cells of the external granular layer (EGL) in the developing cerebellum are highly sensitive to ionizing radiation. We examined the effect of lithium, an inhibitor of intracellular signaling, on the manifestation of radiation-induced apoptosis. Newborn mice were exposed to 0.5 Gy gamma-irradiation alone, or first were treated with lithium (10 μmol/g, SC) then given 0.5 Gy irradiation 2 hr later. The EGL was examined histologically for apoptosis at various times after treatment. Apoptotic cells increased rapidly, peaked (about 14%) 6 hr after irradiation, then decreased gradually to the control level by 24 hr. Prior treatment with lithium delayed the manifestation of apoptosis, the peak appearing at 12 hr. The disappearance of dead cells was delayed for about one day. The lithium concentration in the whole brain increased rapidly, being 30 μg/g at the time of irradiation and remaining at more than 40 μg/g for 40 hr. Lithium is reported to inhibit guanine-nucleotide binding to G proteins as well as phosphoinositide turnover. Of the variety of lesions induced by radiation, DNA double strand breaks are the most important source of cell lethality. The present findings, however, suggest that cyclic AMP-mediated and/or phosphoinositide-mediated signaling systems regulate radiation-induced apoptosis. (author)

  7. Lithium delays the radiation-induced apoptotic process in external granule cells of mouse cerebellum.

    Science.gov (United States)

    Inouye, M; Yamamura, H; Nakano, A

    1995-09-01

    Proliferating cells of the external granular layer (EGL) in the developing cerebellum are highly sensitive to ionizing radiation. We examined the effect of lithium, an inhibitor of intracellular signaling, on the manifestation of radiation-induced apoptosis. Newborn mice were exposed to 0.5 Gy gamma-irradiation alone, or first were treated with lithium (10 mumol/g, SC) then given 0.5 Gy irradiation 2 hr later. The EGL was examined histologically for apoptosis at various times after treatment. Apoptotic cells increased rapidly, peaked (about 14%) 6 hr after irradiation, then decreased gradually to the control level by 24 hr. Prior treatment with lithium delayed the manifestation of apoptosis, the peak appearing at 12 hr. The disappearance of dead cells was delayed for about one day. The lithium concentration in the whole brain increased rapidly, being 30 micrograms/g at the time of irradiation and remaining at more than 40 micrograms/g for 40 hr. Lithium is reported to inhibit guanine-nucleotide binding to G proteins as well as phosphoinositide turnover. Of the variety of lesions induced by radiation, DNA double strand breaks are the most important source of cell lethality. The present findings, however, suggest that cyclic AMP-mediated and/or phosphoinositidemediated signaling systems regulate radiation-induced apoptosis.

  8. Lithium delays the radiation-induced apoptotic process in external granule cells of mouse cerebellum

    Energy Technology Data Exchange (ETDEWEB)

    Inouye, Minoru; Yamamura, Hideki [Nagoya Univ. (Japan). Research Inst. of Environmental Medicine; Nakano, Atsuhiro

    1995-09-01

    Proliferating cells of the external granular layer (EGL) in the developing cerebellum are highly sensitive to ionizing radiation. We examined the effect of lithium, an inhibitor of intracellular signaling, on the manifestation of radiation-induced apoptosis. Newborn mice were exposed to 0.5 Gy gamma-irradiation alone, or first were treated with lithium (10 {mu}mol/g, SC) then given 0.5 Gy irradiation 2 hr later. The EGL was examined histologically for apoptosis at various times after treatment. Apoptotic cells increased rapidly, peaked (about 14%) 6 hr after irradiation, then decreased gradually to the control level by 24 hr. Prior treatment with lithium delayed the manifestation of apoptosis, the peak appearing at 12 hr. The disappearance of dead cells was delayed for about one day. The lithium concentration in the whole brain increased rapidly, being 30 {mu}g/g at the time of irradiation and remaining at more than 40 {mu}g/g for 40 hr. Lithium is reported to inhibit guanine-nucleotide binding to G proteins as well as phosphoinositide turnover. Of the variety of lesions induced by radiation, DNA double strand breaks are the most important source of cell lethality. The present findings, however, suggest that cyclic AMP-mediated and/or phosphoinositide-mediated signaling systems regulate radiation-induced apoptosis. (author).

  9. Glycine tomentella Hayata inhibits IL-1β and IL-6 production, inhibits MMP-9 activity, and enhances RAW264.7 macrophage clearance of apoptotic cells

    Directory of Open Access Journals (Sweden)

    Sun Yu-Shu

    2010-11-01

    Full Text Available Abstract Background To assess the effects of Glycine tomentella Hayata (GTH, a traditional herbal medicine for treatment of rheumatic diseases on the expression of the proinflammatory cytokines and on the clearance of apoptotic cells by macrophages. Methods RAW264.7 cells were cultured with lipopolysaccharide (LPS in the presence or absence of ethanol extract of GTH. The expression of proinflammatory cytokines IL-1β, IL-6, and TNF-α, and inducible nitric oxide synthase (iNOS and transglutaminase 2 (TG2 were assayed by reverse transcriptase-polymerase chain reaction (RT-PCR and enzyme-linked immunosorbent assay (ELISA. Matrix metalloproteinase (MMP-2 and MMP-9 were assayed by gelatin zymography. For detecting uptake of apoptotic cells, RAW264.7 cells were cultured with carboxyfluorescein diacetate (CFDA-stained apoptotic cells and assayed by flow cytometry. Results The major components of GTH analyzed by high-performance liquid chromatography (HPLC chromatogram were daidzein (42.5%, epicatechin (28.8%, and naringin (9.4%. GTH treatment inhibited the expression of proinflammatory cytokines IL-1β, IL-6 and MMP-9 but did not affect the expression of TNF-α and iNOS. GTH significantly enhanced the expression of TG2 and the clearance of apoptotic cells by RAW264.7 macrophages. Conclusions GTH inhibits proinflammatory cytokine secretion and MMP-9 activity, enhances apoptotic cell uptake and up-regulates TG2 expression. Our data show that GTH might have beneficial effects on rheumatic diseases.

  10. Anti-apoptotic peptides protect against radiation-induced cell death

    International Nuclear Information System (INIS)

    McConnell, Kevin W.; Muenzer, Jared T.; Chang, Kathy C.; Davis, Chris G.; McDunn, Jonathan E.; Coopersmith, Craig M.; Hilliard, Carolyn A.; Hotchkiss, Richard S.; Grigsby, Perry W.; Hunt, Clayton R.

    2007-01-01

    The risk of terrorist attacks utilizing either nuclear or radiological weapons has raised concerns about the current lack of effective radioprotectants. Here it is demonstrated that the BH4 peptide domain of the anti-apoptotic protein Bcl-xL can be delivered to cells by covalent attachment to the TAT peptide transduction domain (TAT-BH4) and provide protection in vitro and in vivo from radiation-induced apoptotic cell death. Isolated human lymphocytes treated with TAT-BH4 were protected against apoptosis following exposure to 15 Gy radiation. In mice exposed to 5 Gy radiation, TAT-BH4 treatment protected splenocytes and thymocytes from radiation-induced apoptotic cell death. Most importantly, in vivo radiation protection was observed in mice whether TAT-BH4 treatment was given prior to or after irradiation. Thus, by targeting steps within the apoptosis signaling pathway it is possible to develop post-exposure treatments to protect radio-sensitive tissues

  11. Oxidized low-density lipoprotein-induced apoptotic dendritic cells as a novel therapy for atherosclerosis

    NARCIS (Netherlands)

    Frodermann, Vanessa; van Puijvelde, Gijs H M; Wierts, Laura; Lagraauw, H Maxime; Foks, Amanda C; van Santbrink, Peter J; Bot, Ilze; Kuiper, Johan; de Jager, Saskia C A

    2015-01-01

    Modulation of immune responses may form a powerful approach to treat atherosclerosis. It was shown that clearance of apoptotic cells results in tolerance induction to cleared Ags by dendritic cells (DCs); however, this seems impaired in atherosclerosis because Ag-specific tolerance is lacking. This

  12. Irigenin sensitizes TRAIL-induced apoptosis via enhancing pro-apoptotic molecules in gastric cancer cells.

    Science.gov (United States)

    Xu, Ying; Gao, Cheng-Cheng; Pan, Zhen-Guo; Zhou, Chuan-Wen

    2018-02-12

    Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) holds promising value for cancer therapy due to its capacity to induce apoptosis in cancer cells. Nevertheless, TRAIL therapy is greatly hampered by its resistance. Irigenin (Iri), isoflavonoids, can be isolated from the rhizome of Belamcanda chinensis, and has been shown anti-cancer properties. In this study, we explored if Iri could enhance TRAIL-regulated apoptosis in TRAIL resistant gastric cancer cells. Iri significantly potentiated TRAIL-triggered cytotoxicity. Iri alone and TRAIL alone showed no effective role in apoptosis induction, whereas combined treatment with Iri and TRAIL markedly induced apoptosis in cancer cells, as evidenced by the up-regulation of cleaved Caspase-8/-9/-3 and PARP. Additionally, the sensitization to TRAIL was along with the enhancement of pro-apoptotic proteins, including FAS-associated protein with death domain (FADD), death receptor 5 (DR5) and Bax. And suppressing FADD, DR5 and Bax by si RNA significantly reduced the apoptosis and enhanced the cell viability induced by the co-application of Iri and TRAIL. Moreover, the sensitization to TRAIL was accompanied by the decrease of Cellular-FLICE inhibitory protein (c-FLIP), Bcl-2 and Survivin. Additionally, Iri could sensitize TRAIL to produce reactive oxygen species (ROS). Pre-treatment of N-acetyl-cysteine (NAC), ROS scavenger, attenuated Iri plus TRAIL-induced apoptosis and improved cell viability. Finally, combination of Iri and TRAIL inhibited tumor growth in the xenograft model. Collectively, our present study gave new insights into the effects of Iri on potentiating TRAIL-sensitivity, and suggested that Iri could be a potential candidate for sensitizer of TRAIL-resistant cancer cell treatment. Copyright © 2018. Published by Elsevier Inc.

  13. The apoptotic effect and the plausible mechanism of microwave radiation on rat myocardial cells.

    Science.gov (United States)

    Zhu, Wenhe; Cui, Yan; Feng, Xianmin; Li, Yan; Zhang, Wei; Xu, Junjie; Wang, Huiyan; Lv, Shijie

    2016-08-01

    Microwaves may exert adverse biological effects on the cardiovascular system at the integrated system and cellular levels. However, the mechanism underlying such effects remains poorly understood. Here, we report a previously uncharacterized mechanism through which microwaves damage myocardial cells. Rats were treated with 2450 MHz microwave radiation at 50, 100, 150, or 200 mW/cm(2) for 6 min. Microwave treatment significantly enhanced the levels of various enzymes in serum. In addition, it increased the malondialdehyde content while decreasing the levels of antioxidative stress enzymes, activities of enzyme complexes I-IV, and ATP in myocardial tissues. Notably, irradiated myocardial cells exhibited structural damage and underwent apoptosis. Furthermore, Western blot analysis revealed significant changes in expression levels of proteins involved in oxidative stress regulation and apoptotic signaling pathways, indicating that microwave irradiation could induce myocardial cell apoptosis by interfering with oxidative stress and cardiac energy metabolism. Our findings provide useful insights into the mechanism of microwave-induced damage to the cardiovascular system.

  14. Immune responses of dendritic cells after acquiring antigen from apoptotic hepatocholangioma cells caused by γ-ray

    International Nuclear Information System (INIS)

    Wu Gang; Gu Hongguang; Han Benli; Pei Xuetao

    2002-01-01

    Objective: To investigate the induction of cytotoxic T lymphocytes (CTLs) in antitumor responsiveness and therapeutic effects after dendritic cells (DCs) acquired antigen from apoptotic hepatocholangioma cells. Methods: DCs from blood mononuclear cells that maintain the characteristics of immaturity-anti-gen-capturing and-processing capacity were established in vitro by using granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin-4. Then, apoptosis in hepatocholangioma cells was induced with γ-radiation. The experimental groups included (1) co-culture of DCs, and apoptotic cancer cells and T cells; (2) co-culture of DCs necrotic cancer cells and T cells; (3) co-culture of DCs-cultured cancer cell and T cells. These cells were co-cultured for 7 days. DCs and T cell were enriched separately. Finally, antitumor response test was carried out. Results: These cells had typical dendritic morphology, expressed high levels of CD1a, B7 and acquired antigen from apoptotic cells caused by γ-rays and induced an increased T cell-stimulatory capacity in MLR. Conclusions: DCs obtained from blood mononuclear cells using GM-CSF and IL-4 and DCs can efficiently present antigen driven from apoptotic cells caused by γ-rays and induce T cells increasing obviously. It can probably become an effective approach of DC transduction with antigen

  15. Cytotoxicity and apoptotic effects of nickel oxide nanoparticles in cultured HeLa cells

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    Kezban Ada

    2010-04-01

    Full Text Available The aim of this study was to observe the cytotoxicity and apoptotic effects of nickel oxide nanoparticles on humancervix epithelioid carcinoma cell line (HeLa. Nickel oxide precursors were synthesized by an nickel sulphate-excess ureareaction in boiling aqueous solution. The synthesized NiO nanoparticles (<200 nm were investigated by X-ray diffractionanalysis and transmission electron microscopy techniques. For cytotoxicity experiments, HeLa cells were incubated in50-500 μg/mL NiO for 2, 6, 12 and 16 hours. The viable cells were counted with a haemacytometer using light microscopy.The cytotoxicity was observed low in 50-200 μg/mL concentration for 16 h, but high in 400-500 μg/mL concentration for2-6 h. HeLa cells' cytoplasm membrane was lysed and detached from the well surface in 400 μg/mL concentration NiOnanoparticles. Double staining and M30 immunostaining were performed to quantify the number of apoptotic cells in cultureon the basis of apoptotic cell nuclei scores. The apoptotic effect was observed 20% for 16 h incubation.

  16. Induction of apoptotic death and retardation of neuronal differentiation of human neural stem cells by sodium arsenite treatment

    International Nuclear Information System (INIS)

    Ivanov, Vladimir N.; Hei, Tom K.

    2013-01-01

    Chronic arsenic toxicity is a global health problem that affects more than 100 million people worldwide. Long-term health effects of inorganic sodium arsenite in drinking water may result in skin, lung and liver cancers and in severe neurological abnormalities. We investigated in the present study whether sodium arsenite affects signaling pathways that control cell survival, proliferation and neuronal differentiation of human neural stem cells (NSC). We demonstrated that the critical signaling pathway, which was suppressed by sodium arsenite in NSC, was the protective PI3K–AKT pathway. Sodium arsenite (2–4 μM) also caused down-regulation of Nanog, one of the key transcription factors that control pluripotency and self-renewal of stem cells. Mitochondrial damage and cytochrome-c release induced by sodium arsenite exposure was followed by initiation of the mitochondrial apoptotic pathway in NSC. Beside caspase-9 and caspase-3 inhibitors, suppression of JNK activity decreased levels of arsenite-induced apoptosis in NSC. Neuronal differentiation of NSC was substantially inhibited by sodium arsenite exposure. Overactivation of JNK1 and ERK1/2 and down-regulation of PI3K–AKT activity induced by sodium arsenite were critical factors that strongly affected neuronal differentiation. In conclusion, sodium arsenite exposure of human NSC induces the mitochondrial apoptotic pathway, which is substantially accelerated due to the simultaneous suppression of PI3K–AKT. Sodium arsenite also negatively affects neuronal differentiation of NSC through overactivation of MEK–ERK and suppression of PI3K–AKT. - Highlights: ► Arsenite induces the mitochondrial apoptotic pathway in human neural stem cells. ► Arsenite-induced apoptosis is strongly upregulated by suppression of PI3K–AKT. ► Arsenite-induced apoptosis is strongly down-regulated by inhibition of JNK–cJun. ► Arsenite negatively affects neuronal differentiation by inhibition of PI3K–AKT

  17. Induction of apoptotic death and retardation of neuronal differentiation of human neural stem cells by sodium arsenite treatment

    Energy Technology Data Exchange (ETDEWEB)

    Ivanov, Vladimir N., E-mail: vni3@columbia.edu [Center for Radiological Research, Department of Radiation Oncology, College of Physicians and Surgeons, Columbia University, 630 West 168th Street, NY 10032 (United States); Hei, Tom K. [Center for Radiological Research, Department of Radiation Oncology, College of Physicians and Surgeons, Columbia University, 630 West 168th Street, NY 10032 (United States)

    2013-04-01

    Chronic arsenic toxicity is a global health problem that affects more than 100 million people worldwide. Long-term health effects of inorganic sodium arsenite in drinking water may result in skin, lung and liver cancers and in severe neurological abnormalities. We investigated in the present study whether sodium arsenite affects signaling pathways that control cell survival, proliferation and neuronal differentiation of human neural stem cells (NSC). We demonstrated that the critical signaling pathway, which was suppressed by sodium arsenite in NSC, was the protective PI3K–AKT pathway. Sodium arsenite (2–4 μM) also caused down-regulation of Nanog, one of the key transcription factors that control pluripotency and self-renewal of stem cells. Mitochondrial damage and cytochrome-c release induced by sodium arsenite exposure was followed by initiation of the mitochondrial apoptotic pathway in NSC. Beside caspase-9 and caspase-3 inhibitors, suppression of JNK activity decreased levels of arsenite-induced apoptosis in NSC. Neuronal differentiation of NSC was substantially inhibited by sodium arsenite exposure. Overactivation of JNK1 and ERK1/2 and down-regulation of PI3K–AKT activity induced by sodium arsenite were critical factors that strongly affected neuronal differentiation. In conclusion, sodium arsenite exposure of human NSC induces the mitochondrial apoptotic pathway, which is substantially accelerated due to the simultaneous suppression of PI3K–AKT. Sodium arsenite also negatively affects neuronal differentiation of NSC through overactivation of MEK–ERK and suppression of PI3K–AKT. - Highlights: ► Arsenite induces the mitochondrial apoptotic pathway in human neural stem cells. ► Arsenite-induced apoptosis is strongly upregulated by suppression of PI3K–AKT. ► Arsenite-induced apoptosis is strongly down-regulated by inhibition of JNK–cJun. ► Arsenite negatively affects neuronal differentiation by inhibition of PI3K–AKT.

  18. The Apoptotic Effects of the P300 Activator on Breast Cancer and Lung Fibroblast Cell Lines

    Directory of Open Access Journals (Sweden)

    Mohammad Reza Salahshoor

    2013-10-01

    Full Text Available Background: P300 is an enzyme that acetylates histones during stress. It alsoacetylates several non-histone proteins, including P53 which is the most important tumorsuppressor gene. P53 plays an important role in the apoptosis of tumor cells. Hereby,this study describes the potency of cholera toxin B subunit as a P300 activator to induceapoptosis in a breast cancer cell line (MCF-7 and a lung fibroblast cell line (MRC-5as a non-tumorigenic control sample. Methods: MCF-7 and MRC-5 were cultured in RPMI-1640 and treated with orwithout cholera toxin B subunit at the concentration of 85.43 μmol/L, based on the half-maximal inhibitory concentration index at different times (24, 48 and 72 h. Thepercentage of apoptotic cells was measured by flow cytometry. Real-time quantitativeRT-PCR was performed to estimate the mRNA expression of P300 in MCF-7 and MRC-5 with cholera toxin B subunit at different times. We used the ELISA and Bradford proteintechniques to detect levels of total and acetylated P53 protein generated in MCF-7 andMRC-5. Results: Our findings indicated that the cholera toxin B subunit effectively andsignificantly induced more apoptosis in MCF-7 compared to MRC-5. We showed thatexpression of P300 up-regulated by increasing the time of the cholera toxin B subunittreatment in MCF-7 but not in MRC-5. In addition, the acetylated and total P53protein levels increased more in MCF-7 cells than in MRC-5 cells.Conclusion: Cholera toxin B subunit induced significant cell death in MCF-7, butit could be well tolerated in MRC-5. Therefore, cholera toxin B subunit can besuggested as an anti-cancer agent.

  19. Multicolor fluorescence technique to detect apoptotic cells in advanced coronary atherosclerotic plaques

    Directory of Open Access Journals (Sweden)

    C Soldani

    2009-06-01

    Full Text Available Apoptosis occurring in atherosclerotic lesions has been suggested to be involved in the evolution and the structural stability of the plaques. It is still a matter of debate whether apoptosis mainly involves vascular smooth muscle cells (vSMCs in the fibrous tissue or inflammatory (namely foam cells, thus preferentially affecting the cell-poor lipid core of the atherosclerotic plaques. The aim of the present investigation was to detect the presence of apoptotic cells and to estimate their percentage in a series of atherosclerotic plaques obtained either by autopsy or during surgical atherectomy. Apoptotic cells were identified on paraffinembedded sections on the basis of cell nuclear morphology after DNA staining and/or by cytochemical reactions (TUNEL assay, immunodetection of the proteolytic poly (ADP-ribose polymerase-1 [PARP-1] fragment; biochemical procedures (identifying DNA fragmentation or PARP-1 proteolysis were also used. Indirect immunofluorescence techniques were performed to label specific antigens for either vSMCs or macrophages (i.e., the cells which are most likely prone to apoptosis in atherosclerotic lesions: the proper selection of fluorochrome labeling allowed the simultaneous detection of the cell phenotype and the apoptotic characteristics, by multicolor fluorescence techniques. Apoptotic cells proved to be less than 5% of the whole cell population, in atherosclerotic plaque sections: this is, in fact, a too low cell fraction to be detected by widely used biochemical methods, such as agarose gel electrophoresis of low-molecular-weight DNA or Western-blot analysis of PARP-1 degradation. Most apoptotic cells were of macrophage origin, and clustered in the tunica media, near or within the lipid-rich core; only a few TUNEL-positive cells were labeled for antigens specific for vSMCs. These results confirm that, among the cell populations in atherosclerotic plaques, macrophage foam-cells are preferentially involved in apoptosis

  20. Proteinase 3 on apoptotic cells disrupts immune silencing in autoimmune vasculitis

    Science.gov (United States)

    Millet, Arnaud; Martin, Katherine R.; Bonnefoy, Francis; Saas, Philippe; Mocek, Julie; Alkan, Manal; Terrier, Benjamin; Kerstein, Anja; Tamassia, Nicola; Satyanarayanan, Senthil Kumaran; Ariel, Amiram; Ribeil, Jean-Antoine; Guillevin, Loïc; Cassatella, Marco A.; Mueller, Antje; Thieblemont, Nathalie; Lamprecht, Peter; Mouthon, Luc; Perruche, Sylvain; Witko-Sarsat, Véronique

    2015-01-01

    Granulomatosis with polyangiitis (GPA) is a systemic necrotizing vasculitis that is associated with granulomatous inflammation and the presence of anti-neutrophil cytoplasmic antibodies (ANCAs) directed against proteinase 3 (PR3). We previously determined that PR3 on the surface of apoptotic neutrophils interferes with induction of antiinflammatory mechanisms following phagocytosis of these cells by macrophages. Here, we demonstrate that enzymatically active membrane-associated PR3 on apoptotic cells triggered secretion of inflammatory cytokines, including granulocyte CSF (G-CSF) and chemokines. This response required the IL-1R1/MyD88 signaling pathway and was dependent on the synthesis of NO, as macrophages from animals lacking these pathways did not exhibit a PR3-associated proinflammatory response. The PR3-induced microenvironment facilitated recruitment of inflammatory cells, such as macrophages, plasmacytoid DCs (pDCs), and neutrophils, which were observed in close proximity within granulomatous lesions in the lungs of GPA patients. In different murine models of apoptotic cell injection, the PR3-induced microenvironment instructed pDC-driven Th9/Th2 cell generation. Concomitant injection of anti-PR3 ANCAs with PR3-expressing apoptotic cells induced a Th17 response, revealing a GPA-specific mechanism of immune polarization. Accordingly, circulating CD4+ T cells from GPA patients had a skewed distribution of Th9/Th2/Th17. These results reveal that PR3 disrupts immune silencing associated with clearance of apoptotic neutrophils and provide insight into how PR3 and PR3-targeting ANCAs promote GPA pathophysiology. PMID:26436651

  1. Pro- and anti-apoptotic CD95 signaling in T cells

    Directory of Open Access Journals (Sweden)

    Janssen Ottmar

    2011-04-01

    Full Text Available Abstract The TNF receptor superfamily member CD95 (Fas, APO-1, TNFRSF6 is known as the prototypic death receptor in and outside the immune system. In fact, many mechanisms involved in apoptotic signaling cascades were solved by addressing consequences and pathways initiated by CD95 ligation in activated T cells or other "CD95-sensitive" cell populations. As an example, the binding of the inducible CD95 ligand (CD95L to CD95 on activated T lymphocytes results in apoptotic cell death. This activation-induced cell death was implicated in the control of immune cell homeostasis and immune response termination. Over the past years, however, it became evident that CD95 acts as a dual function receptor that also exerts anti-apoptotic effects depending on the cellular context. Early observations of a potential non-apoptotic role of CD95 in the growth control of resting T cells were recently reconsidered and revealed quite unexpected findings regarding the costimulatory capacity of CD95 for primary T cell activation. It turned out that CD95 engagement modulates TCR/CD3-driven signal initiation in a dose-dependent manner. High doses of immobilized CD95 agonists or cellular CD95L almost completely silence T cells by blocking early TCR-induced signaling events. In contrast, under otherwise unchanged conditions, lower amounts of the same agonists dramatically augment TCR/CD3-driven activation and proliferation. In the present overview, we summarize these recent findings with a focus on the costimulatory capacity of CD95 in primary T cells and discuss potential implications for the T cell compartment and the interplay between T cells and CD95L-expressing cells including antigen-presenting cells.

  2. Cordycepin enhances cisplatin apoptotic effect through caspase/MAPK pathways in human head and neck tumor cells

    Directory of Open Access Journals (Sweden)

    Chen YH

    2013-07-01

    -Jun NH2-terminal kinase, extracellular signal-regulated kinase, and p38 protein phosphorylations. Moreover, cordycepin plus cisplatin cotreatment significantly activated those proteins with much better effects among three cell lines. Conclusion: Cordycepin plus cisplatin have better apoptotic effect by activating caspase activation with possible MAPK pathway involvement in HNSCC cells. Keywords: cordycepin, cisplatin, apoptosis, caspase, MAPK, HNSCC

  3. Apoptotic block in colon cancer cells may be rectified by lentivirus mediated overexpression of caspase-9.

    Science.gov (United States)

    Xu, D; Wang, C; Shen, X; Yu, Y; Rui, Y; Zhang, D; Zhou, Z

    2013-12-01

    At present, the inhibition of apoptosis during pathogenesis of colorectal cancer is widely recognized while the role of caspase-9 in this process remains controversial. We aimed to investigate the differential expression of caspase-9 and evaluate the therapeutic potential of expression intervention in this study. We first examined the different expression of caspase-9 in normal colon mucosa, adenoma and cancer, investigating the relationship between its expression and clinico-pathological characteristics. Secondly, overexpression of caspase-9 was established in colon cancer cell lines by lentivirus infection to study the changes in growth, proliferation and apoptosis. Compared with normal colon mucosa, the expression of caspase-9 was higher in adenoma while lower in cancer both at mRNA and protein level (P expression is more common in poorly differentiated cancers (P expression of caspase-9, poorer colony formation and slower cell proliferation. In terms of apoptosis related indicators, caspase-9 overexpression leads to higher apoptosis rate and GO/G1 arrest, while up-regulating the expression of caspase-3 (P expression from colon mucosa, adenoma to cancer suggested it may be involved in the carcinogenesis of colon cancer. The overexpression of caspase-9 exhibits an inhibitory role in cancer growth and proliferation while promoting apoptosis. However, a non-apoptotic role of caspase-9 facilitating differentiation was also implied.

  4. Nerve growth factor reduces apoptotic cell death in rat facial motor neurons after facial nerve injury.

    Science.gov (United States)

    Hui, Lian; Yuan, Jing; Ren, Zhong; Jiang, Xuejun

    2015-01-01

    To assess the effects of nerve growth factor (NGF) on motor neurons after induction of a facial nerve lesion, and to compare the effects of different routes of NGF injection on motor neuron survival. This study was carried out in the Department of Otolaryngology Head & Neck Surgery, China Medical University, Liaoning, China from October 2012 to March 2013. Male Wistar rats (n = 65) were randomly assigned into 4 groups: A) healthy controls; B) facial nerve lesion model + normal saline injection; C) facial nerve lesion model + NGF injection through the stylomastoid foramen; D) facial nerve lesion model + intraperitoneal injection of NGF. Apoptotic cell death was detected using the terminal deoxynucleotidyl transferase dUTP nick end-labeling assay. Expression of caspase-3 and p53 up-regulated modulator of apoptosis (PUMA) was determined by immunohistochemistry. Injection of NGF significantly reduced cell apoptosis, and also greatly decreased caspase-3 and PUMA expression in injured motor neurons. Group C exhibited better efficacy for preventing cellular apoptosis and decreasing caspase-3 and PUMA expression compared with group D (pfacial nerve injury in rats. The NGF injected through the stylomastoid foramen demonstrated better protective efficacy than when injected intraperitoneally.

  5. In vitro evidence for participation of DEC-205 expressed by thymic cortical epithelial cells in clearance of apoptotic thymocytes.

    NARCIS (Netherlands)

    Small, M; Kraal, G.

    2003-01-01

    Binding of apoptotic cells was compared after incubation of thymocytes with two clones of murine thymic stromal cells to which CD4(+)/CD8(+) thymocytes attach. With the BA/10, but not the BA/2, clone, thymocytes with apoptotic morphology were bound irreversibly. These tightly bound thymocytes were

  6. An in vivo root hair assay for determining rates of apoptotic-like programmed cell death in plants

    Directory of Open Access Journals (Sweden)

    Hogg Bridget V

    2011-12-01

    Full Text Available Abstract In Arabidopsis thaliana we demonstrate that dying root hairs provide an easy and rapid in vivo model for the morphological identification of apoptotic-like programmed cell death (AL-PCD in plants. The model described here is transferable between species, can be used to investigate rates of AL-PCD in response to various treatments and to identify modulation of AL-PCD rates in mutant/transgenic plant lines facilitating rapid screening of mutant populations in order to identify genes involved in AL-PCD regulation.

  7. HSF-1 activates the ubiquitin proteasome system to promote non-apoptotic developmental cell death in C. elegans.

    Science.gov (United States)

    Kinet, Maxime J; Malin, Jennifer A; Abraham, Mary C; Blum, Elyse S; Silverman, Melanie R; Lu, Yun; Shaham, Shai

    2016-03-08

    Apoptosis is a prominent metazoan cell death form. Yet, mutations in apoptosis regulators cause only minor defects in vertebrate development, suggesting that another developmental cell death mechanism exists. While some non-apoptotic programs have been molecularly characterized, none appear to control developmental cell culling. Linker-cell-type death (LCD) is a morphologically conserved non-apoptotic cell death process operating in Caenorhabditis elegans and vertebrate development, and is therefore a compelling candidate process complementing apoptosis. However, the details of LCD execution are not known. Here we delineate a molecular-genetic pathway governing LCD in C. elegans. Redundant activities of antagonistic Wnt signals, a temporal control pathway, and mitogen-activated protein kinase kinase signaling control heat shock factor 1 (HSF-1), a conserved stress-activated transcription factor. Rather than protecting cells, HSF-1 promotes their demise by activating components of the ubiquitin proteasome system, including the E2 ligase LET-70/UBE2D2 functioning with E3 components CUL-3, RBX-1, BTBD-2, and SIAH-1. Our studies uncover design similarities between LCD and developmental apoptosis, and provide testable predictions for analyzing LCD in vertebrates.

  8. Foveolar cells phagocytose apoptotic neutrophils in chronic active Helicobacter pylori gastritis.

    Science.gov (United States)

    Caruso, R A; Fedele, F; Di Bella, C; Mazzon, E; Rigoli, L

    2012-11-01

    The recognition and removal of apoptotic inflammatory cells by tissue macrophages and non-professional phagocytes, in a process called efferocytosis, is required for resolution of inflammation and is actively anti-inflammatory. We have previously demonstrated phagocytosis of apoptotic neutrophils by tumor cells in human gastric carcinoma, but to date, there have been no studies investigating this process in chronic active Helicobacter pylori gastritis. Biopsy specimens from 28 subjects with or without H. pylori infection and active inflammation were examined and graded according to the updated Sydney system. Light microscopy, electron microscopy, and Terminal Deoxynucleotidyltransferase-Mediated UTP End Labeling staining were used to identify apoptosis. H. pylori infection was detected by histology and by molecular assay in 16 out of 28 cases. DNA from paraffin-embedded gastric biopsies was amplified using primers specific for cagA, for the cag "empty site" as well as for the s and m alleles of vacA. The more virulent cagA-positive strains were found in five out of nine patients with chronic active gastritis. The vacA s1/m1 and s2/m1 genotypes were more common in nine patients with chronic active gastritis, while the vacA s2/m2 genotype was more frequent in seven patients with chronic inactive gastritis. Apoptotic neutrophils were also detected within the cytoplasmic vacuoles of the foveolar cells of nine cases with chronic active gastritis. Transmission electron micrographs revealed further apoptotic neutrophils within spacious phagosomes of foveolar cells in a similar manner to those described in late-phase efferocytosis both in vivo and in vitro. These new observations expand the morphological spectrum of gastritis in patients infected with more virulent H. pylori strains, compatible with an anti-inflammatory role for the gastric epithelial cells in their removal of apoptotic neutrophils during active chronic gastritis.

  9. The extrinsic and intrinsic apoptotic pathways are involved in manganese toxicity in rat astrocytoma C6 cells.

    Science.gov (United States)

    Alaimo, Agustina; Gorojod, Roxana M; Kotler, Mónica L

    2011-08-01

    Manganese (Mn) is a trace element known to be essential for maintaining the proper function and regulation of many biochemical and cellular reactions. However, chronic exposure to high levels of Mn in occupational or environmental settings can lead to its accumulation in the brain resulting in a degenerative brain disorder referred to as Manganism. Astrocytes are the main Mn store in the central nervous system and several lines of evidence implicate these cells as major players in the role of Manganism development. In the present study, we employed rat astrocytoma C6 cells as a sensitive experimental model for investigating molecular mechanisms involved in Mn neurotoxicity. Our results show that C6 cells undergo reactive oxygen species-mediated apoptotic cell death involving caspase-8 and mitochondrial-mediated pathways in response to Mn. Exposed cells exhibit typical apoptotic features, such as chromatin condensation, cell shrinkage, membrane blebbing, caspase-3 activation and caspase-specific cleavage of the endogenous substrate poly (ADP-ribose) polymerase. Participation of the caspase-8 dependent pathway was assessed by increased levels of FasL, caspase-8 activation and Bid cleavage. The involvement of the mitochondrial pathway was demonstrated by the disruption of the mitochondrial membrane potential, the opening of the mitochondrial permeability transition pore, cytochrome c release, caspase-9 activation and the increased mitochondrial levels of the pro-apoptotic Bcl-2 family proteins. In addition, our data also shows for the first time that mitochondrial fragmentation plays a relevant role in Mn-induced apoptosis. Taking together, these findings contribute to a deeper elucidation of the molecular signaling mechanisms underlying Mn-induced apoptosis. Copyright © 2011 Elsevier B.V. All rights reserved.

  10. p53 dependent apoptotic cell death induces embryonic malformation in Carassius auratus under chronic hypoxia.

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    Paramita Banerjee Sawant

    Full Text Available Hypoxia is a global phenomenon affecting recruitment as well as the embryonic development of aquatic fauna. The present study depicts hypoxia induced disruption of the intrinsic pathway of programmed cell death (PCD, leading to embryonic malformation in the goldfish, Carrasius auratus. Constant hypoxia induced the early expression of pro-apoptotic/tumor suppressor p53 and concomitant expression of the cell death molecule, caspase-3, leading to high level of DNA damage and cell death in hypoxic embryos, as compared to normoxic ones. As a result, the former showed delayed 4 and 64 celled stages and a delay in appearance of epiboly stage. Expression of p53 efficiently switched off expression of the anti-apoptotic Bcl-2 during the initial 12 hours post fertilization (hpf and caused embryonic cell death. However, after 12 hours, simultaneous downregulation of p53 and Caspase-3 and exponential increase of Bcl-2, caused uncontrolled cell proliferation and prevented essential programmed cell death (PCD, ultimately resulting in significant (p<0.05 embryonic malformation up to 144 hpf. Evidences suggest that uncontrolled cell proliferation after 12 hpf may have been due to downregulation of p53 abundance, which in turn has an influence on upregulation of anti-apoptotic Bcl-2. Therefore, we have been able to show for the first time and propose that hypoxia induced downregulation of p53 beyond 12 hpf, disrupts PCD and leads to failure in normal differentiation, causing malformation in gold fish embryos.

  11. Regulation of apoptotic pathways by Stylophora pistillata (Anthozoa, Pocilloporidae to survive thermal stress and bleaching.

    Directory of Open Access Journals (Sweden)

    Hagit Kvitt

    Full Text Available Elevated seawater temperatures are associated with coral bleaching events and related mortality. Nevertheless, some coral species are able to survive bleaching and recover. The apoptotic responses associated to this ability were studied over 3 years in the coral Stylophora pistillata from the Gulf of Eilat subjected to long term thermal stress. These include caspase activity and the expression profiles of the S. pistillata caspase and Bcl-2 genes (StyCasp and StyBcl-2-like cloned in this study. In corals exposed to thermal stress (32 or 34°C, caspase activity and the expression levels of the StyBcl-2-like gene increased over time (6-48 h and declined to basal levels within 72 h of thermal stress. Distinct transcript levels were obtained for the StyCasp gene, with stimulated expression from 6 to 48 h of 34°C thermal stress, coinciding with the onset of bleaching. Increased cell death was detected in situ only between 6 to 48 h of stress and was limited to the gastroderm. The bleached corals survived up to one month at 32°C, and recovered back symbionts when placed at 24°C. These results point to a two-stage response in corals that withstand thermal stress: (i the onset of apoptosis, accompanied by rapid activation of anti-oxidant/anti-apoptotic mediators that block the progression of apoptosis to other cells and (ii acclimatization of the coral to the chronic thermal stress alongside the completion of symbiosis breakdown. Accordingly, the coral's ability to rapidly curb apoptosis appears to be the most important trait affecting the coral's thermotolerance and survival.

  12. Akebia saponin PA induces autophagic and apoptotic cell death in AGS human gastric cancer cells.

    Science.gov (United States)

    Xu, Mei-Ying; Lee, Dong Hwa; Joo, Eun Ji; Son, Kun Ho; Kim, Yeong Shik

    2013-09-01

    In this study, we investigated the anticancer mechanism of akebia saponin PA (AS), a natural product isolated from Dipsacus asperoides in human gastric cancer cell lines. It was shown that AS-induced cell death is caused by autophagy and apoptosis in AGS cells. The apoptosis-inducing effect of AS was characterized by annexin V/propidium (PI) staining, increase of sub-G1 phase and caspase-3 activation, while the autophagy-inducing effect was indicated by the formation of cytoplasmic vacuoles and microtubule-associated protein 1 light chain-3 II (LC3-II) conversion. The autophagy inhibitor bafilomycin A1 (BaF1) decreased AS-induced cell death and caspase-3 activation, but caspase-3 inhibitor Ac-DEVD-CHO did not affect LC3-II accumulation or AS-induced cell viability, suggesting that AS induces autophagic cell death and autophagy contributes to caspase-3-dependent apoptosis. Furthermore, AS activated p38/c-Jun N-terminal kinase (JNK), which could be inhibited by BaF1, and caspase-3 activation was attenuated by both SB202190 and SP600125, indicating that AS-induced autophagy promotes mitogen-activated protein kinases (MAPKs)-mediated apoptosis. Taken together, these results demonstrate that AS induces autophagic and apoptotic cell death and autophagy plays the main role in akebia saponin PA-induced cell death. Copyright © 2013 Elsevier Ltd. All rights reserved.

  13. Reemergence of apoptotic cells between fractionated doses in irradiated murine tumors

    International Nuclear Information System (INIS)

    Meyn, R.E.; Hunter, N.R.; Milas, L.

    1994-01-01

    The purpose of this investigation was to follow up our previous studies on the development of apoptosis in irradiated murine tumors by testing whether an apoptotic subpopulation of cells reemerges between fractionated exposures. Mice bearing a murine ovarian carcinoma, OCa-I, were treated in vivo with two fractionation protocols: two doses of 12.5 Gy separated by various times out to 5 days and multiple daily fractions of 2.5 Gy. Animals were killed 4 h after the last dose in each protocol, and the percent apoptosis was scored from stained histological sections made from the irradiated tumors according to the specific features characteristic of this mode of cell death. The 12.5+12.5 Gy protocol yielded a net total percent apoptosis of about 45% when the two doses were separated by 5 days (total dose = 25 Gy), whereas the 2.5 Gy per day protocol yielded about 50% net apoptotic cells when given for 5 days (total dose = 12.5 Gy). These values are to be compared to the value of 36% apoptotic cells that is yielded by large single doses (> 25 Gy). Thus, these results indicate that an apoptotic subpopulation of cells reemerged between the fractions in both protocols, but the kinetics appeared to be delayed in the 12.5+12.5 Gy vs. the multiple 2.5 Gy protocol. This reemergence of cells with the propensity for radiation-induced apoptosis between fractionated exposures is consistent with a role for this mode of cell death in the response of tumors to radiotherapy and may represent the priming of a new subpopulation of tumor cells for apoptosis as part of normal tumor homeostasis to counterbalance cell division. 25 refs., 3 figs., 1 tab

  14. PKCη confers protection against apoptosis by inhibiting the pro-apoptotic JNK activity in MCF-7 cells

    International Nuclear Information System (INIS)

    Rotem-Dai, Noa; Oberkovitz, Galia; Abu-Ghanem, Sara; Livneh, Etta

    2009-01-01

    Apoptosis is frequently regulated by different protein kinases including protein kinase C family enzymes. Both inhibitory and stimulatory effects were demonstrated for several of the different PKC isoforms. Here we show that the novel PKC isoform, PKCη, confers protection against apoptosis induced by the DNA damaging agents, UVC irradiation and the anti-cancer drug - Camptothecin, of the breast epithelial adenocarcinoma MCF-7 cells. The induced expression of PKCη in MCF-7 cells, under the control of the tetracycline-responsive promoter, resulted in increased cell survival and inhibition of cleavage of the apoptotic marker PARP-1. Activation of caspase-7 and 9 and the release of cytochrome c were also inhibited by the inducible expression of PKCη. Furthermore, JNK activity, required for apoptosis in MCF-7, as indicated by the inhibition of both caspase-7 cleavage and cytochrome c release from the mitochondria in the presence of the JNK inhibitor SP600125, was also suppressed by PKCη expression. Hence, in contrast to most PKC isoforms enhancing JNK activation, our studies show that PKCη is an anti-apoptotic protein, acting as a negative regulator of JNK activity. Thus, PKCη could represent a target for intervention aimed to reduce resistance to anti-cancer treatments.

  15. Kinetics of apoptotic markers in exogeneously induced apoptosis of EL4 cells.

    Science.gov (United States)

    Jessel, Robert; Haertel, Steffen; Socaciu, Carmen; Tykhonova, Svetlana; Diehl, Horst A

    2002-01-01

    We investigated the time-dependence of apoptotic events in EL4 cells by monitoring plasma membrane changes in correlation to DNA fragmentation and cell shrinkage. We applied three apoptosis inducers (staurosporine, tubericidine and X-rays) and we looked at various markers to follow the early-to-late apoptotic events: phospholipid translocation (identified through annexin V-fluorescein assay and propidium iodide), lipid package (via merocyanine assay), membrane fluidity and anisotropy (via fluorescent measurements), DNA fragmentation by the fluorescence-labeling test and cell size measurements. The different apoptotic inducers caused different reactions of the cells: staurosporine induced apoptosis most rapidly in a high number of cells, tubercidine triggered apoptosis only in the S phase cells, while X-rays caused a G2/M arrest and subsequently apoptosis. Loss of lipid asymmetry is promptly detectable after one hour of incubation time. The phosphatidylserine translocation, decrease of lipid package and anisotropy, and the increase of membrane fluidity appeared to be based on the same process of lipid asymmetry loss. Therefore, the DNA fragmentation and the cell shrinkage appear to be parallel and independent processes running on different time scales but which are kinetically inter-related. The results indicate different signal steps to apoptosis dependent on inducer characteristics but the kinetics of "early-to-late" apoptosis appears to be a fixed program.

  16. Manganese induces mitochondrial dynamics impairment and apoptotic cell death: a study in human Gli36 cells.

    Science.gov (United States)

    Alaimo, Agustina; Gorojod, Roxana M; Miglietta, Esteban A; Villarreal, Alejandro; Ramos, Alberto J; Kotler, Mónica L

    2013-10-25

    Manganese (Mn) is an essential trace element due to its participation in many physiological processes. However, overexposure to this metal leads to a neurological disorder known as Manganism whose clinical manifestations and molecular mechanisms resemble Parkinson's disease. Several lines of evidence implicate astrocytes as an early target of Mn neurotoxicity being the mitochondria the most affected organelles. The aim of this study was to investigate the possible mitochondrial dynamics alterations in Mn-exposed human astrocytes. Therefore, we employed Gli36 cells which express the astrocytic markers GFAP and S100B. We demonstrated that Mn triggers the mitochondrial apoptotic pathway revealed by increased Bax/Bcl-2 ratio, by the loss of mitochondrial membrane potential and by caspase-9 activation. This apoptotic program may be in turn responsible of caspase-3/7 activation, PARP-1 cleavage, chromatin condensation and fragmentation. In addition, we determined that Mn induces deregulation in mitochondria-shaping proteins (Opa-1, Mfn-2 and Drp-1) expression levels in parallel with the disruption of the mitochondrial network toward to an exacerbated fragmentation. Since mitochondrial dynamics is altered in several neurodegenerative diseases, these proteins could become future targets to be considered in Manganism treatment. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  17. Apoptotic Cell Death Induced by Resveratrol Is Partially Mediated by the Autophagy Pathway in Human Ovarian Cancer Cells.

    Directory of Open Access Journals (Sweden)

    Fangfang Lang

    Full Text Available Resveratrol (trans-3,4,5'-trihydroxystilbene is an active compound in food, such as red grapes, peanuts, and berries. Resveratrol exhibits an anticancer effect on various human cancer cells. However, the mechanism of resveratrol-induced anti-cancer effect at the molecular level remains to be elucidated. In this study, the mechanism underlying the anti-cancer effect of resveratrol in human ovarian cancer cells (OVCAR-3 and Caov-3 was investigated using various molecular biology techniques, such as flow cytometry, western blotting, and RNA interference, with a major focus on the potential role of autophagy in resveratrol-induced apoptotic cell death. We demonstrated that resveratrol induced reactive oxygen species (ROS generation, which triggers autophagy and subsequent apoptotic cell death. Resveratrol induced ATG5 expression and promoted LC3 cleavage. The apoptotic cell death induced by resveratrol was attenuated by both pharmacological and genetic inhibition of autophagy. The autophagy inhibitor chloroquine, which functions at the late stage of autophagy, significantly reduced resveratrol-induced cell death and caspase 3 activity in human ovarian cancer cells. We also demonstrated that targeting ATG5 by siRNA also suppressed resveratrol-induced apoptotic cell death. Thus, we concluded that a common pathway between autophagy and apoptosis exists in resveratrol-induced cell death in OVCAR-3 human ovarian cancer cells.

  18. Sesamol induced apoptotic effect in lung adenocarcinoma cells through both intrinsic and extrinsic pathways.

    Science.gov (United States)

    Siriwarin, Boondaree; Weerapreeyakul, Natthida

    2016-07-25

    Sesamol is a phenolic lignan found in sesame seeds (Sesamum indicum L.) and sesame oil. The anticancer effects and molecular mechanisms underlying its apoptosis-inducing effect were investigated in human lung adenocarcinoma (SK-LU-1) cells. Sesamol inhibited SK-LU-1 cell growth with an IC50 value of 2.7 mM and exhibited less toxicity toward normal Vero cells after 48 h of treatment (Selective index = 3). Apoptotic bodies-the hallmark of apoptosis-were observed in sesamol-treated SK-LU-1 cells, stained with DAPI. Sesamol increased the activity of caspase 8, 9, and 3/7, indicating that apoptotic cell death occurred through both extrinsic and intrinsic pathways. Sesamol caused the loss of mitochondrial transmembrane potential signifying intrinsic apoptosis induction. Decreasing Bid expression revealed crosstalk between the intrinsic and extrinsic apoptotic pathways; demonstrating clearly that sesamol induces apoptosis through both pathways in human lung adenocarcinoma (SK-LU-1) cells. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  19. Increased endothelial apoptotic cell density in human diabetic erectile tissue--comparison with clinical data.

    Science.gov (United States)

    Costa, Carla; Soares, Raquel; Castela, Angela; Adães, Sara; Hastert, Véronique; Vendeira, Pedro; Virag, Ronald

    2009-03-01

    Erectile dysfunction (ED) is a common complication of diabetes. Endothelial cell (EC) dysfunction is one of the main mechanisms of diabetic ED. However, loss of EC integrity has never been assessed in human diabetic corpus cavernosum. To identify and quantify apoptotic cells in human diabetic and normal erectile tissue and to compare these results with each patient's clinical data and erection status. Eighteen cavernosal samples were collected, 13 from diabetics with ED and 5 from nondiabetic individuals. Cavernosal structure and cell proliferation status were evaluated by immunohistochemistry. Tissue integrity was assessed by terminal transferase dUTP nick end labeling assay, an index of apoptotic cell density (ACD) established and compared with each patient age, type of diabetes, arterial risk factors number, arterial/veno-occlusive disease, response to intracavernous vasoactive injections (ICI), and penile nitric oxide release test (PNORT). Establish an index of ACD and correlate those results with patient clinical data. Nondiabetic samples presented few scattered cells in apoptosis and an ACD of 7.15 +/- 0.44 (mean apoptotic cells/tissue area mm(2) +/- standard error). The diabetic group showed an increased ACD of 23.82 +/- 1.53, and apoptotic cells were located specifically at vascular sites. Rehabilitation of these endothelial lesions seemed impaired, as no evidence of EC proliferation was observed. Furthermore, higher ACD in diabetic individuals correlated to poor response to PNORT and to ICI. We provided evidence for the first time that loss of cavernosal EC integrity is a crucial event involved in diabetic ED. Furthermore, we were able to establish a threshold between ACD values and cavernosal tissue functionality, as assessed by PNORT and vasoactive ICI.

  20. Ellagic acid radiosensitizes tumor cells by evoking apoptotic pathway

    International Nuclear Information System (INIS)

    Ahire, Vidhula R.; Mishra, K.P.

    2016-01-01

    Cancer causes millions of deaths each year globally. In most patients, the cause of treatment failure is found associated with the resistance to chemotherapy and radiotherapy. The development of tumor cell resistance evokes multiple intracellular molecular pathways. In addition, the limitation in treatment outcome arises due to unintended cytotoxic effects of the synthetic anticancer drugs to normal cells and tissues. Considerable focus of research is, therefore, devoted to examine plant-based herbal compounds which may prove potential anticancer drug for developing effective cancer therapy. Research results from our laboratory have shown that ellagic acid (EA), a natural flavonoid displays enhanced tumor toxicity in combination with gamma radiation to many types of cancers in vitro as well as in vivo. Studies on the underlying mechanisms of toxicity suggest that EA employs the cellular signaling pathways in producing the observed effects. This paper gives an account of molecular mechanisms of EA-induced apoptosis process in tumor cytotoxicity. It is suggested that EA acts as a novel radiosensitizer for tumors and a radioprotector for normal cells which may offer a novel protocol for cancer treatment. (author)

  1. Apoptotic intrinsic pathway proteins predict survival in canine cutaneous mast cell tumours.

    Science.gov (United States)

    Barra, C N; Macedo, B M; Cadrobbi, K G; Pulz, L H; Huete, G C; Kleeb, S R; Xavier, J G; Catão-Dias, J L; Nishiya, A T; Fukumasu, H; Strefezzi, R F

    2018-03-01

    Mast cell tumours (MCTs) are the most frequent canine round cell neoplasms and show variable biological behaviours with high metastatic and recurrence rates. The disease is treated surgically and wide margins are recommended. Adjuvant chemotherapy and radiotherapy used in this disease cause DNA damage in neoplastic cells, which is aimed to induce apoptotic cell death. Resisting cell death is a hallmark of cancer, which contributes to the development and progression of tumours. The aim of this study was to investigate the expression of the proteins involved in the apoptotic intrinsic pathway and to evaluate their potential use as prognostic markers for canine cutaneous MCTs. Immunohistochemistry for BAX, BCL2, APAF1, Caspase-9, and Caspase-3 was performed in 50 canine cases of MCTs. High BAX expression was associated with higher mortality rate and shorter survival. BCL2 and APAF1 expressions offered additional prognostic information to the histopathological grading systems. The present results indicate that variations in the expression of apoptotic proteins are related to malignancy of cutaneous MCTs in dogs. © 2017 John Wiley & Sons Ltd.

  2. ARP2, a novel pro-apoptotic protein expressed in epithelial prostate cancer LNCaP cells and epithelial ovary CHO transformed cells.

    Science.gov (United States)

    Mas-Oliva, Jaime; Navarro-Vidal, Enrique; Tapia-Vieyra, Juana Virginia

    2014-01-01

    Neoplastic epithelial cells generate the most aggressive types of cancers such as those located in the lung, breast, colon, prostate and ovary. During advanced stages of prostate cancer, epithelial cells are associated to the appearance of androgen-independent tumors, an apoptotic-resistant phenotype that ultimately overgrows and promotes metastatic events. We have previously identified and electrophysiologically characterized a novel Ca(2+)-permeable channel activated during apoptosis in the androgen-independent prostate epithelial cancer cell line, LNCaP. In addition, we reported for the first time the cloning and characterization of this channel-like molecule named apoptosis regulated protein 2 (ARP2) associated to a lethal influx of Ca(2+) in Xenopus oocytes. In the present study, LNCaP cells and Chinese hamster ovary cells (CHO cell line) transfected with arp2-cDNA are induced to undergo apoptosis showing an important impact on cell viability and activation of caspases 3 and 7 when compared to serum deprived grown cells and ionomycin treated cells. The subcellular localization of ARP2 in CHO cells undergoing apoptosis was studied using confocal microscopy. While apoptosis progresses, ARP2 initially localized in the peri-nuclear region of cells migrates with time towards the plasma membrane region. Based on the present results and those of our previous studies, the fact that ARP2 constitutes a novel cation channel is supported. Therefore, ARP2 becomes a valuable target to modulate the influx and concentration of calcium in the cytoplasm of epithelial cancer cells showing an apoptotic-resistant phenotype during the onset of an apoptotic event.

  3. Nitric oxide and calcium ions in apoptotic esophageal carcinoma cells induced by arsenite

    Science.gov (United States)

    Shen, Zhong-Ying; Shen, Wen-Ying; Chen, Ming-Hua; Shen, Jian; Cai, Wei-Jie; Yi, Zeng

    2002-01-01

    AIM: To Quantitatively analyze the nitri oxide (NO) and Ca2+ in apoptosis of esophageal carcinoma cells induced by arsenic trioxide (As2O3). METHODS: The cell line SHEEC1, a malignant esophageal epithelial cell induced by HPV in synergy with TPA in our laboratory, was cultured in a serum-free medium and treated with As2O3. Before and after administration of As2O3, NO production in cultured medium was detected quantitatively using the Griess Colorimetric method. Intracellular Ca2+ was labeled by using the fluorescent dye Fluo3-AM and detected under confocal laser scanning microscope (CLSM), which was able to acquire data in real-time enabling Ca2+ dynamics of individual cells in vitro. The apoptotic cells were examined under electron microscopy. RESULTS: Intracellular concentration of Ca2+ increased from 1.00 units to 1.09-1.38 units of fluorescent intensity at As2O3 treatment and NO products subsequently released from As2O3-treated cells increased from 0.98-1.00 × 10-2 μmol·L-1 up to 1.48-1.52 × 10-2 μmol·L-1 and maintained in a high level continuously. Finally apoptosis of cells occurred, chromatin being agglutinated, cells shrunk, nuclei became round and mitochondria swelled. CONCLUSION: Ca2+ and NO increased with cell damage and apoptosis in cells treated by As2O3. The Ca2+ is an initial messenger to the apoptotic pathway. To investigate Ca2+ and NO will be a new direction for studying the apoptotic signaling messenger of the esophageal carcinoma cells induced by As2O3. PMID:11833068

  4. Parallel single-cell analysis of active caspase-3/7 in apoptotic and non-apoptotic cells

    Czech Academy of Sciences Publication Activity Database

    Ledvina, Vojtěch; Janečková, Eva; Matalová, Eva; Klepárník, Karel

    2017-01-01

    Roč. 409, č. 1 (2017), s. 269-274 ISSN 1618-2642 R&D Projects: GA ČR(CZ) GA14-28254S Institutional support: RVO:68081715 ; RVO:67985904 Keywords : single-cell analysis * bioluminescence * apoptosis * caspase-3/7 Subject RIV: CB - Analytical Chemistry , Separation; EB - Genetics ; Molecular Biology (UZFG-Y) OBOR OECD: Analytical chemistry ; Developmental biology (UZFG-Y) Impact factor: 3.431, year: 2016

  5. Single cell analysis of caspase-3 in apoptotic and non-apoptotic cells during mouse limb development

    Czech Academy of Sciences Publication Activity Database

    Adamová, Eva; Klepárník, Karel; Matalová, E.

    2014-01-01

    Roč. 3, - (2014), PP58 ISSN 2052-1219. [European Calcified Tissue Society Congress /41./. 17.05.2014-20.05.2014, Praha] R&D Projects: GA ČR GAP206/11/2377; GA ČR(CZ) GA14-28254S Institutional support: RVO:68081715 Keywords : single cell analysis * caspase-3 * mouse limb development Subject RIV: CB - Analytical Chemistry, Separation

  6. Parallel single-cell analysis of active caspase-3/7 in apoptotic and non-apoptotic cells

    Czech Academy of Sciences Publication Activity Database

    Ledvina, Vojtěch; Janečková, Eva; Matalová, Eva; Klepárník, Karel

    2017-01-01

    Roč. 409, č. 1 (2017), s. 269-274 ISSN 1618-2642 R&D Projects: GA ČR(CZ) GA14-28254S Institutional support: RVO:68081715 ; RVO:67985904 Keywords : single-cell analysis * bioluminescence * apoptosis * caspase-3/7 Subject RIV: CB - Analytical Chemistry, Separation; EB - Genetics ; Molecular Biology (UZFG-Y) OBOR OECD: Analytical chemistry; Developmental biology (UZFG-Y) Impact factor: 3.431, year: 2016

  7. In vitro assays for predicting tumor cell response to radiation by apoptotic pathways

    International Nuclear Information System (INIS)

    Algan, Oe.; Hanks, G.E.; Biade, S.; Chapman, J.D.

    1995-01-01

    Purpose: We had previously shown that the rate of spontaneous and radiation-induced apoptosis was significantly greater in well-differentiated compared to anaplastic Dunning prostate carcinomas. The goal of this study was to define the most useful assay for quantifying radiation-induced apoptotic cell death and to determine if measured rates of radiation-induced apoptosis in tumor cell populations can predict treatment outcome. Materials and Methods: The time course and extent of radiation-induced apoptosis after single doses of Cesium-137 gamma-rays were measured by five different assays. These included gross DNA degradation, nucleosome ladder formation, labeling of 3'-OH ends in DNA with an immunofluorescence probe, immunofluorescence vital stains (LIVE/DEAD[reg] EUKOLIGHT TM ) and trypan blue. The majority of these studies were performed with DU-145 human prostate cells. Data was analyzed to determine the component of cell inactivation resulting from apoptosis with the modified linear quadratic equation, -1n (SF) = (α a + α p ) D + β p D 2 , were α a represents cell inactivation by radiation-induced apoptosis, α p and β p represent cell death by proliferative mechanisms and D represents radiation dose. Results: These studies indicated that DU-145 cell death after radiation occurs over two distinct time periods. The first phase of death begins shortly after irradiation and plateaus within 16-24 hr. This process of cell death has properties consistent with apoptosis as determined by 3'-OH DNA end-labeling and nucleosome ladder assays. The second phase of cell death (determined by viability staining) begins approximately 48 hr after irradiation and continues until the remainder of inactivated cells express their death. This longer phase of cell inactivation probably represents proliferative cell death and other non-apoptotic mechanisms. The five different assays were performed on DU-145 cells 24 hr after irradiation with 10 Gy. Significant nucleosome ladders

  8. Leptin suppresses non-apoptotic cell death in ischemic rat cardiomyocytes by reduction of iPLA{sub 2} activity

    Energy Technology Data Exchange (ETDEWEB)

    Takatani-Nakase, Tomoka, E-mail: nakase@mukogawa-u.ac.jp; Takahashi, Koichi, E-mail: koichi@mukogawa-u.ac.jp

    2015-07-17

    Caspase-independent, non-apoptotic cell death is an important therapeutic target in myocardial ischemia. Leptin, an adipose-derived hormone, is known to exhibit cytoprotective effects on the ischemic heart, but the mechanisms are poorly understood. In this research, we found that pretreatment of leptin strongly suppressed ischemic-augmented nuclear shrinkage and non-apoptotic cell death on cardiomyocytes. Leptin was also shown to significantly inhibit the activity of iPLA{sub 2}, which is considered to play crucial roles in non-apoptotic cell death, resulting in effective prevention of ischemia-induced myocyte death. These findings provide the first evidence of a protective mechanism of leptin against ischemia-induced non-apoptotic cardiomyocyte death. - Highlights: • Myocardial ischemia-model induces in caspase-independent, non-apoptotic cell death. • Leptin strongly inhibits ischemic-augmented non-apoptotic cell death. • Leptin reduces iPLA{sub 2} activity, leading to avoidance of non-apoptotic cell death.

  9. Leptin suppresses non-apoptotic cell death in ischemic rat cardiomyocytes by reduction of iPLA2 activity

    International Nuclear Information System (INIS)

    Takatani-Nakase, Tomoka; Takahashi, Koichi

    2015-01-01

    Caspase-independent, non-apoptotic cell death is an important therapeutic target in myocardial ischemia. Leptin, an adipose-derived hormone, is known to exhibit cytoprotective effects on the ischemic heart, but the mechanisms are poorly understood. In this research, we found that pretreatment of leptin strongly suppressed ischemic-augmented nuclear shrinkage and non-apoptotic cell death on cardiomyocytes. Leptin was also shown to significantly inhibit the activity of iPLA 2 , which is considered to play crucial roles in non-apoptotic cell death, resulting in effective prevention of ischemia-induced myocyte death. These findings provide the first evidence of a protective mechanism of leptin against ischemia-induced non-apoptotic cardiomyocyte death. - Highlights: • Myocardial ischemia-model induces in caspase-independent, non-apoptotic cell death. • Leptin strongly inhibits ischemic-augmented non-apoptotic cell death. • Leptin reduces iPLA 2 activity, leading to avoidance of non-apoptotic cell death

  10. miR-130a activates apoptotic signaling through activation of caspase-8 in taxane-resistant prostate cancer cells.

    Science.gov (United States)

    Fujita, Yasunori; Kojima, Toshio; Kawakami, Kyojiro; Mizutani, Kosuke; Kato, Taku; Deguchi, Takashi; Ito, Masafumi

    2015-10-01

    The acquisition of drug resistance is one of the most malignant phenotypes of cancer and identification of its therapeutic target is a prerequisite for the development of novel therapy. MicroRNAs (miRNAs) have been implicated in various types of cancer and proposed as potential therapeutic targets for patients. In the present study, we aimed to identify miRNA that could serve as a therapeutic target for taxane-resistant prostate cancer. In order to identify miRNAs related to taxane-resistance, miRNA profiling was performed using prostate cancer PC-3 cells and paclitaxel-resistant PC-3 cell lines established from PC-3 cells. Microarray analysis of mRNA expression was also conducted to search for potential target genes of miRNA. Luciferase reporter assay was performed to examine miRNA binding to the 3'-UTR of target genes. The effects of ectopic expression of miRNA on cell growth, tubulin polymerization, drug sensitivity, and apoptotic signaling pathway were investigated in a paclitaxel-resistant PC-3 cell line. The expression of miR-130a was down-regulated in all paclitaxel-resistant cell lines compared with parental PC-3 cells. Based on mRNA microarray analysis and luciferase reporter assay, we identified SLAIN1 as a direct target gene for miR-130a. Transfection of a miR-130a precursor into a paclitaxel-resistant cell line suppressed cell growth and increased the sensitivity to paclitaxel. Lastly, ectopic expression of miR-130a did not affect the polymerized tubulin level, but activated apoptotic signaling through activation of caspase-8. Our results suggested that reduced expression of miR-130a may be involved in the paclitaxel-resistance and that miR-130a could be a therapeutic target for taxane-resistant prostate cancer patients. © 2015 Wiley Periodicals, Inc.

  11. Cytotoxic and Pro-Apoptotic Effects of Honey Bee Venom and Chrysin on Human Ovarian Cancer Cells

    Directory of Open Access Journals (Sweden)

    Elaheh Amini

    2015-06-01

    Full Text Available Background: The anti-cancer effects of honey bee venom (BV and chrysin might open a new window for treatment of chemo-resistant cancers. This study was designed to evaluate cytotoxic and pro-apoptotic effects of BV and chrysin on A2780cp cistplatin- resistant human ovarian cancer cells. Methods: As per the study objectives, A2780cp cells were categorized to 4 groups: 3 experiment groups (treated either with BV or chrysin or BV + chrysin and 1 control group (untreated cells.  Experiment group cells were cultured and treated by different concentrations of BV and chrysin for 24 hours. Then, experiment and control cells were studied with MTT assay, Annexin V-FITC, DAPI and Acridine Orange / Propidium Iodide statining, flow cytometry, caspase-3 and -9 assay, measurement of intracellular level of reactive oxygen species (ROS and RT-PCR. Results: MTT assay showed that 8 μg/mL BV, 40 µg/ml chrysin and 6 + 15 μg/mL BV + chrysin co-treatment induced 50% cell death on A2780cp cells compared with controls (P < 0.001. Morphological observations by inverted and fluorescent microscopy revealed ROS generation and apoptotic cell death under exposure to BV or chrysin or BV + chrysin co-treatment. Caspase-3 and -9 assay demonstrated that BV and chrysin triggered apoptosis through intrinsic pathway and RT-PCR demonstrated down-regulation of Bcl-2. Conclusion: Honey bee venom and chrysin are effective for destroying chemoresistant ovarian cancer cells through activation of intrinsic apoptosis, which propose them as potential candidates to be used in development of improved chemotherapeutic agents in the future.

  12. Reactive oxygen species are key mediators of the nitric oxide apoptotic pathway in anterior pituitary cells.

    Science.gov (United States)

    Machiavelli, Leticia I; Poliandri, Ariel H; Quinteros, Fernanda A; Cabilla, Jimena P; Duvilanski, Beatriz H

    2007-03-01

    We previously showed that long-term exposure of anterior pituitary cells to nitric oxide (NO) induces apoptosis. The intracellular signals underlying this effect remained unclear. In this study, we searched for possible mechanisms involved in the early stages of the NO apoptotic cascade. Caspase 3 was activated by NO with no apparent disruption of mitochondrial membrane potential. NO caused a rapid increase of reactive oxygen species (ROS), and this increase seems to be dependent of mitochondrial electron transport chain. The antioxidant N-acetyl-cysteine avoided ROS increase, prevented the NO-induced caspase 3 activation, and reduced the NO apoptotic effect. Catalase was inactivated by NO, while glutathione peroxidase (GPx) activity and reduced glutathione (GSH) were not modified at first, but increased at later times of NO exposure. The increase of GSH level is important for the scavenging of the NO-induced ROS overproduction. Our results indicate that ROS have an essential role as a trigger of the NO apoptotic cascade in anterior pituitary cells. The permanent inhibition of catalase may strengthen the oxidative damage induced by NO. GPx activity and GSH level augment in response to the oxidative damage, though this increase seems not to be enough to rescue the cells from the NO effect.

  13. Apoptotic-cell-derived membrane microparticles and IFN-α induce an inflammatory immune response.

    Science.gov (United States)

    Niessen, Anna; Heyder, Petra; Krienke, Stefan; Blank, Norbert; Tykocinski, Lars-Oliver; Lorenz, Hanns-Martin; Schiller, Martin

    2015-07-15

    A dysregulation in the clearance of apoptotic material is considered a major pathogenetic factor for the emergence of autoimmune diseases. Apoptotic-cell-derived membrane microparticles (AdMPs), which are released from the cell surface during apoptosis, have been implicated in the pathogenesis of autoimmunity. Also of importance are cytokines, such as interferon-α (IFN-α), which is known to be a major player in patients with systemic lupus erythematosus (SLE). This study investigates the combined effect of AdMPs and IFN-α on professional phagocytes. In the presence of IFN-α, phagocytosis of AdMPs by human monocytes was significantly increased in a dose-dependent manner. The combination of AdMPs and raised IFN-α concentrations resulted in an increase in the secretion of pro-inflammatory cytokines and an upregulation of surface molecule expression involved in antigen uptake. In addition, macrophage polarisation was shifted towards a more inflammatory type of cell. The synergism between IFN-α and AdMPs seemed to be mediated by an upregulation of phosphorylated STAT1. Our results indicate that IFN-α, together with AdMPs, amplify the initiation and maintenance of inflammation. This mechanism might especially play a crucial role in disorders with a defective clearance of apoptotic material. © 2015. Published by The Company of Biologists Ltd.

  14. A study on apoptotic signaling pathway in HL-60 cells induced by radiation

    International Nuclear Information System (INIS)

    Kim, Hye Jung; Moon, Sung Keun; Lee, Jae Hoon; Moon, Sun Rock

    2001-01-01

    The mechanical insights of death at cancer cells by ionizing radiation are not yet clearly defined. Recent evidences have demonstrated that radiation therapy may induce cell death via activation of signaling pathway for apoptosis in target cells. This study is designed whether ionizing radiation may activate the signaling cascades of apoptosis including caspase family cysteine proteases, Bcl2/Bax, cytochrome c and Fas/Fas-L in target cells. HL-60 cells were irradiated in vitro with 6 MV X-ray at dose ranges from 2 Gy to 32 Gy. The cell viability was tested by MTT assay and the extent of apoptosis was determined using agarose gel electrophoresis. The activities of caspase proteases were measured by proteolytic cleavages of substrates. Western blot analysis was used to monitor PARP, caspase-3, Cytochrome-c, BcI-2, Bax, Fas and Fas-L. Ionizing radiation decreases the viability of HL -60 cells in a time and dose dependent manner. Ionizing radiation-induced death in HL- 60 cells is an apoptotic death which is revealed as characteristic ladder-pattern fragmentation at genomic DNA over 16 Gy at 4 hours. Ionizing radiation induces the activation of caspase-2, 3, 6, 8 and 9 of HL --60 cells in a time-dependent manner. The activation of caspase- 3 protease is also evidenced by the digestion of poly (ADP-ribose) polymerase and procaspase 3 with 16Gy ionizing irradiation. Anti-apoptotic Bcl2 expression is decreased but apoptotic Bax expression is increased with mitochondrial cytochrome c release in a time- dependent manner. In addition, expression of Fas and Fas-L is also increased in a time dependent manner. These data suggest that ionizing radiation-induced apoptosis is mediated by the activation of various signaling pathways including caspase family cysteine proteases, BcI 2 /Bax, Fas and Fas-L in a time and dose dependent manner

  15. Extrinsic and Intrinsic Apoptotic Responses Induced by Shiitake Culinary-Medicinal Mushroom Lentinus edodes (Agaricomycetes) Aqueous Extract against a Larynx Carcinoma Cell Line.

    Science.gov (United States)

    Finimundy, Tiane C; Scola, Gustavo; Scariot, Fernando J; Dillon, Aldo J P; Moura, Sidnei; Echeverrigaray, Sérgio; Henriques, João Pegas; Roesch-Ely, Mariana

    2018-01-01

    Cumulative evidence from research studies has shown that the shiitake culinary-medicinal mushroom, Lentinus edodes, is an excellent source of natural antitumor agents and is capable of inhibiting cancer cell growth. However, the cell signaling pathway that leads tumor cells to apoptosis is not well understood because many chemical compounds may be acting. This study investigated the chemopreventive effects of an L. edodes aqueous extract on human HEp-2 epithelial larynx carcinoma cells and normal human MRC-5 lung fibroblasts by identifying proliferative and apoptotic pathways. The chemical characterization of the dry powder was assessed by high-performance liquid chromatography. Antiproliferative and proapoptotic effects induced by the extract were evaluated by assessing proliferative markers, cell sorting through flow cytometry, and expression levels of apoptotic proteins with Western blotting. The results suggest that inhibition of cell proliferation was more prominent in HEp-2 than in MRC-5 cells. Cell death analysis showed the appearance of cell populations in the sub-G1 phase, with late apoptotic signal increased in a dose-dependent manner. In addition, the aqueous extract induced depolarization of mitochondria, activating the generation of intracellular reactive oxygen species in HEp-2 cells. These observations suggest that L. edodes extract may exert a chemopreventive effect, regulating mitotic induction of apoptogenic signals. These findings highlight the mushroom's pharmacological potential in cancer treatment.

  16. Mis-targeting of the mitochondrial protein LIPT2 leads to apoptotic cell death.

    Directory of Open Access Journals (Sweden)

    Emanuele Bernardinelli

    Full Text Available Lipoyl(Octanoyl Transferase 2 (LIPT2 is a protein involved in the post-translational modification of key energy metabolism enzymes in humans. Defects of lipoic acid synthesis and transfer start to emerge as causes of fatal or severe early-onset disease. We show that the first 31 amino acids of the N-terminus of LIPT2 represent a mitochondrial targeting sequence and inhibition of the transit of LIPT2 to the mitochondrion results in apoptotic cell death associated with activation of the apoptotic volume decrease (AVD current in normotonic conditions, as well as over-activation of the swelling-activated chloride current (IClswell, mitochondrial membrane potential collapse, caspase-3 cleavage and nuclear DNA fragmentation. The findings presented here may help elucidate the molecular mechanisms underlying derangements of lipoic acid biosynthesis.

  17. 4β-Hydroxywithanolide E Modulates Alternative Splicing of Apoptotic Genes in Human Hepatocellular Carcinoma Huh-7 Cells.

    Science.gov (United States)

    Lee, Chien-Chin; Chang, Wen-Hsin; Chang, Ya-Sian; Liu, Ting-Yuan; Chen, Yu-Chia; Wu, Yang-Chang; Chang, Jan-Gowth

    2017-08-04

    Alternative splicing is a mechanism for increasing protein diversity from a limited number of genes. Studies have demonstrated that aberrant regulation in the alternative splicing of apoptotic gene transcripts may contribute to the development of cancer. In this study, we isolated 4β-Hydroxywithanolide E (4bHWE) from the traditional herb Physalis peruviana and investigated its biological effect in cancer cells. The results demonstrated that 4bHWE modulates the alternative splicing of various apoptotic genes, including HIPK3, SMAC/DIABLO, and SURVIVIN. We also discovered that the levels of SRSF1 phospho-isoform were decreased and the levels of H3K36me3 were increased in 4bHWE treatment. Knockdown experiments revealed that the splicing site selection of SMAC/DIABLO could be mediated by changes in the level of H3K36me3 in 4bHWE-treated cells. Furthermore, we extended our study to apoptosis-associated molecules, and detected increased levels of poly ADP-ribose polymerase cleavage and the active form of CASPASE-3 in 4bHWE-induced apoptosis. In vivo experiments indicated that the treatment of tumor-bearing mice with 4bHWE resulted in a marked decrease in tumor size. This study is the first to demonstrate that 4bHWE affects alternative splicing by modulating splicing factors and histone modifications, and provides a novel view of the antitumor mechanism of 4bHWE.

  18. Apoptotic activity and gene responses in Drosophila melanogaster S2 cells, induced by azadirachtin A.

    Science.gov (United States)

    Xu, Lin; Li, Sheng; Ran, Xueqin; Liu, Chang; Lin, Rutao; Wang, Jiafu

    2016-09-01

    Azadirachtin has been used as an antifeedant and growth disruption agent for many insect species. Previous investigations have reported the apoptotic effects of azadirachtin on some insect cells, but the molecular mechanisms are still not clear. This study investigated the underlying molecular mechanisms for the apoptotic effects induced by azadirachtin on Drosophila melanogaster S2 cells in vitro. The results of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay demonstrated that azadirachtin exhibited significant cytotoxicity to S2 cells in a time- and dose-dependent manner. The changes in cellular morphology and the DNA fragmentation demonstrated that azadirachtin induced remarkable apoptosis of S2 cells. Expression levels of 276 genes were found to be significantly changed in S2 cells after exposure to azadirachtin, as detected by Drosophila genome array. Among these genes, calmodulin (CaM) was the most highly upregulated gene. Azadirachtin was further demonstrated to trigger intracellular Ca(2+) release in S2 cells. The genes related to the apoptosis pathway, determined from chip data, were validated by the real-time quantitative polymerase chain reaction method. The results showed that azadirachtin-mediated intracellular Ca(2+) release was the primary event that triggered apoptosis in Drosophila S2 cells through both pathways of the Ca(2+) -CaM and EcR/Usp signalling cascade. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.

  19. Primula auriculata Extracts Exert Cytotoxic and Apoptotic Effects against HT-29 Human Colon Adenocarcinoma Cells.

    Science.gov (United States)

    Behzad, Sahar; Ebrahim, Karim; Mosaddegh, Mahmoud; Haeri, Ali

    2016-01-01

    Primula auriculata (Tootia) is one of the most important local medicinal plants in Hamedan district, Iran. To investigate cytotoxicity and apoptosis induction of crude methanolic extract and different fraction of it, we compared several methods on HT-29 human colon Adenocarcinoma cells. Cancer cell proliferation was measured by 3-(4, 5‑dimethylthiazolyl)2, 5‑diphenyl‑tetrazolium bromide (MTT) assay and apoptosis induction was analyzed by fluorescence microscopy (acridin orange/ethidium bromide, annexin V/propidium iodide staining, TUNEL assay and Caspase-3 activity assay). Crude methanolic extract (CM) inhibited the growth of malignant cells in a dose-dependent manner. Among solvent fractions, the dichloromethane fraction (CF) was found to be the most toxic compared to other fractions. With double staining methods, high percentage of 40 µg/mL of (CM) and (CF) treated cells exhibited typical characteristics of apoptotic cells. Apoptosis induction was also revealed by apoptotic fragmentation of nuclear DNA and activation of caspas-3 in treated cells. These findings indicate that crude methanolic extract and dichloromethan fraction of P.auriculata induced apoptosis and inhibited proliferation in colon cancer cells and could be used as a source for new lead structures in drug design to combat colon cancer.

  20. Orphan Nuclear Receptor NR4A1 Binds a Novel Protein Interaction Site on Anti-apoptotic B Cell Lymphoma Gene 2 Family Proteins.

    Science.gov (United States)

    Godoi, Paulo H C; Wilkie-Grantham, Rachel P; Hishiki, Asami; Sano, Renata; Matsuzawa, Yasuko; Yanagi, Hiroko; Munte, Claudia E; Chen, Ya; Yao, Yong; Marassi, Francesca M; Kalbitzer, Hans R; Matsuzawa, Shu-Ichi; Reed, John C

    2016-07-01

    B cell lymphoma gene 2 (Bcl-2) family proteins are key regulators of programmed cell death and important targets for drug discovery. Pro-apoptotic and anti-apoptotic Bcl-2 family proteins reciprocally modulate their activities in large part through protein interactions involving a motif known as BH3 (Bcl-2 homology 3). Nur77 is an orphan member of the nuclear receptor family that lacks a BH3 domain but nevertheless binds certain anti-apoptotic Bcl-2 family proteins (Bcl-2, Bfl-1, and Bcl-B), modulating their effects on apoptosis and autophagy. We used a combination of NMR spectroscopy-based methods, mutagenesis, and functional studies to define the interaction site of a Nur77 peptide on anti-apoptotic Bcl-2 family proteins and reveal a novel interaction surface. Nur77 binds adjacent to the BH3 peptide-binding crevice, suggesting the possibility of cross-talk between these discrete binding sites. Mutagenesis of residues lining the identified interaction site on Bcl-B negated the interaction with Nur77 protein in cells and prevented Nur77-mediated modulation of apoptosis and autophagy. The findings establish a new protein interaction site with the potential to modulate the apoptosis and autophagy mechanisms governed by Bcl-2 family proteins. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Melatonin pre-treatment mitigates SHSY-5Y cells against oxaliplatin induced mitochondrial stress and apoptotic cell death

    Science.gov (United States)

    Choudhury, Arnab; Kar, Sudeshna; Tabassum, Heena

    2017-01-01

    Oxaliplatin (Oxa) treatment to SH-SY5Y human neuroblastoma cells has been shown by previous studies to induce oxidative stress, which in turn modulates intracellular signaling cascades resulting in cell death. While this phenomenon of Oxa-induced neurotoxicity is known, the underlying mechanisms involved in this cell death cascade must be clarified. Moreover, there is still little known regarding the roles of neuronal mitochondria and cytosolic compartments in mediating Oxa-induced neurotoxicity. With a better grasp of the mechanisms driving neurotoxicity in Oxa-treated SH-SY5Y cells, we can then identify certain pathways to target in protecting against neurotoxic cell damage. Therefore, the purpose of this study was to determine whether one such agent, melatonin (Mel), could confer protection against Oxa-induced neurotoxicity in SH-SY5Y cells. Results from the present study found Oxa to significantly reduce SH-SY5Y cell viability in a dose-dependent manner. Alternatively, we found Mel pre-treatment to SH-SY5Y cells to attenuate Oxa-induced toxicity, resulting in a markedly increased cell viability. Mel exerted its protective effects by regulating reactive oxygen species (ROS) production and reducing superoxide radicals inside Oxa-exposed. In addition, we observed pre-treatment with Mel to rescue Oxa-treated cells by protecting mitochondria. As Oxa-treatment alone decreases mitochondrial membrane potential (Δψm), resulting in an altered Bcl-2/Bax ratio and release of sequestered cytochrome c, so Mel was shown to inhibit these pathways. Mel was also found to inhibit proteolytic activation of caspase 3, inactivation of Poly (ADP Ribose) polymerase, and DNA damage, thereby allowing SH-SY5Y cells to resist apoptotic cell death. Collectively, our results suggest a role for melatonin in reducing Oxa induced neurotoxicity. Further studies exploring melatonin’s protective effects may prove successful in eliciting pathways to further alter the neurotoxic pathways of

  2. Melatonin pre-treatment mitigates SHSY-5Y cells against oxaliplatin induced mitochondrial stress and apoptotic cell death.

    Directory of Open Access Journals (Sweden)

    Mohammad Waseem

    Full Text Available Oxaliplatin (Oxa treatment to SH-SY5Y human neuroblastoma cells has been shown by previous studies to induce oxidative stress, which in turn modulates intracellular signaling cascades resulting in cell death. While this phenomenon of Oxa-induced neurotoxicity is known, the underlying mechanisms involved in this cell death cascade must be clarified. Moreover, there is still little known regarding the roles of neuronal mitochondria and cytosolic compartments in mediating Oxa-induced neurotoxicity. With a better grasp of the mechanisms driving neurotoxicity in Oxa-treated SH-SY5Y cells, we can then identify certain pathways to target in protecting against neurotoxic cell damage. Therefore, the purpose of this study was to determine whether one such agent, melatonin (Mel, could confer protection against Oxa-induced neurotoxicity in SH-SY5Y cells. Results from the present study found Oxa to significantly reduce SH-SY5Y cell viability in a dose-dependent manner. Alternatively, we found Mel pre-treatment to SH-SY5Y cells to attenuate Oxa-induced toxicity, resulting in a markedly increased cell viability. Mel exerted its protective effects by regulating reactive oxygen species (ROS production and reducing superoxide radicals inside Oxa-exposed. In addition, we observed pre-treatment with Mel to rescue Oxa-treated cells by protecting mitochondria. As Oxa-treatment alone decreases mitochondrial membrane potential (Δψm, resulting in an altered Bcl-2/Bax ratio and release of sequestered cytochrome c, so Mel was shown to inhibit these pathways. Mel was also found to inhibit proteolytic activation of caspase 3, inactivation of Poly (ADP Ribose polymerase, and DNA damage, thereby allowing SH-SY5Y cells to resist apoptotic cell death. Collectively, our results suggest a role for melatonin in reducing Oxa induced neurotoxicity. Further studies exploring melatonin's protective effects may prove successful in eliciting pathways to further alter the neurotoxic

  3. Decidual activin: its role in the apoptotic process and its regulation by prolactin.

    Science.gov (United States)

    Tessier, Christian; Prigent-Tessier, Anne; Bao, Lei; Telleria, Carlos M; Ferguson-Gottschall, Susan; Gibori, Gil B; Gu, Yan; Bowen-Shauver, Jennifer M; Horseman, Nelson D; Gibori, Geula

    2003-05-01

    Successful pregnancy requires profound differentiation and reorganization of the uterine tissues including, as pregnancy progresses, extensive apoptosis of decidual tissue to accommodate the developing conceptus. We have previously shown a positive correlation between expression of activin A and apoptosis in the decidua and have also shown that expression of activin A occurs at the time when prolactin (PRL) receptors disappear from decidual cells. The goals of this study were to examine whether activin A plays a role in decidual apoptosis and whether expression of activin A in the decidua is regulated by PRL and placental lactogens. Studies were carried out using primary rat decidual cells, a decidual cell line (GG-AD), and PRL null mice. Treatment of decidual cells with activin A significantly increased DNA degradation, caspase 3 activity, and caspase 3 mRNA expression. However, this effect was observed only in the absence of endogenous activin production by these cells. Addition of follistatin to decidual cells that were producing activin A decreased both caspase 3 activity and mRNA expression. Similarly, addition of activin-blocking antibodies to cultures of GG-AD cells, which also produce activin A, caused a reduction in both DNA degradation and caspase 3 activity. PRL and placental lactogens caused an inhibition of activin A mRNA expression in primary decidual cells. Even more convincingly, decidua of PRL null mice expressed abundant activin A at a time when no expression of this hormone is detected in wild-type mice and treatment of PRL null mice with PRL caused a profound inhibition of activin A mRNA expression. In summary, our investigations into the role and regulation of decidual activin have revealed that activin A can induce cell death in the decidua and that its expression is under tight regulation by PRL and placental lactogens.

  4. Distinct apoptotic blocks mediate resistance to panHER inhibitors in HER2+ breast cancer cells.

    Science.gov (United States)

    Karakas, Bahriye; Ozmay, Yeliz; Basaga, Huveyda; Gul, Ozgur; Kutuk, Ozgur

    2018-05-04

    Despite the development of novel targeted therapies, de novo or acquired chemoresistance remains a significant factor for treatment failure in breast cancer therapeutics. Neratinib and dacomitinib are irreversible panHER inhibitors, which block their autophosphorylation and downstream signaling. Moreover, neratinib and dacomitinib have been shown to activate cell death in HER2-overexpressing cell lines. Here we showed that increased MCL1 and decreased BIM and PUMA mediated resistance to neratinib in ZR-75-30 and SKBR3 cells while increased BCL-XL and BCL-2 and decreased BIM and PUMA promoted neratinib resistance in BT474 cells. Cells were also cross-resistant to dacomitinib. BH3 profiles of HER2+ breast cancer cells efficiently predicted antiapoptotic protein dependence and development of resistance to panHER inhibitors. Reactivation of ERK1/2 was primarily responsible for acquired resistance in SKBR3 and ZR-75-30 cells. Adding specific ERK1/2 inhibitor SCH772984 to neratinib or dacomitinib led to increased apoptotic response in neratinib-resistant SKBR3 and ZR-75-30 cells, but we did not detect a similar response in neratinib-resistant BT474 cells. Accordingly, suppression of BCL-2/BCL-XL by ABT-737 was required in addition to ERK1/2 inhibition for neratinib- or dacomitinib-induced apoptosis in neratinib-resistant BT474 cells. Our results showed that different mitochondrial apoptotic blocks mediated acquired panHER inhibitor resistance in HER2+ breast cancer cell lines as well as highlighted the potential of BH3 profiling assay in prediction of panHER inhibitor resistance in breast cancer cells. Copyright © 2018 Elsevier B.V. All rights reserved.

  5. Apoptotic effects of non-edible parts of Punica granatum on human multiple myeloma cells.

    Science.gov (United States)

    Kiraz, Yağmur; Neergheen-Bhujun, Vidushi S; Rummun, Nawraj; Baran, Yusuf

    2016-02-01

    Multiple myeloma is of great concern since existing therapies are unable to cure this clinical condition. Alternative therapeutic approaches are mandatory, and the use of plant extracts is considered interesting. Punica granatum and its derived products were suggested as potential anticancer agents due to the presence of bioactive compounds. Thus, polypenolic-rich extracts of the non-edible parts of P. granatum were investigated for their antiproliferative and apoptotic effects on U266 multiple myeloma cells. We demonstrated that there were dose-dependent decreases in the proliferation of U266 cells in response to P. granatum extracts. Also, exposure to the extracts triggered apoptosis with significant increases in loss of mitochondrial membrane potential in U266 cells exposed to the leaves and stem extracts, while the flower extract resulted in slight increases in loss of MMP. These results were confirmed by Annexin-V analysis. These results documented the cytotoxic and apoptotic effects of P. granatum extracts on human U266 multiple myeloma cells via disruption of mitochondrial membrane potential and increasing cell cycle arrest. The data suggest that the extracts can be envisaged in cancer chemoprevention and call for further exploration into the potential application of these plant parts.

  6. A neurodegenerative disease mutation that accelerates the clearance of apoptotic cells.

    Science.gov (United States)

    Kao, Aimee W; Eisenhut, Robin J; Martens, Lauren Herl; Nakamura, Ayumi; Huang, Anne; Bagley, Josh A; Zhou, Ping; de Luis, Alberto; Neukomm, Lukas J; Cabello, Juan; Farese, Robert V; Kenyon, Cynthia

    2011-03-15

    Frontotemporal lobar degeneration is a progressive neurodegenerative syndrome that is the second most common cause of early-onset dementia. Mutations in the progranulin gene are a major cause of familial frontotemporal lobar degeneration [Baker M, et al. (2006) Nature 442:916-919 and Cruts M, et al. (2006) Nature 442:920-924]. Although progranulin is involved in wound healing, inflammation, and tumor growth, its role in the nervous system and the mechanism by which insufficient levels result in neurodegeneration are poorly understood [Eriksen and Mackenzie (2008) J Neurochem 104:287-297]. We have characterized the normal function of progranulin in the nematode Caenorhabditis elegans. We found that mutants lacking pgrn-1 appear grossly normal, but exhibit fewer apoptotic cell corpses during development. This reduction in corpse number is not caused by reduced apoptosis, but instead by more rapid clearance of dying cells. Likewise, we found that macrophages cultured from progranulin KO mice displayed enhanced rates of apoptotic-cell phagocytosis. Although most neurodegenerative diseases are thought to be caused by the toxic effects of aggregated proteins, our findings suggest that susceptibility to neurodegeneration may be increased by a change in the kinetics of programmed cell death. We propose that cells that might otherwise recover from damage or injury are destroyed in progranulin mutants, which in turn facilitates disease progression.

  7. Effect of Transient Maternal Hypotension on Apoptotic Cell Death in Foetal Rat Brain

    Directory of Open Access Journals (Sweden)

    Hamit Özyürek

    2014-03-01

    Full Text Available Background: Intrauterine perfusion insufficiency induced by transient maternal hypotension has been reported to be associated with foetal brain malformations. However, the effects of maternal hypotension on apoptotic processes in the foetal brain have not been investigated experimentally during the intrauterine period. Aims: The aim of this study was to investigate the effects of transient maternal hypotension on apoptotic cell death in the intrauterine foetal brain. Study Design: Animal experimentation. Methods: Three-month-old female Wistar albino rats were allocated into four groups (n=5 each. The impact of hypoxic/ischemic injury induced by transient maternal hypotension on the 15th day of pregnancy (late gestation in rats was investigated at 48 (H17 group or 96 hours (H19 group after the insult. Control groups underwent the same procedure except for induction of hypotension (C17 and H17 groups. Brain sections of one randomly selected foetus from each pregnant rat were histopathologically evaluated for hypoxic/ischemic injury in the metencephalon, diencephalon, and telencephalon by terminal transferase-mediated dUTP nick end labelling and active cysteine-dependent aspartate-directed protease-3 (caspase-3 positivity for cell death. Results: The number of terminal transferase-mediated dUTP nick end labelling (+ cells in all the areas examined was comparable in both hypotension and control groups. The H17 group had active caspase-3 (+ cells in the metencephalon and telencephalon, sparing diencephalon, whereas the C19 and H19 groups had active caspase-3 (+ cells in all three regions. The number of active caspase-3 (+ cells in the telencephalon in the H19 group was higher compared with the metencephalon and diencephalon and compared with H17 group (p<0.05. Conclusion: Our results suggest that prenatal hypoxic/ischemic injury triggers apoptotic mechanisms. Therefore, blockade of apoptotic pathways, considering the time pattern of the insult, may

  8. Apoptotic-like programmed cell death in fungi: the benefits in filamentous species

    Directory of Open Access Journals (Sweden)

    Neta eShlezinger

    2012-08-01

    Full Text Available Studies conducted in the early 1990's showed for the first time that Saccahromyces cerevisiae can undergo cell death with hallmarks of animal apoptosis. These findings came as a surprise, since suicide machinery was unexpected in unicellular organisms. Today, apoptosis in yeast is well documented. Apoptotic death of yeast cells has been described under various conditions and S. cerevisiae homologues of human apoptotic genes have been identified and characterized. These studies also revealed fundamental differences between yeast and animal apoptosis; in S. cerevisiae apoptosis is mainly associated with ageing and stress adaptation, unlike animal apoptosis, which is essential for proper development. Further, many apoptosis regulatory genes are either missing, or highly divergent in S. cerevisiae. Therefore, in this review we will use the term apoptosis-like programmed cell death (PCD instead of apoptosis. Despite these significant differences, S. cerevisiae has been instrumental in promoting the study of heterologous apoptotic proteins, particularly from human. Work in fungi other than S. cerevisiae revealed differences in the manifestation of PCD in single cell (yeasts and multi-cellular (filamentous species. Such differences may reflect the higher complexity level of filamentous species, and hence the involvement of PCD in a wider range of processes and life styles. It is also expected that differences might be found in the apoptosis apparatus of yeast and filamentous species. In this review we focus on aspects of PCD that are unique or can be better studied in filamentous species. We will highlight the similarities and differences of the PCD machinery between yeast and filamentous species and show the value of using S. cerevisiae along with filamentous species to study apoptosis.

  9. Apoptotic-like programed cell death in fungi: the benefits in filamentous species

    International Nuclear Information System (INIS)

    Shlezinger, Neta; Goldfinger, Nir; Sharon, Amir

    2012-01-01

    Studies conducted in the early 1990s showed for the first time that Saccharomyces cerevisiae can undergo cell death with hallmarks of animal apoptosis. These findings came as a surprise, since suicide machinery was unexpected in unicellular organisms. Today, apoptosis in yeast is well-documented. Apoptotic death of yeast cells has been described under various conditions and S. cerevisiae homologs of human apoptotic genes have been identified and characterized. These studies also revealed fundamental differences between yeast and animal apoptosis; in S. cerevisiae apoptosis is mainly associated with aging and stress adaptation, unlike animal apoptosis, which is essential for proper development. Further, many apoptosis regulatory genes are either missing, or highly divergent in S. cerevisiae. Therefore, in this review we will use the term apoptosis-like programed cell death (PCD) instead of apoptosis. Despite these significant differences, S. cerevisiae has been instrumental in promoting the study of heterologous apoptotic proteins, particularly from human. Work in fungi other than S. cerevisiae revealed differences in the manifestation of PCD in single cell (yeasts) and multicellular (filamentous) species. Such differences may reflect the higher complexity level of filamentous species, and hence the involvement of PCD in a wider range of processes and life styles. It is also expected that differences might be found in the apoptosis apparatus of yeast and filamentous species. In this review we focus on aspects of PCD that are unique or can be better studied in filamentous species. We will highlight the similarities and differences of the PCD machinery between yeast and filamentous species and show the value of using S. cerevisiae along with filamentous species to study apoptosis.

  10. Apoptotic-like programed cell death in fungi: the benefits in filamentous species

    Energy Technology Data Exchange (ETDEWEB)

    Shlezinger, Neta; Goldfinger, Nir; Sharon, Amir, E-mail: amirsh@ex.tau.ac.il [Department of Molecular Biology and Ecology of Plants, Tel Aviv University,, Tel Aviv (Israel)

    2012-08-07

    Studies conducted in the early 1990s showed for the first time that Saccharomyces cerevisiae can undergo cell death with hallmarks of animal apoptosis. These findings came as a surprise, since suicide machinery was unexpected in unicellular organisms. Today, apoptosis in yeast is well-documented. Apoptotic death of yeast cells has been described under various conditions and S. cerevisiae homologs of human apoptotic genes have been identified and characterized. These studies also revealed fundamental differences between yeast and animal apoptosis; in S. cerevisiae apoptosis is mainly associated with aging and stress adaptation, unlike animal apoptosis, which is essential for proper development. Further, many apoptosis regulatory genes are either missing, or highly divergent in S. cerevisiae. Therefore, in this review we will use the term apoptosis-like programed cell death (PCD) instead of apoptosis. Despite these significant differences, S. cerevisiae has been instrumental in promoting the study of heterologous apoptotic proteins, particularly from human. Work in fungi other than S. cerevisiae revealed differences in the manifestation of PCD in single cell (yeasts) and multicellular (filamentous) species. Such differences may reflect the higher complexity level of filamentous species, and hence the involvement of PCD in a wider range of processes and life styles. It is also expected that differences might be found in the apoptosis apparatus of yeast and filamentous species. In this review we focus on aspects of PCD that are unique or can be better studied in filamentous species. We will highlight the similarities and differences of the PCD machinery between yeast and filamentous species and show the value of using S. cerevisiae along with filamentous species to study apoptosis.

  11. Anti-Apoptotic Signature in Thymic Squamous Cell Carcinomas - Functional Relevance of Anti-Apoptotic BIRC3 Expression in the Thymic Carcinoma Cell Line 1889c.

    Science.gov (United States)

    Huang, Bei; Belharazem, Djeda; Li, Li; Kneitz, Susanne; Schnabel, Philipp A; Rieker, Ralf J; Körner, Daniel; Nix, Wilfred; Schalke, Berthold; Müller-Hermelink, Hans Konrad; Ott, German; Rosenwald, Andreas; Ströbel, Philipp; Marx, Alexander

    2013-01-01

    The molecular pathogenesis of thymomas and thymic carcinomas (TCs) is poorly understood and results of adjuvant therapy are unsatisfactory in case of metastatic disease and tumor recurrence. For these clinical settings, novel therapeutic strategies are urgently needed. Recently, limited sequencing efforts revealed that a broad spectrum of genes that play key roles in various common cancers are rarely affected in thymomas and TCs, suggesting that other oncogenic principles might be important. This made us re-analyze historic expression data obtained in a spectrum of thymomas and thymic squamous cell carcinomas (TSCCs) with a custom-made cDNA microarray. By cluster analysis, different anti-apoptotic signatures were detected in type B3 thymoma and TSCC, including overexpression of BIRC3 in TSCCs. This was confirmed by qRT-PCR in the original and an independent validation set of tumors. In contrast to several other cancer cell lines, the BIRC3-positive TSCC cell line, 1889c showed spontaneous apoptosis after BIRC3 knock-down. Targeting apoptosis genes is worth testing as therapeutic principle in TSCC.

  12. Anti-Apoptotic Signature in Thymic Squamous Cell Carcinomas – Functional Relevance of Anti-Apoptotic BIRC3 Expression in the Thymic Carcinoma Cell Line 1889c

    Science.gov (United States)

    Huang, Bei; Belharazem, Djeda; Li, Li; Kneitz, Susanne; Schnabel, Philipp A.; Rieker, Ralf J.; Körner, Daniel; Nix, Wilfred; Schalke, Berthold; Müller-Hermelink, Hans Konrad; Ott, German; Rosenwald, Andreas; Ströbel, Philipp; Marx, Alexander

    2013-01-01

    The molecular pathogenesis of thymomas and thymic carcinomas (TCs) is poorly understood and results of adjuvant therapy are unsatisfactory in case of metastatic disease and tumor recurrence. For these clinical settings, novel therapeutic strategies are urgently needed. Recently, limited sequencing efforts revealed that a broad spectrum of genes that play key roles in various common cancers are rarely affected in thymomas and TCs, suggesting that other oncogenic principles might be important. This made us re-analyze historic expression data obtained in a spectrum of thymomas and thymic squamous cell carcinomas (TSCCs) with a custom-made cDNA microarray. By cluster analysis, different anti-apoptotic signatures were detected in type B3 thymoma and TSCC, including overexpression of BIRC3 in TSCCs. This was confirmed by qRT-PCR in the original and an independent validation set of tumors. In contrast to several other cancer cell lines, the BIRC3-positive TSCC cell line, 1889c showed spontaneous apoptosis after BIRC3 knock-down. Targeting apoptosis genes is worth testing as therapeutic principle in TSCC. PMID:24427739

  13. Anti-apoptotic signature in thymic squamous cell carcinomas - functional relevance of anti-apoptotic BIRC3 expression in the thymic carcinoma cell line 1889c

    Directory of Open Access Journals (Sweden)

    Bei eHuang

    2013-12-01

    Full Text Available The molecular pathogenesis of thymomas and thymic carcinomas (TCs is poorly understood and results of adjuvant therapy are unsatisfactory in case of metastatic disease and tumor recurrence. For these clinical settings, novel therapeutic strategies are urgently needed. Recently, limited sequencing efforts revealed that a broad spectrum of genes that play key roles in various common cancers are rarely affected in thymomas and thymic carcinomas, suggesting that other oncogenic principles might be important. This made us re-analyze historic expression data obtained in a spectrum of thymomas and thymic squamous cell carcinomas (TSCC with a custom made cDNA microarray. By cluster analysis, different anti-apoptotic signatures were detected in type B3 thymoma and TSCC, including overexpression of BIRC3 in TSCCs. This was confirmed by qRT-PCR in the original and an independent validation set of tumors. In contrast to several other cancer cell lines, the BIRC3-positive TSCC cell line, 1889c showed spontaneous apoptosis after BIRC3 knock-down. Targeting apoptosis genes is worth testing as therapeutic principle in TSCC.

  14. Ultrastructural Complexity of Nuclear Components During Early Apoptotic Phases in Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Christian Castelli

    2001-01-01

    Full Text Available Fractal morphometry was used to investigate the ultrastructural features of the plasma membrane, perinuclear membrane and nuclear chromatin in SK‐BR‐3 human breast cancer cells undergoing apoptosis. Cells were incubated with 1 μM calcimycin (A23187 for 24 h. Cells in the early stage of apoptosis had fractal dimension (FD values indicating that their plasma membranes were less rough (lower FD than those of control cells, while their perinuclear membranes were unaffected. Changes of the chromatin texture within the entire nucleus and in selected nuclear domains were more pronounced in treated cells. This confirms that the morphological reorganization imputable to a loss of structural complexity (reduced FD occurs in the early stage of apoptosis, is accompanied by the inhibition of distinct enzymatic events and precedes the onset of conventional cellular markers, which can only be detected during the active phases of the apoptotic process.

  15. FoxP3 inhibits proliferation and induces apoptosis of gastric cancer cells by activating the apoptotic signaling pathway

    International Nuclear Information System (INIS)

    Ma, Gui-Fen; Chen, Shi-Yao; Sun, Zhi-Rong; Miao, Qing; Liu, Yi-Mei; Zeng, Xiao-Qing; Luo, Tian-Cheng; Ma, Li-Li; Lian, Jing-Jing; Song, Dong-Li

    2013-01-01

    Highlights: ► The article revealed FoxP3 gene function in gastric cancer firstly. ► Present the novel roles of FoxP3 in inhibiting proliferation and promoting apoptosis in gastric cancer cells. ► Overexpression of FoxP3 increased proapoptotic molecules and repressed antiapoptotic molecules. ► Silencing of FoxP3 reduced the expression of proapoptotic genes, such as PARP, caspase-3 and caspase-9. ► FoxP3 is sufficient for activating the apoptotic signaling pathway. -- Abstract: Forkhead Box Protein 3 (FoxP3) was identified as a key transcription factor to the occurring and function of the regulatory T cells (Tregs). However, limited evidence indicated its function in tumor cells. To elucidate the precise roles and underlying molecular mechanism of FoxP3 in gastric cancer (GC), we examined the expression of FoxP3 and the consequences of interfering with FoxP3 gene in human GC cell lines, AGS and MKN45, by multiple cellular and molecular approaches, such as immunofluorescence, gene transfection, CCK-8 assay, clone formation assay, TUNEL assay, Flow cytometry, immunoassay and quantities polymerase chain reaction (PCR). As a result, FoxP3 was expressed both in nucleus and cytoplasm of GC cells. Up-regulation of FoxP3 inhibited cell proliferation and promoted cell apoptosis. Overexpression of FoxP3 increased the protein and mRNA levels of proapoptotic molecules, such as poly ADP-ribose polymerase1 (PARP), caspase-3 and caspase-9, and repressed the expression of antiapoptotic molecules, such as cellular inhibitor of apoptosis-1 (c-IAP1) and the long isoform of B cell leukemia/lymphoma-2 (Bcl-2). Furthermore, silencing of FoxP3 by siRNA in GC cells reduced the expression of proapoptotic genes, such as PARP, caspase-3 and caspase-9. Collectively, our findings identify the novel roles of FoxP3 in inhibiting proliferation and inducing apoptosis in GC cells by regulating apoptotic signaling, which could be a promising therapeutic approach for gastric cancer.

  16. FoxP3 inhibits proliferation and induces apoptosis of gastric cancer cells by activating the apoptotic signaling pathway

    Energy Technology Data Exchange (ETDEWEB)

    Ma, Gui-Fen [Department of Gastroenterology, Zhongshan Hospital, Fudan University, Shanghai (China); Chen, Shi-Yao, E-mail: shiyao_chen@163.com [Department of Gastroenterology, Zhongshan Hospital, Fudan University, Shanghai (China); Endoscopy Center, Zhongshan Hospital, Fudan University, Shanghai (China); Sun, Zhi-Rong [Department of Anesthesiology, Cancer Center, Fudan University, Shanghai (China); Miao, Qing; Liu, Yi-Mei; Zeng, Xiao-Qing; Luo, Tian-Cheng [Department of Gastroenterology, Zhongshan Hospital, Fudan University, Shanghai (China); Ma, Li-Li; Lian, Jing-Jing [Endoscopy Center, Zhongshan Hospital, Fudan University, Shanghai (China); Song, Dong-Li [Biomedical Research Center, Zhongshan Hospital, Fudan University, Shanghai (China)

    2013-01-11

    Highlights: Black-Right-Pointing-Pointer The article revealed FoxP3 gene function in gastric cancer firstly. Black-Right-Pointing-Pointer Present the novel roles of FoxP3 in inhibiting proliferation and promoting apoptosis in gastric cancer cells. Black-Right-Pointing-Pointer Overexpression of FoxP3 increased proapoptotic molecules and repressed antiapoptotic molecules. Black-Right-Pointing-Pointer Silencing of FoxP3 reduced the expression of proapoptotic genes, such as PARP, caspase-3 and caspase-9. Black-Right-Pointing-Pointer FoxP3 is sufficient for activating the apoptotic signaling pathway. -- Abstract: Forkhead Box Protein 3 (FoxP3) was identified as a key transcription factor to the occurring and function of the regulatory T cells (Tregs). However, limited evidence indicated its function in tumor cells. To elucidate the precise roles and underlying molecular mechanism of FoxP3 in gastric cancer (GC), we examined the expression of FoxP3 and the consequences of interfering with FoxP3 gene in human GC cell lines, AGS and MKN45, by multiple cellular and molecular approaches, such as immunofluorescence, gene transfection, CCK-8 assay, clone formation assay, TUNEL assay, Flow cytometry, immunoassay and quantities polymerase chain reaction (PCR). As a result, FoxP3 was expressed both in nucleus and cytoplasm of GC cells. Up-regulation of FoxP3 inhibited cell proliferation and promoted cell apoptosis. Overexpression of FoxP3 increased the protein and mRNA levels of proapoptotic molecules, such as poly ADP-ribose polymerase1 (PARP), caspase-3 and caspase-9, and repressed the expression of antiapoptotic molecules, such as cellular inhibitor of apoptosis-1 (c-IAP1) and the long isoform of B cell leukemia/lymphoma-2 (Bcl-2). Furthermore, silencing of FoxP3 by siRNA in GC cells reduced the expression of proapoptotic genes, such as PARP, caspase-3 and caspase-9. Collectively, our findings identify the novel roles of FoxP3 in inhibiting proliferation and inducing apoptosis

  17. Prolonged sulforaphane treatment activates survival signaling in nontumorigenic NCM460 colon cells but apoptotic signaling in tumorigenic HCT116 colon cells.

    Science.gov (United States)

    Zeng, Huawei; Trujillo, Olivia N; Moyer, Mary P; Botnen, James H

    2011-01-01

    Sulforaphane (SFN) is a naturally occurring chemopreventive agent; the induction of cell cycle arrest and apoptosis is a key mechanism by which SFN exerts its colon cancer prevention. However, little is known about the differential effects of SFN on colon cancer and normal cells. In this study, we demonstrated that SFN (15 μmol/L) exposure (72 h) inhibited cell proliferation by up to 95% in colon cancer cells (HCT116) and by 52% in normal colon mucosa-derived (NCM460) cells. Our data also showed that SFN exposure (5 and 10 μmol/L) led to the reduction of G1 phase cell distribution and an induction of apoptosis in HCT116 cells, but to a much lesser extent in NCM460 cells. Furthermore, the examination of mitogen-activated protein kinase (MAPK) signaling status revealed that SFN upregulated the phosphorylation of extracellular-regulated kinase 1/2 (ERK1/2) in NCM460 cells but not in HCT116 cells. In contrast, SFN enhanced the phosphorylation of stress-activated protein kinase (SAPK) and decreased cellular myelocytomatosis oncogene (c-Myc) expression in HCT116 cells but not NCM460 cells. Taken together, the activation of survival signaling in NCM460 cells and apoptotic signaling in HCT116 cells may play a critical role in SFN's stronger potential of inhibiting cell proliferation in colon cancer cells than in normal colon cells. Copyright © 2011, Taylor & Francis Group, LLC

  18. SorCS2 Regulates Dopaminergic Wiring and Is Processed into an Apoptotic Two-Chain Receptor in Peripheral Glia

    DEFF Research Database (Denmark)

    Glerup, Simon; Olsen, Ditte; Vægter, Christian Bjerggaard

    2014-01-01

    Balancing trophic and apoptotic cues is critical for development and regeneration of neuronal circuits. Here we identify SorCS2 as a proneurotrophin (proNT) receptor, mediating both trophic and apoptotic signals in conjunction with p75NTR. CNS neurons, but not glia, express SorCS2 as a single-chain...... behavioral response to amphetamine reminiscent of ADHD. Contrary, in PNS glia, but not in neurons, proteolytic processing produced a two-chain SorCS2 isoform that mediated proNT-dependent Schwann cell apoptosis. Sciatic nerve injury triggered generation of two-chain SorCS2 in p75NTR-positive dying Schwann...... cells, with apoptosis being profoundly attenuated in Sorcs2−/− mice. In conclusion, we have demonstrated that two-chain processing of SorCS2 enables neurons and glia to respond differently to proneurotrophins....

  19. The Study of Apoptotic Effect of p-Coumaric Acid on Breast Cancer Cells MCF-7

    Directory of Open Access Journals (Sweden)

    M Kolahi

    2016-06-01

    Full Text Available Introduction: Polyphenolic compounds have anti proliferative and induced apoptotic features on cancer cells. p-Coumaric acid can be abundantly found in fruits, vegetables, plant production and honey. .  Breast cancer is the most frequently diagnosed cancer among women in the world. This study aimed to investigate the effect and mechanism of p- coumaric acid on apoptosis of MCF-7 breast cancer cells. Methods: In order to study appoptic effect of p- coumaric acid, MCF-7 breast cancer cells were treated with different concentrations of p- coumaric acid (10, 37, 70, 150 and 300 mM for 24 h. Cell viability was determined using MTT assay. Apoptosis markers including phosphatidylserine exposure at the outer leaflet of the plasma membrane were measured using flow cytometery for Annexin V affinity. Results: Cell viability of MCF-7 cells was decreased with increasing of p- coumaric acid concentration. Maximal effect of p- coumaric acid was observed in cells that treated with 300 mM for 24h (p< 0.05. Viability assay showed that the IC50 of p- coumaric acid in MCF-7 cells was about 40 mM. p- coumaric acid at dose of 300 mM significantly increased the late apoptotic cells with Annexin V+ and propium iodide (PI+ features after 24 h treatment. Conclusion: The results of this study showed that p- coumaric acid had effective appoptic activity against MCF-7 cells. The results can be helpful in understanding the anticancer mechanism of p- coumaric acid and using it was suggested as an alternative or complementary drug in cancer chemotherapy.

  20. Freezing and post-thaw apoptotic behaviour of cells in the presence of palmitoyl nanogold particles

    International Nuclear Information System (INIS)

    Thirumala, Sreedhar; Forman, Julianne M; Monroe, W Todd; Devireddy, Ram V

    2007-01-01

    The aim of this study was to evaluate the freezing response of HeLa and Jurkat cells in the presence of commercially available nanoparticles, NPs (Palmitoyl Nanogold[reg], Nanoprobes). The cells were incubated with NPs for either 5 min or 3 h, and a calorimeter technique was then used to generate the volumetric shrinkage response during freezing at 20 deg. C min -1 . Concomitantly, we also examined the effect of a commonly used cryoprotectant, dimethylsulfoxide, DMSO (10% v/v ratio) on the freezing response of HeLa and Jurkat cells. By fitting a model of water transport to the experimentally determined volumetric shrinkage data, the reference hydraulic conductivity, L pg (μm/min-atm) and activation energy, E Lp (kcal mol -1 ) were obtained. For HeLa cells, the values of L pg ranged from 0.08 to 0.23 μm/min-atm, while E Lp ranged from 10.9 to 37.4 kcal mol -1 . For Jurkat cells these parameter values ranged from 0.05 to 0.16 μm/min-atm and 9.5 to 35.9 kcal mol -1 . A generic optimal cooling rate equation was then used to predict the optimal rates of freezing HeLa and Jurkat cells in the presence and absence of DMSO and NPs. The post-thaw viability and apoptotic response of HeLa and Jurkat cells was further investigated by cooling cells at three rates in the presence and absence of DMSO and NPs using a commercially available controlled rate freezer. Jurkat cells treated in this manner demonstrated an increase in their adhesive properties after 18 h incubation and adhered strongly to the bottom of the culture plate. This observation prevented further analysis of Jurkat apoptotic and necrotic post-thaw responses. There was no significant effect of NPs or DMSO alone on HeLa cell viability prior to freezing. The post-thaw results from HeLa cells show that the NPs increased the measured post-freeze apoptotic response when cooled at 1 deg. C min -1 , suggesting a possible therapeutic use of NPs in cryodestructive procedures

  1. Exocytosis of macrophage lysosomes leads to digestion of apoptotic adipocytes and foam cell formation[S

    Science.gov (United States)

    Haka, Abigail S.; Barbosa-Lorenzi, Valéria C.; Lee, Hyuek Jong; Falcone, Domenick J.; Hudis, Clifford A.; Dannenberg, Andrew J.

    2016-01-01

    Many types of apoptotic cells are phagocytosed and digested by macrophages. Adipocytes can be hundreds of times larger than macrophages, so they are too large to be digested by conventional phagocytic processes. The nature of the interaction between macrophages and apoptotic adipocytes has not been studied in detail. We describe a cellular process, termed exophagy, that is important for macrophage clearance of dead adipocytes and adipose tissue homeostasis. Using mouse models of obesity, human tissue, and a cell culture model, we show that macrophages form hydrolytic extracellular compartments at points of contact with dead adipocytes using local actin polymerization. These compartments are acidic and contain lysosomal enzymes delivered by exocytosis. Uptake and complete degradation of adipocyte fragments, which are released by extracellular hydrolysis, leads to macrophage foam cell formation. Exophagy-mediated foam cell formation is a highly efficient means by which macrophages internalize large amounts of lipid, which may ultimately overwhelm the metabolic capacity of the macrophage. This process provides a mechanism for degradation of objects, such as dead adipocytes, that are too large to be phagocytosed by macrophages. PMID:27044658

  2. Lactadherin inhibits secretory phospholipase A2 activity on pre-apoptotic leukemia cells.

    Directory of Open Access Journals (Sweden)

    Steffen Nyegaard

    Full Text Available Secretory phospholipase A2 (sPLA2 is a critical component of insect and snake venoms and is secreted by mammalian leukocytes during inflammation. Elevated secretory PLA2 concentrations are associated with autoimmune diseases and septic shock. Many sPLA2's do not bind to plasma membranes of quiescent cells but bind and digest phospholipids on the membranes of stimulated or apoptotic cells. The capacity of these phospholipases to digest membranes of stimulated or apoptotic cells correlates to the exposure of phosphatidylserine. In the present study, the ability of the phosphatidyl-L-serine-binding protein, lactadherin to inhibit phospholipase enzyme activity has been assessed. Inhibition of human secretory phospholipase A2-V on phospholipid vesicles exceeded 90%, whereas inhibition of Naja mossambica sPLA2 plateaued at 50-60%. Lactadherin inhibited 45% of activity of Naja mossambica sPLA2 and >70% of human secretory phospholipase A2-V on the membranes of human NB4 leukemia cells treated with calcium ionophore A23187. The data indicate that lactadherin may decrease inflammation by inhibiting sPLA2.

  3. Branchial lesions associated with abundant apoptotic cells in oysters Ostrea edulis of Galicia (NW Spain).

    Science.gov (United States)

    Mirella da Silva, P; Villalba, Antonio; Sunila, Inke

    2006-06-12

    An experiment to evaluate differences in growth, mortality and disease susceptibility among Ostrea edulis stocks was performed. Five families were produced from each of 4 oyster populations (Irish, Greek and 2 Galician). The spat were transferred to a raft in the Ria de Arousa (Galicia, Spain) for grow-out. Monthly samples of each family were histologically processed from 2001 to 2003. One of the pathological conditions discovered by this study was the occurrence of extensive branchial lesions characterized by haemocytic infiltration and loss of branchial architecture. Furthermore, abundant atypical cells occurred among the haemocytes in the lesions in the branchial connective and epithelial tissues, but rarely in the mantle. These cells were contracted in size with nuclei showing chromatin condensation and fragmentation. Some nuclear chromatin aggregated under the nuclear membranes into crescent shapes, whereas others were uniformly dense. Those characteristics suggested that the cells were apoptotic haemocytes, which was confirmed by transmission electron microscopy (TEM) and by a terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labelling (TUNEL) assay using the Apoptag Kit on paraffin sections. A low prevalence of gill lesions was detected in some, but not all, families of every origin peaking in July 2002 and April 2003. No etiologic agent was identified by either histology or TEM; thus, the cause of the abundance of apoptotic cells remains unclear.

  4. Metallothionein treatment reduces proinflammatory cytokines IL-6 and TNF-alpha and apoptotic cell death during experimental autoimmune encephalomyelitis (EAE)

    DEFF Research Database (Denmark)

    Penkowa, M; Hidalgo, J

    2001-01-01

    cytokines and apoptosis during EAE could contribute to the reported diminution of clinical symptoms and mortality in EAE-immunized rats receiving Zn-MT-II treatment. Our results demonstrate that MT-II reduces the CNS expression of proinflammatory cytokines and the number of apoptotic neurons during EAE......, which is characterized by significant inflammation and neuroglial damage. We have recently shown that the exogenous administration of the antioxidant protein zinc-metallothionein-II (Zn-MT-II) significantly decreased the clinical symptoms, mortality, and leukocyte infiltration of the CNS during EAE....... However, it is not known how EAE progression is regulated nor how cytokine production and cell death can be reduced. We herewith demonstrate that treatment with Zn-MT-II significantly decreased the CNS expression of IL-6 and TNF-alpha during EAE. Zn-MT-II treatment could also significantly reduce...

  5. Cytokine regulation of pro- and anti-apoptotic genes in rat hepatocytes: NF-kappaB-regulated inhibitor of apoptosis protein 2 (cIAP2) prevents apoptosis

    NARCIS (Netherlands)

    Schoemaker, Marieke H.; Ros, Jenny E.; Homan, Manon; Trautwein, Christian; Liston, Peter; Poelstra, Klaas; van Goor, Harry; Jansen, Peter L. M.; Moshage, Han

    2002-01-01

    BACKGROUND/AIMS: In acute liver failure, hepatocytes are exposed to various cytokines that activate both cell survival and apoptotic pathways. NF-kappaB is a central transcription factor in these responses. Recent studies indicate that blocking NF-kappaB causes apoptosis, indicating the existence of

  6. Synthesis and Characterization of a New Benzoindole Derivative with Apoptotic Activity Against Colon Cancer Cells.

    Science.gov (United States)

    Hajiaghaalipour, Fatemeh; Faraj, Fadhil L; Bagheri, Elham; Ali, Hapipah M; Abdulla, Mahmood Ameen; Majid, Nazia A

    2017-01-01

    Colorectal cancer is the third most common form of cancer in both men and women around the world. The chemistry and biological study of heterocyclic compounds have been an interesting area for a long time in pharmaceutical and medicinal chemistry. A new synthetic compound, 2-(1,1-dimethyl-1H-benzo[e]indol-2-yl)-3-((2-hydroxyphenyl)amino) acrylaldehyde, abbreviated as DBID, was prepared through the reaction of 2-(diformylmethylidene)-1,1- dimethylbenzo[e]indole with 2-aminophenol. The chemical structure of the synthesized compound was characterized by 1H NMR, 13C NMR and APT-NMR spectroscopy and confirmed by elemental analysis (CHN). The compound was screened for the antiproliferation effect against colorectal cancer cell line, HCT 116 and its possible mechanism of action was elucidated. To determine the IC50 value, the MTT assay was used and its apoptosisinducing effect was investigated. DBID inhibited the proliferation of HCT 116 cells with an IC50 of 9.32 µg/ml and significantly increased the levels of caspase -8, -9 and -3/7 in the treated cells compared to untreated cells. Apoptosis features in HCT 116 cell was detected in treated cells by using the AO/PI staining that confirmed that the cells had undergone remarkable morphological changes in apoptotic bodies. Furthermore, this changes in expression of caspase -8, -9 and -3 were confirmed by gene and protein quantification using RT-PCR and western blot analysis, respectively. The current study showed that the DBID compound has demonstrated chemotherapeutic activity which was evidenced by significant increases in the expression and activation of caspase and exploit the apoptotic signaling pathways to trigger cancer cell death. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  7. Agmatine Attenuates Brain Edema and Apoptotic Cell Death after Traumatic Brain Injury.

    Science.gov (United States)

    Kim, Jae Young; Lee, Yong Woo; Kim, Jae Hwan; Lee, Won Taek; Park, Kyung Ah; Lee, Jong Eun

    2015-07-01

    Traumatic brain injury (TBI) is associated with poor neurological outcome, including necrosis and brain edema. In this study, we investigated whether agmatine treatment reduces edema and apoptotic cell death after TBI. TBI was produced by cold injury to the cerebral primary motor cortex of rats. Agmatine was administered 30 min after injury and once daily until the end of the experiment. Animals were sacrificed for analysis at 1, 2, or 7 days after the injury. Various neurological analyses were performed to investigate disruption of the blood-brain barrier (BBB) and neurological dysfunction after TBI. To examine the extent of brain edema after TBI, the expression of aquaporins (AQPs), phosphorylation of mitogen-activated protein kinases (MAPKs), and nuclear translocation of nuclear factor-κB (NF-κB) were investigated. Our findings demonstrated that agmatine treatment significantly reduces brain edema after TBI by suppressing the expression of AQP1, 4, and 9. In addition, agmatine treatment significantly reduced apoptotic cell death by suppressing the phosphorylation of MAPKs and by increasing the nuclear translocation of NF-κB after TBI. These results suggest that agmatine treatment may have therapeutic potential for brain edema and neural cell death in various central nervous system diseases.

  8. Phagocytosis (cannibalism) of apoptotic neutrophils by tumor cells in gastric micropapillary carcinomas.

    Science.gov (United States)

    Barresi, Valeria; Branca, Giovanni; Ieni, Antonio; Rigoli, Luciana; Tuccari, Giovanni; Caruso, Rosario Alberto

    2015-05-14

    To identify those with a micropapillary pattern, ascertain relative frequency and document clinicopathological characteristics by reviewing gastric carcinomas. One hundred and fifty-one patients diagnosed with gastric cancer who underwent gastrectomy were retrospectively studied and the presence of a regional invasive micropapillary component was evaluated by light microscopy. All available hematoxylin-eosin (HE)-stained slides were histologically reviewed and 5 tumors were selected as putative micropapillary carcinoma when cancer cell clusters without a vascular core within empty lymphatic-like space comprised at least 5% of the tumor. Tumor tissues from these 5 invasive gastric carcinomas were immunostained using an anti-mucin 1 (MUC1) antibody (clone MA695) to detect the characteristic inside-out pattern and with D2-40 antibody to determine the presence of intratumoral lymph vessels. Detection of intraepithelial neutrophil apoptosis was evaluated in consecutive histological tissue sections by three independent methods, namely light microscopy with HE staining, the conventional terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) method and immunohistochemistry for activated caspase-3 (clone C92-605). Among 151 gastric cancers resected for cure, 5 (3.3%) were adenocarcinomas with a micropapillary component. Four of the patients died of disease from 6 to 23 mo and one patient was alive with metastases at 9 mo. All patients had advanced-stage cancer (≥ pT2) and lymph node metastasis. Positive MUC1 immunostaining on the stroma-facing surface (inside-out pattern) of the carcinomatous cluster cells, together with negative immunostaining for D2-40 in the cells limiting lymphatic-like spaces, confirmed the true micropapillary pattern in these gastric neoplasms. In all five cases, several micropapillae were infiltrated by neutrophils. HE staining, TUNEL assay and immunostaining for caspase-3 demonstrated apoptotic neutrophils within

  9. Metformin combined with aspirin significantly inhibit pancreatic cancer cell growth in vitro and in vivo by suppressing anti-apoptotic proteins Mcl-1 and Bcl-2

    Science.gov (United States)

    Yue, Wen; Zheng, Xi; Lin, Yong; Yang, Chung S.; Xu, Qing; Carpizo, Darren; Huang, Huarong; DiPaola, Robert S.; Tan, Xiang-Lin

    2015-01-01

    Metformin and aspirin have been studied extensively as cancer preventive or therapeutic agents. However, the effects of their combination on pancreatic cancer cells have not been investigated. Herein, we evaluated the effects of metformin and aspirin, alone or in combination, on cell viability, migration, and apoptosis as well as the molecular changes in mTOR, STAT3 and apoptotic signaling pathways in PANC-1 and BxPC3 cells. Metformin and aspirin, at relatively low concentrations, demonstrated synergistically inhibitory effects on cell viability. Compared to the untreated control or individual drug, the combination of metformin and aspirin significantly inhibited cell migration and colony formation of both PANC-1 and BxPC-3 cells. Metformin combined with aspirin significantly inhibited the phosphorylation of mTOR and STAT3, and induced apoptosis as measured by caspase-3 and PARP cleavage. Remarkably, metformin combined with aspirin significantly downregulated the anti-apoptotic proteins Mcl-1 and Bcl-2, and upregulated the pro-apoptotic proteins Bim and Puma, as well as interrupted their interactions. The downregulation of Mcl-1 and Bcl-2 was independent of AMPK or STAT3 pathway but partially through mTOR signaling and proteasome degradation. In a PANC-1 xenograft mouse model, we demonstrated that the combination of metformin and aspirin significantly inhibited tumor growth and downregulated the protein expression of Mcl-1 and Bcl-2 in tumors. Taken together, the combination of metformin and aspirin significantly inhibited pancreatic cancer cell growth in vitro and in vivo by regulating the pro- and anti-apoptotic Bcl-2 family members, supporting the continued investigation of this two drug combination as chemopreventive or chemotherapeutic agents for pancreatic cancer. PMID:26056043

  10. Inhibition of Autophagy Potentiates Atorvastatin-Induced Apoptotic Cell Death in Human Bladder Cancer Cells in Vitro

    Science.gov (United States)

    Kang, Minyong; Jeong, Chang Wook; Ku, Ja Hyeon; Kwak, Cheol; Kim, Hyeon Hoe

    2014-01-01

    Statins are cholesterol reduction agents that exhibit anti-cancer activity in several human cancers. Because autophagy is a crucial survival mechanism for cancer cells under stress conditions, cooperative inhibition of autophagy acts synergistically with other anti-cancer drugs. Thus, this study investigates whether combined treatment of atorvastatin and autophagy inhibitors results in enhancing the cytotoxic effects of atorvastatin, upon human bladder cancer cells, T24 and J82, in vitro. To measure cell viability, we performed the EZ-Cytox cell viability assay. We examined apoptosis by flow cytometry using annexin-V/propidium iodide (PI and western blot using procaspase-3 and poly (ADP-ribose) polymerase (PARP) antibodies. To examine autophagy activation, we evaluated the co-localization of LC3 and LysoTracker by immunocytochemistry, as well as the expression of LC3 and p62/sequestosome-1 (SQSTM1) by western blot. In addition, we assessed the survival and proliferation of T24 and J82 cells by a clonogenic assay. We found that atorvastatin reduced the cell viability of T24 and J82 cells via apoptotic cell death and induced autophagy activation, shown by the co-localization of LC3 and LysoTracker. Moreover, pharmacologic inhibition of autophagy significantly enhanced atorvastatin-induced apoptosis in T24 and J82 cells. In sum, inhibition of autophagy potentiates atorvastatin-induced apoptotic cell death in human bladder cancer cells in vitro, providing a potential therapeutic approach to treat bladder cancer. PMID:24815071

  11. Transglutaminase 2 is needed for the formation of an efficient phagocyte portal in macrophages engulfing apoptotic cells.

    Science.gov (United States)

    Tóth, Beáta; Garabuczi, Eva; Sarang, Zsolt; Vereb, György; Vámosi, György; Aeschlimann, Daniel; Blaskó, Bernadett; Bécsi, Bálint; Erdõdi, Ferenc; Lacy-Hulbert, Adam; Zhang, Ailiang; Falasca, Laura; Birge, Raymond B; Balajthy, Zoltán; Melino, Gerry; Fésüs, László; Szondy, Zsuzsa

    2009-02-15

    Transglutaminase 2 (TG2), a protein cross-linking enzyme with many additional biological functions, acts as coreceptor for integrin beta(3). We have previously shown that TG2(-/-) mice develop an age-dependent autoimmunity due to defective in vivo clearance of apoptotic cells. Here we report that TG2 on the cell surface and in guanine nucleotide-bound form promotes phagocytosis. Besides being a binding partner for integrin beta(3), a receptor known to mediate the uptake of apoptotic cells via activating Rac1, we also show that TG2 binds MFG-E8 (milk fat globulin EGF factor 8), a protein known to bridge integrin beta(3) to apoptotic cells. Finally, we report that in wild-type macrophages one or two engulfing portals are formed during phagocytosis of apoptotic cells that are characterized by accumulation of integrin beta(3) and Rac1. In the absence of TG2, integrin beta(3) cannot properly recognize the apoptotic cells, is not accumulated in the phagocytic cup, and its signaling is impaired. As a result, the formation of the engulfing portals, as well as the portals formed, is much less efficient. We propose that TG2 has a novel function to stabilize efficient phagocytic portals.

  12. Chronic MDMA induces neurochemical changes in the hippocampus of adolescent and young adult rats: Down-regulation of apoptotic markers.

    Science.gov (United States)

    García-Cabrerizo, Rubén; García-Fuster, M Julia

    2015-07-01

    While hippocampus is a brain region particularly susceptible to the effects of MDMA, the cellular and molecular changes induced by MDMA are still to be fully elucidated, being the dosage regimen, the species and the developmental stage under study great variables. This study compared the effects of one and four days of MDMA administration following a binge paradigm (3×5 mg/kg, i.p., every 2 h) on inducing hippocampal neurochemical changes in adolescent (PND 37) and young adult (PND 58) rats. The results showed that chronic MDMA caused hippocampal protein deficits in adolescent and young adult rats at different levels: (1) impaired serotonergic (5-HT2A and 5-HT2C post-synaptic receptors) and GABAergic (GAD2 enzyme) signaling, and (2) decreased structural cytoskeletal neurofilament proteins (NF-H, NF-M and NF-L). Interestingly, these effects were not accompanied by an increase in apoptotic markers. In fact, chronic MDMA inhibited proteins of the apoptotic pathway (i.e., pro-apoptotic FADD, Bax and cytochrome c) leading to an inhibition of cell death markers (i.e., p-JNK1/2, cleavage of PARP-1) and suggesting regulatory mechanisms in response to the neurochemical changes caused by the drug. The data, together with the observed lack of GFAP activation, support the view that chronic MDMA effects, regardless of the rat developmental age, extends beyond neurotransmitter systems to impair other hippocampal structural cell markers. Interestingly, inhibitory changes in proteins from the apoptotic pathway might be taking place to overcome the protein deficits caused by MDMA. Copyright © 2015 Elsevier Inc. All rights reserved.

  13. Hypothesis for thermal activation of the caspase cascade in apoptotic cell death at elevated temperatures

    Science.gov (United States)

    Pearce, John A.

    2013-02-01

    Apoptosis is an especially important process affecting disease states from HIV-AIDS to auto-immune disease to cancer. A cascade of initiator and executioner capsase functional proteins is the hallmark of apoptosis. When activated the various caspases activate other caspases or cleave structural proteins of the cytoskeleton, resulting in "blebbing" of the plasma membrane forming apoptotic bodies that completely enclose the disassembled cellular components. Containment of the cytosolic components within the apoptotic bodies differentiates apoptosis from necroptosis and necrosis, both of which release fragmented cytosol and other cellular constituents into the intracellular space. Biochemical models of caspase activation reveal the extensive feedback loops characteristic of apoptosis. They clearly explain the failure of Arrhenius models to give accurate predictions of cell survival curves in hyperthermic heating protocols. Nevertheless, each of the individual reaction velocities can reasonably be assumed to follow Arrhenius kinetics. If so, the thermal sensitivity of the reaction velocity to temperature elevation is: ∂k/∂T = Ea [k/RT2]. Particular reaction steps described by higher activation energies, Ea, are likely more thermally-sensitive than lower energy reactions and may initiate apoptosis in the absence of other stress signals. Additionally, while the classical irreversible Arrhenius formulation fails to accurately represent many cell survival and/or dye uptake curves - those that display an early stage shoulder region - an expanded reversible model of the law of mass action equation seems to prove effective and is directly based on a firm theoretical thermodynamic foundation.

  14. 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.

  15. Matrix metalloproteinase-10 promotes tumor progression through regulation of angiogenic and apoptotic pathways in cervical tumors

    International Nuclear Information System (INIS)

    Zhang, Ge; Miyake, Makito; Lawton, Adrienne; Goodison, Steve; Rosser, Charles J

    2014-01-01

    Cancer invasion and metastasis develops through a series of steps that involve the loss of cell to cell and cell to matrix adhesion, degradation of extracellular matrix and induction of angiogenesis. Different protease systems (e.g., matrix metalloproteinases, MMPs) are involved in these steps. MMP-10, one of the lesser studied MMPs, is limited to epithelial cells and can facilitate tumor cell invasion by targeting collagen, elastin and laminin. Enhanced MMP-10 expression has been linked to poor clinical prognosis in some cancers, however, mechanisms underlying a role for MMP-10 in tumorigenesis and progression remain largely unknown. Here, we report that MMP-10 expression is positively correlated with the invasiveness of human cervical and bladder cancers. Using commercial tissue microarray (TMA) of cervical and bladder tissues, MMP-10 immunohistochemical staining was performed. Furthermore using a panel of human cells (HeLa and UROtsa), in vitro and in vivo experiments were performed in which MMP-10 was overexpressed or silenced and we noted phenotypic and genotypic changes. Experimentally, we showed that MMP-10 can regulate tumor cell migration and invasion, and endothelial cell tube formation, and that MMP-10 effects are associated with a resistance to apoptosis. Further investigation revealed that increasing MMP-10 expression stimulates the expression of HIF-1α and MMP-2 (pro-angiogenic factors) and PAI-1 and CXCR2 (pro-metastatic factors), and accordingly, targeting MMP-10 with siRNA in vivo resulted in diminution of xenograft tumor growth with a concomitant reduction of angiogenesis and a stimulation of apoptosis. Taken together, our findings show that MMP-10 can play a significant role in tumor growth and progression, and that MMP-10 perturbation may represent a rational strategy for cancer treatment

  16. Reduced IRE1α mediates apoptotic cell death by disrupting calcium homeostasis via the InsP3 receptor.

    Science.gov (United States)

    Son, S M; Byun, J; Roh, S-E; Kim, S J; Mook-Jung, I

    2014-04-17

    The endoplasmic reticulum (ER) is not only a home for folding and posttranslational modifications of secretory proteins but also a reservoir for intracellular Ca(2+). Perturbation of ER homeostasis contributes to the pathogenesis of various neurodegenerative diseases, such as Alzheimer's and Parkinson diseases. One key regulator that underlies cell survival and Ca(2+) homeostasis during ER stress responses is inositol-requiring enzyme 1α (IRE1α). Despite extensive studies on this ER membrane-associated protein, little is known about the molecular mechanisms by which excessive ER stress triggers cell death and Ca(2+) dysregulation via the IRE1α-dependent signaling pathway. In this study, we show that inactivation of IRE1α by RNA interference increases cytosolic Ca(2+) concentration in SH-SY5Y cells, leading to cell death. This dysregulation is caused by an accelerated ER-to-cytosolic efflux of Ca(2+) through the InsP3 receptor (InsP3R). The Ca(2+) efflux in IRE1α-deficient cells correlates with dissociation of the Ca(2+)-binding InsP3R inhibitor CIB1 and increased complex formation of CIB1 with the pro-apoptotic kinase ASK1, which otherwise remains inactivated in the IRE1α-TRAF2-ASK1 complex. The increased cytosolic concentration of Ca(2+) induces mitochondrial production of reactive oxygen species (ROS), in particular superoxide, resulting in severe mitochondrial abnormalities, such as fragmentation and depolarization of membrane potential. These Ca(2+) dysregulation-induced mitochondrial abnormalities and cell death in IRE1α-deficient cells can be blocked by depleting ROS or inhibiting Ca(2+) influx into the mitochondria. These results demonstrate the importance of IRE1α in Ca(2+) homeostasis and cell survival during ER stress and reveal a previously unknown Ca(2+)-mediated cell death signaling between the IRE1α-InsP3R pathway in the ER and the redox-dependent apoptotic pathway in the mitochondrion.

  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. Gallic acid induced apoptotic events in HCT-15 colon cancer cells

    Science.gov (United States)

    Subramanian, Aruna Priyadharshni; Jaganathan, Saravana Kumar; Mandal, Mahitosh; Supriyanto, Eko; Muhamad, Ida Idayu

    2016-01-01

    AIM: To investigate the inhibitory action of diet-derived phenolic compound gallic acid (GA) against HCT-15 colon cancer cells. METHODS: The antiproliferative effect of GA against colon cancer cells was determined by performing thiazolyl blue tetrazolium bromide (MTT) assay. The colony forming ability of GA treated colon cancer cells was evaluated using the colony forming assay. The cell cycle changes induced by GA in HCT-15 cells were analyzed by propidium iodide staining. Levels of reactive oxygen species (ROS) and mitochondrial membrane potential of HCT-15 exposed to GA was assessed using 2’,7’-dichlorfluorescein-diacetate and rhodamine-123 respectively, with the help of flow cytometry. Morphological changes caused by GA treatment in the colon cancer cells were identified by scanning electron microscope and photomicrograph examination. Apoptosis was confirmed using flow cytometric analysis of GA treated HCT-15 cells after staining with Yo-Pro-1. RESULTS: MTT assay results illustrated that GA has an inhibitory effect on HCT-15 cells with IC50 value of 740 μmol/L. A time-dependent inhibition of colony formation was evident with GA treatment. Cell cycle arrest was evident from the accumulation of GA treated HCT-15 cells at sub-G1 phase (0.98 ± 1.03 vs 58.01 ± 2.05) with increasing exposure time. Flow cytometric analysis of GA treated HCT-15 cells depicted early events associated with apoptosis like lipid layer breakage and fall in mitochondrial membrane potential apart from an increase in the generation of ROS which were in a time dependent manner. SEM and photomicrograph images of the GA-treated cells displayed membrane blebbing and cell shrinking characteristics of apoptosis. Further apoptosis confirmation by Yo-Pro-1 staining also showed the time-dependent increase of apoptotic cells after treatment. CONCLUSION: These results show that GA induced ROS dependent apoptosis and inhibited the growth of colon cancer cells. PMID:27099438

  19. The Acetone Extract of Sclerocarya birrea (Anacardiaceae) Possesses Antiproliferative and Apoptotic Potential against Human Breast Cancer Cell Lines (MCF-7)

    Science.gov (United States)

    Tanih, Nicoline Fri; Ndip, Roland Ndip

    2013-01-01

    Interesting antimicrobial data from the stem bark of Sclerocarya birrea, which support its use in traditional medicine for the treatment of many diseases, have been delineated. The current study was aimed to further study some pharmacological and toxicological properties of the plant to scientifically justify its use. Anticancer activity of water and acetone extracts of S. birrea was evaluated on three different cell lines, HT-29, HeLa, and MCF-7 using the cell titre blue viability assay in 96-well plates. Apoptosis was evaluated using the acridine orange and propidium iodide staining method, while morphological structure of treated cells was examined using SEM. The acetone extract exhibited remarkable antiproliferative activities on MCF-7 cell lines at dose- and time-dependent manners (24 h and 48 h of incubation). The extract also exerted apoptotic programmed cell death in MCF-7 cells with significant effect on the DNA. Morphological examination also displayed apoptotic characteristics in the treated cells, including clumping, condensation, and culminating to budding of the cells to produce membrane-bound fragmentation, as well as formation of apoptotic bodies. The acetone extract of S. birrea possesses antiproliferative and apoptotic potential against MCF-7-treated cells and could be further exploited as a potential lead in anticancer therapy. PMID:23576913

  20. Terminalia Chebula provides protection against dual modes of necroptotic and apoptotic cell death upon death receptor ligation.

    Science.gov (United States)

    Lee, Yoonjung; Byun, Hee Sun; Seok, Jeong Ho; Park, Kyeong Ah; Won, Minho; Seo, Wonhyoung; Lee, So-Ra; Kang, Kidong; Sohn, Kyung-Cheol; Lee, Ill Young; Kim, Hyeong-Geug; Son, Chang Gue; Shen, Han-Ming; Hur, Gang Min

    2016-04-27

    Death receptor (DR) ligation elicits two different modes of cell death (necroptosis and apoptosis) depending on the cellular context. By screening a plant extract library from cells undergoing necroptosis or apoptosis, we identified a water extract of Terminalia chebula (WETC) as a novel and potent dual inhibitor of DR-mediated cell death. Investigation of the underlying mechanisms of its anti-necroptotic and anti-apoptotic action revealed that WETC or its constituents (e.g., gallic acid) protected against tumor necrosis factor-induced necroptosis via the suppression of TNF-induced ROS without affecting the upstream signaling events. Surprisingly, WETC also provided protection against DR-mediated apoptosis by inhibition of the caspase cascade. Furthermore, it activated the autophagy pathway via suppression of mTOR. Of the WETC constituents, punicalagin and geraniin appeared to possess the most potent anti-apoptotic and autophagy activation effect. Importantly, blockage of autophagy with pharmacological inhibitors or genetic silencing of Atg5 selectively abolished the anti-apoptotic function of WETC. These results suggest that WETC protects against dual modes of cell death upon DR ligation. Therefore, WETC might serve as a potential treatment for diseases characterized by aberrantly sensitized apoptotic or non-apoptotic signaling cascades.

  1. Co-culture of apoptotic breast cancer cells with immature dendritic cells: a novel approach for DC-based vaccination in breast cancer

    International Nuclear Information System (INIS)

    Zheng, Jin; Liu, Qiang; Yang, Jiandong; Ren, Qinyou; Cao, Wei; Yang, Jingyue; Yu, Zhaocai; Yu, Fang; Wu, Yanlan; Shi, Hengjun; Liu, Wenchao

    2012-01-01

    A dendritic cell (DC)-based vaccine strategy could reduce the risk of recurrence and improve the survival of breast cancer patients. However, while therapy-induced apoptosis of hepatocellular and colorectal carcinoma cells can enhance maturation and antigen presentation of DCs, whether this effect occurs in breast cancer is currently unknown. In the present study, we investigated the effect of doxorubicin (ADM)-induced apoptotic MCF-7 breast cancer cells on the activation of DCs. ADM-induced apoptotic MCF-7 cells could effectively induce immature DC (iDC) maturation. The mean fluorescence intensity (MFI) of DC maturity marker CD83 was 23.3 in the ADM-induced apoptotic MCF-7 cell group compared with 8.5 in the MCF-7 cell group. The MFI of DC co-stimulatory marker CD86 and HLA-DR were also increased after iDCs were treated with ADM-induced apoptotic MCF-7 cells. Furthermore, the proliferating autologous T-lymphocytes increased from 14.2 to 40.3% after incubated with DCs induced by apoptotic MCF-7 cells. The secretion of interferon-γ by these T-lymphocytes was also increased. In addition, cell-cell interaction between apoptotic MCF-7 cells and iDCs, but not soluble factors released by apoptotic MCF-7 cells, was crucial for the maturation of iDCs. These findings constitute a novel in vitro DC-based vaccine strategy for the treatment of breast cancer by ADM-induced apoptotic MCF-7 cells

  2. Co-culture of apoptotic breast cancer cells with immature dendritic cells: a novel approach for DC-based vaccination in breast cancer

    Energy Technology Data Exchange (ETDEWEB)

    Zheng, Jin [Department of Oncology, State Key Discipline of Cell Biology, Xijing Hospital, the Fourth Military Medical University, Xi' an, Shaanxi (China); Department of Traditional Chinese and Western Medicine of Oncology, Tangdu Hospital, the Fourth Military Medical University, Xi' an, Shaanxi (China); Liu, Qiang [Department of Hematology, Tangdu Hospital, the Fourth Military Medical University, Xi' an, Shaanxi (China); Yang, Jiandong [Department of Hepatobiliary Surgery, Xijing Hospital, the Fourth Military Medical University, Xi' an, Shaanxi (China); Ren, Qinyou [Department of Traditional Chinese and Western Medicine of Oncology, Tangdu Hospital, the Fourth Military Medical University, Xi' an, Shaanxi (China); Cao, Wei [Department of Interventional Radiology, Tangdu Hospital, the Fourth Military Medical University, Xi' an, Shaanxi (China); Yang, Jingyue; Yu, Zhaocai [Department of Oncology, State Key Discipline of Cell Biology, Xijing Hospital, the Fourth Military Medical University, Xi' an, Shaanxi (China); Yu, Fang [Department of Gastrointestinal Surgery, Xijing Hospital of Digestive Diseases, the Fourth Military Medical University, Xi' an, Shaanxi (China); Wu, Yanlan [Department of Infectious Diseases, Tangdu Hospital, the Fourth Military Medical University, Xi' an, Shaanxi (China); Shi, Hengjun [Department of Traditional Chinese and Western Medicine of Oncology, Tangdu Hospital, the Fourth Military Medical University, Xi' an, Shaanxi (China); Liu, Wenchao [Department of Oncology, State Key Discipline of Cell Biology, Xijing Hospital, the Fourth Military Medical University, Xi' an, Shaanxi (China)

    2012-04-27

    A dendritic cell (DC)-based vaccine strategy could reduce the risk of recurrence and improve the survival of breast cancer patients. However, while therapy-induced apoptosis of hepatocellular and colorectal carcinoma cells can enhance maturation and antigen presentation of DCs, whether this effect occurs in breast cancer is currently unknown. In the present study, we investigated the effect of doxorubicin (ADM)-induced apoptotic MCF-7 breast cancer cells on the activation of DCs. ADM-induced apoptotic MCF-7 cells could effectively induce immature DC (iDC) maturation. The mean fluorescence intensity (MFI) of DC maturity marker CD83 was 23.3 in the ADM-induced apoptotic MCF-7 cell group compared with 8.5 in the MCF-7 cell group. The MFI of DC co-stimulatory marker CD86 and HLA-DR were also increased after iDCs were treated with ADM-induced apoptotic MCF-7 cells. Furthermore, the proliferating autologous T-lymphocytes increased from 14.2 to 40.3% after incubated with DCs induced by apoptotic MCF-7 cells. The secretion of interferon-γ by these T-lymphocytes was also increased. In addition, cell-cell interaction between apoptotic MCF-7 cells and iDCs, but not soluble factors released by apoptotic MCF-7 cells, was crucial for the maturation of iDCs. These findings constitute a novel in vitro DC-based vaccine strategy for the treatment of breast cancer by ADM-induced apoptotic MCF-7 cells.

  3. Simulated hypogravity impairs the angiogenic response of endothelium by up-regulating apoptotic signals

    International Nuclear Information System (INIS)

    Morbidelli, Lucia; Monici, Monica; Marziliano, Nicola; Cogoli, Augusto; Fusi, Franco; Waltenberger, Johannes; Ziche, Marina

    2005-01-01

    Health hazards in astronauts are represented by cardiovascular problems and impaired bone healing. These disturbances are characterized by a common event, the loss of function by vascular endothelium, leading to impaired angiogenesis. We investigated whether the exposure of cultured endothelial cells to hypogravity condition could affect their behaviour in terms of functional activity, biochemical responses, morphology, and gene expression. Simulated hypogravity conditions for 72 h produced a reduction of cell number. Genomic analysis of endothelial cells exposed to hypogravity revealed that proapoptotic signals increased, while antiapoptotic and proliferation/survival genes were down-regulated by modelled low gravity. Activation of apoptosis was accompanied by morphological changes with mitochondrial disassembly and organelles/cytoplasmic NAD(P)H redistribution, as evidenced by autofluorescence analysis. In this condition cells were not able to respond to angiogenic stimuli in terms of migration and proliferation. Our study documents functional, morphological, and transcription alterations in vascular endothelium exposed to simulated low gravity conditions, thus providing insights on the occurrence of vascular tissue dysregulation in crewmen during prolonged space flights. Moreover, the alteration of vascular endothelium can intervene as a concause in other systemic effects, like bone remodelling, observed in weightlessness

  4. Developmental programming: impact of prenatal testosterone excess on ovarian cell proliferation and apoptotic factors in sheep.

    Science.gov (United States)

    Salvetti, Natalia R; Ortega, Hugo H; Veiga-Lopez, Almudena; Padmanabhan, Vasantha

    2012-07-01

    Prenatal testosterone (T) excess leads to reproductive dysfunctions in sheep, which include increased ovarian follicular recruitment and persistence. To test the hypothesis that follicular disruptions in T sheep stem from changes in the developmental ontogeny of ovarian proliferation and apoptotic factors, pregnant Suffolk sheep were injected twice weekly with T propionate or dihydrotestosterone propionate (DHT; a nonaromatizable androgen) from Days 30 to 90 of gestation. Changes in developmental expression of proliferating cell nuclear antigen (PCNA), BCL2, BAX, activated CASP3, and FAS/FASLG were determined at Fetal Days 90 and 140, 22 wk, 10 mo, and 21 mo of age by immunocytochemisty. Prenatal T treatment induced changes in expression of proliferative and apoptotic markers in a follicle-, age-, and steroid-specific manner. Changes in BAX were evident only during fetal life and PCNA, BCL2, and CASP3 only postnatally. Prenatal T and not DHT increased PCNA and decreased BCL2 in granulosa/theca cells of antral follicles at 10 and 21 mo but decreased CASP3 in granulosa/theca cells of antral follicles at 22 wk (prepubertal) and 10 and 21 mo. Both treatments decreased BAX immunostaining in granulosa cells of Fetal Day 90 primordial/primary follicles. Neither treatment affected FAS expression at any developmental time point in any follicular compartment. Effects on BAX appear to be programmed by androgenic actions and PCNA, BCL2, and CASP3 by estrogenic actions of T. Overall, the findings demonstrate that fetal exposure to excess T disrupts the ovarian proliferation/apoptosis balance, thus providing a basis for the follicular disruptions evidenced in these females.

  5. TNF alpha induces ABCA1 through NF-kappa B in macrophages and in phagocytes ingesting apoptotic cells

    NARCIS (Netherlands)

    Gerbod-Giannone, Marie-Christine; Li, Yankun; Holleboom, Adriaan; Han, Seongah; Hsu, Li-Chung; Tabas, Ira; Tall, Alan R.

    2006-01-01

    Recent evidence suggests that tumor necrosis factor alpha (TNF alpha) signaling in vascular cells can have antiatherogenic consequences, but the mechanisms are poorly understood. TNFa is released by free cholesterol loaded apoptotic macrophages, and the clearance of these cells by phagocytic

  6. Mast cells dysregulate apoptotic and cell cycle genes in mucosal squamous cell carcinoma

    Directory of Open Access Journals (Sweden)

    Davis Paul

    2006-12-01

    Full Text Available Abstract Background Mucosal squamous cell carcinoma of the head and neck is a disease of high mortality and morbidity. Interactions between the squamous cell carcinoma and the host's local immunity, and how the latter contributes to the biological behavior of the tumor are unclear. In vivo studies have demonstrated sequential mast cell infiltration and degranulation during squamous cell carcinogenesis. The degree of mast cell activation correlates closely with distinct phases of hyperkeratosis, dysplasia, carcinoma in-situ and invasive carcinoma. However, the role of mast cells in carcinogenesis is unclear. Aim This study explores the effects of mast cells on the proliferation and gene expression profile of mucosal squamous cell carcinoma using human mast cell line (HMC-1 and human glossal squamous cell carcinoma cell line (SCC25. Methods HMC-1 and SCC25 were co-cultured in a two-compartment chamber, separated by a polycarbonate membrane. HMC-1 was stimulated to degranulate with calcium ionophore A23187. The experiments were done in quadruplicate. Negative controls were established where SCC25 were cultured alone without HMC-1. At 12, 24, 48 and 72 hours, proliferation and viability of SCC25 were assessed with MTT colorimetric assay. cDNA microarray was employed to study differential gene expression between co-cultured and control SCC25. Results HMC-1/SCC25 co-culture resulted in suppression of growth rate for SCC-25 (34% compared with 110% for the control by 72 hours, p Conclusion We show that mast cells have a direct inhibitory effect on the proliferation of mucosal squamous cell carcinoma in vitro by dysregulating key genes in apoptosis and cell cycle control.

  7. Anti-mutagenic and Pro-apoptotic Effects of Apigenin on Human Chronic Lymphocytic Leukemia Cells

    Directory of Open Access Journals (Sweden)

    Mehrdad Hashemi

    2010-09-01

    Full Text Available "nDiet can play a vital role in cancer prevention. Nowadays the scientists are looking for food materials which can potentially prevent the cancer occurrence. The purpose of this research is to examine anti-mutagenic and apoptotic effects of apigenin in human lymphoma cells. In present study human chronic lymphocytic leukemia (Eheb cell line were cultured in RPMI 1640 (Sigma, supplemented with 10% fetal calf serum, penicillin-streptomycin, L-glutamine and incubated at 37 ºC for 2 days. In addition cancer cell line was treated by and apigenin and cellular vital capacity was determined by MTT assay. Then effect of apigenin in human lymphoma B cells was examined by flow cytometry techniques. The apigenin was subsequently evaluated in terms of anti-mutagenic properties by a standard reverse mutation assay (Ames test. This was performed with histidine auxotroph strain of Salmonella typhimurium (TA100. Thus, it requires histidine from a foreign supply to ensure its growth. The aforementioned strain gives rise to reverted colonies when expose to sodium azide as a carcinogen substance. During MTT assay, human chronic lymphocytic leukemia revealed to have a meaningful cell death when compared with controls (P<0.01 Apoptosis was induced suitably after 48 hours by flow cytometry assay. In Ames test apigenin prevented the reverted mutations and the hindrance percent of apigenin was 98.17%.These results have revealed apigenin induced apoptosis in human lymphoma B cells in vitro.

  8. Exploration of intrinsic and extrinsic apoptotic pathways in zearalenone-treated rat sertoli cells.

    Science.gov (United States)

    Xu, Ming-Long; Hu, Jin; Guo, Bao-Ping; Niu, Ya-Ru; Xiao, Cheng; Xu, Yin-Xue

    2016-12-01

    Zearalenone (ZEA) is a nonsteroidal estrogenic mycotoxin produced mainly by Fusarium. ZEA causes reproductive disorders and is both cytotoxic and genotoxic in animals; however, little is known regarding the molecular mechanism(s) leading to ZEA toxicity. Sertoli cells are somatic cells that support the development of spermatogenic cells. The objective of this study was to explore the effects of ZEA on the proliferation, apoptosis, and necrosis of rat Sertoli cells to uncover signaling pathways underlying ZEA cytotoxicity. ZEA reduced the proliferation of rat Sertoli cells in a dose-dependent manner, as indicated by a CCK8 assay, while flow cytometry revealed that ZEA caused both apoptosis and necrosis. Immunoblotting revealed that ZEA treatment increased the ratio of Bax/Bcl-2, as well as the expression of FasL and caspases-3, -8, and -9, in a dose-dependent manner. Collectively, these data suggest that ZEA induced apoptosis and necrosis in rat Sertoli cells via extrinsic and intrinsic apoptotic pathways. This study provides new insights into the molecular mechanisms by which ZEA exhibits cytotoxicity. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1731-1739, 2016. © 2015 Wiley Periodicals, Inc.

  9. Calreticulin Release at an Early Stage of Death Modulates the Clearance by Macrophages of Apoptotic Cells

    Science.gov (United States)

    Osman, Rim; Tacnet-Delorme, Pascale; Kleman, Jean-Philippe; Millet, Arnaud; Frachet, Philippe

    2017-01-01

    Calreticulin (CRT) is a well-known “eat-me” signal harbored by dying cells participating in their recognition by phagocytes. CRT is also recognized to deeply impact the immune response to altered self-cells. In this study, we focus on the role of the newly exposed CRT following cell death induction. We show that if CRT increases at the outer face of the plasma membrane and is well recognized by C1q even when phosphatidylserine is not yet detected, CRT is also released in the surrounding milieu and is able to interact with phagocytes. We observed that exogenous CRT is endocytosed by THP1 macrophages through macropinocytosis and that internalization is associated with a particular phenotype characterized by an increase of cell spreading and migration, an upregulation of CD14, an increase of interleukin-8 release, and a decrease of early apoptotic cell uptake. Importantly, CRT-induced pro-inflammatory phenotype was confirmed on human monocytes-derived macrophages by the overexpression of CD40 and CD274, and we found that monocyte-derived macrophages exposed to CRT display a peculiar polarization notably associated with a downregulation of the histocompatibility complex of class II molecules hampering its description through the classical M1/M2 dichotomy. Altogether our results highlight the role of soluble CRT with strong possible consequences on the macrophage-mediated immune response to dying cell. PMID:28878781

  10. Calreticulin Release at an Early Stage of Death Modulates the Clearance by Macrophages of Apoptotic Cells

    Directory of Open Access Journals (Sweden)

    Rim Osman

    2017-08-01

    Full Text Available Calreticulin (CRT is a well-known “eat-me” signal harbored by dying cells participating in their recognition by phagocytes. CRT is also recognized to deeply impact the immune response to altered self-cells. In this study, we focus on the role of the newly exposed CRT following cell death induction. We show that if CRT increases at the outer face of the plasma membrane and is well recognized by C1q even when phosphatidylserine is not yet detected, CRT is also released in the surrounding milieu and is able to interact with phagocytes. We observed that exogenous CRT is endocytosed by THP1 macrophages through macropinocytosis and that internalization is associated with a particular phenotype characterized by an increase of cell spreading and migration, an upregulation of CD14, an increase of interleukin-8 release, and a decrease of early apoptotic cell uptake. Importantly, CRT-induced pro-inflammatory phenotype was confirmed on human monocytes-derived macrophages by the overexpression of CD40 and CD274, and we found that monocyte-derived macrophages exposed to CRT display a peculiar polarization notably associated with a downregulation of the histocompatibility complex of class II molecules hampering its description through the classical M1/M2 dichotomy. Altogether our results highlight the role of soluble CRT with strong possible consequences on the macrophage-mediated immune response to dying cell.

  11. Apoptotic Effect of the Urtica Dioica Plant Extracts on Breast Cancer Cell Line (MDA- MB- 468

    Directory of Open Access Journals (Sweden)

    A Mohammadi

    2015-09-01

    Full Text Available Background & objectives: Cancer is one of the most causes of mortality in worldwide. Components derived from natural plants that induce apoptosis are used for cancer treatment. Therefore investigation of different herbal components for new anti-cancer drug is one of the main research activities throughout the world. According to low cost, oral consumption and easy access to the public extracts of Urtica dioica, in this study we aimed to investigate the effectiveness of this herb on MDA-MB-468 breast cancer cells.   Methods: Cytotoxic effect of Urtica dioica extract was measured using MTT assays. To show induction of apoptosis by this plant TUNEL and DNA Fragmentation test were performed.   Results: In the present study dichloromethane extracts noticeably killed cancer cells. IC50 values related to human breast adenocarcinoma cell line MDA-MB-468 were 29.46±1.05 µg/ml in 24 hours and 15.54±1.04 µg/ml in 48 hours. TUNEL test and DNA Fragmentation assay showed apoptotic characteristic in the extract treated cells.   Conclusion: The results showed that MDA-MB-468 cells after treatment with dichloromethane extract of Urtica dioica, induces apoptosis in MDA-MB-468 cancer cells which may be useful in the treatment of cancer.

  12. Interaction of DNA-lesions induced by sodium fluoride and radiation and its influence in apoptotic induction in cancer cell lines

    Directory of Open Access Journals (Sweden)

    Santosh Podder

    2015-01-01

    Full Text Available Fluoride is an essential trace element but also an environmental contaminant with major sources of exposure being drinking water, food and pesticides. Previous studies showed that sodium fluoride (NaF at 5 mM or more is required to induce apoptosis and chromosome aberrations and proposed that DNA damage and apoptosis play an important role in toxicity of excessive fluoride. The aim of this study is directed to understand the nature of DNA-lesions induced by NaF by allowing its interaction with radiation induced DNA-lesions. NaF 5 mM was used after observing inability to induce DNA damages and apoptosis by single exposure with 50 μM or 1 mM NaF. Co-exposure to NaF and radiation significantly increased the frequency of aberrant metaphases and exchange aberrations in human lymphocytes and arrested the cells in G1 stage instead of apoptotic death. Flow cytometric analysis, DNA fragmentation and PARP-cleavage analysis clearly indicated that 5 mM NaF together with radiation (1 Gy induced apoptosis in both U87 and K562 cells due to down regulation of expression of anti-apoptotic proteins, like Bcl2 in U87 and inhibitors of apoptotic proteins like survivin and cIAP in K562 cells. This study herein suggested that single exposure with extremely low concentration of NaF unable to induce DNA lesions whereas higher concentration induced DNA lesions interact with the radiation-induced DNA lesions. Both are probably repaired rapidly thus showed increased interactive effect. Coexposure to NaF and radiation induces more apoptosis in cancer cell lines which could be due to increased exchange aberrations through lesions interaction and downregulating anti-apoptotic genes.

  13. Involvement of apoptotic cell death and cell cycle perturbation in retinoic acid-induced cleft palate in mice

    International Nuclear Information System (INIS)

    Okano, Junko; Suzuki, Shigehiko; Shiota, Kohei

    2007-01-01

    Retinoic acid (RA), a metabolite of vitamin A, plays a key role in a variety of biological processes and is essential for normal embryonic development. On the other hand, exogenous RA could cause cleft palate in offspring when it is given to pregnant animals at either the early or late phases of palatogenesis, but the pathogenetic mechanism of cleft palate caused by excess RA remains not fully elucidated. The aim of the present study was to investigate the effects of excess of RA on early palatogenesis in mouse fetuses and analyze the teratogenic mechanism, especially at the stage prior to palatal shelf elevation. We gave all-trans RA (100 mg/kg) orally to E11.5 ICR pregnant mice and observed the changes occurring in the palatal shelves of their fetuses. It was found that apoptotic cell death increased not only in the epithelium of the palatal shelves but also in the tongue primordium, which might affect tongue withdrawal movement during palatogenesis and impair the horizontal elevation of palatal shelves. In addition, RA was found to prevent the G 1 /S progression of palatal mesenchymal cells through upregulation of p21 Cip1 , leading to Rb hypophospholylation. Thus, RA appears to cause G 1 arrest in palatal mesenchymal cells in a similar manner as in various cancer and embryonic cells. It is likely that apoptotic cell death and cell cycle disruption are involved in cleft palate formation induced by RA

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

    Directory of Open Access Journals (Sweden)

    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.

  15. TRPV2 activation induces apoptotic cell death in human T24 bladder cancer cells: a potential therapeutic target for bladder cancer.

    Science.gov (United States)

    Yamada, Takahiro; Ueda, Takashi; Shibata, Yasuhiro; Ikegami, Yosuke; Saito, Masaki; Ishida, Yusuke; Ugawa, Shinya; Kohri, Kenjiro; Shimada, Shoichi

    2010-08-01

    To investigate the functional expression of the transient receptor potential vanilloid 2 (TRPV2) channel protein in human urothelial carcinoma (UC) cells and to determine whether calcium influx into UC cells through TRPV2 is involved in apoptotic cell death. The expression of TRPV2 mRNA in bladder cancer cell lines (T24, a poorly differentiated UC cell line and RT4, a well-differentiated UC cell line) was analyzed using reverse transcriptase-polymerase chain reaction. The calcium permeability of TRPV2 channels in T24 cells was investigated using a calcium imaging assay that used cannabidiol (CBD), a relatively selective TRPV2 agonist, and ruthenium red (RuR), a nonselective TRPV channel antagonist. The death of T24 or RT4 cells in the presence of CBD was evaluated using a cellular viability assay. Apoptosis of T24 cells caused by CBD was confirmed using an annexin-V assay and small interfering RNA (siRNA) silencing of TRPV2. TRPV2 mRNA was abundantly expressed in T24 cells. The expression level in UC cells was correlated with high-grade disease. The administration of CBD increased intracellular calcium concentrations in T24 cells. In addition, the viability of T24 cells progressively decreased with increasing concentrations of CBD, whereas RT4 cells were mostly unaffected. Cell death occurred via apoptosis caused by continuous influx of calcium through TRPV2. TRPV2 channels in UC cells are calcium-permeable and the regulation of calcium influx through these channels leads directly to the death of UC cells. TRPV2 channels in UC cells may be a potential new therapeutic target, especially in higher-grade UC cells. Copyright 2010 Elsevier Inc. All rights reserved.

  16. Synergistic apoptotic response between valproic acid and fludarabine in chronic lymphocytic leukaemia (CLL) cells involves the lysosomal protease cathepsin B

    International Nuclear Information System (INIS)

    Yoon, J-Y; Szwajcer, D; Ishdorj, G; Benjaminson, P; Xiao, W; Kumar, R; Johnston, J B; Gibson, S B

    2013-01-01

    Fludarabine, a nucleoside analogue, is commonly used in combination with other agents for the treatment of chronic lymphocytic leukaemia (CLL). In previous studies, valproic acid (VPA), an inhibitor of histone deacetylases, combined with fludarabine to synergistically increase apoptotic cell death in CLL cells. In the present study, we found that the combination of fludarabine and VPA decreases the level of the anti-apoptotic proteins Mcl-1 and XIAP in primary CLL cells. Treatment with fludarabine alone, or in combination with VPA, led to the loss of lysosome integrity, and chemical inhibition of the lysosomal protease cathepsin B, using CA074-Me, was sufficient to reduce apoptosis. VPA treatment increased cathepsin B levels and activities in primary CLL cells, thereby priming CLL cells for lysosome-mediated cell death. Six previously treated patients with relapsed CLL were treated with VPA, followed by VPA/fludarabine combination. The combined therapy resulted in reduced lymphocyte count in five out of six and reduced lymph node sizes in four out of six patients. In vivo VPA treatment increased histone-3 acetylation and cathepsin B expression levels. Thus, the synergistic apoptotic response with VPA and fludarabine in CLL is mediated by cathepsin B activation leading to a decrease in the anti-apoptotic proteins

  17. Anti-mutagenic and Pro-apoptotic Effects of Apigenin on Human Chronic Lymphocytic Leukemia Cells

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    Mehrdad Hashemi

    2010-10-01

    Full Text Available Diet can play a vital role in cancer prevention. Nowadays the scientists are looking for food materials which can potentially prevent the cancer occurrence. The purpose of this research is to examine anti-mutagenic and apoptotic effects of apigenin in human lymphoma cells. In present study human chronic lymphocytic leukemia (Eheb cell line were cultured in RPMI 1640 (Sigma, supplemented with 10% fetal calf serum, penicillin-streptomycin, L-glutamine and incubated at 37 ºC for 2 days. In addition cancer cell line was treated by and apigenin and cellular vital capacity was determined by MTT assay. Then effect of apigenin in human lymphoma B cells was examined by flow cytometry techniques. The apigenin was subsequently evaluated in terms of anti-mutagenic properties by a standard reverse mutation assay (Ames test. This was performed with histidine auxotroph strain of Salmonella typhimurium (TA100. Thus, it requires histidine from a foreign supply to ensure its growth. The aforementioned strain gives rise to reverted colonies when expose to sodium azide as a carcinogen substance. During MTT assay, human chronic lymphocytic leukemia revealed to have a meaningful cell death when compared with controls (P

  18. Trichostatin A Enhances the Apoptotic Potential of Palladium Nanoparticles in Human Cervical Cancer Cells

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    Xi-Feng Zhang

    2016-08-01

    Full Text Available Cervical cancer ranks seventh overall among all types of cancer in women. Although several treatments, including radiation, surgery and chemotherapy, are available to eradicate or reduce the size of cancer, many cancers eventually relapse. Thus, it is essential to identify possible alternative therapeutic approaches for cancer. We sought to identify alternative and effective therapeutic approaches, by first synthesizing palladium nanoparticles (PdNPs, using a novel biomolecule called saponin. The synthesized PdNPs were characterized by several analytical techniques. They were significantly spherical in shape, with an average size of 5 nm. Recently, PdNPs gained much interest in various therapies of cancer cells. Similarly, histone deacetylase inhibitors are known to play a vital role in anti-proliferative activity, gene expression, cell cycle arrest, differentiation and apoptosis in various cancer cells. Therefore, we selected trichostatin A (TSA and PdNPs and studied their combined effect on apoptosis in cervical cancer cells. Cells treated with either TSA or PdNPs showed a dose-dependent effect on cell viability. The combinatorial effect, tested with 50 nM TSA and 50 nMPdNPs, had a more dramatic inhibitory effect on cell viability, than either TSA or PdNPs alone. The combination of TSA and PdNPs had a more pronounced effect on cytotoxicity, oxidative stress, mitochondrial membrane potential (MMP, caspase-3/9 activity and expression of pro- and anti-apoptotic genes. Our data show a strong synergistic interaction between TSA and PdNPs in cervical cancer cells. The combinatorial treatment increased the therapeutic potential and demonstrated relevant targeted therapy for cervical cancer. Furthermore, we provide the first evidence for the combinatory effect and cytotoxicity mechanism of TSA and PdNPs in cervical cancer cells.

  19. Cytotoxic and apoptotic activities of black widow spiderling extract against HeLa cells.

    Science.gov (United States)

    Peng, Xiaozhen; Dai, Zhipan; Lei, Qian; Liang, Long; Yan, Shuai; Wang, Xianchun

    2017-06-01

    Black widow spiders contain toxic components not only in the venom glands but also in other parts of the spider body, including the legs and abdomen. Additionally, both the eggs and newborn spiderlings of the black widow spider contain venom. It is important to investigate their potential effects on cancer cells. In the present study, the effects of newborn black widow spiderling extract on human HeLa cells were evaluated in vitro . When applied at different concentrations, the total extract decreased HeLa cell viability in a dose-dependent manner, with an IC 50 value of 158 µg/ml. Flow cytometry indicated that treatment of HeLa cells with the total extract of the spiderlings induced apoptosis in HeLa cells in a dose-dependent manner and led to cell cycle arrest in the S-phase. Additionally, application of the total extract at different concentrations increased apoptosis-related caspase 3 activity in a dose-dependent manner. HeLa cells treated with the total extract appeared to be morphologically changed, exhibiting membrane blebbing, nuclear fragmentation and condensation of chromatin. Further separation and activity screening demonstrated that the cytotoxic and apoptotic activities of the total extract were attributable mainly to its high molecular mass proteins, one of which was purified and characterized to determine its anti-tumor activities on HeLa cells. The results of the present study therefore have expanded understanding regarding the effect of spider toxins on cancer cells and suggested that components of black widow spiderlings may be developed as a promising novel agent to treat cancer.

  20. p85α recruitment by the CD300f phosphatidylserine receptor mediates apoptotic cell clearance required for autoimmunity suppression

    Science.gov (United States)

    Tian, Linjie; Choi, Seung-Chul; Murakami, Yousuke; Allen, Joselyn; Morse, Herbert C., III; Qi, Chen-Feng; Krzewski, Konrad; Coligan, John E.

    2014-01-01

    Apoptotic cell (AC) clearance is essential for immune homeostasis. Here we show that mouse CD300f (CLM-1) recognizes outer membrane-exposed phosphatidylserine, and regulates the phagocytosis of ACs. CD300f accumulates in phagocytic cups at AC contact sites. Phosphorylation within CD300f cytoplasmic tail tyrosine-based motifs initiates signals that positively or negatively regulate AC phagocytosis. Y276 phosphorylation is necessary for enhanced CD300f-mediated phagocytosis through the recruitment of the p85α regulatory subunit of phosphatidylinositol-3-kinase (PI3K). CD300f-PI3K association leads to activation of downstream Rac/Cdc42 GTPase and mediates changes of F-actin that drive AC engulfment. Importantly, primary macrophages from CD300f-deficient mice have impaired phagocytosis of ACs. The biological consequence of CD300f deficiency is predisposition to autoimmune disease development, as FcγRIIB-deficient mice develop a systemic lupus erythematosus-like disease at a markedly accelerated rate if CD300f is absent. In this report we identify the mechanism and role of CD300f in AC phagocytosis and maintenance of immune homeostasis.

  1. Curcumin induces apoptotic cell death of activated human CD4+ T cells via increasing endoplasmic reticulum stress and mitochondrial dysfunction.

    Science.gov (United States)

    Zheng, Min; Zhang, Qinggao; Joe, Yeonsoo; Lee, Bong Hee; Ryu, Do Gon; Kwon, Kang Beom; Ryter, Stefan W; Chung, Hun Taeg

    2013-03-01

    Curcumin, a natural polyphenolic antioxidant compound, exerts well-known anti-inflammatory and immunomodulatory effects, the latter which can influence the activation of immune cells including T cells. Furthermore, curcumin can inhibit the expression of pro-inflammatory cytokines and chemokines, through suppression of the NF-κB signaling pathway. The beneficial effects of curcumin in diseases such as arthritis, allergy, asthma, atherosclerosis, diabetes and cancer may be due to its immunomodulatory properties. We studied the potential of curcumin to modulate CD4+ T cells-mediated autoimmune disease, by examining the effects of this compound on human CD4+ lymphocyte activation. Stimulation of human T cells with PHA or CD3/CD28 induced IL-2 mRNA expression and activated the endoplasmic reticulum (ER) stress response. The treatment of T cells with curcumin induced the unfolded protein response (UPR) signaling pathway, initiated by the phosphorylation of PERK and IRE1. Furthermore, curcumin increased the expression of the ER stress associated transcriptional factors XBP-1, cleaved p50ATF6α and C/EBP homologous protein (CHOP) in human CD4+ and Jurkat T cells. In PHA-activated T cells, curcumin further enhanced PHA-induced CHOP expression and reduced the expression of the anti-apoptotic protein Bcl-2. Finally, curcumin treatment induced apoptotic cell death in activated T cells via eliciting an excessive ER stress response, which was reversed by the ER-stress inhibitor 4-phenylbutyric acid or transfection with CHOP-specific siRNA. These results suggest that curcumin can impact both ER stress and mitochondria functional pathways, and thereby could be used as a promising therapy in the context of Th1-mediated autoimmune diseases. Copyright © 2013 Elsevier B.V. All rights reserved.

  2. JNK Controls the Onset of Mitosis in Planarian Stem Cells and Triggers Apoptotic Cell Death Required for Regeneration and Remodeling

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    Almuedo-Castillo, María; Crespo, Xenia; Seebeck, Florian; Bartscherer, Kerstin; Salò, Emili; Adell, Teresa

    2014-01-01

    Regeneration of lost tissues depends on the precise interpretation of molecular signals that control and coordinate the onset of proliferation, cellular differentiation and cell death. However, the nature of those molecular signals and the mechanisms that integrate the cellular responses remain largely unknown. The planarian flatworm is a unique model in which regeneration and tissue renewal can be comprehensively studied in vivo. The presence of a population of adult pluripotent stem cells combined with the ability to decode signaling after wounding enable planarians to regenerate a complete, correctly proportioned animal within a few days after any kind of amputation, and to adapt their size to nutritional changes without compromising functionality. Here, we demonstrate that the stress-activated c-jun–NH2–kinase (JNK) links wound-induced apoptosis to the stem cell response during planarian regeneration. We show that JNK modulates the expression of wound-related genes, triggers apoptosis and attenuates the onset of mitosis in stem cells specifically after tissue loss. Furthermore, in pre-existing body regions, JNK activity is required to establish a positive balance between cell death and stem cell proliferation to enable tissue renewal, remodeling and the maintenance of proportionality. During homeostatic degrowth, JNK RNAi blocks apoptosis, resulting in impaired organ remodeling and rescaling. Our findings indicate that JNK-dependent apoptotic cell death is crucial to coordinate tissue renewal and remodeling required to regenerate and to maintain a correctly proportioned animal. Hence, JNK might act as a hub, translating wound signals into apoptotic cell death, controlled stem cell proliferation and differentiation, all of which are required to coordinate regeneration and tissue renewal. PMID:24922054

  3. Vitamins K2, K3 and K5 exert antitumor effects on established colorectal cancer in mice by inducing apoptotic death of tumor cells.

    Science.gov (United States)

    Ogawa, Mutsumi; Nakai, Seiji; Deguchi, Akihiro; Nonomura, Takako; Masaki, Tsutomu; Uchida, Naohito; Yoshiji, Hitoshi; Kuriyama, Shigeki

    2007-08-01

    Although a number of studies have shown that vitamin K possesses antitumor activities on various neoplastic cell lines, there are few reports demonstrating in vivo antitumor effects of vitamin K, and the antitumor effect on colorectal cancer (CRC) remains to be examined. Therefore, antitumor effects of vitamin K on CRC were examined both in vitro and in vivo. Vitamins K2, K3 and K5 suppressed the proliferation of colon 26 cells in a dose-dependent manner, while vitamin K1 did not. On flow cytometry, induction of apoptosis by vitamins K2, K3 and K5 was suggested by population in sub-G1 phase of the cell cycle. Hoechst 33342 staining and a two-color flow cytometric assay using fluorescein isothiocyanate-conjugated annexin V and propidium iodide confirmed that vitamins K2, K3 and K5 induced apoptotic death of colon 26 cells. Enzymatic activity of caspase-3 in colon 26 cells was significantly up-regulated by vitamins K2, K3 and K5. The pan-caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, substantially prevented vitamin K-mediated apoptosis. In vivo study using syngeneic mice with subcutaneously established colon 26 tumors demonstrated that intravenous administration of vitamins K2, K3 and K5 significantly suppressed the tumor growth. The number of apoptotic tumor cells was significantly larger in the vitamin K-treated groups than in the control group. These results suggest that vitamins K2, K3 and K5 exerted effective antitumor effects on CRC in vitro and in vivo by inducing caspase-dependent apoptotic death of tumor cells, suggesting that these K vitamins may be promising agents for the treatment of patients with CRC.

  4. Cytotoxic, genotoxic and apoptotic effects of naringenin-oxime relative to naringenin on normal and cancer cell lines

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    Abdurrahim Kocyigit

    2016-10-01

    Conclusions: This study showed that both NG-Ox and NG possess cytotoxic, genotoxic and apoptotic activities through the production of ROS on cells, NG-Ox being the more effective one. Therefore, derived compound of NG might be used as antiproliferative agents for the treatment of cancer.

  5. Preparation of anastrozole loaded PEG-PLA nanoparticles: evaluation of apoptotic response of breast cancer cell lines.

    Science.gov (United States)

    Alyafee, Yusra A; Alaamery, Manal; Bawazeer, Shahad; Almutairi, Mansour S; Alghamdi, Badr; Alomran, Nawaf; Sheereen, Atia; Daghestani, Maha; Massadeh, Salam

    2018-01-01

    Anastrozole (ANS) is an aromatase inhibitor that is widely used as a treatment for breast cancer in postmenopausal women. Despite the wide use of ANS, it is associated with serious side effects due to uncontrolled delivery. In addition, ANS exhibits low solubility and short plasma half-life. Nanotechnology-based drug delivery has the potential to enhance the efficacy of drugs and overcome undesirable side effects. In this study, we aimed to prepare novel ANS-loaded PLA-PEG-PLA nanoparticles (ANS-NPs) and to compare the apoptotic response of MCF-7 cell line to both ANS and ANS-loaded NPs. ANS-NPs were synthesized using double emulsion method and characterized using different methods. The apoptotic response was evaluated by assessing cell viability, morphology, and studying changes in the expression of MAPK3 , MCL1 , and c-MYC apoptotic genes in MCF-7 cell lines. ANS was successfully encapsulated within PLA-PEG-PLA, forming monodisperse therapeutic NPs with an encapsulation efficiency of 67%, particle size of 186±27.13, and a polydispersity index of 0.26±0.11 with a sustained release profile extended over 144 hours. In addition, results for cell viability and for gene expression represent a similar apoptotic response between the free ANS and ANS-NPs. The synthesized ANS-NPs showed a similar therapeutic effect as the free ANS, which provides a rationale to pursue pre-clinical evaluation of ANS-NPs on animal models.

  6. Kidney stone matrix proteins ameliorate calcium oxalate monohydrate induced apoptotic injury to renal epithelial cells.

    Science.gov (United States)

    Narula, Shifa; Tandon, Simran; Singh, Shrawan Kumar; Tandon, Chanderdeep

    2016-11-01

    Kidney stone formation is a highly prevalent disease, affecting 8-10% of the human population worldwide. Proteins are the major constituents of human kidney stone's organic matrix and considered to play critical role in the pathogenesis of disease but their mechanism of modulation still needs to be explicated. Therefore, in this study we investigated the effect of human kidney stone matrix proteins on the calcium oxalate monohydrate (COM) mediated cellular injury. The renal epithelial cells (MDCK) were exposed to 200μg/ml COM crystals to induce injury. The effect of proteins isolated from human kidney stone was studied on COM injured cells. The alterations in cell-crystal interactions were examined by phase contrast, polarizing, fluorescence and scanning electron microscopy. Moreover, its effect on the extent of COM induced cell injury, was quantified by flow cytometric analysis. Our study indicated the antilithiatic potential of human kidney stone proteins on COM injured MDCK cells. Flow cytometric analysis and fluorescence imaging ascertained that matrix proteins decreased the extent of apoptotic injury caused by COM crystals on MDCK cells. Moreover, the electron microscopic studies of MDCK cells revealed that matrix proteins caused significant dissolution of COM crystals, indicating cytoprotection against the impact of calcium oxalate injury. The present study gives insights into the mechanism implied by urinary proteins to restrain the pathogenesis of kidney stone disease. This will provide a better understanding of the formation of kidney stones which can be useful for the proper management of the disease. Copyright © 2016 Elsevier Inc. All rights reserved.

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

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    Leonie Harmse

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

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

    Science.gov (United States)

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

    2015-01-01

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

  9. Apoptotic markers in protozoan parasites

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    Fasel Nicolas

    2010-11-01

    Full Text Available Abstract The execution of the apoptotic death program in metazoans is characterized by a sequence of morphological and biochemical changes that include cell shrinkage, presentation of phosphatidylserine at the cell surface, mitochondrial alterations, chromatin condensation, nuclear fragmentation, membrane blebbing and the formation of apoptotic bodies. Methodologies for measuring apoptosis are based on these markers. Except for membrane blebbing and formation of apoptotic bodies, all other events have been observed in most protozoan parasites undergoing cell death. However, while techniques exist to detect these markers, they are often optimised for metazoan cells and therefore may not pick up subtle differences between the events occurring in unicellular organisms and multi-cellular organisms. In this review we discuss the markers most frequently used to analyze cell death in protozoan parasites, paying special attention to changes in cell morphology, mitochondrial activity, chromatin structure and plasma membrane structure/permeability. Regarding classical regulators/executors of apoptosis, we have reviewed the present knowledge of caspase-like and nuclease activities.

  10. The role of baculovirus apoptotic suppressors in AcMNPV-mediated translation arrest in Ld652Y cells

    International Nuclear Information System (INIS)

    Thiem, Suzanne M.; Chejanovsky, Nor

    2004-01-01

    Infecting the insect cell line IPLB-Ld652Y with the baculovirus Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV) results in global translation arrest, which correlates with the presence of the AcMNPV apoptotic suppressor, p35. In this study, we investigated the role of apoptotic suppression on AcMNPV-induced translation arrest. Infecting cells with AcMNPV bearing nonfunctional mutant p35 did not result in global translation arrest. In contrast, global translation arrest was observed in cells infected with AcMNPV in which p35 was replaced with Opiap, Cpiap, or p49, baculovirus apoptotic suppressors that block apoptosis by different mechanisms than p35. These results indicated that suppressing apoptosis triggered translation arrest in AcMNPV-infected Ld652Y cells. Experiments using the DNA synthesis inhibitor aphidicolin and temperature shift experiments, using the AcMNPV replication mutants ts8 and ts8Δp35, indicated that translation arrest initiated during the early phase of infection, but events during the late phase were required for global translation arrest. Peptide caspase inhibitors could not substitute for baculovirus apoptotic suppressors to induce translation arrest in Ld652Y cells infected with a p35-null virus. However, if the p35-null-AcMNPV also carried hrf-1, a novel baculovirus host range gene, progeny virus was produced and treatment with peptide caspase inhibitors enhanced translation of a late viral gene transcript. Together, these results indicate that translation arrest in AcMNPV-infected Ld652Y cells is due to the anti-apoptotic function of p35, but suggests that rather than simply preventing caspase activation, its activity enhances signaling to a separate translation arrest pathway, possibly by stimulating the late stages of the baculovirus infection cycle

  11. The anti-apoptotic BAG3 protein is involved in BRAF inhibitor resistance in melanoma cells.

    Science.gov (United States)

    Guerriero, Luana; Palmieri, Giuseppe; De Marco, Margot; Cossu, Antonio; Remondelli, Paolo; Capunzo, Mario; Turco, Maria Caterina; Rosati, Alessandra

    2017-10-06

    BAG3 protein, a member of BAG family of co-chaperones, has a pro-survival role in several tumour types. BAG3 anti-apoptotic properties rely on its characteristic to bind several intracellular partners, thereby modulating crucial events such as apoptosis, differentiation, cell motility, and autophagy. In human melanomas, BAG3 positivity is correlated with the aggressiveness of the tumour cells and can sustain IKK-γ levels, allowing a sustained activation of NF-κB. Furthermore, BAG3 is able to modulate BRAFV600E levels and activity in thyroid carcinomas. BRAFV600E is the most frequent mutation detected in malignant melanomas and is targeted by Vemurafenib, a specific inhibitor found to be effective in the treatment of advanced melanoma. However, patients with BRAF-mutated melanoma may result insensitive ab initio or, mostly, develop acquired resistance to the treatment with this molecule. Here we show that BAG3 down-modulation interferes with BRAF levels in melanoma cells and sensitizes them to Vemurafenib treatment. Furthermore, the down-modulation of BAG3 protein in an in vitro model of acquired resistance to Vemurafenib can induce sensitization to the BRAFV600E specific inhibition by interfering with BRAF pathway through reduction of ERK phosphorylation, but also on parallel survival pathways. Future studies on BAG3 molecular interactions with key proteins responsible of acquired BRAF inhibitor resistance may represent a promising field for novel multi-drugs treatment design.

  12. Characterization of the apoptotic response of human leukemia cells to organosulfur compounds

    International Nuclear Information System (INIS)

    Wong, W Wei-Lynn; Langler, Richard F; Penn, Linda Z; Boutros, Paul C; Wasylishen, Amanda R; Guckert, Kristal D; O'Brien, Erin M; Griffiths, Rebecca; Martirosyan, Anna R; Bros, Christina; Jurisica, Igor

    2010-01-01

    Novel therapeutic agents that selectively induce tumor cell death are urgently needed in the clinical management of cancers. Such agents would constitute effective adjuvant approaches to traditional chemotherapy regimens. Organosulfur compounds (OSCs), such as diallyl disulfide, have demonstrated anti-proliferative effects on cancer cells. We have previously shown that synthesized relatives of dysoxysulfone, a natural OSC derived from the Fijian medicinal plant, Dysoxylum richi, possess tumor-specific antiproliferative effects and are thus promising lead candidates. Because our structure-activity analyses showed that regions flanking the disulfide bond mediated specificity, we synthesized 18 novel OSCs by structural modification of the most promising dysoxysulfone derivatives. These compounds were tested for anti-proliferative and apoptotic activity in both normal and leukemic cells. Six OSCs exhibited tumor-specific killing, having no effect on normal bone marrow, and are thus candidates for future toxicity studies. We then employed mRNA expression profiling to characterize the mechanisms by which different OSCs induce apoptosis. Using Gene Ontology analysis we show that each OSC altered a unique set of pathways, and that these differences could be partially rationalized from a transcription factor binding site analysis. For example, five compounds altered genes with a large enrichment of p53 binding sites in their promoter regions (p < 0.0001). Taken together, these data establish OSCs derivatized from dysoxysulfone as a novel group of compounds for development as anti-cancer agents

  13. Apoptotic effect of cordycepin combined with cisplatin and/or paclitaxel on MA-10 mouse Leydig tumor cells

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    Kang FC

    2015-09-01

    Full Text Available Fu-Chi Kang,1 Pei-Jung Chen,2 Bo-Syong Pan,2,3 Meng-Shao Lai,2,3 Yung-Chia Chen,4 Bu-Miin Huang2,31Department of Anesthesia, Chi Mei Medical Center, Chiali, 2Department of Cell Biology and Anatomy, 3Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan, 4Department of Anatomy, School of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan, Republic of China Background: Chemotherapy is not limited to a single treatment, and the evidence demonstrates that different drug combinations can have positive results in patients. In this study, we sought to determine whether cordycepin combined with cisplatin and/or paclitaxel would have an additive effective on inducing apoptosis in mouse Leydig tumor cells, and the mechanisms were also briefly examined.Methods: The additive effects of cordycepin combined with cisplatin and/or paclitaxel on apoptosis in MA-10 cells were investigated by monitoring changes in morphological characteristics and examining cell viability, flow cytometry assays, and Western blot analyses.Results: Combination of cordycepin plus cisplatin and/or paclitaxel for 12 and 24 hours induced apoptotic features in MA-10 cells. The MTT assay showed that the combination treatment reduced the viability of MA-10 cells in a dose-dependent manner, with additive effects. Cell cycle analysis showed that combination treatment significantly increased subG1 phase cell numbers in MA-10 cells, indicating apoptosis. Moreover, cordycepin plus cisplatin and/or paclitaxel significantly induced cleavage of caspase-8, caspase-9, caspase-3, and poly ADP-ribose polymerase, and phosphorylation of c-Jun NH2-terminal kinase, extracellular signal-regulated kinase, p38, and p53 proteins in MA-10 cells.Conclusion: Cordycepin plus cisplatin and/or paclitaxel can have an additive effect on apoptosis in MA-10 cells, with activation of caspase, mitogen-activated protein kinase, and p53 signal pathways. Keywords: cordycepin

  14. Anti-Proliferative and Apoptotic Effects of Beta-Ionone in Human Leukemia Cell Line K562

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    Zohreh Faezizadeh

    2016-06-01

    Full Text Available Background Beta-ionone is an aroma compound found in the Rosaceae family. Some evidence supported that beta-ionone has a great potential for cancer prevention. To date, the anti-proliferative and apoptotic effects of beta-ionone in human leukemia cell line K562 were not studied. Objectives Hence, we investigated whether beta-ionone could inhibit cell growth and induce apoptosis in the K562 cells. Materials and Methods In this experimental study, human leukemia cell line K562 was cultured and anti-proliferation effect of beta-ionone with different doses (25 - 400 µm at different times (24 - 96 hours on treated cells was evaluated by the MTT assay. To determine apoptosis rate, the Hoechst 33342 staining and flow cytometry was performed. Results The MTT assay showed that beta-ionone inhibited proliferation of K562 cells in a dose-dependent manner significantly (P = 0.0008. Moreover, the increased apoptotic rate was found after incubation of K562 cells with 200 µm beta-ionone. The Hoechst staining and flow cytometry analysis indicated that beta-ionone could increase apoptosis of K562 cells in a dose-dependent manner. Conclusions The results demonstrated that beta-ionone has anti-proliferative and apoptotic effects on K562 cells, and in the future may be used in the treatment of some leukemia sub-types.

  15. Bifidobacterium breve reduces apoptotic epithelial cell shedding in an exopolysaccharide and MyD88-dependent manner.

    Science.gov (United States)

    Hughes, K R; Harnisch, L C; Alcon-Giner, C; Mitra, S; Wright, C J; Ketskemety, J; van Sinderen, D; Watson, A J M; Hall, L J

    2017-01-01

    Certain members of the microbiota genus Bifidobacterium are known to positively influence host well-being. Importantly, reduced bifidobacterial levels are associated with inflammatory bowel disease (IBD) patients, who also have impaired epithelial barrier function, including elevated rates of apoptotic extrusion of small intestinal epithelial cells (IECs) from villi-a process termed 'cell shedding'. Using a mouse model of pathological cell shedding, we show that mice receiving Bifidobacterium breve UCC2003 exhibit significantly reduced rates of small IEC shedding. Bifidobacterial-induced protection appears to be mediated by a specific bifidobacterial surface exopolysaccharide and interactions with host MyD88 resulting in downregulation of intrinsic and extrinsic apoptotic responses to protect epithelial cells under highly inflammatory conditions. Our results reveal an important and previously undescribed role for B. breve, in positively modulating epithelial cell shedding outcomes via bacterial- and host-dependent factors, supporting the notion that manipulation of the microbiota affects intestinal disease outcomes. © 2017 The Authors.

  16. Echinacoside induces apoptotic cancer cell death by inhibiting the nucleotide pool sanitizing enzyme MTH1

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    Dong L

    2015-12-01

    Full Text Available Liwei Dong,1 Hongge Wang,1 Jiajing Niu,1 Mingwei Zou,2 Nuoting Wu,1 Debin Yu,1 Ye Wang,1 Zhihua Zou11Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education, National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun, Jilin Province, People’s Republic of China; 2Department of Psychology, College of Liberal Arts and Social Sciences, University of Houston, Houston, TX, USA Abstract: Inhibition of the nucleotide pool sanitizing enzyme MTH1 causes extensive oxidative DNA damages and apoptosis in cancer cells and hence may be used as an anticancer strategy. As natural products have been a rich source of medicinal chemicals, in the present study, we used the MTH1-catalyzed enzymatic reaction as a high-throughput in vitro screening assay to search for natural compounds capable of inhibiting MTH1. Echinacoside, a compound derived from the medicinal plants Cistanche and Echinacea, effectively inhibited the catalytic activity of MTH1 in an in vitro assay. Treatment of various human cancer cell lines with Echinacoside resulted in a significant increase in the cellular level of oxidized guanine (8-oxoguanine, while cellular reactive oxygen species level remained unchanged, indicating that Echinacoside also inhibited the activity of cellular MTH1. Consequently, Echinacoside treatment induced an immediate and dramatic increase in DNA damage markers and upregulation of the G1/S-CDK inhibitor p21, which were followed by marked apoptotic cell death and cell cycle arrest in cancer but not in noncancer cells. Taken together, these studies identified a natural compound as an MTH1 inhibitor and suggest that natural products can be an important source of anticancer agents. Keywords: Echinacoside, MTH1, 8-oxoG, DNA damage, apoptosis, cell cycle arrest

  17. Apoptotic potential of two Caryophyllaceae species in MCF-7 and MDA-MB-468 cell lines

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    M. Mosaddegh

    2018-01-01

    Full Text Available Background and objectives: Plants have been used to treat diseases like cancer for many years and today the trend towards their use is increasing. One of the most effective mechanisms of plants against cancer is inducing apoptosis. Apoptosis is a programmed cell death which acts opposite to cell division. It starts in response to some stimuli. Despite the effectiveness of apoptosis inducing agents, their use has been limited due to side effects and resistance to these treatments; so, applying medicinal herbs due to their lower cost and toxicity has drawn attentions. Recent research at the Traditional Medicine and Materia Medica Research Center, Shahid Beheshti University of Medical Sciences on two medicinal plants Acanthophyllum bracteatum and A. microcephalum has shown cytotoxic effects of these two species, but the mechanism of their toxicity has remained unknown; thus, the present study was designed to evaluate the apoptotic potential of Acanthophyllum bracteatum and A. microcephalum. Methods: In the present study, the cytotoxic effects of the methanol extract of Acanthophyllum bracteatum and A. microcephalum was evaluated against MCF-7 and MDA-MB-468 cells by MTT assay; furthermore, their apoptosis potential has been evaluated by annexin-V/propidium iodide assay and Hoechst 33258 staining in the same cell lines. Results: The methanol extract of A. microcephalum and A. bracteatum showed cytotoxic effects against MCF-7 and MDA-MB-468 cell lines with IC50 values of 64, 159 and 102, 250 μg/mL, respectively. The results of the apoptosis assays confirmed the potential of the two plants extracts to induce apoptosis in both cell lines while A. microcephalum demonstrated more considerable results. Conclusion: A. microcephalum could be a suitable choice for further breast cancer studies.

  18. Pre-apoptotic response to therapeutic DNA damage involves protein modulation of Mcl-1, Hdm2 and Flt3 in acute myeloid leukemia cells

    Directory of Open Access Journals (Sweden)

    Hovland Randi

    2007-05-01

    Full Text Available Abstract Background Acute myeloid leukemia (AML cells are characterized by non-mutated TP53, high levels of Hdm2, and frequent mutation of the Flt3 receptor tyrosine kinase. The juxtamembrane mutation of FLT3 is the strongest independent marker for disease relapse and is associated with elevated Bcl-2 protein and p53 hyper-phosphorylation in AML. DNA damage forms the basic mechanism of cancer cell eradication in current therapy of AML. Hdm2 and pro-apoptotic Bcl-2 members are among the most intensely induced genes immediately after chemotherapy and Hdm2 is proposed a role in receptor tyrosine kinase regulation. Thus we examined the DNA damage related modulation of these proteins in relation to FLT3 mutational status and induction of apoptosis. Results Within one hour after exposure to ionizing radiation (IR, the AML cells (NB4, MV4-11, HL-60, primary AML cells showed an increase in Flt3 protein independent of mRNA levels, while the Hdm2 protein decreased. The FLT3 mutant MV4-11 cells were resistant to IR accompanied by presence of both Mcl-1 and Hdm2 protein three hours after IR. In contrast, the FLT3 wild type NB4 cells responded to IR with apoptosis and pre-apoptotic Mcl-1 down regulation. Daunorubicin (DNR induced continuing down regulation of Hdm2 and Mcl-1 in both cell lines followed by apoptosis. Conclusion Both IR and DNR treatment resulted in concerted protein modulations of Mcl-1, Hdm2 and Flt3. Cell death induction was associated with persistent attenuation of Mcl-1 and Hdm2. These observations suggest that defining the pathway(s modulating Flt3, Hdm2 and Mcl-1 may propose new strategies to optimize therapy for the relapse prone FLT3 mutated AML patients.

  19. Straw blood cell count, growth, inhibition and comparison to apoptotic bodies

    Directory of Open Access Journals (Sweden)

    Tomkins Jeffrey P

    2008-05-01

    Full Text Available Abstract Background Mammalian cells transform into individual tubular straw cells naturally in tissues and in response to desiccation related stress in vitro. The transformation event is characterized by a dramatic cellular deformation process which includes: condensation of certain cellular materials into a much smaller tubular structure, synthesis of a tubular wall and growth of filamentous extensions. This study continues the characterization of straw cells in blood, as well as the mechanisms of tubular transformation in response to stress; with specific emphasis placed on investigating whether tubular transformation shares the same signaling pathway as apoptosis. Results There are approximately 100 billion, unconventional, tubular straw cells in human blood at any given time. The straw blood cell count (SBC is 45 million/ml, which accounts for 6.9% of the bloods dry weight. Straw cells originating from the lungs, liver and lymphocytes have varying nodules, hairiness and dimensions. Lipid profiling reveals severe disruption of the plasma membrane in CACO cells during transformation. The growth rates for the elongation of filaments and enlargement of rabbit straw cells is 0.6~1.1 (μm/hr and 3.8 (μm3/hr, respectively. Studies using apoptosis inhibitors and a tubular transformation inhibitor in CACO2 cells and in mice suggested apoptosis produced apoptotic bodies are mediated differently than tubular transformation produced straw cells. A single dose of 0.01 mg/kg/day of p38 MAPK inhibitor in wild type mice results in a 30% reduction in the SBC. In 9 domestic animals SBC appears to correlate inversely with an animal's average lifespan (R2 = 0.7. Conclusion Straw cells are observed residing in the mammalian blood with large quantities. Production of SBC appears to be constant for a given animal and may involve a stress-inducible protein kinase (P38 MAPK. Tubular transformation is a programmed cell survival process that diverges from apoptosis

  20. Phosphatidylserine-Liposomes Promote Tolerogenic Features on Dendritic Cells in Human Type 1 Diabetes by Apoptotic Mimicry

    Directory of Open Access Journals (Sweden)

    Silvia Rodriguez-Fernandez

    2018-02-01

    Full Text Available Type 1 diabetes (T1D is a metabolic disease caused by the autoimmune destruction of insulin-producing β-cells. With its incidence increasing worldwide, to find a safe approach to permanently cease autoimmunity and allow β-cell recovery has become vital. Relying on the inherent ability of apoptotic cells to induce immunological tolerance, we demonstrated that liposomes mimicking apoptotic β-cells arrested autoimmunity to β-cells and prevented experimental T1D through tolerogenic dendritic cell (DC generation. These liposomes contained phosphatidylserine (PS—the main signal of the apoptotic cell membrane—and β-cell autoantigens. To move toward a clinical application, PS-liposomes with optimum size and composition for phagocytosis were loaded with human insulin peptides and tested on DCs from patients with T1D and control age-related subjects. PS accelerated phagocytosis of liposomes with a dynamic typical of apoptotic cell clearance, preserving DCs viability. After PS-liposomes phagocytosis, the expression pattern of molecules involved in efferocytosis, antigen presentation, immunoregulation, and activation in DCs concurred with a tolerogenic functionality, both in patients and control subjects. Furthermore, DCs exposed to PS-liposomes displayed decreased ability to stimulate autologous T cell proliferation. Moreover, transcriptional changes in DCs from patients with T1D after PS-liposomes phagocytosis pointed to an immunoregulatory prolife. Bioinformatics analysis showed 233 differentially expressed genes. Genes involved in antigen presentation were downregulated, whereas genes pertaining to tolerogenic/anti-inflammatory pathways were mostly upregulated. In conclusion, PS-liposomes phagocytosis mimics efferocytosis and leads to phenotypic and functional changes in human DCs, which are accountable for tolerance induction. The herein reported results reinforce the potential of this novel immunotherapy to re-establish immunological

  1. Alterations in glutathione levels and apoptotic regulators are associated with acquisition of arsenic trioxide resistance in multiple myeloma.

    Directory of Open Access Journals (Sweden)

    Shannon M Matulis

    Full Text Available Arsenic trioxide (ATO has been tested in relapsed/refractory multiple myeloma with limited success. In order to better understand drug mechanism and resistance pathways in myeloma we generated an ATO-resistant cell line, 8226/S-ATOR05, with an IC50 that is 2-3-fold higher than control cell lines and significantly higher than clinically achievable concentrations. Interestingly we found two parallel pathways governing resistance to ATO in 8226/S-ATOR05, and the relevance of these pathways appears to be linked to the concentration of ATO used. We found changes in the expression of Bcl-2 family proteins Bfl-1 and Noxa as well as an increase in cellular glutathione (GSH levels. At low, clinically achievable concentrations, resistance was primarily associated with an increase in expression of the anti-apoptotic protein Bfl-1 and a decrease in expression of the pro-apoptotic protein Noxa. However, as the concentration of ATO increased, elevated levels of intracellular GSH in 8226/S-ATOR05 became the primary mechanism of ATO resistance. Removal of arsenic selection resulted in a loss of the resistance phenotype, with cells becoming sensitive to high concentrations of ATO within 7 days following drug removal, indicating changes associated with high level resistance (elevated GSH are dependent upon the presence of arsenic. Conversely, not until 50 days without arsenic did cells once again become sensitive to clinically relevant doses of ATO, coinciding with a decrease in the expression of Bfl-1. In addition we found cross-resistance to melphalan and doxorubicin in 8226/S-ATOR05, suggesting ATO-resistance pathways may also be involved in resistance to other chemotherapeutic agents used in the treatment of multiple myeloma.

  2. Pro-apoptotic effects of the flavonoid luteolin in rat H4IIE cells

    International Nuclear Information System (INIS)

    Michels, G.; Waetjen, W.; Niering, P.; Steffan, B.; Thi, Q.-H. Tran; Chovolou, Y.; Kampkoetter, A.; Bast, A.; Proksch, P.; Kahl, R.

    2005-01-01

    Polyphenols are ubiquitous substances in the diet. Their anti-oxidative, anti-inflammatory and anti-viral effects are of interest for human health, and polyphenols such as luteolin are used at high concentrations in food supplements. The aim of this project was to determine the intrinsic effects of luteolin in H4IIE rat hepatoma cells. Luteolin is relatively toxic, cell death was caused via induction of apoptosis as detected by DNA-ladder formation, by nuclear fragmentation and activation of apoptotic enzymes (caspase-2, -3/7, -9 and -8/10). Luteolin (250 μM, 24 h) increased the caspase-3/7 activity four-fold and the caspase-9 activity six-fold. In a time course experiment caspase-9 is activated after 6 h, while caspase-2 and -3/7 are activated after 12 h. After 24 h, caspase-8/10 also displays activation. We found a concentration-dependent increase in malondialdehyde release suggesting a prooxidative effect of luteolin. Furthermore, we analysed DNA strand break formation by luteolin and found a distinct increase of DNA strand breaks after incubation for 3 h with 100 μM luteolin, a concentration which induces oligonucleosomal DNA cleavage at 24 h. In conclusion, the sequence of events is compatible with the assumption that luteolin triggers the mitochondrial pathway of apoptosis, probably by inducing DNA damage

  3. Titanium dioxide induces apoptotic cell death through reactive oxygen species-mediated Fas upregulation and Bax activation

    Directory of Open Access Journals (Sweden)

    Yoon TH

    2012-03-01

    Full Text Available Ki-Chun Yoo1, Chang-Hwan Yoon1, Dongwook Kwon2, Kyung-Hwan Hyun1, Soo Jung Woo1, Rae-Kwon Kim1, Eun-Jung Lim1, Yongjoon Suh1, Min-Jung Kim1, Tae Hyun Yoon2, Su-Jae Lee11Laboratory of Molecular Biochemistry, 2Laboratory of Nanoscale Characterization and Environmental Chemistry, Department of Chemistry, Hanyang University, Seoul, Republic of KoreaBackground: Titanium dioxide (TiO2 has been widely used in many areas, including biomedicine, cosmetics, and environmental engineering. Recently, it has become evident that some TiO2 particles have a considerable cytotoxic effect in normal human cells. However, the molecular basis for the cytotoxicity of TiO2 has yet to be defined.Methods and results: In this study, we demonstrated that combined treatment with TiO2 nanoparticles sized less than 100 nm and ultraviolet A irradiation induces apoptotic cell death through reactive oxygen species-dependent upregulation of Fas and conformational activation of Bax in normal human cells. Treatment with P25 TiO2 nanoparticles with a hydrodynamic size distribution centered around 70 nm (TiO2P25–70 together with ultraviolet A irradiation-induced caspase-dependent apoptotic cell death, accompanied by transcriptional upregulation of the death receptor, Fas, and conformational activation of Bax. In line with these results, knockdown of either Fas or Bax with specific siRNA significantly inhibited TiO2-induced apoptotic cell death. Moreover, inhibition of reactive oxygen species with an antioxidant, N-acetyl-L-cysteine, clearly suppressed upregulation of Fas, conformational activation of Bax, and subsequent apoptotic cell death in response to combination treatment using TiO2P25–70 and ultraviolet A irradiation.Conclusion: These results indicate that sub-100 nm sized TiO2 treatment under ultraviolet A irradiation induces apoptotic cell death through reactive oxygen species-mediated upregulation of the death receptor, Fas, and activation of the preapoptotic protein

  4. Association of anti-apoptotic Mcl-1L isoform expression with radioresistance of oral squamous carcinoma cells

    International Nuclear Information System (INIS)

    Palve, Vinayak C; Teni, Tanuja R

    2012-01-01

    Oral cancer is a common cancer and a major health problem in the Indian subcontinent. At our laboratory Mcl-1, an anti-apoptotic member of the Bcl-2 family has been demonstrated to be overexpressed in oral cancers and to predict outcome in oral cancer patients treated with definitive radiotherapy. To study the role of Mcl-1 isoforms in radiation response of oral squamous carcinoma cells (OSCC), we investigated in the present study, the association of Mcl-1 isoform expression with radiosensitivity of OSCC, using siRNA strategy. The time course expression of Mcl-1 splice variants (Mcl-1L, Mcl-1S & Mcl-1ES) was studied by RT-PCR, western blotting & immunofluorescence, post-irradiation in oral cell lines [immortalized FBM (radiosensitive) and tongue cancer AW8507 & AW13516 (radioresistant)]of relatively differing radiosensitivities. The effect of Mcl-1L knockdown alone or in combination with ionizing radiation (IR) on cell proliferation, apoptosis & clonogenic survival, was investigated in AW8507 & AW13516 cells. Further the expression of Mcl-1L protein was assessed in radioresistant sublines generated by fractionated ionizing radiation (FIR). Three to six fold higher expression of anti-apoptotic Mcl-1L versus pro-apoptotic Mcl-1S was observed at mRNA & protein levels in all cell lines, post-irradiation. Sustained high levels of Mcl-1L, downregulation of pro-apoptotic Bax & Bak and a significant (P < 0.05) reduction in apoptosis was observed in the more radioresistant AW8507, AW13516 versus FBM cells, post-IR. The ratios of anti to pro-apoptotic proteins were high in AW8507 as compared to FBM. Treatment with Mcl-1L siRNA alone or in combination with IR significantly (P < 0.01) increased apoptosis viz. 17.3% (IR), 25.3% (siRNA) and 46.3% (IR plus siRNA) and upregulated pro-apoptotic Bax levels in AW8507 cells. Combination of siRNA & IR treatment significantly (P < 0.05) reduced cell proliferation and clonogenic survival of radioresistant AW8507 & AW13516 cells

  5. Externalization and recognition by macrophages of large subunit of eukaryotic translation initiation factor 3 in apoptotic cells

    International Nuclear Information System (INIS)

    Nakai, Yuji; Shiratsuchi, Akiko; Manaka, Junko; Nakayama, Hiroshi; Takio, Koji; Zhang Jianting; Suganuma, Tatsuo; Nakanishi, Yoshinobu

    2005-01-01

    We previously isolated a monoclonal antibody named PH2 that inhibits phosphatidylserine-mediated phagocytosis of apoptotic cells by macrophages [C. Fujii, A. Shiratsuchi, J. Manaka, S. Yonehara, Y. Nakanishi. Cell Death Differ. 8 (2001) 1113-1122]. We report here the identification of the cognate antigen. A protein bound by PH2 in Western blotting was identified as the 170-kDa subunit of eukaryotic translation initiation factor 3 (eIF3 p170/eIF3a). When eIF3a was expressed in a culture cell line as a protein fused to green fluorescence protein, the fusion protein was detected at the cell surface only after the induction of apoptosis. The same phenomenon was seen when the localization of endogenous eIF3a was determined using anti-eIF3a antibody, and eIF3a seemed to be partially degraded during apoptosis. Furthermore, bacterially expressed N-terminal half of eIF3a fused to glutathione S-transferase bound to the surface of macrophages and inhibited phagocytosis of apoptotic cells by macrophages when it was added to phagocytosis reactions. These results collectively suggest that eIF3a translocates to the cell surface upon apoptosis, probably after partial degradation, and bridges apoptotic cells and macrophages to enhance phagocytosis

  6. Visualization of proteolytic activity associated with the apoptotic response in cancer cells

    Science.gov (United States)

    Tice, Brian George

    Caspases execute programmed cell death, where low levels of caspase activity are linked to cancer. Chemotherapies utilize induction of apoptosis as a key mechanism for cancer treatment, where caspase-3 is a major player involved in dismantling these aberrant cells. The ability to sensitively measure the initial caspase-3 cleavage events during apoptosis is important for understanding the initiation of this complex cellular process, however, current ensemble methods are not sensitive enough to measure single cleavage events in cells. By utilizing the optical properties of plasmon coupling, peptide-linked gold nanoparticles were developed to enable single molecule imaging of caspase-3 activity in two different cancer systems. Au crown nanoparticles were assembled in a multimeric fashion to overcome the high and heterogeneous background scattering of live cells. In a colon cancer (SW620) cell line challenged with tumor necrosis factor-alpha (TNF-alpha), single molecule trajectories show early stage caspase-3 activation within minutes, which was not detectable by ensemble assays until 23 hours. Variability in caspase-3 activation among the population of cells was identified and likely a result of each cell's specific resistance to death receptor-induced apoptosis. Following these studies, improvements by way of sensitivity and selectivity were tailored into an improved nanosensor construct. Au nanoshell dimers were prepared as a comparably bright construct with 1) reduced heterogeneity compared to the synthesis of the crown nanoparticles and 2) a peptide sequence highly selective for caspase-3. Chronic myeloid leukemia (CML) K562 cells were assessed for their early apoptotic response upon treatment with dasatinib, a clinically approved tyrosine kinase inhibitor that specifically targets BCR-ABL. It has been demonstrated that inhibition of BCR-ABL by dasatinib commits K562 cells to apoptosis. Single molecule experiments with Au nanoshell dimers show caspase-3 activation

  7. Metabolism and apoptotic properties of elevated ceramide in HT29(rev) cells

    NARCIS (Netherlands)

    Veldman, R J; Klappe, K; Hoekstra, D; Kok, J W

    1998-01-01

    Ceramide (Cer) has been implicated in the regulation of apoptosis. In this study, we elevated cellular Cer levels in human colon-carcinoma (HT29(rev)) cells by incubating the cells in the presence of bacterial sphingomyelinase (bSMase) or, alternatively, in the presence of C2-Cer, a short-chain

  8. Inhibition of neutral sphingomyelinase decreases elevated levels of inducible nitric oxide synthase and apoptotic cell death in ocular hypertensive rats

    Energy Technology Data Exchange (ETDEWEB)

    Aslan, Mutay, E-mail: mutayaslan@akdeniz.edu.tr [Department of Medical Biochemistry, Akdeniz University Faculty of Medicine, Antalya (Turkey); Basaranlar, Goksun [Department of Biophysics, Akdeniz University Faculty of Medicine, Antalya (Turkey); Unal, Mustafa [Department of Ophthalmology, Akdeniz University Faculty of Medicine, Antalya (Turkey); Ciftcioglu, Akif [Department of Pathology, Akdeniz University Faculty of Medicine, Antalya (Turkey); Derin, Narin [Department of Biophysics, Akdeniz University Faculty of Medicine, Antalya (Turkey); Mutus, Bulent [Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario (Canada)

    2014-11-01

    Endoplasmic reticulum (ER) stress and excessive nitric oxide production via induction of inducible nitric oxide synthase (NOS2) have been implicated in the pathogenesis of neuronal retinal cell death in ocular hypertension. Neutral sphingomyelinase (N-SMase)/ceramide pathway can regulate NOS2 expression, hence this study determined the role of selective neutral sphingomyelinase (N-SMase) inhibition on retinal NOS2 levels, ER stress, apoptosis and visual evoked potentials (VEPs) in a rat model of elevated intraocular pressure (EIOP). NOS2 expression and retinal protein nitration were significantly greater in EIOP and significantly decreased with N-SMase inhibition. A significant increase was observed in retinal ER stress markers pPERK, CHOP and GRP78 in EIOP, which were not significantly altered by N-SMase inhibition. Retinal TUNEL staining showed increased apoptosis in all EIOP groups; however N-SMase inhibition significantly decreased the percent of apoptotic cells in EIOP. Caspase-3, -8 and -9 activities were significantly increased in EIOP and returned to baseline levels following N-SMase inhibition. Latencies of all VEP components were significantly prolonged in EIOP and shortened following N-SMase inhibition. Data confirm the role of nitrative injury in EIOP and highlight the protective effect of N-SMase inhibition in EIOP via down-regulation of NOS2 levels and nitrative stress. - Highlights: • Inhibition of N-SMase decreases NOS2 levels in ocular hypertension. • Inhibition of N-SMase decreases protein nitration in ocular hypertension. • Inhibition of N-SMase decreases caspase activation in ocular hypertension. • Inhibition of N-SMase decreases apoptosis in ocular hypertension.

  9. Lovastatin prevents cisplatin-induced activation of pro-apoptotic DNA damage response (DDR) of renal tubular epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Krüger, Katharina; Ziegler, Verena; Hartmann, Christina; Henninger, Christian [Institute of Toxicology, Medical Faculty, Heinrich Heine University Düsseldorf, 40225 Düsseldorf (Germany); Thomale, Jürgen [Institute of Cell Biology, University Duisburg-Essen, 45122 Essen (Germany); Schupp, Nicole [Institute of Toxicology, Medical Faculty, Heinrich Heine University Düsseldorf, 40225 Düsseldorf (Germany); Fritz, Gerhard, E-mail: fritz@uni-duesseldorf.de [Institute of Toxicology, Medical Faculty, Heinrich Heine University Düsseldorf, 40225 Düsseldorf (Germany)

    2016-02-01

    The platinating agent cisplatin (CisPt) is commonly used in the therapy of various types of solid tumors. The anticancer efficacy of CisPt largely depends on the formation of bivalent DNA intrastrand crosslinks, which stimulate mechanisms of the DNA damage response (DDR), thereby triggering checkpoint activation, gene expression and cell death. The clinically most relevant adverse effect associated with CisPt treatment is nephrotoxicity that results from damage to renal tubular epithelial cells. Here, we addressed the question whether the HMG-CoA-reductase inhibitor lovastatin affects the DDR of renal cells by employing rat renal proximal tubular epithelial (NRK-52E) cells as in vitro model. The data show that lovastatin has extensive inhibitory effects on CisPt-stimulated DDR of NRK-52E cells as reflected on the levels of phosphorylated ATM, Chk1, Chk2, p53 and Kap1. Mitigation of CisPt-induced DDR by lovastatin was independent of the formation of DNA damage as demonstrated by (i) the analysis of Pt-(GpG) intrastrand crosslink formation by Southwestern blot analyses and (ii) the generation of DNA strand breaks as analyzed on the level of nuclear γH2AX foci and employing the alkaline comet assay. Lovastatin protected NRK-52E cells from the cytotoxicity of high CisPt doses as shown by measuring cell viability, cellular impedance and flow cytometry-based analyses of cell death. Importantly, the statin also reduced the level of kidney DNA damage and apoptosis triggered by CisPt treatment of mice. The data show that the lipid-lowering drug lovastatin extensively counteracts pro-apoptotic signal mechanisms of the DDR of tubular epithelial cells following CisPt injury. - Highlights: • Lovastatin blocks ATM/ATR-regulated DDR of tubular cells following CisPt treatment. • Lovastatin attenuates CisPt-induced activation of protein kinase ATM in vitro. • Statin-mediated DDR inhibition is independent of initial DNA damage formation. • Statin-mediated blockage of Cis

  10. Lovastatin prevents cisplatin-induced activation of pro-apoptotic DNA damage response (DDR) of renal tubular epithelial cells

    International Nuclear Information System (INIS)

    Krüger, Katharina; Ziegler, Verena; Hartmann, Christina; Henninger, Christian; Thomale, Jürgen; Schupp, Nicole; Fritz, Gerhard

    2016-01-01

    The platinating agent cisplatin (CisPt) is commonly used in the therapy of various types of solid tumors. The anticancer efficacy of CisPt largely depends on the formation of bivalent DNA intrastrand crosslinks, which stimulate mechanisms of the DNA damage response (DDR), thereby triggering checkpoint activation, gene expression and cell death. The clinically most relevant adverse effect associated with CisPt treatment is nephrotoxicity that results from damage to renal tubular epithelial cells. Here, we addressed the question whether the HMG-CoA-reductase inhibitor lovastatin affects the DDR of renal cells by employing rat renal proximal tubular epithelial (NRK-52E) cells as in vitro model. The data show that lovastatin has extensive inhibitory effects on CisPt-stimulated DDR of NRK-52E cells as reflected on the levels of phosphorylated ATM, Chk1, Chk2, p53 and Kap1. Mitigation of CisPt-induced DDR by lovastatin was independent of the formation of DNA damage as demonstrated by (i) the analysis of Pt-(GpG) intrastrand crosslink formation by Southwestern blot analyses and (ii) the generation of DNA strand breaks as analyzed on the level of nuclear γH2AX foci and employing the alkaline comet assay. Lovastatin protected NRK-52E cells from the cytotoxicity of high CisPt doses as shown by measuring cell viability, cellular impedance and flow cytometry-based analyses of cell death. Importantly, the statin also reduced the level of kidney DNA damage and apoptosis triggered by CisPt treatment of mice. The data show that the lipid-lowering drug lovastatin extensively counteracts pro-apoptotic signal mechanisms of the DDR of tubular epithelial cells following CisPt injury. - Highlights: • Lovastatin blocks ATM/ATR-regulated DDR of tubular cells following CisPt treatment. • Lovastatin attenuates CisPt-induced activation of protein kinase ATM in vitro. • Statin-mediated DDR inhibition is independent of initial DNA damage formation. • Statin-mediated blockage of Cis

  11. NCAM regulates cell motility

    DEFF Research Database (Denmark)

    Prag, Søren; Lepekhin, Eugene A; Kolkova, Kateryna

    2002-01-01

    Cell migration is required during development of the nervous system. The regulatory mechanisms for this process, however, are poorly elucidated. We show here that expression of or exposure to the neural cell adhesion molecule (NCAM) strongly affected the motile behaviour of glioma cells...... independently of homophilic NCAM interactions. Expression of the transmembrane 140 kDa isoform of NCAM (NCAM-140) caused a significant reduction in cellular motility, probably through interference with factors regulating cellular attachment, as NCAM-140-expressing cells exhibited a decreased attachment...... to a fibronectin substratum compared with NCAM-negative cells. Ectopic expression of the cytoplasmic part of NCAM-140 also inhibited cell motility, presumably via the non-receptor tyrosine kinase p59(fyn) with which NCAM-140 interacts. Furthermore, we showed that the extracellular part of NCAM acted as a paracrine...

  12. ANTIPROLIFERATIVE AND APOPTOTIC EFFECTS OF THE ESSENTIAL OIL OF ORIGANUM ONITES AND CARVACROL ON HEP-G2 CELLS

    Directory of Open Access Journals (Sweden)

    Özlem TOMSUK

    2011-08-01

    Full Text Available The essential oil Origanum onites L. and its phenolic constituent carvacrol were examined for their cytotoxic and apoptotic effects in a human hepatocellular carcinoma cells Hep-G2. WST-1 and neutral red uptake assays were performed to determine the inhibitory effects of the oil and carvacrol on the growth of the cells. Possible induction of apoptosis by Origanum oil and carvacrol was further investigated by acridine orange/ethidium bromide (AO/EB staining. Results showed that the Ori- ganum oil and carvacrol was significantly cytotoxic and induced apoptosis in Hep-G2 cells. IC₅₀ value of essential oil and carvacrol was found about 0,009% (v/v and 500 μM, respectively. After incuba- tion of the cells with Origanum oil and carvacrol, characteristics of apoptotic morphology such as chromatin condensation, shrinkage of the cells and cytoplasmic blebbing was observed. In conclusion, both essential oil and its major constituent carvacrol significantly exhibited cytotoxic and apoptotic activities in hepatocellular carcinoma cells, indicating its potential for use as an anticancer agent.

  13. Dapoxetine attenuates testosterone-induced prostatic hyperplasia in rats by the regulation of inflammatory and apoptotic proteins

    International Nuclear Information System (INIS)

    Sayed, Rabab H.; Saad, Muhammed A.; El-Sahar, Ayman E.

    2016-01-01

    Serotonin level plays a role in suppressing the pathological findings of benign prostatic hyperplasia (BPH). Thus a new selective serotonin reuptake inhibitor, dapoxetine was used to test its ability to ameliorate the pathological changes in the rat prostate. A dose response curve was constructed between the dose of dapoxetine and prostate weight as well as relative prostate weight, then a 5 mg/kg dose was used as a representative dose for dapoxetine administration. Rats were divided into four groups; the control group that received the vehicle; the BPH-induced group received daily s.c injection of 3 mg/kg testosterone propionate dissolved in olive oil for four weeks; BPH-induced group treated with finasteride 5 mg/kg/day p.o and BPH-induced group treated with dapoxetine 5 mg/kg/day p.o. Injection of testosterone increased prostate weight and relative prostate weight which were both returned back to the normal value after treatment with dapoxetine as well as finasteride. Testosterone also upregulated androgen receptor (AR) and proliferating cell nuclear antigen gene expression. Furthermore, testosterone injection elevated cyclooxygenase-II (COX II), inducible nitric oxide synthase (iNOS), B-cell lymphoma-2 (Bcl2) expression and tumor necrosis factor alpha content and reduced caspase-3 activity, Bcl-2-associated X protein (Bax) expression and Bax/Bcl2 ratio. Dapoxetine and finasteride administration reverted most of the changes made by testosterone injection. In conclusion, the current study provides an evidence for the protective effects of dapoxetine against testosterone-induced BPH in rats. This can be attributed, at least in part, to decreasing AR expression, and the anti-proliferative, anti-inflammatory and pro-apoptotic activities of dapoxetine in BPH. - Highlights: • Dapoxetine attenuates testosterone-induced prostatic hyperplasia in rats. • Dapoxetine decreased androgen receptor gene expression in rat prostate. • Dapoxetine possess anti

  14. Dapoxetine attenuates testosterone-induced prostatic hyperplasia in rats by the regulation of inflammatory and apoptotic proteins

    Energy Technology Data Exchange (ETDEWEB)

    Sayed, Rabab H., E-mail: rabab.sayed@pharma.cu.edu.eg; Saad, Muhammed A.; El-Sahar, Ayman E.

    2016-11-15

    Serotonin level plays a role in suppressing the pathological findings of benign prostatic hyperplasia (BPH). Thus a new selective serotonin reuptake inhibitor, dapoxetine was used to test its ability to ameliorate the pathological changes in the rat prostate. A dose response curve was constructed between the dose of dapoxetine and prostate weight as well as relative prostate weight, then a 5 mg/kg dose was used as a representative dose for dapoxetine administration. Rats were divided into four groups; the control group that received the vehicle; the BPH-induced group received daily s.c injection of 3 mg/kg testosterone propionate dissolved in olive oil for four weeks; BPH-induced group treated with finasteride 5 mg/kg/day p.o and BPH-induced group treated with dapoxetine 5 mg/kg/day p.o. Injection of testosterone increased prostate weight and relative prostate weight which were both returned back to the normal value after treatment with dapoxetine as well as finasteride. Testosterone also upregulated androgen receptor (AR) and proliferating cell nuclear antigen gene expression. Furthermore, testosterone injection elevated cyclooxygenase-II (COX II), inducible nitric oxide synthase (iNOS), B-cell lymphoma-2 (Bcl2) expression and tumor necrosis factor alpha content and reduced caspase-3 activity, Bcl-2-associated X protein (Bax) expression and Bax/Bcl2 ratio. Dapoxetine and finasteride administration reverted most of the changes made by testosterone injection. In conclusion, the current study provides an evidence for the protective effects of dapoxetine against testosterone-induced BPH in rats. This can be attributed, at least in part, to decreasing AR expression, and the anti-proliferative, anti-inflammatory and pro-apoptotic activities of dapoxetine in BPH. - Highlights: • Dapoxetine attenuates testosterone-induced prostatic hyperplasia in rats. • Dapoxetine decreased androgen receptor gene expression in rat prostate. • Dapoxetine possess anti

  15. Antiproliferative activity and apoptotic effects of Filipendula ulmaria pollen against C26 mice colon tumour cells

    Directory of Open Access Journals (Sweden)

    Mărgăoan Rodica

    2016-06-01

    Full Text Available Honeybee collected pollen exhibits high nutritional and pharmaceutical benefits for the human diet and medicine. Pollen’s antioxidant, anti-ageing, anti-inflammatory, anti-atherosclerosis, and cardioprotective activity, depending on the floral origin, are well known. Recent studies proposed that pollen may also be an excellent cancer-fighting candidate, as pollen harbours high amounts of phenolic substances. In our study, Filipendula ulmaria pollen (bee collected was methanol-water extracted and used to verify its in vitro pharmacological activities on C26 mice cancer tumour cells. Three different concentrations of the extract were tested in antitumour assays. Monitoring was done after 6, 12, 24, and 48 hours. Promising results were obtained for antiproliferative and apoptotic activity of the pollen extracts, with high efficiency for the highest concentration (1 mg/mL. For both activities, time and concentration-dependent effects were observed. Pollen extracts or bee collected pollen has a high potential as an antitumour agent for use in human medicine, because they are both rich in bioactive compounds.

  16. Evaluation of apoptotic cell death in normal and chondrodystrophic canine intervertebral discs

    Directory of Open Access Journals (Sweden)

    Marie Klauser

    2012-02-01

    Full Text Available Disc degeneration occurs commonly in dogs. A variety of factors is thought to contribute an inappropriate disc matrix that isolate cells in the disc and lead to apoptosis. Disc herniation with radiculopathy and discogenic pain are the results of the degenerative process. The objective of this prospective study was to determine the extent of apoptosis in intact and herniated intervertebral discs of chondrodystrophic dogs and non-chondrodystrophic dogs. In addition, the nucleus pulposus (NP was histologically compared between non-chondrodystrophic and chondrodystrophic dogs. Thoracolumbar intervertebral discs and parts of the extruded nucleus pulposus were harvested from 45 dogs. Samples were subsequently stained with haematoxylin-eosin and processed to detect cleaved caspase-3 and poly(ADP-ribose polymerase. A significant greater degree of apoptosis was observed in herniated NPs of chondrodystrophic dogs compared to non- chondrodystrophic dogs with poly (ADP-ribose polymerase and cleaved caspase- 3 detection. Within the group of chondrodystrophic dogs, dogs with an intact disc and younger than 6 years showed a significant lower incidence of apoptosis in the NP compared to the herniated NP of chondrodystrophic dogs. The extent of apoptosis in the annulus fibrosus was not different between the intact disc from chondrodystrophic and non- chondrodystrophic dogs. An age-related increase of apoptotic cells in NP and annulus fibrosus was found in the intact non-herniated intervertebral discs. Histologically, absence of notochordal cells and occurrence of chondroid metaplasia were observed in the nucleus pulposus of chondrodystrophic dogs. As a result, we found that apoptosis plays a role in disc degeneration in chondrodystrophic dogs.

  17. 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.

  18. Biliverdin reductase/bilirubin mediates the anti-apoptotic effect of hypoxia in pulmonary arterial smooth muscle cells through ERK1/2 pathway

    International Nuclear Information System (INIS)

    Song, Shasha; Wang, Shuang; Ma, Jun; Yao, Lan; Xing, Hao; Zhang, Lei; Liao, Lin; Zhu, Daling

    2013-01-01

    Inhibition of pulmonary arterial smooth muscle cell (PASMC) apoptosis induced by hypoxia plays an important role in pulmonary arterial remodeling leading to aggravate hypoxic pulmonary arterial hypertension. However, the mechanisms of hypoxia acting on PASMC apoptosis remain exclusive. Biliverdin reductase (BVR) has many essential biologic roles in physiological and pathological processes. Nevertheless, it is unclear whether the hypoxia-induced inhibition on PASMC apoptosis is mediated by BVR. In the present work, we found BVR majorly localized in PASMCs and was up-regulated in levels of protein and mRNA by hypoxia. Then we studied the contribution of BVR to anti-apoptotic response of hypoxia in PASMCs. Our results showed that siBVR, blocking generation of bilirubin, reversed the effect of hypoxia on enhancing cell survival and apoptotic protein (Bcl-2, procasepase-9, procasepase-3) expression, preventing nuclear shrinkage, DNA fragmentation and mitochondrial depolarization in starved PASMCs, which were recovered by exogenous bilirubin. Moreover, the inhibitory effect of bilirubin on PASMC apoptosis under hypoxic condition was blocked by the inhibitor of ERK1/2 pathway. Taken together, our data indicate that BVR contributes to the inhibitory process of hypoxia on PASMC apoptosis, which is mediated by bilirubin through ERK1/2 pathway. Highlights: • BVR expresses in PASMC and is up-regulated by hypoxia in protein and mRNA levels. • BVR/bilirubin contribute to the inhibitive process of hypoxia on PASMC apoptosis. • Bilirubin protects PASMC from apoptosis under hypoxia via ERK1/2 pathway

  19. Biliverdin reductase/bilirubin mediates the anti-apoptotic effect of hypoxia in pulmonary arterial smooth muscle cells through ERK1/2 pathway

    Energy Technology Data Exchange (ETDEWEB)

    Song, Shasha [Department of Biopharmaceutical Sciences, College of Pharmacy, Harbin Medical, University (Daqing), Daqing 163319 (China); Wang, Shuang [Department of Biopharmaceutical Sciences, College of Pharmacy, Harbin Medical, University (Daqing), Daqing 163319 (China); Biopharmaceutical Key Laboratory of Heilongjiang Province, Harbin 150081 (China); Ma, Jun; Yao, Lan; Xing, Hao; Zhang, Lei; Liao, Lin [Department of Biopharmaceutical Sciences, College of Pharmacy, Harbin Medical, University (Daqing), Daqing 163319 (China); Zhu, Daling, E-mail: dalingz@yahoo.com [Department of Biopharmaceutical Sciences, College of Pharmacy, Harbin Medical, University (Daqing), Daqing 163319 (China); Biopharmaceutical Key Laboratory of Heilongjiang Province, Harbin 150081 (China)

    2013-08-01

    Inhibition of pulmonary arterial smooth muscle cell (PASMC) apoptosis induced by hypoxia plays an important role in pulmonary arterial remodeling leading to aggravate hypoxic pulmonary arterial hypertension. However, the mechanisms of hypoxia acting on PASMC apoptosis remain exclusive. Biliverdin reductase (BVR) has many essential biologic roles in physiological and pathological processes. Nevertheless, it is unclear whether the hypoxia-induced inhibition on PASMC apoptosis is mediated by BVR. In the present work, we found BVR majorly localized in PASMCs and was up-regulated in levels of protein and mRNA by hypoxia. Then we studied the contribution of BVR to anti-apoptotic response of hypoxia in PASMCs. Our results showed that siBVR, blocking generation of bilirubin, reversed the effect of hypoxia on enhancing cell survival and apoptotic protein (Bcl-2, procasepase-9, procasepase-3) expression, preventing nuclear shrinkage, DNA fragmentation and mitochondrial depolarization in starved PASMCs, which were recovered by exogenous bilirubin. Moreover, the inhibitory effect of bilirubin on PASMC apoptosis under hypoxic condition was blocked by the inhibitor of ERK1/2 pathway. Taken together, our data indicate that BVR contributes to the inhibitory process of hypoxia on PASMC apoptosis, which is mediated by bilirubin through ERK1/2 pathway. Highlights: • BVR expresses in PASMC and is up-regulated by hypoxia in protein and mRNA levels. • BVR/bilirubin contribute to the inhibitive process of hypoxia on PASMC apoptosis. • Bilirubin protects PASMC from apoptosis under hypoxia via ERK1/2 pathway.

  20. Manganese nanoparticle activates mitochondrial dependent apoptotic signaling and autophagy in dopaminergic neuronal cells

    International Nuclear Information System (INIS)

    Afeseh Ngwa, Hilary; Kanthasamy, Arthi; Gu, Yan; Fang, Ning; Anantharam, Vellareddy; Kanthasamy, Anumantha G.

    2011-01-01

    The production of man-made nanoparticles for various modern applications has increased exponentially in recent years, but the potential health effects of most nanoparticles are not well characterized. Unfortunately, in vitro nanoparticle toxicity studies are extremely limited by yet unresolved problems relating to dosimetry. In the present study, we systematically characterized manganese (Mn) nanoparticle sizes and examined the nanoparticle-induced oxidative signaling in dopaminergic neuronal cells. Differential interference contrast (DIC) microscopy and transmission electron microscopy (TEM) studies revealed that Mn nanoparticles range in size from single nanoparticles (∼ 25 nM) to larger agglomerates when in treatment media. Manganese nanoparticles were effectively internalized in N27 dopaminergic neuronal cells, and they induced a time-dependent upregulation of the transporter protein transferrin. Exposure to 25–400 μg/mL Mn nanoparticles induced cell death in a time- and dose-dependent manner. Mn nanoparticles also significantly increased ROS, accompanied by a caspase-mediated proteolytic cleavage of proapoptotic protein kinase Cδ (PKCδ), as well as activation loop phosphorylation. Blocking Mn nanoparticle-induced ROS failed to protect against the neurotoxic effects, suggesting the involvement of other pathways. Further mechanistic studies revealed changes in Beclin 1 and LC3, indicating that Mn nanoparticles induce autophagy. Primary mesencephalic neuron exposure to Mn nanoparticles induced loss of TH positive dopaminergic neurons and neuronal processes. Collectively, our results suggest that Mn nanoparticles effectively enter dopaminergic neuronal cells and exert neurotoxic effects by activating an apoptotic signaling pathway and autophagy, emphasizing the need for assessing possible health risks associated with an increased use of Mn nanoparticles in modern applications. -- Highlights: ► Mn nanoparticles activate mitochondrial cell death signaling

  1. The anti-apoptotic members of the Bcl-2 family are attractive tumor-associated antigens

    DEFF Research Database (Denmark)

    Straten, Per thor; Andersen, Mads Hald; Andersen, Mads Hald

    2010-01-01

    Anti-apoptotic members of the Bcl-2 family (Bcl-2, Bcl-X(L) and Mcl-2) are pivotal regulators of apoptotic cell death. They are all highly overexpressed in cancers of different origin in which they enhance the survival of the cancer cells. Consequently, they represent prime candidates for anti-ca...

  2. P53-dependent antiproliferative and pro-apoptotic effects of trichostatin A (TSA) in glioblastoma cells.

    Science.gov (United States)

    Bajbouj, K; Mawrin, C; Hartig, R; Schulze-Luehrmann, J; Wilisch-Neumann, A; Roessner, A; Schneider-Stock, R

    2012-05-01

    Glioblastomas are known to be highly chemoresistant, but HDAC inhibitors (HDACi) have been shown to be of therapeutic relevance for this aggressive tumor type. We treated U87 glioblastoma cells with trichostatin A (TSA) to define potential epigenetic targets for HDACi-mediated antitumor effects. Using a cDNA array analysis covering 96 cell cycle genes, cyclin-dependent kinase inhibitor p21(WAF1) was identified as the major player in TSA-induced cell cycle arrest. TSA slightly inhibited proliferation and viability of U87 cells, cumulating in a G1/S cell cycle arrest. This effect was accompanied by a significant up-regulation of p53 and its transcriptional target p21(WAF1) and by down-regulation of key G1/S regulators, such as cdk4, cdk6, and cyclin D1. Nevertheless, TSA did not induce apoptosis in U87 cells. As expected, TSA promoted the accumulation of total acetylated histones H3 and H4 and a decrease in endogenous HDAC activity. Characterizing the chromatin modulation around the p21(WAF1) promoter after TSA treatment using chromatin immunoprecipitation, we found (1) a release of HDAC1, (2) an increase of acetylated H4 binding, and (3) enhanced recruitment of p53. p53-depleted U87 cells showed an abrogation of the G1/S arrest and re-entered the cell cycle. Immunofluorescence staining revealed that TSA induced the nuclear translocation of p21(WAF1) verifying a cell cycle arrest. On the other hand, a significant portion of p21(WAF1) was present in the cytoplasmic compartment causing apoptosis resistance. Furthermore, TSA-treated p53-mutant cell line U138 failed to show an induction in p21(WAF1), showed a deficient G2/M checkpoint, and underwent mitotic catastrophe. We suggest that HDAC inhibition in combination with other clinically used drugs may be considered an effective strategy to overcome chemoresistance in glioblastoma cells.

  3. Caffeine Induces Cell Death via Activation of Apoptotic Signal and Inactivation of Survival Signal in Human Osteoblasts

    Directory of Open Access Journals (Sweden)

    Wen-Hsiung Chan

    2008-05-01

    Full Text Available Caffeine consumption is a risk factor for osteoporosis, but the precise regulatory mechanisms are currently unknown. Here, we show that cell viability decreases in osteoblasts treated with caffeine in a dose-dependent manner. This cell death is attributed primarily to apoptosis and to a smaller extent, necrosis. Moreover, caffeine directly stimulates intracellular oxidative stress. Our data support caffeine-induced apoptosis in osteoblasts via a mitochondria-dependent pathway. The apoptotic biochemical changes were effectively prevented upon pretreatment with ROS scavengers, indicating that ROS plays a critical role as an upstream controller in the caffeine-induced apoptotic cascade. Additionally, p21-activated protein kinase 2 (PAK2 and c-Jun N-terminal kinase (JNK were activated in caffeine-treated osteoblasts. Experiments further found that PAK2 activity is required for caffeine-induced JNK activation and apoptosis. Importantly, our data also show that caffeine triggers cell death via inactivation of the survival signal, including the ERK- and Akt-mediated anti-apoptotic pathways. Finally, exposure of rats to dietary water containing 10~20 μM caffeine led to bone mineral density loss. These results demonstrate for the first time that caffeine triggers apoptosis in osteoblasts via activation of mitochondria-dependent cell death signaling and inactivation of the survival signal, and causes bone mineral density loss in vivo.

  4. Nanosecond pulsed electric fields induce poly(ADP-ribose) formation and non-apoptotic cell death in HeLa S3 cells.

    Science.gov (United States)

    Morotomi-Yano, Keiko; Akiyama, Hidenori; Yano, Ken-ichi

    2013-08-30

    Nanosecond pulsed electric fields (nsPEFs) have recently gained attention as effective cancer therapy owing to their potency for cell death induction. Previous studies have shown that apoptosis is a predominant mode of nsPEF-induced cell death in several cell lines, such as Jurkat cells. In this study, we analyzed molecular mechanisms for cell death induced by nsPEFs. When nsPEFs were applied to Jurkat cells, apoptosis was readily induced. Next, we used HeLa S3 cells and analyzed apoptotic events. Contrary to our expectation, nsPEF-exposed HeLa S3 cells exhibited no molecular signs of apoptosis execution. Instead, nsPEFs induced the formation of poly(ADP-ribose) (PAR), a hallmark of necrosis. PAR formation occurred concurrently with a decrease in cell viability, supporting implications of nsPEF-induced PAR formation for cell death. Necrotic PAR formation is known to be catalyzed by poly(ADP-ribose) polymerase-1 (PARP-1), and PARP-1 in apoptotic cells is inactivated by caspase-mediated proteolysis. Consistently, we observed intact and cleaved forms of PARP-1 in nsPEF-exposed and UV-irradiated cells, respectively. Taken together, nsPEFs induce two distinct modes of cell death in a cell type-specific manner, and HeLa S3 cells show PAR-associated non-apoptotic cell death in response to nsPEFs. Copyright © 2013 Elsevier Inc. All rights reserved.

  5. Role of wild-type p53 in apoptotic and non-apoptotic cell death induced by X-irradiation and heat treatment in p53-mutated mouse M10 cells

    International Nuclear Information System (INIS)

    Ito, Atsushi; Nakano, Hisako; Shinohara, Kunio

    2010-01-01

    The sensitizing effects of wild-type p53 on X-ray-induced cell death and on heat-induced apoptosis in M10, a radiosensitive and Trp53 (mouse p53 gene)-mutated lymphoma cell line which dies through necrosis by X-irradiation, were investigated using three M10 derived transfectants with wild-type TP53 (human p53 gene). Cell death was determined by colony formation and/or dye exclusion test, and apoptosis was detected as the changes in nuclear morphology by Giemsa staining. Expression of wild-type p53 protein increased radiosensitivity of cell death as determined by both clonogenic and dye exclusion assays. This increase in radiosensitivity was attributable largely to apoptosis induction in addition to a small enhancement of necrosis. Interestingly neither pathway to cell death was accompanied by caspase-3 activation. On the other hand, heat-induced caspase-3 dependent apoptotic cell death without transfection was further increased by the transfection of wild-type p53. In conclusion, the introduction of wild-type p53 enhanced apoptotic cell death by X-rays or heat via different mechanisms that do or do not activate caspase-3, respectively. In addition, p53 also enhanced the X-ray-induced necrosis in M10 cells. (author)

  6. Serratia marcescens Induces Apoptotic Cell Death in Host Immune Cells via a Lipopolysaccharide- and Flagella-dependent Mechanism*

    Science.gov (United States)

    Ishii, Kenichi; Adachi, Tatsuo; Imamura, Katsutoshi; Takano, Shinya; Usui, Kimihito; Suzuki, Kazushi; Hamamoto, Hiroshi; Watanabe, Takeshi; Sekimizu, Kazuhisa

    2012-01-01

    Injection of Serratia marcescens into the blood (hemolymph) of the silkworm, Bombyx mori, induced the activation of c-Jun NH2-terminal kinase (JNK), followed by caspase activation and apoptosis of blood cells (hemocytes). This process impaired the innate immune response in which pathogen cell wall components, such as glucan, stimulate hemocytes, leading to the activation of insect cytokine paralytic peptide. S. marcescens induced apoptotic cell death of silkworm hemocytes and mouse peritoneal macrophages in vitro. We searched for S. marcescens transposon mutants with attenuated ability to induce apoptosis of silkworm hemocytes. Among the genes identified, disruption mutants of wecA (a gene involved in lipopolysaccharide O-antigen synthesis), and flhD and fliR (essential genes in flagella synthesis) showed reduced motility and impaired induction of mouse macrophage cell death. These findings suggest that S. marcescens induces apoptosis of host immune cells via lipopolysaccharide- and flagella-dependent motility, leading to the suppression of host innate immunity. PMID:22859304

  7. E-Cigarette Vapor Induces an Apoptotic Response in Human Gingival Epithelial Cells Through the Caspase-3 Pathway.

    Science.gov (United States)

    Rouabhia, Mahmoud; Park, Hyun Jin; Semlali, Abdelhabib; Zakrzewski, Andrew; Chmielewski, Witold; Chakir, Jamila

    2017-06-01

    Electronic cigarettes represent an increasingly significant proportion of today's consumable tobacco products. E-cigarettes contain several chemicals which may promote oral diseases. The aim of this study was to investigate the effect of e-cigarette vapor on human gingival epithelial cells. Results show that e-cigarette vapor altered the morphology of cells from small cuboidal form to large undefined shapes. Both single and multiple exposures to e-cigarette vapor led to a bulky morphology with large faint nuclei and an enlarged cytoplasm. E-cigarette vapor also increased L-lactate dehydrogenase (LDH) activity in the targeted cells. This activity was greater with repeated exposures. Furthermore, e-cigarette vapor increased apoptotic/necrotic epithelial cell percentages compared to that observed in the control. Epithelial cell apoptosis was confirmed by TUNEL assay showing that exposure to e-cigarette vapor increased apoptotic cell numbers, particularly after two and three exposures. This negative effect involved the caspase-3 pathway, the activity of which was greater with repeated exposure and which decreased following the use of caspase-3 inhibitor. The adverse effects of e-cigarette vapor on gingival epithelial cells may lead to dysregulated gingival cell function and result in oral disease. J. Cell. Physiol. 232: 1539-1547, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  8. Increase in the fraction of necrotic, not apoptotic, cells in SiHa xenograft tumours shortly after irradiation

    International Nuclear Information System (INIS)

    Olive, P.L.; Vikse, C.M.; Vanderbyl, S.

    1999-01-01

    Background and purpose: Approximately 18% of the cells recovered by rapid mechanical dissociation of SiHa xenograft tumours contain large numbers of DNA strand breaks. The number of damaged cells increases to 30-40% 4-6 h after exposure to 5 or 15 Gy, returning to normal levels by 12 h. This observation is reminiscent of the rate of production of apoptotic cells in other murine and human xenograft tumours. The nature of this damage, rate of development and relation to cell proliferation rate were therefore examined in detail.Materials and methods: SiHa human cervical carcinoma cells were grown as xenograft tumours in SCID mice. Single-cell suspensions were prepared as a function of time after irradiation of the mouse and examined for DNA damage using the alkaline comet assay. Cell cycle progression was measured by flow cytometry evaluation of anti-bromodeoxyuridine-labelled tumour cells.Results: Significant numbers of apoptotic cells could not be detected in irradiated SiHa tumours using an end-labelling assay, electron microscopy, or histological examination of thin sections. Instead, xenograft cells exhibiting extensive DNA damage in the comet assay were predominantly necrotic cells. The increase in the proportion of heavily damaged cells 4-6 h after irradiation could be the result of an interplay between several factors including loss of viable cells and change in production or loss of necrotic cells. Analysis of the progression of BrdUrd-labelled cells confirmed that while 35% of cells from untreated SiHa tumours had divided and entered G 1 phase by 6 h after BrdUrd injection, none of the labelled cells from tumours exposed to 5 or 15 Gy had progressed to G 1 .Conclusions: The increase in the percentage of SiHa tumour cells with extensive DNA damage 4-6 h after irradiation is attributable to necrosis, not apoptosis. Cell cycle progression and cell loss are likely to influence the kinetics of appearance of both apoptotic and necrotic cells in irradiated tumours

  9. Normal and malignant epithelial cells with stem-like properties have an extended G2 cell cycle phase that is associated with apoptotic resistance

    Directory of Open Access Journals (Sweden)

    Biddle Adrian

    2010-04-01

    Full Text Available Abstract Background Subsets of cells with stem-like properties have been previously isolated from human epithelial cancers and their resistance to apoptosis-inducing stimuli has been related to carcinoma recurrence and treatment failure. The aim of this study was to investigate the mechanisms of resistance to apoptosis-inducing agents of cells with stem-like properties in both normal and malignant human epithelia. Methods Cells isolated from fresh human head and neck carcinomas (n = 11, cell lines derived from head and neck, prostate and breast human carcinomas (n = 7, and from normal human oral mucosa (n = 5, were exposed to various apoptosis-inducing stimuli (UV, Tumour Necrosis Factor, Cisplatin, Etoposide, and Neocarzinostatin. Flow cytometry for CD44 and epithelial-specific antigen (ESA expression, colony morphology, tumour sphere formation and rapid adherence assays were used to identify the subset of cells with stem-like properties. Apoptosis, cell cycle and expression of various cell cycle checkpoint proteins were assessed (Western Blot, qPCR. The role of G2-checkpoint regulators Chk1 and Chk2 was investigated by use of debromohymenialdisine (DBH and siRNA. Results In both cancer biopsies and carcinoma cell lines a subset of CD44high cells showed increased clonogenicity, a significantly lower rate of apoptosis, and a significantly higher proportion of cells in the G2-phase of the cell cycle. An inverse correlation between the percentage of cells in G2-phase and the rate of apoptosis was found. Pulse-chase with iododeoxyuridine (IdU demonstrated that CD44high carcinoma cells spent longer time in G2, even in un-treated controls. These cells expressed higher levels of G2 checkpoint proteins, and their release from G2 with BDH or Chk1 siRNA increased their rate of apoptosis. Low passage cultures of normal keratinocytes were also found to contain a subset of CD44high cells showing increased clonogenicity, and a similar pattern of G2-block

  10. Normal and malignant epithelial cells with stem-like properties have an extended G2 cell cycle phase that is associated with apoptotic resistance

    International Nuclear Information System (INIS)

    Harper, Lisa J; Costea, Daniela Elena; Gammon, Luke; Fazil, Bilal; Biddle, Adrian; Mackenzie, Ian C

    2010-01-01

    Subsets of cells with stem-like properties have been previously isolated from human epithelial cancers and their resistance to apoptosis-inducing stimuli has been related to carcinoma recurrence and treatment failure. The aim of this study was to investigate the mechanisms of resistance to apoptosis-inducing agents of cells with stem-like properties in both normal and malignant human epithelia. Cells isolated from fresh human head and neck carcinomas (n = 11), cell lines derived from head and neck, prostate and breast human carcinomas (n = 7), and from normal human oral mucosa (n = 5), were exposed to various apoptosis-inducing stimuli (UV, Tumour Necrosis Factor, Cisplatin, Etoposide, and Neocarzinostatin). Flow cytometry for CD44 and epithelial-specific antigen (ESA) expression, colony morphology, tumour sphere formation and rapid adherence assays were used to identify the subset of cells with stem-like properties. Apoptosis, cell cycle and expression of various cell cycle checkpoint proteins were assessed (Western Blot, qPCR). The role of G2-checkpoint regulators Chk1 and Chk2 was investigated by use of debromohymenialdisine (DBH) and siRNA. In both cancer biopsies and carcinoma cell lines a subset of CD44 high cells showed increased clonogenicity, a significantly lower rate of apoptosis, and a significantly higher proportion of cells in the G2-phase of the cell cycle. An inverse correlation between the percentage of cells in G2-phase and the rate of apoptosis was found. Pulse-chase with iododeoxyuridine (IdU) demonstrated that CD44 high carcinoma cells spent longer time in G2, even in un-treated controls. These cells expressed higher levels of G2 checkpoint proteins, and their release from G2 with BDH or Chk1 siRNA increased their rate of apoptosis. Low passage cultures of normal keratinocytes were also found to contain a subset of CD44 high cells showing increased clonogenicity, and a similar pattern of G2-block associated with apoptotic resistance. These data

  11. Synsepalum dulcificum extracts exhibit cytotoxic activity on human colorectal cancer cells and upregulate c-fos and c-jun early apoptotic gene expression

    Directory of Open Access Journals (Sweden)

    Jichang Seong

    2018-01-01

    Full Text Available Objective: To explore cytotoxicity of Synsepalum dulcificum (S. dulcificum Daniell (Sapotaceae on human colon cancer (HCT-116 and HT-29, human monocytic leukemia (THP-1 and normal (HDFn cell lines, and its effect on the expression of early apoptotic genes, c-fos and c-jun. Methods: Leaf, stem and berry of S. dulcificum were separately extracted by using 2 solvents, 10% ethanol (EtOH and 80% methanol (MeOH. PrestoBlue® cell viability assay and qRT-PCR assay were conducted to examine the above objectives respectively. Results: Stem MeOH, stem EtOH, and berry EtOH extracts of S. dulcificum were cytotoxic to HCT-116 and HT-29 human colon cancer cells. For HCT-116, IC50 values of these 3 extracts were not significantly different (P>0.05 from that of the positive control bleomycin (IC50 of 33.57 μg/mL, while for HT-29, IC50 values of these 3 extracts were significantly lower (P<0.05 than that of bleomycin (IC50 of 25.24 μg/mL. None of the extracts were cytotoxic to the THP-1 monocytic leukemia cells and HDFn normal human dermal fibroblasts. For both HCT-116 and HT-29, these extracts significantly up-regulated (P<0.05 the expression of c-fos and c-jun compared to the untreated negative control. Conclusions: The results of this study suggest that cytotoxicity of stem MeOH, stem EtOH, and berry EtOH extracts of S. dulcificum on HCT-116 and HT-29 colon cancer cells is due to the induced apoptosis which is caused by the up-regulation of the expression of early apoptotic genes, c-fos and c-jun.

  12. Neuroprotective effects of corn silk maysin via inhibition of H2O2-induced apoptotic cell death in SK-N-MC cells.

    Science.gov (United States)

    Choi, Doo Jin; Kim, Sun-Lim; Choi, Ji Won; Park, Yong Il

    2014-07-25

    Neuroprotective effects of maysin, which is a flavone glycoside that was isolated from the corn silk (CS, Zea mays L.) of a Korean hybrid corn Kwangpyeongok, against oxidative stress (H2O2)-induced apoptotic cell death of human neuroblastoma SK-N-MC cells were investigated. Maysin cytotoxicity was determined by measuring cell viability using MTT and lactate dehydrogenase (LDH) assays. Intracellular reactive oxygen species (ROS) were measured using a 2,7-dichlorofluorescein diacetate (DCF-DA) assay. Apoptotic cell death was monitored by annexin V-FITC/PI double staining and by a TUNEL assay. Antioxidant enzyme mRNA levels were determined by real-time PCR. The cleavage of poly (ADP-ribose) polymerase (PARP) was measured by western blotting. Maysin pretreatment reduced the cytotoxic effect of H2O2 on SK-N-MC cells, as shown by the increase in cell viability and by reduced LDH release. Maysin pretreatment also dose-dependently reduced the intracellular ROS level and inhibited PARP cleavage. In addition, DNA damage and H2O2-induced apoptotic cell death were significantly attenuated by maysin pretreatment. Moreover, maysin pretreatment (5-50 μg/ml) for 2h significantly and dose-dependently increased the mRNA levels of antioxidant enzymes (CAT, GPx-1, SOD-1, SOD-2 and HO-1) in H2O2 (200 μM)-insulted cells. These results suggest that CS maysin has neuroprotective effects against oxidative stress (H2O2)-induced apoptotic death of human brain SK-N-MC cells through its antioxidative action. This report is the first regarding neuroprotective health benefits of corn silk maysin by its anti-apoptotic action and by triggering the expression of intracellular antioxidant enzyme systems in SK-N-MC cells. Copyright © 2014 Elsevier Inc. All rights reserved.

  13. Acrolein activates cell survival and apoptotic death responses involving the endoplasmic reticulum in A549 lung cells.

    Science.gov (United States)

    Tanel, André; Pallepati, Pragathi; Bettaieb, Ahmed; Morin, Patrick; Averill-Bates, Diana A

    2014-05-01

    Acrolein, a highly reactive α,β-unsaturated aldehyde, is a product of endogenous lipid peroxidation. It is a ubiquitous environmental pollutant that is generated mainly by smoke, overheated cooking oil and vehicle exhaust. Acrolein damages cellular proteins, which could lead to accumulation of aberrantly-folded proteins in the endoplasmic reticulum (ER). This study determines the mechanisms involved in acrolein-induced apoptosis mediated by the ER and possible links with the ER stress response in human A549 lung cells. The exposure of cells to acrolein (15-50μM) for shorter times of 15 to 30min activated several ER stress markers. These included the ER chaperone protein BiP and the three ER sensors: (i) the survival/rescue molecules protein kinase RNA (PKR)-like ER kinase (PERK) and eukaryotic initiation factor 2 alpha (eIF2α) were phosphorylated; (ii) cleavage of activating transcription factor 6 (ATF6) occurred, and (iii) inositol-requiring protein-1 alpha (IRE1α) was phosphorylated. Acrolein (25-50μM) caused apoptotic cell death mediated by the ER after 2h, which was characterised by the induction of CHOP and activation of ER proteases calpain and caspase-4. Calpain and caspase-7 were the initiating factors for caspase-4 activation in acrolein-induced apoptosis. These results increase our knowledge about cellular responses to acrolein in lung cells, which have implications for human health. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. Mycobacterium tuberculosis infection induces non-apoptotic cell death of human dendritic cells

    LENUS (Irish Health Repository)

    Ryan, Ruth CM

    2011-10-24

    Abstract Background Dendritic cells (DCs) connect innate and adaptive immunity, and are necessary for an efficient CD4+ and CD8+ T cell response after infection with Mycobacterium tuberculosis (Mtb). We previously described the macrophage cell death response to Mtb infection. To investigate the effect of Mtb infection on human DC viability, we infected these phagocytes with different strains of Mtb and assessed viability, as well as DNA fragmentation and caspase activity. In parallel studies, we assessed the impact of infection on DC maturation, cytokine production and bacillary survival. Results Infection of DCs with live Mtb (H37Ra or H37Rv) led to cell death. This cell death proceeded in a caspase-independent manner, and without nuclear fragmentation. In fact, substrate assays demonstrated that Mtb H37Ra-induced cell death progressed without the activation of the executioner caspases, 3\\/7. Although the death pathway was triggered after infection, the DCs successfully underwent maturation and produced a host-protective cytokine profile. Finally, dying infected DCs were permissive for Mtb H37Ra growth. Conclusions Human DCs undergo cell death after infection with live Mtb, in a manner that does not involve executioner caspases, and results in no mycobactericidal effect. Nonetheless, the DC maturation and cytokine profile observed suggests that the infected cells can still contribute to TB immunity.

  15. Synergistic effect of aluminum and ionizing radiation upon ultrastructure, oxidative stress and apoptotic alterations in Paneth cells of rat intestine.

    Science.gov (United States)

    Eltahawy, N A; Elsonbaty, S M; Abunour, S; Zahran, W E

    2017-03-01

    Environmental and occupational exposure to aluminum along with ionizing radiation results in serious health problems. This study was planned to investigate the impact of oxidative stress provoked by exposure to ionizing radiation with aluminum administration upon cellular ultra structure and apoptotic changes in Paneth cells of rat small intestine . Animals received daily aluminum chloride by gastric gavage at a dose 0.5 mg/Kg BW for 4 weeks. Whole body gamma irradiation was applied at a dose 2 Gy/week up to 8 Gy. Ileum malondialdehyde, advanced oxidative protein products, protein carbonyl and tumor necrosis factor-alpha were assessed as biomarkers of lipid peroxidation, protein oxidation and inflammation respectively along with superoxide dismutase, catalase, and glutathione peroxidase activities as enzymatic antioxidants. Moreover, analyses of cell cycle division and apoptotic changes were evaluated by flow cytometry. Intestinal cellular ultra structure was investigated using transmission electron microscope.Oxidative and inflammatory stresses assessment in the ileum of rats revealed that aluminum and ionizing radiation exposures exhibited a significant effect upon the increase in oxidative stress biomarkers along with the inflammatory marker tumor necrosis factor-α accompanied by a significant decreases in the antioxidant enzyme activities. Flow cytometric analyses showed significant alterations in the percentage of cells during cell cycle division phases along with significant increase in apoptotic cells. Ultra structurally, intestinal cellular alterations with marked injury in Paneth cells at the sites of bacterial translocation in the crypt of lumens were recorded. The results of this study have clearly showed that aluminum and ionizing radiation exposures induced apoptosis with oxidative and inflammatory disturbance in the Paneth cells of rat intestine, which appeared to play a major role in the pathogenesis of cellular damage. Furthermore, the

  16. Effects of Downregulation of MicroRNA-181a on H2O2-Induced H9c2 Cell Apoptosis via the Mitochondrial Apoptotic Pathway

    Directory of Open Access Journals (Sweden)

    Lei Wang

    2014-01-01

    Full Text Available Glutathione peroxidase-1 (GPx1 is a pivotal intracellular antioxidant enzyme that enzymatically reduces hydrogen peroxide to water to limit its harmful effects. This study aims to identify a microRNA (miRNA that targets GPx1 to maintain redox homeostasis. Dual luciferase assays combined with mutational analysis and immunoblotting were used to validate the bioinformatically predicted miRNAs. We sought to select miRNAs that were responsive to oxidative stress induced by hydrogen peroxide (H2O2 in the H9c2 rat cardiomyocyte cell line. Quantitative real-time PCR (qPCR demonstrated that the expression of miR-181a in H2O2-treated H9c2 cells was markedly upregulated. The downregulation of miR-181a significantly inhibited H2O2-induced cellular apoptosis, ROS production, the increase in malondialdehyde (MDA levels, the disruption of mitochondrial structure, and the activation of key signaling proteins in the mitochondrial apoptotic pathway. Our results suggest that miR-181a plays an important role in regulating the mitochondrial apoptotic pathway in cardiomyocytes challenged with oxidative stress. MiR-181a may represent a potential therapeutic target for the treatment of oxidative stress-associated cardiovascular diseases.

  17. Classification of Normal and Apoptotic Cells from Fluorescence Microscopy Images Using Generalized Polynomial Chaos and Level Set Function.

    Science.gov (United States)

    Du, Yuncheng; Budman, Hector M; Duever, Thomas A

    2016-06-01

    Accurate automated quantitative analysis of living cells based on fluorescence microscopy images can be very useful for fast evaluation of experimental outcomes and cell culture protocols. In this work, an algorithm is developed for fast differentiation of normal and apoptotic viable Chinese hamster ovary (CHO) cells. For effective segmentation of cell images, a stochastic segmentation algorithm is developed by combining a generalized polynomial chaos expansion with a level set function-based segmentation algorithm. This approach provides a probabilistic description of the segmented cellular regions along the boundary, from which it is possible to calculate morphological changes related to apoptosis, i.e., the curvature and length of a cell's boundary. These features are then used as inputs to a support vector machine (SVM) classifier that is trained to distinguish between normal and apoptotic viable states of CHO cell images. The use of morphological features obtained from the stochastic level set segmentation of cell images in combination with the trained SVM classifier is more efficient in terms of differentiation accuracy as compared with the original deterministic level set method.

  18. 99mTc rh-Annexin V for imaging apoptotic cells: Clinical experience

    International Nuclear Information System (INIS)

    Abdel-Dayem, H.M.; Sadek, S.; Heiba, S.; Steinmetz, N.; Cho, J.; Cook, W.; Klein, P.

    2002-01-01

    Introduction: 99m Tc rh-Annexin V (Apomate, Theseus Imaging Corp., Boston, MA), an investigational nuclear medicine agent for imaging apoptotic and necrotic cell death, is currently in Phase II-III clinical trials. Potential applications being evaluated include identification and localization of acute myocardial infarction, non-invasive assessment of cardiac transplant rejection, and early prediction of cancer chemotherapy response. The purposes of this presentation are: 1) To familiarize attendees with the biological basis of 99m Tc-Annexin V localization, the labeling procedure, biodistribution following I.V. injection, and dosimetric data; 2) In patients with acute myocardial infarction (AMI), dual isotope SPECT using 99m Tc-Annexin V and Thallium-201 was performed within 4 days from onset of AMI and 6 weeks later. It demonstrates the increased uptake of 99m Tc-Annexin V in the recent MI area correlating with the perfusion defect in the Thallium study. 99m Tc-Annexin V disappears in the follow-up scan 6 weeks later indicating that 99m Tc-Annexin V will be a clinically useful agent for determining the size and location of recent AMI and for differentiating AMI from myocardial scar. 3) To demonstrate examples of current applications in oncology for predicting chemo- and radiotherapy response in patients with small and non-small cell lung cancer, Hodgkin's and non-Hodgkin's malignant lymphoma and recurrent, metastatic or inoperable breast carcinoma. Early results show significant correlation with treatment response and patient survival. 4) To demonstrate unusual site of 99m Tc-Annexin V uptake in subdural hematoma and non-lactating breast 4 weeks post partum. In light of the current clinical studies of 99m Tc-Annexin V (Apomate) imaging and the potential approval for clinical use in the future, it is important for practicing nuclear medicine physicians to be aware of the normal distribution, expected patterns of uptake in various pathologic conditions, and unusual

  19. Activation of the intrinsic-apoptotic pathway in LNCaP prostate cancer cells by genistein- topotecan combination treatments

    Directory of Open Access Journals (Sweden)

    Vanessa Hörmann

    2013-03-01

    Full Text Available ABSTRACTBackground: Prostate cancer is the second most common cancer in American men. The development of alternative preventative and/or treatment options utilizing a combination of phytochemicals and chemotherapeutic drugs could be an attractive alternative compared to conventional carcinoma treatments. Genistein isoflavone is the primary dietary phytochemical found in soy and has demonstrated anti-tumor activities in LNCaP prostate cancer cells. Topotecan Hydrochloride (Hycamtin is an FDA-approved chemotherapy for secondary treatment of lung, ovarian and cervical cancers. The purpose of this study was to detail the potential activation of the intrinsic apoptotic pathway in LNCaP prostate cancer cells through genistein-topotecan combination treatments.Methods: LNCaP cells were cultured in complete RPMI medium in a monolayer (70-80% confluency at 37ºC and 5% CO2. Treatment consisted of single and combination groups of genistein and topotecan for 24 hours. The treated cells were assayed for i growth inhibition through trypan blue exclusion assay and microphotography , ii classification of cellular death through acridine/ ethidium bromide fluorescent staining, and iii activation of the intrinsic apoptotic pathway through Jc-1: mitochondrial membrane potential assay, cytochrome c release and Bcl-2 protein expression.Results: The overall data indicated that genistein-topotecan combination was significantly more efficacious in reducing the prostate carcinoma’s viability compared to the single treatment options. In all treatment groups, cell death occurred primarily through the activation of the intrinsic apoptotic pathway.Conclusion: The combination of topotecan and genistein has the potential to lead to treatment options with equal therapeutic efficiency as traditional chemo- and radiation therapies, but lower cell cytotoxicity and fewer side effects in patients.

  20. Human surfactant protein D alters oxidative stress and HMGA1 expression to induce p53 apoptotic pathway in eosinophil leukemic cell line.

    Directory of Open Access Journals (Sweden)

    Lakshna Mahajan

    Full Text Available Surfactant protein D (SP-D, an innate immune molecule, has an indispensable role in host defense and regulation of inflammation. Immune related functions regulated by SP-D include agglutination of pathogens, phagocytosis, oxidative burst, antigen presentation, T lymphocyte proliferation, cytokine secretion, induction of apoptosis and clearance of apoptotic cells. The present study unravels a novel ability of SP-D to reduce the viability of leukemic cells (eosinophilic leukemic cell line, AML14.3D10; acute myeloid leukemia cell line, THP-1; acute lymphoid leukemia cell lines, Jurkat, Raji; and human breast epithelial cell line, MCF-7, and explains the underlying mechanisms. SP-D and a recombinant fragment of human SP-D (rhSP-D induced G2/M phase cell cycle arrest, and dose and time-dependent apoptosis in the AML14.3D10 eosinophilic leukemia cell line. Levels of various apoptotic markers viz. activated p53, cleaved caspase-9 and PARP, along with G2/M checkpoints (p21 and Tyr15 phosphorylation of cdc2 showed significant increase in these cells. We further attempted to elucidate the underlying mechanisms of rhSP-D induced apoptosis using proteomic analysis. This approach identified large scale molecular changes initiated by SP-D in a human cell for the first time. Among others, the proteomics analysis highlighted a decreased expression of survival related proteins such as HMGA1, overexpression of proteins to protect the cells from oxidative burst, while a drastic decrease in mitochondrial antioxidant defense system. rhSP-D mediated enhanced oxidative burst in AML14.3D10 cells was confirmed, while antioxidant, N-acetyl-L-cysteine, abrogated the rhSP-D induced apoptosis. The rhSP-D mediated reduced viability was specific to the cancer cell lines and viability of human PBMCs from healthy controls was not affected. The study suggests involvement of SP-D in host's immunosurveillance and therapeutic potential of rhSP-D in the eosinophilic leukemia and

  1. Aurora A kinase RNAi and small molecule inhibition of Aurora kinases with VE-465 induce apoptotic death in multiple myeloma cells.

    Science.gov (United States)

    Evans, Robert; Naber, Claudia; Steffler, Tara; Checkland, Tamara; Keats, Jonathan; Maxwell, Christopher; Perry, Troy; Chau, Heidi; Belch, Andrew; Pilarski, Linda; Reiman, Tony

    2008-03-01

    The expression of RHAMM and other centrosome-associated genes are known to correlate with the extent of centrosome amplification in multiple myeloma, and with poor prognosis. RHAMM has a significant interaction with TPX2, a protein which regulates the localization and action of Aurora A kinase (AURKA) at the spindle poles. AURKA is known to be a central determinant of centrosome and spindle function and is a target for cancer therapy. Given these observations, we investigated the role of Aurora kinases as therapeutic targets in myeloma. Here we report that AURKA is expressed ubiquitously in myeloma, to varying degrees, in both cell lines and patients' bone marrow plasma cells. siRNA targeting AURKA induces apoptotic cell death in myeloma cell lines. The Aurora kinase inhibitor VE-465 also induces apoptosis and death in myeloma cell lines and primary myeloma plasma cells. The combination of VE-465 and dexamethasone improves cell killing compared with the use of either agent alone, even in cells resistant to the single agents. The phenotype of myeloma cells treated with VE-465 is consistent with published reports on the effects of Aurora kinase inhibition. Aurora kinase inhibitors should be pursued as potential treatments for myeloma.

  2. Isoegomaketone induces apoptosis in SK-MEL-2 human melanoma cells through mitochondrial apoptotic pathway via activating the PI3K/Akt pathway.

    Science.gov (United States)

    Kwon, Soon-Jae; Lee, Ju-Hye; Moon, Kwang-Deog; Jeong, Il-Yun; Yee, Sung-Tae; Lee, Mi-Kyung; Seo, Kwon-Il

    2014-11-01

    Isoegomaketone (IK) is a major biologically active component of Perilla frutescens. In this study, we investigated the contribution of reactive oxygen species (ROS) to IK-induced apoptosis in human melanoma SK-MEL-2 cells. We found that IK inhibited the proliferation of SK-MEL-2 human melanoma cells in a dose-dependent manner. IK also induced sub-G1 DNA accumulation, formation of apoptotic bodies, nuclear condensation, and a DNA ladder in SK-MEL-2 cells. IK also induced activation of caspase-3 and -9, whereas caspase‑8 was unaffected. Further, N-acetyl-L-cysteine (NAC, ROS scavenger) treatment to SK-MEL-2 cells significantly reduced IK-induced cell death. Pretreatment of NAC to SK-MEL-2 cells followed by 100 µM IK reduced the protein levels of Bax and cytochrome c as well as PARP cleavage, whereas the protein level of Bcl-2 increased. Moreover, IK inhibited the phosphorylation of AKT/mTOR protein and cell proliferation induced by LY294002, a PI3K inhibitor. In conclusion, IK-induced ROS generation regulates cell growth inhibition and it induces apoptosis through caspase‑dependent and -independent pathways via modulation of PI3K/AKT signaling in SK-MEL-2 cells.

  3. The antimicrobial peptide nisin Z induces selective toxicity and apoptotic cell death in cultured melanoma cells.

    Science.gov (United States)

    Lewies, Angélique; Wentzel, Johannes Frederik; Miller, Hayley Christy; Du Plessis, Lissinda Hester

    2018-01-01

    Reprogramming of cellular metabolism is now considered one of the hallmarks of cancer. Most malignant cells present with altered energy metabolism which is associated with elevated reactive oxygen species (ROS) generation. This is also evident for melanoma, the leading cause of skin cancer related deaths. Altered mechanisms affecting mitochondrial bioenergetics pose attractive targets for novel anticancer therapies. Antimicrobial peptides have been shown to exhibit selective anticancer activities. In this study, the anti-melanoma potential of the antimicrobial peptide, nisin Z, was evaluated in vitro. Nisin Z was shown to induce selective toxicity in melanoma cells compared to non-malignant keratinocytes. Furthermore, nisin Z was shown to negatively affect the energy metabolism (glycolysis and mitochondrial respiration) of melanoma cells, increase reactive oxygen species generation and cause apoptosis. Results also indicate that nisin Z can decrease the invasion and proliferation of melanoma cells demonstrating its potential use against metastasis associated with melanoma. As nisin Z seems to place a considerable extra burden on the energy metabolism of melanoma cells, combination therapies with known anti-melanoma agents may be effective treatment options. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  4. Structurally related hydrazone-based metal complexes with different antitumor activities variably induce apoptotic cell death.

    Science.gov (United States)

    Megger, Dominik A; Rosowski, Kristin; Radunsky, Christian; Kösters, Jutta; Sitek, Barbara; Müller, Jens

    2017-04-05

    Three new complexes bearing the tridentate hydrazone-based ligand 2-(2-(1-(pyridin-2-yl)ethylidene)hydrazinyl)pyridine (L) were synthesized and structurally characterized. Biological tests indicate that the Zn(ii) complex [ZnCl 2 (L)] is of low cytotoxicity against the hepatocellular carcinoma cell line HepG2. In contrast, the Cu(ii) and Mn(ii) complexes [CuCl 2 (L)] and [MnCl 2 (L)] are highly cytotoxic with EC 50 values of 1.25 ± 0.01 μM and 20 ± 1 μM, respectively. A quantitative proteome analysis reveals that treatment of the cells with the Cu(ii) complex leads to a significantly altered abundance of 102 apoptosis-related proteins, whereas 38 proteins were up- or down-regulated by the Mn(ii) complex. A closer inspection of those proteins regulated only by the Cu(ii) complex suggests that the superior cytotoxic activity of this complex is likely to be related to an initiation of the caspase-independent cell death (CICD). In addition, an increased generation of reactive oxygen species (ROS) and a strong up-regulation of proteins responsive to oxidative stress suggest that alterations of the cellular redox metabolism likely contribute to the cytotoxicity of the Cu(ii) complex.

  5. Hibiscus anthocyanins rich extract-induced apoptotic cell death in human promyelocytic leukemia cells

    International Nuclear Information System (INIS)

    Chang, Y.-C.; Huang, H.-P.; Hsu, J.-D.; Yang, S.-F.; Wang, C.-J.

    2005-01-01

    Hibiscus sabdariffa Linne (Malvaceae), an attractive plant believed to be native to Africa, is cultivated in the Sudan and Eastern Taiwan. Anthocyanins exist widely in many vegetables and fruits. Some reports demonstrated that anthocyanins extracted from H. sabdariffa L., Hibiscus anthocyanins (HAs) (which are a group of natural pigments existing in the dried calyx of H. sabdariffa L.) exhibited antioxidant activity and liver protection. Therefore, in this study, we explored the effect of HAs on human cancer cells. The result showed that HAs could cause cancer cell apoptosis, especially in HL-60 cells. Using flow cytometry, we found that HAs treatment (0-4 mg/ml) markedly induced apoptosis in HL-60 cells in a dose- and time-dependent manner. The result also revealed increased phosphorylation in p38 and c-Jun, cytochrome c release, and expression of tBid, Fas, and FasL in the HAs-treated HL-60 cells. We further used SB203580 (p38 inhibitor), PD98059 (MEK inhibitor), SP600125 (JNK inhibitor), and wortmannin (phosphatidylinositol 3-kinase; PI-3K inhibitor) to evaluate their effect on the HAs-induced HL-60 death. The data showed that only SB203580 had strong potential in inhibiting HL-60 cell apoptosis and related protein expression and phosphorylation. Therefore, we suggested that HAs mediated HL-60 apoptosis via the p38-FasL and Bid pathway. According to these results, HAs could be developed as chemopreventive agents. However, further investigations into the specificity and mechanism(s) of HAs are needed

  6. dp53 Restrains ectopic neural stem cell formation in the Drosophila brain in a non-apoptotic mechanism involving Archipelago and cyclin E.

    Directory of Open Access Journals (Sweden)

    Yingshi Ouyang

    Full Text Available Accumulating evidence suggests that tumor-initiating stem cells or cancer stem cells (CSCs possibly originating from normal stem cells may be the root cause of certain malignancies. How stem cell homeostasis is impaired in tumor tissues is not well understood, although certain tumor suppressors have been implicated. In this study, we use the Drosophila neural stem cells (NSCs called neuroblasts as a model to study this process. Loss-of-function of Numb, a key cell fate determinant with well-conserved mammalian counterparts, leads to the formation of ectopic neuroblasts and a tumor phenotype in the larval brain. Overexpression of the Drosophila tumor suppressor p53 (dp53 was able to suppress ectopic neuroblast formation caused by numb loss-of-function. This occurred in a non-apoptotic manner and was independent of Dacapo, the fly counterpart of the well-characterized mammalian p53 target p21 involved in cellular senescence. The observation that dp53 affected Edu incorporation into neuroblasts led us to test the hypothesis that dp53 acts through regulation of factors involved in cell cycle progression. Our results show that the inhibitory effect of dp53 on ectopic neuroblast formation was mediated largely through its regulation of Cyclin E (Cyc E. Overexpression of Cyc E was able to abrogate dp53's ability to rescue numb loss-of-function phenotypes. Increasing Cyc E levels by attenuating Archipelago (Ago, a recently identified transcriptional target of dp53 and a negative regulator of Cyc E, had similar effects. Conversely, reducing Cyc E activity by overexpressing Ago blocked ectopic neuroblast formation in numb mutant. Our results reveal an intimate connection between cell cycle progression and NSC self-renewal vs. differentiation control, and indicate that p53-mediated regulation of ectopic NSC self-renewal through the Ago/Cyc E axis becomes particularly important when NSC homeostasis is perturbed as in numb loss-of-function condition. This has

  7. A chalcone-related small molecule that induces methuosis, a novel form of non-apoptotic cell death, in glioblastoma cells.

    Science.gov (United States)

    Overmeyer, Jean H; Young, Ashley M; Bhanot, Haymanti; Maltese, William A

    2011-06-06

    Methuosis is a unique form of non-apoptotic cell death triggered by alterations in the trafficking of clathrin-independent endosomes, ultimately leading to extreme vacuolization and rupture of the cell. Here we describe a novel chalcone-like molecule, 3-(2-methyl-1H- indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one (MIPP) that induces cell death with the hallmarks of methuosis. MIPP causes rapid accumulation of vacuoles derived from macropinosomes, based on time-lapse microscopy and labeling with extracellular fluid phase tracers. Vacuolization can be blocked by the cholesterol-interacting compound, filipin, consistent with the origin of the vacuoles from non-clathrin endocytic compartments. Although the vacuoles rapidly acquire some characteristics of late endosomes (Rab7, LAMP1), they remain distinct from lysosomal and autophagosomal compartments, suggestive of a block at the late endosome/lysosome boundary. MIPP appears to target steps in the endosomal trafficking pathway involving Rab5 and Rab7, as evidenced by changes in the activation states of these GTPases. These effects are specific, as other GTPases (Rac1, Arf6) are unaffected by the compound. Cells treated with MIPP lose viability within 2-3 days, but their nuclei show no evidence of apoptotic changes. Inhibition of caspase activity does not protect the cells, consistent with a non-apoptotic death mechanism. U251 glioblastoma cells selected for temozolomide resistance showed sensitivity to MIPP-induced methuosis that was comparable to the parental cell line. MIPP might serve as a prototype for new drugs that could be used to induce non-apoptotic death in cancers that have become refractory to agents that work through DNA damage and apoptotic mechanisms.

  8. A chalcone-related small molecule that induces methuosis, a novel form of non-apoptotic cell death, in glioblastoma cells

    Directory of Open Access Journals (Sweden)

    Bhanot Haymanti

    2011-06-01

    Full Text Available Abstract Background Methuosis is a unique form of non-apoptotic cell death triggered by alterations in the trafficking of clathrin-independent endosomes, ultimately leading to extreme vacuolization and rupture of the cell. Results Here we describe a novel chalcone-like molecule, 3-(2-methyl-1H- indol-3-yl-1-(4-pyridinyl-2-propen-1-one (MIPP that induces cell death with the hallmarks of methuosis. MIPP causes rapid accumulation of vacuoles derived from macropinosomes, based on time-lapse microscopy and labeling with extracellular fluid phase tracers. Vacuolization can be blocked by the cholesterol-interacting compound, filipin, consistent with the origin of the vacuoles from non-clathrin endocytic compartments. Although the vacuoles rapidly acquire some characteristics of late endosomes (Rab7, LAMP1, they remain distinct from lysosomal and autophagosomal compartments, suggestive of a block at the late endosome/lysosome boundary. MIPP appears to target steps in the endosomal trafficking pathway involving Rab5 and Rab7, as evidenced by changes in the activation states of these GTPases. These effects are specific, as other GTPases (Rac1, Arf6 are unaffected by the compound. Cells treated with MIPP lose viability within 2-3 days, but their nuclei show no evidence of apoptotic changes. Inhibition of caspase activity does not protect the cells, consistent with a non-apoptotic death mechanism. U251 glioblastoma cells selected for temozolomide resistance showed sensitivity to MIPP-induced methuosis that was comparable to the parental cell line. Conclusions MIPP might serve as a prototype for new drugs that could be used to induce non-apoptotic death in cancers that have become refractory to agents that work through DNA damage and apoptotic mechanisms.

  9. SIGN-R1 and complement factors are involved in the systemic clearance of radiation-induced apoptotic cells in whole-body irradiated mice

    Energy Technology Data Exchange (ETDEWEB)

    Park, Jin-Yeon; Loh, SoHee; Cho, Eun-hee [Department of Biomedical Science & Technology, Konkuk University, 1 Hwayang-dong, Gwangjin-gu, Seoul, 143-701 (Korea, Republic of); Choi, Hyeong-Jwa [Division of Radiation Effect, Korea Institute of Radiological and Medical Sciences, 215-4, 75 Nowon gil Nowon-Gu, Seoul, 139-706 (Korea, Republic of); Na, Tae-Young [College of Pharmacy, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 151-741 (Korea, Republic of); Nemeno, Judee Grace E.; Lee, Jeong Ik [Regenerative Medicine Laboratory, Department of Veterinary Medicine, College of Veterinary Medicine, Konkuk University, Seoul, 143-701 (Korea, Republic of); Yoon, Taek Joon [Department of Food and Nutrition, Yuhan College, Bucheon, Gyeonggi-do, 422-749 (Korea, Republic of); Choi, In-Soo [Department of Infectious Diseases, College of Veterinary Medicine, Konkuk University, 1 Hwayang-dong, Gwangjin-gu, Seoul, 143-701 (Korea, Republic of); Lee, Minyoung [Division of Radiation Effect, Korea Institute of Radiological and Medical Sciences, 215-4, 75 Nowon gil Nowon-Gu, Seoul, 139-706 (Korea, Republic of); Lee, Jae-Seon [Department of Biomedical Sciences, College of Medicine, Inha University, Incheon, 400-712 (Korea, Republic of); Kang, Young-Sun, E-mail: kangys1967@naver.com [Department of Biomedical Science & Technology, Konkuk University, 1 Hwayang-dong, Gwangjin-gu, Seoul, 143-701 (Korea, Republic of); Department of Veterinary Pharmacology and Toxicology, College of Veterinary Medicine, Konkuk University, 1 Hwayang-dong, Gwangjin-gu, Seoul, 143-701 (Korea, Republic of)

    2015-08-07

    Although SIGN-R1-mediated complement activation pathway has been shown to enhance the systemic clearance of apoptotic cells, the role of SIGN-R1 in the clearance of radiation-induced apoptotic cells has not been characterized and was investigated in this study. Our data indicated that whole-body γ-irradiation of mice increased caspase-3{sup +} apoptotic lymphocyte numbers in secondary lymphoid organs. Following γ-irradiation, SIGN-R1 and complements (C4 and C3) were simultaneously increased only in the mice spleen tissue among the assessed tissues. In particular, C3 was exclusively activated in the spleen. The delayed clearance of apoptotic cells was markedly prevalent in the spleen and liver of SIGN-R1 KO mice, followed by a significant increase of CD11b{sup +} cells. These results indicate that SIGN-R1 and complement factors play an important role in the systemic clearance of radiation-induced apoptotic innate immune cells to maintain tissue homeostasis after γ-irradiation. - Highlights: • Splenic SIGN-R1{sup +} macrophages are activated after γ-irradiation. • C3 and C4 levels increased and C3 was activated in the spleen after γ-irradiation. • SIGN-R1 mediated the systemic clearance of radiation-induced apoptotic cells in spleen and liver.

  10. To the Large Nucleolar Bodies in Apoptotic Leukaemic Granulocytic Progenitors without Further Differentiation. Are Large Nucleoli Always Present in Proliferating Cells?

    Science.gov (United States)

    Smetana, K; Kuželová, K; Zápotocký, M; Hrkal, Z

    2017-01-01

    Large nucleoli have generally been believed to be present in less differentiated and proliferating cells including the malignant ones. Such nucleoli have also been considered to be active in the biosynthetic process and major cell developmental activities. In contrast, after cytostatic treatment, apoptotic leukaemic progenitors still containing nuclei did not exhibit substantial reduction of the nucleolar size but displayed decreased nucleolar biosynthetic activity. The present study was undertaken to provide more information on the large nucleoli in spontaneously occurring apoptotic leukaemic progenitors without further differentiation. Leukaemic progenitors of established cell lineages originating from leukaemic patients represented a very convenient model for such study. Some of them exhibit morphological signs of the spontaneously occurring apoptotic process. Since such signs are expressed by nuclear and cytoplasmic morphological variability, the present study dealt with spontaneously occurring apoptotic progenitors with preserved nuclei characterized by heavy chromatin condensation and occasional fragmentation. Based of nucleolar body and nuclear maximal diameter measurements it seems to be clear that the nucleolar size in these cells was not substantially reduced, contrary to that of the nucleus. However, large nucleolar bodies in spontaneously occurring apoptotic cells were characterized by markedly reduced biosynthetic activity, as expressed by the decreased number of nucleolar transcription markers such as nucleolar fibrillar centres. In conclusion, large nucleoli may be present not only in proliferating, but also in spontaneously occurring apoptotic cells.

  11. SIGN-R1 and complement factors are involved in the systemic clearance of radiation-induced apoptotic cells in whole-body irradiated mice

    International Nuclear Information System (INIS)

    Park, Jin-Yeon; Loh, SoHee; Cho, Eun-hee; Choi, Hyeong-Jwa; Na, Tae-Young; Nemeno, Judee Grace E.; Lee, Jeong Ik; Yoon, Taek Joon; Choi, In-Soo; Lee, Minyoung; Lee, Jae-Seon; Kang, Young-Sun

    2015-01-01

    Although SIGN-R1-mediated complement activation pathway has been shown to enhance the systemic clearance of apoptotic cells, the role of SIGN-R1 in the clearance of radiation-induced apoptotic cells has not been characterized and was investigated in this study. Our data indicated that whole-body γ-irradiation of mice increased caspase-3 + apoptotic lymphocyte numbers in secondary lymphoid organs. Following γ-irradiation, SIGN-R1 and complements (C4 and C3) were simultaneously increased only in the mice spleen tissue among the assessed tissues. In particular, C3 was exclusively activated in the spleen. The delayed clearance of apoptotic cells was markedly prevalent in the spleen and liver of SIGN-R1 KO mice, followed by a significant increase of CD11b + cells. These results indicate that SIGN-R1 and complement factors play an important role in the systemic clearance of radiation-induced apoptotic innate immune cells to maintain tissue homeostasis after γ-irradiation. - Highlights: • Splenic SIGN-R1 + macrophages are activated after γ-irradiation. • C3 and C4 levels increased and C3 was activated in the spleen after γ-irradiation. • SIGN-R1 mediated the systemic clearance of radiation-induced apoptotic cells in spleen and liver

  12. Hypoxia-response plasmid vector producing bcl-2 shRNA enhances the apoptotic cell death of mouse rectum carcinoma.

    Science.gov (United States)

    Fujioka, Takashi; Matsunaga, Naoya; Okazaki, Hiroyuki; Koyanagi, Satoru; Ohdo, Shigehiro

    2010-01-01

    Hypoxia-induced gene expression frequently occurs in malignant solid tumors because they often have hypoxic areas in which circulation is compromised due to structurally disorganized blood vessels. Hypoxia-response elements (HREs) are responsible for activating gene transcription in response to hypoxia. In this study, we constructed a hypoxia-response plasmid vector producing short hairpin RNA (shRNA) against B-cell leukemia/lymphoma-2 (bcl-2), an anti-apoptotic factor. The hypoxia-response promoter was made by inserting tandem repeats of HREs upstream of cytomegalovirus (CMV) promoter (HRE-CMV). HRE-CMV shbcl-2 vector consisted of bcl-2 shRNA under the control of HRE-CMV promoter. In hypoxic mouse rectum carcinoma cells (colon-26), the production of bcl-2 shRNA driven by HRE-CMV promoter was approximately 2-fold greater than that driven by CMV promoter. A single intratumoral (i.t.) injection of 40 microg HRE-CMV shbcl-2 to colon-26 tumor-bearing mice caused apoptotic cell death, and repetitive treatment with HRE-CMV shbcl-2 (40 microg/mouse, i.t.) also significantly suppressed the growth of colon-26 tumor cells implanted in mice. Apoptotic and anti-tumor effects were not observed in tumor-bearing mice treated with CMV shbcl-2. These results reveal the ability of HRE-CMV shbcl-2 vector to suppress the expression of bcl-2 in hypoxic tumor cells and suggest the usefulness of our constructed hypoxia-response plasmid vector to treat malignant tumors. [Supplementary Figures: available only at http://dx.doi.org/10.1254/jphs.10054FP].

  13. Andrographolide potentiates the antitumor effect of topotecan in acute myeloid leukemia cells through an intrinsic apoptotic pathway.

    Science.gov (United States)

    Hodroj, Mohammad Hassan; Jardaly, Achraf; Abi Raad, Sarah; Zouein, Annalise; Rizk, Sandra

    2018-01-01

    Topotecan (TP) is an anticancer drug acting as topoisomerase I inhibitor that is used in the treatment of many types of cancers including leukemia, but it has significant side effects. Andrographolide, a compound extracted from Andrographis paniculata , was recently proven to inhibit the growth of cancer cells and can induce apoptosis. The aim of this study is to investigate the possible synergism between TP and andrographolide in acute myeloid cells in vitro. U937 acute myeloid leukemic cells were cultured using Roswell Park Memorial Institute (RPMI) medium and then treated for 24 h with TP and andrographolide prepared through the dilution of dimethyl sulfoxide (DMSO) stocks with RPMI on the day of treatment. Cell proliferation was assessed using cell proliferation assay upon treatment with both compounds separately and in combination. Cell-cycle study and apoptosis detection were performed by staining the cells with propidium iodide (PI) stain and Annexin V/PI stain, respectively, followed by flow cytometry analysis. Western blotting was used to assess the expression of various proteins involved in apoptotic pathways. Both TP and andrographolide showed an antiproliferative effect in a dose-dependent manner when applied on U937 cells separately; however, pretreating the cells with andrographolide before applying TP exhibited a synergistic effect with lower inhibitory concentrations (half-maximal inhibitory concentration). Treating the cells with TP alone led to specific cell-cycle arrest at S phase that was more prominent upon pretreatment combination with andrographolide. Using Annexin V/PI staining to assess the proapoptotic effect following the pretreatment combination showed an increase in the number of apoptotic cells, which was supported by the Western blot results that manifested an upregulation of several proapoptotic proteins expression. The pretreatment of U937 with andrographolide followed by low doses of TP showed an enhancement in inducing apoptosis

  14. 1-Nitropyrene (1-NP) induces apoptosis and apparently a non-apoptotic programmed cell death (paraptosis) in Hepa1c1c7 cells

    International Nuclear Information System (INIS)

    Asare, Nana; Landvik, Nina E.; Lagadic-Gossmann, Dominique; Rissel, Mary; Tekpli, Xavier; Ask, Kjetil; Lag, Marit; Holme, Jorn A.

    2008-01-01

    Mechanistic studies of nitro-PAHs (polycyclic aromatic hydrocarbons) of interest might help elucidate which chemical characteristics are most important in eliciting toxic effects. 1-Nitropyrene (1-NP) is the predominant nitrated PAH emitted in diesel exhaust. 1-NP-exposed Hepa1c1c7 cells exhibited marked changes in cellular morphology, decreased proliferation and different forms of cell death. A dramatic increase in cytoplasmic vacuolization was observed already after 6 h of exposure and the cells started to round up at 12 h. The rate of cell proliferation was markedly reduced at 24 h and apoptotic as well as propidium iodide (PI)-positive cells appeared. Electron microscopic examination revealed that the vacuolization was partly due to mitochondria swelling. The caspase inhibitor Z-VAD-FMK inhibited only the apoptotic cell death and Nec-1 (an inhibitor of necroptosis) exhibited no inhibitory effects on either cell death or vacuolization. In contrast, cycloheximide markedly reduced both the number of apoptotic and PI-positive cells as well as the cytoplasmic vacuolization, suggesting that 1-NP induced paraptotic cell death. All the MAPKs; ERK1/2, p38 and JNK, appear to be involved in the death process since marked activation was observed upon 1-NP exposure, and their inhibitors partly reduced the induced cell death. The ERK1/2 inhibitor PD 98057 completely blocked the induced vacuolization, whereas the other MAPKs inhibitors only had minor effects on this process. These findings suggest that 1-NP may cause apoptosis and paraptosis. In contrast, the corresponding amine (1-aminopyrene) elicited only minor apoptotic and necrotic cell death, and cells with characteristics typical of paraptosis were absent

  15. GTSE1 expression represses apoptotic signaling and confers cisplatin resistance in gastric cancer cells

    International Nuclear Information System (INIS)

    Subhash, Vinod Vijay; Tan, Shi Hui; Tan, Woei Loon; Yeo, Mei Shi; Xie, Chen; Wong, Foong Ying; Kiat, Zee Ying; Lim, Robert; Yong, Wei Peng

    2015-01-01

    Platinum based therapy is commonly used in the treatment of advanced gastric cancer. However, resistance to chemotherapy is a major challenge that causes marked variation in individual response rate and survival rate. In this study, we aimed to identify the expression of GTSE1 and its correlation with cisplatin resistance in gastric cancer cells. Methylation profiling was carried out in tissue samples from gastric cancer patients before undergoing neoadjuvent therapy using docetaxel, cisplatin and 5FU (DCX) and in gastric cancer cell lines. The correlation between GTSE1 expression and methylation in gastric cancer cells was determined by RT-PCR and MSP respectively. GTSE1 expression was knocked-down using shRNA’s and its effects on cisplatin cytotoxicity and cell survival were detected by MTS, proliferation and clonogenic survival assays. Additionally, the effect of GTSE1 knock down in drug induced apoptosis was determined by western blotting and apoptosis assays. GTSE1 exhibited a differential methylation index in gastric cancer patients and in cell lines that correlated with DCX treatment response and cisplatin sensitivity, respectively. In-vitro, GTSE1 expression showed a direct correlation with hypomethylation. Interestingly, Cisplatin treatment induced a dose dependent up regulation as well as nuclear translocation of GTSE1 expression in gastric cancer cells. Knock down of GTSE1 enhanced cisplatin cytotoxity and led to a significant reduction in cell proliferation and clonogenic survival. Also, loss of GTSE1 expression caused a significant increase in P53 mediated apoptosis in cisplatin treated cells. Our study identifies GTSE1 as a biomarker for cisplatin resistance in gastric cancer cells. This study also suggests the repressive role of GTSE1 in cisplatin induced apoptosis and signifies its potential utility as a therapeutic target for better clinical management of gastric cancer patients. The online version of this article (doi:10.1186/s12885

  16. Loss of functional E-cadherin renders cells more resistant to the apoptotic agent taxol in vitro

    International Nuclear Information System (INIS)

    Ferreira, Paulo; Oliveira, Maria Jose; Beraldi, Eliana; Mateus, Ana Rita; Nakajima, Takashi; Gleave, Martin; Yokota, Jun; Carneiro, Fatima; Huntsman, David; Seruca, Raquel; Suriano, Gianpaolo

    2005-01-01

    Experimental evidence supports a role for E-cadherin in suppressing invasion, metastasis, and proliferation. Germline mutations of the E-cadherin represent the genetic cause of hereditary diffuse gastric cancer (HDGC). In this type of tumor, isolated cancer cells permeate the basal membrane and paradoxically survive in the gastric wall in the absence of contact with neighbor epithelial cells or with the extracellular matrix. This suggests that upon E-cadherin deregulation, cells acquired resistance to apoptosis. To test this hypothesis, CHO cells stably expressing either wild-type E-cadherin or the HDGC-related germline mutations T340A and V832M were seeded either on a thin layer of collagen type I or on plastic and then subjected to the apoptotic agent taxol. We found that in vitro functional E-cadherin renders cells more sensitive to the effect of taxol. Our results also indicate that this effect is associated to decreased level of the anti-apoptotic bcl-2 protein

  17. Fibered confocal fluorescence microscopy for imaging apoptotic DNA fragmentation at the single-cell level in vivo

    International Nuclear Information System (INIS)

    Al-Gubory, Kais H.

    2005-01-01

    The major characteristic of cell death by apoptosis is the loss of nuclear DNA integrity by endonucleases, resulting in the formation of small DNA fragments. The application of confocal imaging to in vivo monitoring of dynamic cellular events, like apoptosis, within internal organs and tissues has been limited by the accessibility to these sites. Therefore, the aim of the present study was to test the feasibility of fibered confocal fluorescence microscopy (FCFM) to image in situ apoptotic DNA fragmentation in surgically exteriorized sheep corpus luteum in the living animal. Following intra-luteal administration of a fluorescent DNA-staining dye, YO-PRO-1, DNA cleavage within nuclei of apoptotic cells was serially imaged at the single-cell level by FCFM. This imaging technology is sufficiently simple and rapid to allow time series in situ detection and visualization of cells undergoing apoptosis in the intact animal. Combined with endoscope, this approach can be used for minimally invasive detection of fluorescent signals and visualization of cellular events within internal organs and tissues and thereby provides the opportunity to study biological processes in the natural physiological environment of the cell in living animals

  18. Regulation of beta cell replication

    DEFF Research Database (Denmark)

    Lee, Ying C; Nielsen, Jens Høiriis

    2008-01-01

    Beta cell mass, at any given time, is governed by cell differentiation, neogenesis, increased or decreased cell size (cell hypertrophy or atrophy), cell death (apoptosis), and beta cell proliferation. Nutrients, hormones and growth factors coupled with their signalling intermediates have been...... suggested to play a role in beta cell mass regulation. In addition, genetic mouse model studies have indicated that cyclins and cyclin-dependent kinases that determine cell cycle progression are involved in beta cell replication, and more recently, menin in association with cyclin-dependent kinase...... inhibitors has been demonstrated to be important in beta cell growth. In this review, we consider and highlight some aspects of cell cycle regulation in relation to beta cell replication. The role of cell cycle regulation in beta cell replication is mostly from studies in rodent models, but whether...

  19. ROS-mediated apoptotic cell death in prostate cancer LNCaP cells induced by biosurfactant stabilized CdS quantum dots.

    Science.gov (United States)

    Singh, Braj R; Singh, Brahma N; Khan, W; Singh, H B; Naqvi, A H

    2012-08-01

    Cadmium sulfide (CdS) quantum dots (QDs) have raised great attention because of their superior optical properties and wide utilization in biological and biomedical studies. However, little is known about the cell death mechanisms of CdS QDs in human cancer cells. This study was designed to investigate the possible mechanisms of apoptosis induced by biosurfactant stabilized CdS QDs (denoted as "bsCdS QDs") in human prostate cancer LNCaP cells. It was also noteworthy that apoptosis correlated with reactive oxygen species (ROS) production, mitochondrial damage, oxidative stress and chromatin condensation in a dose- and time-dependent manner. Results also showed involvement of caspases, Bcl-2 family proteins, heat shock protein 70, and a cell-cycle checkpoint protein p53 in apoptosis induction by bsCdS QDs in LNCaP cells. Moreover, pro-apoptotic protein Bax was upregulated and the anti-apoptotic proteins, survivin and NF-κB were downregulated in bsCdS QDs exposed cells. Protection of N-acetyl cysteine (NAC) against ROS clearly suggested the implication of ROS in hyper-activation of apoptosis and cell death. It is encouraging to conclude that biologically stabilized CdS QDs bear the potential of its applications in biomedicine, such as tumor therapy specifically by inducing caspase-dependent apoptotic cell death of human prostate cancer LNCaP cells. Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.

  20. Antioxidant and apoptotic effects of an aqueous extract of Urtica dioica on the MCF-7 human breast cancer cell line.

    Science.gov (United States)

    Fattahi, Sadegh; Ardekani, Ali Motevalizadeh; Zabihi, Ebrahim; Abedian, Zeinab; Mostafazadeh, Amrollah; Pourbagher, Roghayeh; Akhavan-Niaki, Haleh

    2013-01-01

    Breast cancer is the most prevalent cancer and one of the leading causes of death among women in the world. Plants and herbs may play an important role in complementary or alternative treatment. The aim of this study was to evaluate the antioxidant and anti-proliferative potential of Urtica dioica. The anti oxidant activity of an aqueous extract of Urtica dioica leaf was measured by MTT assay and the FRAP method while its anti-proliferative activity on the human breast cancer cell line (MCF-7) and fibroblasts isolated from foreskin tissue was evaluated using MTT assay. Mechanisms leading to apoptosis were also investigated at the molecular level by measuring the amount of anti and pro-apoptotic proteins and at the cellular level by studying DNA fragmentation and annexin V staining by flow cytometry. The aqueous extract of Urtica dioica showed antioxidant effects with a correlation coefficient of r(2)=0.997. Dose-dependent and anti-proliferative effects of the extract were observed only on MCF-7 cells after 72 hrs with an IC50 value of 2 mg/ml. This anti proliferative activity was associated with an increase of apoptosis as demonstrated by DNA fragmentation, the appearance of apoptotic cells in flow cytometry analysis and an increase of the amount of calpain 1, calpastatin, caspase 3, caspase 9, Bax and Bcl-2, all proteins involved in the apoptotic pathway. This is the first time such in vitro antiproliferative effect of aqueous extract of Urtica dioica leaf has been described for a breast cancer cell line. Our findings warrant further research on Urtica dioica as a potential chemotherapeutic agent for breast cancer.

  1. Effects of Cigarette Smoke Condensate on Oxidative Stress, Apoptotic Cell Death, and HIV Replication in Human Monocytic Cells.

    Directory of Open Access Journals (Sweden)

    Pss Rao

    Full Text Available While cigarette smoking is prevalent amongst HIV-infected patients, the effects of cigarette smoke constituents in cells of myeloid lineage are poorly known. Recently, we have shown that nicotine induces oxidative stress through cytochrome P450 (CYP 2A6-mediated pathway in U937 monocytic cells. The present study was designed to examine the effect of cigarette smoke condensate (CSC, which contains majority of tobacco constituents, on oxidative stress, cytotoxicity, expression of CYP1A1, and/or HIV-1 replication in HIV-infected (U1 and uninfected U937 cells. The effects of CSC on induction of CYP1 enzymes in HIV-infected primary macrophages were also analyzed. The results showed that the CSC-mediated increase in production of reactive oxygen species (ROS in U937 cells is dose- and time-dependent. Moreover, CSC treatment was found to induce cytotoxicity in U937 cells through the apoptotic pathway via activation of caspase-3. Importantly, pretreatment with vitamin C blocked the CSC-mediated production of ROS and induction of caspase-3 activity. In U1 cells, acute treatment of CSC increased ROS production at 6H (>2-fold and both ROS (>2 fold and HIV-1 replication (>3-fold after chronic treatment. The CSC mediated effects were associated with robust induction in the expression of CYP1A1 mRNA upon acute CSC treatment of U937 and U1 cells (>20-fold, and upon chronic CSC treatment to U1 cells (>30-fold. In addition, the CYP1A1 induction in U937 cells was mediated through the aromatic hydrocarbon receptor pathway. Lastly, CSC, which is known to increase viral replication in primary macrophages, was also found to induce CYP1 enzymes in HIV-infected primary macrophages. While mRNA levels of both CYP1A1 and CYP1B1 were elevated following CSC treatment, only CYP1B1 protein levels were increased in HIV-infected primary macrophages. In conclusion, these results suggest a possible association between oxidative stress, CYP1 expression, and viral replication in

  2. A Novel Combination RNAi toward Warburg Effect by Replacement with miR-145 and Silencing of PTBP1 Induces Apoptotic Cell Death in Bladder Cancer Cells

    Directory of Open Access Journals (Sweden)

    Tomoaki Takai

    2017-01-01

    Full Text Available Bladder cancer is one of the most difficult malignancies to control. We explored the use of a novel RNA-interference method for a driver oncogene regulating cancer specific energy metabolism by the combination treatment with a small interfering RNA (siRNA and a microRNA. After transfection of T24 and 253JB-V cells with miR-145 and/or siR-PTBP1, we examined the effects of cell growth and gene expression by performing the trypan blue dye exclusion test, Western blot, Hoechst 33342 staining, reverse transcription polymerase chain reaction (RT-PCR, and electron microscopy. The anti-cancer effects of xenograft model mice with miR-145 and/or siR-PTBP1 were then assessed. The combination treatment induced the deeper and longer growth inhibition and reduced the levels of both mRNA and protein expression of c-Myc and polypyrimidine tract-binding protein 1 (PTBP1 more than each single treatment. Notably, the combination treatment not only impaired the cancer specific energy metabolism by inhibiting c-Myc/PTBP1/PKMs axis but also inactivated MAPK/ERK and PI3K/AKT pathways examined in vitro and in vivo. Furthermore, the combination treatment induced apoptosis or autophagy; but, in some cells, apoptotic cell death was accompanied by autophagy, because the condensation of chromatin and many autophagosomes were coexistent. This combination treatment could be a novel RNA-interference strategy through the systemic silencing of the Warburg effect-promoting driver oncogene PTBP1 in bladder cancer cells.

  3. Apoptotic-like tumor cells and apoptotic neutrophils in mitochondrion-rich gastric adenocarcinomas: a comparative study with light and electronmicroscopy between these two forms of cell death

    Directory of Open Access Journals (Sweden)

    Antonio Venuti

    2013-04-01

    Full Text Available Mitochondrion-rich adenocarcinomas represent a rare variant of gastric adenocarcinomas composed predominantly of columnar adenocarcinoma cells with eosinophilic cytoplasm, a strong supranuclear immunoreactivity for antimitochondrial antibody, and a marked neutrophil infiltration associated to tumor cell death. The purpose of this work is to investigate, using correlated light and electron microscopy, mitochondrion-rich gastric adenocarcinomas focusing on the nature of the death in neoplastic cells and in infiltrating neutrophils. Adenocarcinoma cells, single or in small clusters, showed convoluted nuclei, irregularly condensed chromatin, loss of microvilli, and nuclear envelope dilatation. No nuclear fragmentation was observed in these dying cells and the plasma membrane did not show signs of disruption. These ultrastructural findings represent intermediate aspects between apoptosis and necrosis and are compatible with apoptosis-like programmed cell death. By contrast, some infiltrating neutrophils showed ultrastructural signs of classic apoptosis such as chromatin condensation into compact geometric (globular, crescent-shaped figures, tightly packed cytoplasmic granules and intact cell membrane. Our study provides ultrastructural evidence of apoptosis-like tumour cell death in mitochondrion-rich gastric carcinomas and confirms that stereotyped outcome either as apoptosis or necrosis of tumor cells cannot always be expected in human neoplasms.

  4. siRNA - Mediated LRP/LR knock-down reduces cellular viability of malignant melanoma cells through the activation of apoptotic caspases.

    Science.gov (United States)

    Rebelo, Thalia M; Vania, Leila; Ferreira, Eloise; Weiss, Stefan F T

    2018-07-01

    The 37 kDa/67 kDa laminin receptor (LRP/LR) is over-expressed in tumor cells and has been implicated in several tumourigenic processes such as metastasis and telomerase activation, however, more importantly the focus of the present study is on the maintenance of cellular viability and the evasion of apoptosis. The aim of the study was to investigate the role of LRP/LR on the cellular viability of early (A375) and late stage (A375SM) malignant melanoma cells. Flow cytometry and western blot analysis revealed that A375SM cells contain more cell-surface and total LRP/LR levels in comparison to the A375 cells, respectively. In order to determine the effect of LRP/LR on cell viability and apoptosis, LRP was down-regulated via siRNA technology. MTT assays revealed that LRP knock-down led to significant reductions in the viability of A375 and A375SM cells. Confocal microscopy indicated nuclear morphological changes suggestive of apoptotic induction in both cell lines and Annexin-V FITC/PI assays confirmed this observation. Additionally, caspase-3 activity assays revealed that apoptosis was induced in both cell lines after siRNA-mediated down-regulation of LRP. Caspase-8 and -9 activity assays suggested that post LRP knock-down; A375 cells undergo apoptosis solely via the extrinsic pathway, while A375SM cells undergo apoptosis via the intrinsic pathway. siRNAs mediated LRP knock-down might represent a powerful alternative therapeutic strategy for the treatment of malignant melanoma through the induction of apoptosis. Copyright © 2018. Published by Elsevier Inc.

  5. Involvement of DNA-PK and ATM in radiation- and heat-induced DNA damage recognition and apoptotic cell death

    International Nuclear Information System (INIS)

    Tomita, Masanori

    2010-01-01

    Exposure to ionizing radiation and hyperthermia results in important biological consequences, e.g. cell death, chromosomal aberrations, mutations, and DNA strand breaks. There is good evidence that the nucleus, specifically cellular DNA, is the principal target for radiation-induced cell lethality. DNA double-strand breaks (DSBs) are considered to be the most serious type of DNA damage induced by ionizing radiation. On the other hand, verifiable mechanisms which can lead to heat-induced cell death are damage to the plasma membrane and/or inactivation of heat-labile proteins caused by protein denaturation and subsequent aggregation. Recently, several reports have suggested that DSBs can be induced after hyperthermia because heat-induced phosphorylated histone H2AX (γ-H2AX) foci formation can be observed in several mammalian cell lines. In mammalian cells, DSBs are repaired primarily through two distinct and complementary mechanisms: non-homologous end joining (NHEJ), and homologous recombination (HR) or homology-directed repair (HDR). DNA-dependent protein kinase (DNA-PK) and ataxia-telangiectasia mutated (ATM) are key players in the initiation of DSB repair and phosphorylate and/or activate many substrates, including themselves. These phosphorylated substrates have important roles in the functioning of cell cycle checkpoints and in cell death, as well as in DSB repair. Apoptotic cell death is a crucial cell suicide mechanism during development and in the defense of homeostasis. If DSBs are unrepaired or misrepaired, apoptosis is a very important system which can protect an organism against carcinogenesis. This paper reviews recently obtained results and current topics concerning the role of DNA-PK and ATM in heat- or radiation-induced apoptotic cell death. (author)

  6. Cannabidiol restores intestinal barrier dysfunction and inhibits the apoptotic process induced by Clostridium difficile toxin A in Caco-2 cells.

    Science.gov (United States)

    Gigli, Stefano; Seguella, Luisa; Pesce, Marcella; Bruzzese, Eugenia; D'Alessandro, Alessandra; Cuomo, Rosario; Steardo, Luca; Sarnelli, Giovanni; Esposito, Giuseppe

    2017-12-01

    Clostridium difficile toxin A is responsible for colonic damage observed in infected patients. Drugs able to restore Clostridium difficile toxin A-induced toxicity have the potential to improve the recovery of infected patients. Cannabidiol is a non-psychotropic component of Cannabis sativa, which has been demonstrated to protect enterocytes against chemical and/or inflammatory damage and to restore intestinal mucosa integrity. The purpose of this study was to evaluate (a) the anti-apoptotic effect and (b) the mechanisms by which cannabidiol protects mucosal integrity in Caco-2 cells exposed to Clostridium difficile toxin A. Caco-2 cells were exposed to Clostridium difficile toxin A (30 ng/ml), with or without cannabidiol (10 -7 -10 -9  M), in the presence of the specific antagonist AM251 (10 -7  M). Cytotoxicity assay, transepithelial electrical resistence measurements, immunofluorescence analysis and immunoblot analysis were performed in the different experimental conditions. Clostridium difficile toxin A significantly decreased Caco-2 cells' viability and reduced transepithelial electrical resistence values and RhoA guanosine triphosphate (GTP), bax, zonula occludens-1 and occludin protein expression, respectively. All these effects were significantly and concentration-dependently inhibited by cannabidiol, whose effects were completely abolished in the presence of the cannabinoid receptor type 1 (CB1) antagonist, AM251. Cannabidiol improved Clostridium difficile toxin A-induced damage in Caco-2 cells, by inhibiting the apoptotic process and restoring the intestinal barrier integrity, through the involvement of the CB1 receptor.

  7. Sub-apoptotic dosages of pro-oxidant vitamin cocktails sensitize human melanoma cells to NK cell lysis.

    Science.gov (United States)

    Tremante, Elisa; Santarelli, Lory; Lo Monaco, Elisa; Sampaoli, Camilla; Ingegnere, Tiziano; Guerrieri, Roberto; Tomasetti, Marco; Giacomini, Patrizio

    2015-10-13

    Alpha-tocopheryl succinate (αTOS), vitamin K3 (VK3) and vitamin C (ascorbic acid, AA) were previously shown to synergistically promote different death pathways in carcinoma cells, depending on their concentrations and combinations. Similar effects were observed herein in melanoma cells, although αTOS behaved as an antagonist. Interestingly, suboptimal cell death-inducing concentrations (1.5 μM αTOS/20 μM AA/0.2 μM VK3) effectively up-regulated activating Natural Killer (NK) cell ligands, including MICA (the stress-signaling ligand of the NKG2D receptor), and/or the ligands of at least one of the natural cytotoxicity receptors (NKp30, NKp44 and NKp46) in 5/6 melanoma cell lines. Only an isolated MICA down-regulation was seen. HLA class I, HLA class II, ULBP1, ULBP2, ULBP3, Nectin-2, and PVR displayed little, if any, change in expression. Ligand up-regulation resulted in improved lysis by polyclonal NK cells armed with the corresponding activating receptors. These results provide the first evidence for concerted induction of cell death by cell-autonomous and extrinsic (immune) mechanisms. Alarming the immune system much below the cell damage threshold may have evolved as a sensitive readout of neoplastic transformation and oxidative stress. Cocktails of vitamin analogues at slightly supra-physiological dosages may find application as mild complements of melanoma treatment, and in chemoprevention.

  8. Novel Phosphorylation and Ubiquitination Sites Regulate Reactive Oxygen Species-dependent Degradation of Anti-apoptotic c-FLIP Protein*

    Science.gov (United States)

    Wilkie-Grantham, Rachel P.; Matsuzawa, Shu-Ichi; Reed, John C.

    2013-01-01

    The cytosolic protein c-FLIP (cellular Fas-associated death domain-like interleukin 1β-converting enzyme inhibitory protein) is an inhibitor of death receptor-mediated apoptosis that is up-regulated in a variety of cancers, contributing to apoptosis resistance. Several compounds found to restore sensitivity of cancer cells to TRAIL, a TNF family death ligand with promising therapeutic potential, act by targeting c-FLIP ubiquitination and degradation by the proteasome. The generation of reactive oxygen species (ROS) has been implicated in c-FLIP protein degradation. However, the mechanism by which ROS post-transcriptionally regulate c-FLIP protein levels is not well understood. We show here that treatment of prostate cancer PPC-1 cells with the superoxide generators menadione, paraquat, or buthionine sulfoximine down-regulates c-FLIP long (c-FLIPL) protein levels, which is prevented by the proteasome inhibitor MG132. Furthermore, pretreatment of PPC-1 cells with a ROS scavenger prevented ubiquitination and loss of c-FLIPL protein induced by menadione or paraquat. We identified lysine 167 as a novel ubiquitination site of c-FLIPL important for ROS-dependent degradation. We also identified threonine 166 as a novel phosphorylation site and demonstrate that Thr-166 phosphorylation is required for ROS-induced Lys-167 ubiquitination. The mutation of either Thr-166 or Lys-167 was sufficient to stabilize c-FLIP protein levels in PPC-1, HEK293T, and HeLa cancer cells treated with menadione or paraquat. Accordingly, expression of c-FLIP T166A or K167R mutants protected cells from ROS-mediated sensitization to TRAIL-induced cell death. Our findings reveal novel ROS-dependent post-translational modifications of the c-FLIP protein that regulate its stability, thus impacting sensitivity of cancer cells to TRAIL. PMID:23519470

  9. Cyclopamine and jervine induce COX-2 overexpression in human erythroleukemia cells but only cyclopamine has a pro-apoptotic effect

    International Nuclear Information System (INIS)

    Ghezali, Lamia; Leger, David Yannick; Limami, Youness; Cook-Moreau, Jeanne; Beneytout, Jean-Louis; Liagre, Bertrand

    2013-01-01

    Erythroleukemia is generally associated with a very poor response and survival to current available therapeutic agents. Cyclooxygenase-2 (COX-2) has been described to play a crucial role in the proliferation and differentiation of leukemia cells, this enzyme seems to play an important role in chemoresistance in different cancer types. Previously, we demonstrated that diosgenin, a plant steroid, induced apoptosis in HEL cells with concomitant COX-2 overexpression. In this study, we investigated the antiproliferative and apoptotic effects of cyclopamine and jervine, two steroidal alkaloids with similar structures, on HEL and TF1a human erythroleukemia cell lines and, for the first time, their effect on COX-2 expression. Cyclopamine, but not jervine, inhibited cell proliferation and induced apoptosis in these cells. Both compounds induced COX-2 overexpression which was responsible for apoptosis resistance. In jervine-treated cells, COX-2 overexpression was NF-κB dependent. Inhibition of NF-κB reduced COX-2 overexpression and induced apoptosis. In addition, cyclopamine induced apoptosis and COX-2 overexpression via PKC activation. Inhibition of the PKC pathway reduced both apoptosis and COX-2 overexpression in both cell lines. Furthermore, we demonstrated that the p38/COX-2 pathway was involved in resistance to cyclopamine-induced apoptosis since p38 inhibition reduced COX-2 overexpression and increased apoptosis in both cell lines. - Highlights: ► Cyclopamine alone but not jervine induces apoptosis in human erythroleukemia cells. ► Cyclopamine and jervine induce COX-2 overexpression. ► COX-2 overexpression is implicated in resistance to cyclopamine-induced apoptosis. ► Apoptotic potential of jervine is restrained by NF-κB pathway activation. ► PKC is involved in cyclopamine-induced apoptosis and COX-2 overexpression

  10. Cyclopamine and jervine induce COX-2 overexpression in human erythroleukemia cells but only cyclopamine has a pro-apoptotic effect

    Energy Technology Data Exchange (ETDEWEB)

    Ghezali, Lamia; Leger, David Yannick; Limami, Youness [Université de Limoges, FR 3503 GEIST, EA 1069 “Laboratoire de Chimie des Substances Naturelles”, GDR CNRS 3049, Faculté de Pharmacie, Laboratoire de Biochimie et Biologie Moléculaire, 2 rue du Docteur Marcland, 87025 Limoges Cedex (France); Cook-Moreau, Jeanne [Université de Limoges, FR 3503 GEIST, UMR CNRS 7276 “Contrôle de la réponse immune B et lymphoproliférations”, Faculté de Médecine, 2 rue du Docteur Marcland, 87025 Limoges Cedex (France); Beneytout, Jean-Louis [Université de Limoges, FR 3503 GEIST, EA 1069 “Laboratoire de Chimie des Substances Naturelles”, GDR CNRS 3049, Faculté de Pharmacie, Laboratoire de Biochimie et Biologie Moléculaire, 2 rue du Docteur Marcland, 87025 Limoges Cedex (France); Liagre, Bertrand, E-mail: bertrand.liagre@unilim.fr [Université de Limoges, FR 3503 GEIST, EA 1069 “Laboratoire de Chimie des Substances Naturelles”, GDR CNRS 3049, Faculté de Pharmacie, Laboratoire de Biochimie et Biologie Moléculaire, 2 rue du Docteur Marcland, 87025 Limoges Cedex (France)

    2013-04-15

    Erythroleukemia is generally associated with a very poor response and survival to current available therapeutic agents. Cyclooxygenase-2 (COX-2) has been described to play a crucial role in the proliferation and differentiation of leukemia cells, this enzyme seems to play an important role in chemoresistance in different cancer types. Previously, we demonstrated that diosgenin, a plant steroid, induced apoptosis in HEL cells with concomitant COX-2 overexpression. In this study, we investigated the antiproliferative and apoptotic effects of cyclopamine and jervine, two steroidal alkaloids with similar structures, on HEL and TF1a human erythroleukemia cell lines and, for the first time, their effect on COX-2 expression. Cyclopamine, but not jervine, inhibited cell proliferation and induced apoptosis in these cells. Both compounds induced COX-2 overexpression which was responsible for apoptosis resistance. In jervine-treated cells, COX-2 overexpression was NF-κB dependent. Inhibition of NF-κB reduced COX-2 overexpression and induced apoptosis. In addition, cyclopamine induced apoptosis and COX-2 overexpression via PKC activation. Inhibition of the PKC pathway reduced both apoptosis and COX-2 overexpression in both cell lines. Furthermore, we demonstrated that the p38/COX-2 pathway was involved in resistance to cyclopamine-induced apoptosis since p38 inhibition reduced COX-2 overexpression and increased apoptosis in both cell lines. - Highlights: ► Cyclopamine alone but not jervine induces apoptosis in human erythroleukemia cells. ► Cyclopamine and jervine induce COX-2 overexpression. ► COX-2 overexpression is implicated in resistance to cyclopamine-induced apoptosis. ► Apoptotic potential of jervine is restrained by NF-κB pathway activation. ► PKC is involved in cyclopamine-induced apoptosis and COX-2 overexpression.

  11. Evaluation of chemical composition, antioxidant, antibacterial, cytotoxic and apoptotic effects of Aloysia citrodora extract on colon cancer cell line

    Directory of Open Access Journals (Sweden)

    Amir Mirzaie

    2016-06-01

    Full Text Available Background: Aloysia citrodora belongs to the Verbenaceae family of plants, a well-known herbal medicine in Iran. The aim of the present study was to investigate the chemical composition, antioxidant, antibacterial, cytotoxic and apoptotic effect of A. citrodora extract against human colon cancer (HT29 cells by using real-time polymerase chain reaction and flow-cytometry methods. Methods: This experimental study was carried out in Islamic Azad University, East Tehran Branch, from March to September of 2014. At first, the A. citrodora chemical constituents were analyzed by gas chromatography-mass spectrometry (GC-MS technique. In addition, antioxidant assay, antibacterial and anti-cancer effect was performed using 1,1-diphenyl-2-picrylhydrazyl (DPPH, disk diffusion and 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT methods, respectively. The half maximal inhibitory concentration (IC50 value was calculated. We extracted total RNA molecules by using RNX solution, after which cDNA was synthesized. Finally, the pro-apoptotic (Bax and anti-apoptotic (Bcl2 gene expression was performed by real-time polymerase chain reaction and apoptotic effects were analyzed using Flow-cytometry method. Results: GC-MS analysis of Aloysia citrodora extract was shown 37 major components and the most frequent component was belonged to Spathulenol (17.57% and Caryophyllene oxide (15.15% The antioxidant activity of the extract was IC50= 0.6±0.03 mg/ml. The maximum and minimum antibacterial effects of extract were belonged to gram-negative and gram-positive bacteria, respectively. Cytotoxic results revealed that the A.citrodora extract have IC50= 20.1±0.78 mg/ml against colon cancer (HT29 cell line and real-time polymerase chain reaction results showed the expression level of Bax and Bcl2 was increased and decreased respectively in colon cancer cell line (3.470±0.72 (P< 0.05, 0.43±0.35 (P< 0.05. In addition, the flow-cytometry results indicated the 38

  12. A histochemical comparison of methyl green-pyronin, and hematoxylin and eosin for detecting apoptotic cells in oral squamous cell carcinoma, oral leukoplakia, oral submucous fibrosis and normal oral mucosa.

    Science.gov (United States)

    Sumedha, S; Kotrashetti, V S; Somannavar, P; Nayak, R; Babji, D

    2015-05-01

    Analysis of apoptotic cells in oral pathological states could be useful for determining the rates of tissue turnover, which would help determine prognosis. The use of histochemical stains such as hematoxylin and eosin (H & E) and methyl green-pyronin (MGP) can provide a simple and cost-effective method for detecting apoptotic cells. We compared the efficacy of MGP and H & E for detecting apoptotic cells in oral squamous cell carcinoma (OSCC), oral leukoplakia (OL), oral submucous fibrosis (OSMF) and normal oral mucosa (NOM). Ten cases each of OSCC, OSMF, OL and NOM were retrieved from the archives and two serial sections were stained, one with H & E and the other with MGP. Apoptotic cells were identified at 100 x magnification and the apoptotic index was calculated. Apoptotic cells were distinguished more readily in MGP stained sections than in those stained with H & E. Also, the apoptotic cell count was greater in OSCC compared to OL, OSMF and NOM. We concluded that MGP staining can be used as a routine, cost-effective method for detecting apoptotic cells.

  13. Repeated Exposure of Epithelial Cells to Apoptotic Cells Induces the Specific Selection of an Adaptive Phenotype: Implications for Tumorigenesis.

    Science.gov (United States)

    Feng, Lanfei; Vujicic, Snezana; Dietrich, Michael E; Litbarg, Natalia; Setty, Suman; Antoni, Angelika; Rauch, Joyce; Levine, Jerrold S

    2018-05-16

    The consequences of apoptosis extend beyond mere death of the cell. We have shown that receptor-mediated recognition of apoptotic target cells by viable kidney proximal tubular epithelial cells (PTECs) inhibits PTEC proliferation, growth, and survival. Here we tested the hypothesis that continual exposure to apoptotic targets can induce a phenotypic change in responding PTECs, as in other instances of natural selection. In particular, we demonstrate that repeated exposure to apoptotic targets leads to emergence of a PTEC line (denoted BU.MPT SEL ) resistant to apoptotic target-induced death. Resistance is exquisitely specific. Not only are BU.MPT SEL responders fully resistant to apoptotic target-induced death (~85% survival versus exposure in selected versus non-selected responders indicated that the acquired resistance of BU.MPT SEL cells lies in a regulatory step affecting the generation of the pro-apoptotic protein, truncated BH3 interacting-domain death agonist (tBID), most likely at the level of BID cleavage by caspase-8. This specific adaptation has especial relevance for cancer, in which the prominence and persistence of cell death entail magnification of the post-mortem effects of apoptotic cells. Just as cancer cells acquire specific resistance to chemotherapeutic agents, we propose that cancer cells may also adapt to their ongoing exposure to apoptotic targets. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Atractylenolide-I Protects Human SH-SY5Y Cells from 1-Methyl-4-Phenylpyridinium-Induced Apoptotic Cell Death

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    Sandeep Vasant More

    2017-05-01

    Full Text Available Oxidative stress and apoptosis are the major mechanisms that induce dopaminergic cell death. Our study investigates the protective effects of atractylenolide-I (ATR-I on 1-methyl-4-phenylpyridinium (MPP+-induced cytotoxicity in human dopaminergic SH-SY5Y cells, as well as its underlying mechanism. Our experimental data indicates that ATR-I significantly inhibits the loss of cell viability induced by MPP+ in SH-SY5Y cells. To further unravel the mechanism, we examined the effect of ATR-I on MPP+-induced apoptotic cell death characterized by an increase in the Bax/Bcl-2 mRNA ratio, the release of cytochrome-c, and the activation of caspase-3 leading to elevated levels of cleaved poly(ADP-ribose polymerase (PARP resulting in SH-SY5Y cell death. Our results demonstrated that ATR-I decreases the level of pro-apoptotic proteins induced by MPP+ and also restored Bax/Bcl-2 mRNA levels, which are critical for inducing apoptosis. In addition, ATR-I demonstrated a significant increase in the protein expression of heme-oxygenase in MPP+-treated SH-SY5Y cells. These results suggest that the pharmacological effect of ATR-I may be, at least in part, caused by the reduction in pro-apoptotic signals and also by induction of anti-oxidant protein.

  15. Cytotoxic and Apoptotic Effect of Structurally Similar Flavonoids on Parental and Drug-Resistant Cells of a Human Cervical Carcinoma

    Directory of Open Access Journals (Sweden)

    Ksenija Durgo

    2009-01-01

    Full Text Available Flavonoids are phytochemicals characterized by a wide range of biological activities, including antioxidant activity, the ability to modulate enzyme or cell receptor activity patterns, and to interfere with essential biochemical pathways. Using HeLa cells of a human cervical carcinoma, and their drug-resistant HeLa CK subline, the effects of three structurally related flavonoids (quercetin, fisetin and luteolin have been examined, in terms of their: (i cytotoxicity, (ii influence on intracellular glutathione (GSH level, (iii influence on glutathione S-transferase (GST activity, and (iv influence on the expression of apoptosis-related genes (PARP, Bcl-2, survivin. Fisetin was more toxic to resistant HeLa CK cell line than to parental cell line, causing decreased expression of survivin in the same cell line. Concentrations of 5 μM of the examined flavonoids caused PARP degradation in parental cell line, leading HeLa cell line into apoptotic cell death. The same event was not determined in the resistant cell line. Fisetin and luteolin induce glutathione and GST in the resistant cell line, pointing to complex cellular effects which could be responsible for higher sensitivity of the resistant cell line in comparison with the parental cell line. Prooxidative nature of the investigated flavonoids was not detected, so free radical formation is not responsible for the induction of GSH, GST and proapoptotic enzymes.

  16. Macrophage-expressed IFN-β contributes to apoptotic alveolar epithelial cell injury in severe influenza virus pneumonia.

    Directory of Open Access Journals (Sweden)

    Katrin Högner

    2013-02-01

    Full Text Available Influenza viruses (IV cause pneumonia in humans with progression to lung failure and fatal outcome. Dysregulated release of cytokines including type I interferons (IFNs has been attributed a crucial role in immune-mediated pulmonary injury during severe IV infection. Using ex vivo and in vivo IV infection models, we demonstrate that alveolar macrophage (AM-expressed IFN-β significantly contributes to IV-induced alveolar epithelial cell (AEC injury by autocrine induction of the pro-apoptotic factor TNF-related apoptosis-inducing ligand (TRAIL. Of note, TRAIL was highly upregulated in and released from AM of patients with pandemic H1N1 IV-induced acute lung injury. Elucidating the cell-specific underlying signalling pathways revealed that IV infection induced IFN-β release in AM in a protein kinase R- (PKR- and NF-κB-dependent way. Bone marrow chimeric mice lacking these signalling mediators in resident and lung-recruited AM and mice subjected to alveolar neutralization of IFN-β and TRAIL displayed reduced alveolar epithelial cell apoptosis and attenuated lung injury during severe IV pneumonia. Together, we demonstrate that macrophage-released type I IFNs, apart from their well-known anti-viral properties, contribute to IV-induced AEC damage and lung injury by autocrine induction of the pro-apoptotic factor TRAIL. Our data suggest that therapeutic targeting of the macrophage IFN-β-TRAIL axis might represent a promising strategy to attenuate IV-induced acute lung injury.

  17. Differential Control of Growth, Apoptotic Activity, and Gene Expression in Human Breast Cancer Cells by Extracts Derived from Medicinal Herbs Zingiber officinale

    Directory of Open Access Journals (Sweden)

    Ayman I. Elkady

    2012-01-01

    Full Text Available The present study aimed to examine the antiproliferative potentiality of an extract derived from the medicinal plant ginger (Zingiber officinale on growth of breast cancer cells. Ginger treatment suppressed the proliferation and colony formation in breast cancer cell lines, MCF-7 and MDA-MB-231. Meanwhile, it did not significantly affect viability of nontumorigenic normal mammary epithelial cell line (MCF-10A. Treatment of MCF-7 and MDA-MB-231 with ginger resulted in sequences of events marked by apoptosis, accompanied by loss of cell viability, chromatin condensation, DNA fragmentation, activation of caspase 3, and cleavage of poly(ADP-ribose polymerase. At the molecular level, the apoptotic cell death mediated by ginger could be attributed in part to upregulation of Bax and downregulation of Bcl-2 proteins. Ginger treatment downregulated expression of prosurvival genes, such as NF-κB, Bcl-X, Mcl-1, and Survivin, and cell cycle-regulating proteins, including cyclin D1 and cyclin-dependent kinase-4 (CDK-4. On the other hand, it increased expression of CDK inhibitor, p21. It also inhibited the expression of the two prominent molecular targets of cancer, c-Myc and the human telomerase reverse transcriptase (hTERT. These findings suggested that the ginger may be a promising candidate for the treatment of breast carcinomas.

  18. Targeting Toll-like receptor 7/8 enhances uptake of apoptotic leukemic cells by monocyte-derived dendritic cells but interferes with subsequent cytokine-induced maturation.

    Science.gov (United States)

    van den Ancker, Willemijn; van Luijn, Marvin M; Ruben, Jurjen M; Westers, Theresia M; Bontkes, Hetty J; Ossenkoppele, Gert J; de Gruijl, Tanja D; van de Loosdrecht, Arjan A

    2011-01-01

    Therapeutic vaccination with dendritic cells (DC) is an emerging investigational therapy for eradication of minimal residual disease in acute myeloid leukemia. Various strategies are being explored in manufacturing DC vaccines ex vivo, e.g., monocyte-derived DC (MoDC) loaded with leukemia-associated antigens (LAA). However, the optimal source of LAA and the choice of DC-activating stimuli are still not well defined. Here, loading with leukemic cell preparations (harboring both unknown and known LAA) was explored in combination with a DC maturation-inducing cytokine cocktail (CC; IL-1β, IL-6, TNF-α, and PGE(2)) and Toll-like receptor ligands (TLR-L) to optimize uptake. Since heat shock induced apoptotic blasts were more efficiently taken up than lysates, we focused on uptake of apoptotic leukemic cells. Uptake of apoptotic blast was further enhanced by the TLR7/8-L R848 (20-30%); in contrast, CC-induced maturation inhibited uptake. CC, and to a lesser extent R848, enhanced the ability of MoDC to migrate and stimulate T cells. Furthermore, class II-associated invariant chain peptide expression was down-modulated after R848- or CC-induced maturation, indicating enhanced processing and presentation of antigenic peptides. To improve both uptake and maturation, leukemic cells and MoDC were co-incubated with R848 for 24 h followed by addition of CC. However, this approach interfered with CC-mediated MoDC maturation as indicated by diminished migratory and T cell stimulatory capacity, and the absence of IL-12 production. Taken together, our data demonstrate that even though R848 improved uptake of apoptotic leukemic cells, the sequential use of R848 and CC is counter-indicated due to its adverse effects on MoDC maturation.

  19. Viral DNA Sensors IFI16 and Cyclic GMP-AMP Synthase Possess Distinct Functions in Regulating Viral Gene Expression, Immune Defenses, and Apoptotic Responses during Herpesvirus Infection

    Directory of Open Access Journals (Sweden)

    Benjamin A. Diner

    2016-11-01

    Full Text Available The human interferon-inducible protein IFI16 is an important antiviral factor that binds nuclear viral DNA and promotes antiviral responses. Here, we define IFI16 dynamics in space and time and its distinct functions from the DNA sensor cyclic dinucleotide GMP-AMP synthase (cGAS. Live-cell imaging reveals a multiphasic IFI16 redistribution, first to viral entry sites at the nuclear periphery and then to nucleoplasmic puncta upon herpes simplex virus 1 (HSV-1 and human cytomegalovirus (HCMV infections. Optogenetics and live-cell microscopy establish the IFI16 pyrin domain as required for nuclear periphery localization and oligomerization. Furthermore, using proteomics, we define the signature protein interactions of the IFI16 pyrin and HIN200 domains and demonstrate the necessity of pyrin for IFI16 interactions with antiviral proteins PML and cGAS. We probe signaling pathways engaged by IFI16, cGAS, and PML using clustered regularly interspaced short palindromic repeat (CRISPR/Cas9-mediated knockouts in primary fibroblasts. While IFI16 induces cytokines, only cGAS activates STING/TBK-1/IRF3 and apoptotic responses upon HSV-1 and HCMV infections. cGAS-dependent apoptosis upon DNA stimulation requires both the enzymatic production of cyclic dinucleotides and STING. We show that IFI16, not cGAS or PML, represses HSV-1 gene expression, reducing virus titers. This indicates that regulation of viral gene expression may function as a greater barrier to viral replication than the induction of antiviral cytokines. Altogether, our findings establish coordinated and distinct antiviral functions for IFI16 and cGAS against herpesviruses.

  20. Corosolic Acid Induces Non-Apoptotic Cell Death through Generation of Lipid Reactive Oxygen Species Production in Human Renal Carcinoma Caki Cells

    Directory of Open Access Journals (Sweden)

    Seon Min Woo

    2018-04-01

    Full Text Available Corosolic acid is one of the pentacyclic triterpenoids isolated from Lagerstroemia speciose and has been reported to exhibit anti-cancer and anti-proliferative activities in various cancer cells. In the present study, we investigated the molecular mechanisms of corosolic acid in cancer cell death. Corosolic acid induces a decrease of cell viability and an increase of cell cytotoxicity in human renal carcinoma Caki cells. Corosolic acid-induced cell death is not inhibited by apoptosis inhibitor (z-VAD-fmk, a pan-caspase inhibitor, necroptosis inhibitor (necrostatin-1, or ferroptosis inhibitors (ferrostatin-1 and deferoxamine (DFO. Furthermore, corosolic acid significantly induces reactive oxygen species (ROS levels, but antioxidants (N-acetyl-l-cysteine (NAC and trolox do not inhibit corosolic acid-induced cell death. Interestingly, corosolic acid induces lipid oxidation, and α-tocopherol markedly prevents corosolic acid-induced lipid peroxidation and cell death. Anti-chemotherapeutic effects of α-tocopherol are dependent on inhibition of lipid oxidation rather than inhibition of ROS production. In addition, corosolic acid induces non-apoptotic cell death in other renal cancer (ACHN and A498, breast cancer (MDA-MB231, and hepatocellular carcinoma (SK-Hep1 and Huh7 cells, and α-tocopherol markedly inhibits corosolic acid-induced cell death. Therefore, our results suggest that corosolic acid induces non-apoptotic cell death in cancer cells through the increase of lipid peroxidation.

  1. Research advances in sorafenib-induced apoptotic signaling pathways in liver cancer cells

    Directory of Open Access Journals (Sweden)

    ZHANG Chaoya

    2016-04-01

    Full Text Available Currently, sorafenib is the multi-target inhibitor for the treatment of advanced primary liver cancer, and can effectively prolong the progression-free survival and overall survival in patients with advanced primary liver cancer. The application of sorafenib in the targeted therapy for liver cancer has become a hot topic. Major targets or signaling pathways include Raf/Mek/Erk, Jak/Stat, PI3K/Akt/mTOR, VEGFR and PDGFR, STAT, microRNA, Wnt/β-catenin, autolysosome, and tumor-related proteins, and sorafenib can regulate the proliferation, differentiation, metastasis, and apoptosis of liver cancer cells through these targets. This article reviews the current research on the action of sorafenib on these targets or signaling pathways to provide useful references for further clinical research on sorafenib.

  2. Study of morphological changes in breast cancer cells MCF-7 under the action of pro-apoptotic agents with laser modulation interference microscope MIM-340

    Science.gov (United States)

    Nebogatikov, V.; Nikitiuk, A.; Konysheva, A.; Ignatyev, P.; Grishko, V.; Naimark, O.

    2017-09-01

    Quantitative phase microscopy is a new method to measure and evaluate the microlevel processes characterized by the high resolution and providing ample opportunities to quantitatively analyze various parameters, including specimens from biological matter. In this study, a laser interference microscope was used to evaluate the state of cancer cells (living and apoptotic). Apoptotic cancer cells were obtained by treatment of MCF-7 cells with the use of betulin-based α-bromomethyl ketone (BMK) derivative. When using the microscope, the main differences in the morphometric parameters of living and apoptotic cells such as height, diameter, perimeter, area and volume were appraised. The criteria that can be used as markers of apoptosis activation were identified.

  3. [Cell-penetrating chimeric apoptotic peptide AVPI-LMWP/DNA co-delivery system for cancer therapy].

    Science.gov (United States)

    Tan, Jiao; Wang, Ya-Ping; Wang, Hui-Xin; Liang, Jian-Ming; Zhang, Meng; Sun, Xun; Huang, Yong-Zhuo

    2014-12-01

    To develop a cell-penetrating chimeric apoptotic peptide AVPI-LMWP/DNA co-delivery system for cancer therapy, we prepared the AVPI-LMWP/pTRAIL self-assembled complexes containing a therapeutic combination of peptide drug AVPI and DNA drug TRAIL. The chimeric apoptotic peptide AVPI-LMWP was synthesized using the standard solid-phase synthesis. The cationic AVPI-LMWP could condense pTRAIL by electrostatic interaction. The physical-chemical properties of the AVPI-LMWP/pTRAIL complexes were characterized. The cellular uptake efficiency and the inhibitory activity of the AVPI-LMWP/pTRAIL complexes on tumor cell were also performed. The results showed that the AVPI-LMWP/pTRAIL complexes were successfully prepared by co-incubation. With the increase of mass ratio (AVPI-LMWP/DNA), the particle size was decreased and the zeta potential had few change. Agarose gel electrophoresis showed that AVPI-LMWP could fully bind and condense pTRAIL at a mass ratio above 15:1. Cellular uptake efficiency was improved along with the increased ratio of W(AVPI-LMWP)/WpTRAIL. The in vitro cytotoxicity experiments demonstrated that the AVPI-LMWP/pTRAIL (W:W = 20:1) complexes was significantly more effective than the pTRAIL, AVPI-LMWP alone or LMWP/pTRAIL complexes on inhibition of HeLa cell growth. Our studies indicated that the AVPI-LMWP/pTRAIL co-delivery system could deliver plasmid into HeLa cell and induce tumor cell apoptosis efficiently, which showed its potential in cancer therapy using combination of apoptoic peptide and gene drugs.

  4. Bee venom protects SH-SY5Y human neuroblastoma cells from 1-methyl-4-phenylpyridinium-induced apoptotic cell death.

    Science.gov (United States)

    Doo, Ah-Reum; Kim, Seung-Nam; Kim, Seung-Tae; Park, Ji-Yeun; Chung, Sung-Hyun; Choe, Bo-Young; Chae, Younbyoung; Lee, Hyejung; Yin, Chang-Shik; Park, Hi-Joon

    2012-01-06

    Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by progressive selective loss of dopaminergic neurons in the substantia nigra. Recently, bee venom was reported to protect dopaminergic neurons in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine induced mice PD model, however, the underlying mechanism is not fully understood. The objective of the present study is to investigate the neuroprotective mechanism of bee venom against Parkinsonian toxin, 1-methyl-4-phenylpyridine (MPP(+)), in SH-SY5Y human neuroblastoma cells. Our results revealed that bee venom pretreatment (1-100 ng/ml) increased the cell viability and decreased apoptosis assessed by DNA fragmentation and caspase-3 activity assays in MPP(+)-induced cytotoxicity in SH-SY5Y cells. Bee venom increased the anti-apoptotic Bcl-2 expression and decreased the pro-apoptotic Bax, cleaved PARP expressions. In addition, bee venom prevented the MPP(+)-induced suppression of Akt phosphorylation, and the neuroprotective effect of bee venom against MPP(+)-induced cytotoxicity was inhibited by a phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002. These results suggest that the anti-apoptotic effect of bee venom is mediated by the cell survival signaling, the PI3K/Akt pathway. These results provide new evidence for elucidating the mechanism of neuroprotection of bee venom against PD. Copyright © 2011 Elsevier B.V. All rights reserved.

  5. Cell death triggered by alpha-emitting 213Bi-immunoconjugates in HSC45-M2 gastric cancer cells is different from apoptotic cell death

    International Nuclear Information System (INIS)

    Seidl, Christof; Schroeck, Hedwig; Seidenschwang, Sabine; Beck, Roswitha; Schwaiger, Markus; Senekowitsch-Schmidtke, Reingard; Schmid, Ernst; Abend, Michael; Becker, Karl-Friedrich; Apostolidis, Christos; Nikula, Tuomo K.; Kremmer, Elisabeth

    2005-01-01

    Radioimmunotherapy with α-particle-emitting nuclides, such as 213 Bi, is a promising concept for the elimination of small tumour nodules or single disseminated tumour cells. The aim of this study was to investigate cellular damage and the mode of cell death triggered by 213 Bi-immunoconjugates. Human gastric cancer cells (HSC45-M2) expressing d9-E-cadherin were incubated with different levels of activity of 213 Bi-d9MAb targeting d9-E-cadherin and 213 Bi-d8MAb, which does not bind to d9-E-cadherin. Micronucleated (M) cells, abnormal (A) cells and apoptotic (A) [(MAA)] cells were scored microscopically in the MAA assay following fluorescent staining of nuclei and cytoplasm. Chromosomal aberrations were analysed microscopically following Giemsa staining. The effect of z-VAD-fmk, known to inhibit apoptosis, on the prevention of cell death was investigated following treatment of HSC45-M2 cells with sorbitol as well as 213 Bi-d9MAb. Activation of caspase 3 after incubation of HSC45-M2 cells with both sorbitol and 213 Bi-d9MAb was analysed via Western blotting. Following incubation of HSC45-M2 human gastric cancer cells expressing d9-E-cadherin with 213 Bi-d9MAb the number of cells killed increased proportional to the applied activity concentration. Microscopically visible effects of α-irradiation of HSC45-M2 cells were formation of micronuclei and severe chromosomal aberrations. Preferential induction of these lesions with specific 213 Bi-d9MAb compared with unspecific 213 Bi-d8MAb (not targeting d9-E-cadherin) was not observed if the number of floating, i.e. unbound 213 Bi-immunoconjugates per cell exceeded 2 x 10 4 , most likely due to intense crossfire. In contrast to sorbitol-induced cell death, cell death triggered by 213 Bi-immunoconjugates was independent of caspase 3 activation and could not be inhibited by z-VAD-fmk, known to suppress the apoptotic pathway. 213 Bi-immunoconjugates seem to induce a mode of cell death different from apoptosis in HSC45-M2 cells

  6. Cell death triggered by alpha-emitting {sup 213}Bi-immunoconjugates in HSC45-M2 gastric cancer cells is different from apoptotic cell death

    Energy Technology Data Exchange (ETDEWEB)

    Seidl, Christof; Schroeck, Hedwig; Seidenschwang, Sabine; Beck, Roswitha; Schwaiger, Markus; Senekowitsch-Schmidtke, Reingard [Technische Universitaet Muenchen, Department of Nuclear Medicine, Munich (Germany); Schmid, Ernst [National Research Center for Environment and Health, Institute of Radiation Biology, GSF, Neuherberg (Germany); Abend, Michael [German Armed Forces, Institute of Radiobiology, Munich (Germany); Becker, Karl-Friedrich [Technische Universitaet Muenchen, Institute of Pathology, Munich (Germany); National Research Center for Environment and Health, Institute of Pathology, GSF, Neuherberg (Germany); National Research Center for Environment and Health, Institute of Molecular Immunology, GSF, Munich (Germany); Apostolidis, Christos; Nikula, Tuomo K. [European Commission, Institute for Transuranium Elements, Karlsruhe (Germany); Kremmer, Elisabeth [National Research Center for Environment and Health, Institute of Molecular Immunology, GSF, Munich (Germany)

    2005-03-01

    Radioimmunotherapy with {alpha}-particle-emitting nuclides, such as{sup 213}Bi, is a promising concept for the elimination of small tumour nodules or single disseminated tumour cells. The aim of this study was to investigate cellular damage and the mode of cell death triggered by {sup 213}Bi-immunoconjugates. Human gastric cancer cells (HSC45-M2) expressing d9-E-cadherin were incubated with different levels of activity of {sup 213}Bi-d9MAb targeting d9-E-cadherin and {sup 213}Bi-d8MAb, which does not bind to d9-E-cadherin. Micronucleated (M) cells, abnormal (A) cells and apoptotic (A) [(MAA)] cells were scored microscopically in the MAA assay following fluorescent staining of nuclei and cytoplasm. Chromosomal aberrations were analysed microscopically following Giemsa staining. The effect of z-VAD-fmk, known to inhibit apoptosis, on the prevention of cell death was investigated following treatment of HSC45-M2 cells with sorbitol as well as {sup 213}Bi-d9MAb. Activation of caspase 3 after incubation of HSC45-M2 cells with both sorbitol and {sup 213}Bi-d9MAb was analysed via Western blotting. Following incubation of HSC45-M2 human gastric cancer cells expressing d9-E-cadherin with {sup 213}Bi-d9MAb the number of cells killed increased proportional to the applied activity concentration. Microscopically visible effects of {alpha}-irradiation of HSC45-M2 cells were formation of micronuclei and severe chromosomal aberrations. Preferential induction of these lesions with specific {sup 213}Bi-d9MAb compared with unspecific {sup 213}Bi-d8MAb (not targeting d9-E-cadherin) was not observed if the number of floating, i.e. unbound {sup 213}Bi-immunoconjugates per cell exceeded 2 x 10{sup 4}, most likely due to intense crossfire. In contrast to sorbitol-induced cell death, cell death triggered by {sup 213}Bi-immunoconjugates was independent of caspase 3 activation and could not be inhibited by z-VAD-fmk, known to suppress the apoptotic pathway. {sup 213}Bi-immunoconjugates seem

  7. Cytotoxicity and apoptotic effects of tea polyphenol-loaded chitosan nanoparticles on human hepatoma HepG2 cells

    Energy Technology Data Exchange (ETDEWEB)

    Liang, Jin [Key Laboratory of Tea Biochemistry and Biotechnology of Ministry of Education and Ministry of Agriculture, Anhui Agricultural University, Hefei 230036 (China); College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095 (China); Li, Feng [College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095 (China); Fang, Yong; Yang, Wenjian [College of Food Science and Engineering, Nanjing University of Finance and Economics, Nanjing 210023 (China); An, Xinxin; Zhao, Liyan; Xin, Zhihong; Cao, Lin [College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095 (China); Hu, Qiuhui, E-mail: qiuhuihu@njau.edu.cn [College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095 (China); College of Food Science and Engineering, Nanjing University of Finance and Economics, Nanjing 210023 (China)

    2014-03-01

    Tea polyphenols have strong antioxidant and antitumor activities. However, these health benefits are limited due to their poor in vivo stability and low bioavailability. Chitosan nanoparticles as delivery systems may provide an alternative approach for enhancing bioavailability of poorly absorbed drugs. In this study, tea polyphenol-loaded chitosan nanoparticles have been prepared using two different chitosan biomaterials, and their antitumor effects were evaluated in HepG2 cells, including cell cytotoxicity comparison, cell morphology analysis, cell apoptosis and cell cycle detection. The results indicated that the tea polyphenol-loaded chitosan nanoparticles showed a branch shape and heterogeneous distribution in prepared suspension. MTT assay suggested that tea polyphenol-loaded chitosan nanoparticles could inhibit the proliferation of HepG2 cells, and the cytotoxicity rates were increased gradually and appeared an obvious dose-dependent relationship. Transmission electron microscope images showed that the HepG2 cells treated with tea polyphenol-loaded chitosan nanoparticles exhibited some typical apoptotic features, such as microvilli disappearance, margination of nuclear chromatin, intracytoplasmic vacuoles and the mitochondrial swelling. In addition, the tea polyphenol-loaded chitosan nanoparticles had relatively weak inhibitory effects on HepG2 cancer cells compared with tea polyphenols. Tea polyphenols not only induced cancer cell apoptosis, but also promoted their necrosis. However, tea polyphenol-loaded chitosan nanoparticles exhibited their antitumor effects mainly through inducing cell apoptosis. Our results revealed that the inhibition effects of tea polyphenol-loaded chitosan nanoparticles on tumor cells probably depended on their controlled drug release and effective cell delivery. The chitosan nanoparticles themselves as the delivery carrier showed limited antitumor effects compared with their encapsulated drugs. - Highlights: • Tea polyphenol

  8. Cytotoxicity and apoptotic effects of tea polyphenol-loaded chitosan nanoparticles on human hepatoma HepG2 cells

    International Nuclear Information System (INIS)

    Liang, Jin; Li, Feng; Fang, Yong; Yang, Wenjian; An, Xinxin; Zhao, Liyan; Xin, Zhihong; Cao, Lin; Hu, Qiuhui

    2014-01-01

    Tea polyphenols have strong antioxidant and antitumor activities. However, these health benefits are limited due to their poor in vivo stability and low bioavailability. Chitosan nanoparticles as delivery systems may provide an alternative approach for enhancing bioavailability of poorly absorbed drugs. In this study, tea polyphenol-loaded chitosan nanoparticles have been prepared using two different chitosan biomaterials, and their antitumor effects were evaluated in HepG2 cells, including cell cytotoxicity comparison, cell morphology analysis, cell apoptosis and cell cycle detection. The results indicated that the tea polyphenol-loaded chitosan nanoparticles showed a branch shape and heterogeneous distribution in prepared suspension. MTT assay suggested that tea polyphenol-loaded chitosan nanoparticles could inhibit the proliferation of HepG2 cells, and the cytotoxicity rates were increased gradually and appeared an obvious dose-dependent relationship. Transmission electron microscope images showed that the HepG2 cells treated with tea polyphenol-loaded chitosan nanoparticles exhibited some typical apoptotic features, such as microvilli disappearance, margination of nuclear chromatin, intracytoplasmic vacuoles and the mitochondrial swelling. In addition, the tea polyphenol-loaded chitosan nanoparticles had relatively weak inhibitory effects on HepG2 cancer cells compared with tea polyphenols. Tea polyphenols not only induced cancer cell apoptosis, but also promoted their necrosis. However, tea polyphenol-loaded chitosan nanoparticles exhibited their antitumor effects mainly through inducing cell apoptosis. Our results revealed that the inhibition effects of tea polyphenol-loaded chitosan nanoparticles on tumor cells probably depended on their controlled drug release and effective cell delivery. The chitosan nanoparticles themselves as the delivery carrier showed limited antitumor effects compared with their encapsulated drugs. - Highlights: • Tea polyphenol

  9. Determination of glutathione in apoptotic SMMC-7221 cells induced by xylitol selenite using capillary electrophoresis.

    Science.gov (United States)

    Wu, Xue; Cao, Yu; Zhang, Jian; Lei, Ming; Deng, Xiaojie; Zahid, Kashif Rafiq; Liu, Yanli; Liu, Ke; Yang, Jihong; Xiong, Guomei; Yao, Hanchao; Qi, Chao

    2016-05-01

    To determine the glutathione (GSH) content in a human hepatoma cell line (SMMC-7221) treated with xylitol/selenite, providing a part of an investigation of its anti-cancer mechanisms. The nuclei of SMMC-7221 cells were stained with Hoechst 33258 in an apoptosis assay, and their morphology subsequently changed from circular to crescent shape. The calibration curve (r(2) = 0.992) was established, and GSH content markedly decreased after treated with 0.5 and 1 mg xylitol/selenite l(-1) for 12, 36 and 60 h (12 h: from 95.57 ± 19.57 to 29.09 ± 7.74 and 24.27 ± 11.15; 36 h: from 70.73 ± 11.35 to 19.54 ± 6.39 and 9.35 ± 6.69; 60 h: from 72.63 ± 16.94 to 7.432 ± 3.84 and 0). The depletion rate of GSH was more related to the concentration of xylitol/selenite than the treatment time (from 69.95 ± 1.87 to 100 % vs. 0.22 ± 0.2 to 100 %). Xylitol/selenite is a promising anti-cancer drug to induce apoptosis in SMMC-7221 cells. It may regulate the apoptosis through the co-action of multiple mechanisms related to GSH depletion.

  10. Andrographolide potentiates the antitumor effect of topotecan in acute myeloid leukemia cells through an intrinsic apoptotic pathway

    Directory of Open Access Journals (Sweden)

    Hodroj MH

    2018-05-01

    Full Text Available Mohammad Hassan Hodroj, Achraf Jardaly, Sarah Abi Raad, Annalise Zouein, Sandra Rizk Department of Natural Sciences, Lebanese American University, Beirut, Lebanon Background: Topotecan (TP is an anticancer drug acting as topoisomerase I inhibitor that is used in the treatment of many types of cancers including leukemia, but it has significant side effects. Andrographolide, a compound extracted from Andrographis paniculata, was recently proven to inhibit the growth of cancer cells and can induce apoptosis. The aim of this study is to investigate the possible synergism between TP and andrographolide in acute myeloid cells in vitro. Materials and methods: U937 acute myeloid leukemic cells were cultured using Roswell Park Memorial Institute (RPMI medium and then treated for 24 h with TP and andrographolide prepared through the dilution of dimethyl sulfoxide (DMSO stocks with RPMI on the day of treatment. Cell proliferation was assessed using cell proliferation assay upon treatment with both compounds separately and in combination. Cell-cycle study and apoptosis detection were performed by staining the cells with propidium iodide (PI stain and Annexin V/PI stain, respectively, followed by flow cytometry analysis. Western blotting was used to assess the expression of various proteins involved in apoptotic pathways. Results: Both TP and andrographolide showed an antiproliferative effect in a dose-dependent manner when applied on U937 cells separately; however, pretreating the cells with andrographolide before applying TP exhibited a synergistic effect with lower inhibitory concentrations (half-maximal inhibitory concentration. Treating the cells with TP alone led to specific cell-cycle arrest at S phase that was more prominent upon pretreatment combination with andrographolide. Using Annexin V/PI staining to assess the proapoptotic effect following the pretreatment combination showed an increase in the number of apoptotic cells, which was supported by

  11. Myotonic dystrophy protein kinase (DMPK) induces actin cytoskeletal reorganization and apoptotic-like blebbing in lens cells

    Science.gov (United States)

    Jin, S.; Shimizu, M.; Balasubramanyam, A.; Epstein, H. F.

    2000-01-01

    DMPK, the product of the DM locus, is a member of the same family of serine-threonine protein kinases as the Rho-associated enzymes. In DM, membrane inclusions accumulate in lens fiber cells producing cataracts. Overexpression of DMPK in cultured lens epithelial cells led to apoptotic-like blebbing of the plasma membrane and reorganization of the actin cytoskeleton. Enzymatically active DMPK was necessary for both effects; inactive mutant DMPK protein did not produce either effect. Active RhoA but not constitutive GDP-state mutant protein produced similar effects as DMPK. The similar actions of DMPK and RhoA suggest that they may function in the same regulatory network. The observed effects of DMPK may be relevant to the removal of membrane organelles during normal lens differentiation and the retention of intracellular membranes in DM lenses. Copyright 2000 Wiley-Liss, Inc.

  12. Anti-apoptotic BFL-1 is the major effector in activation-induced human mast cell survival.

    Directory of Open Access Journals (Sweden)

    Maria Ekoff

    Full Text Available Mast cells are best known for their role in allergic reactions, where aggregation of FcεRI leads to the release of mast cell mediators causing allergic symptoms. The activation also induces a survival program in the cells, i.e., activation-induced mast cell survival. The aim of the present study was to investigate how the activation-induced survival is mediated. Cord blood-derived mast cells and the mast cell line LAD-2 were activated through FcεRI crosslinking, with or without addition of chemicals that inhibit the activity or expression of selected Bcl-2 family members (ABT-737; roscovitine. Cell viability was assessed using staining and flow cytometry. The expression and function of Bcl-2 family members BFL-1 and MCL-1 were investigated using real-time quantitative PCR and siRNA treatment. The mast cell expression of Bfl-1 was investigated in skin biopsies. FcεRI crosslinking promotes activation-induced survival of human mast cells and this is associated with an upregulation of the anti-apoptotic Bcl-2 family member Bfl-1. ABT-737 alone or in combination with roscovitine decreases viability of human mast cells although activation-induced survival is sustained, indicating a minor role for Bcl-X(L, Bcl-2, Bcl-w and Mcl-1. Reducing BFL-1 but not MCL-1 levels by siRNA inhibited activation-induced mast cell survival. We also demonstrate that mast cell expression of Bfl-1 is elevated in birch-pollen-provocated skin and in lesions of atopic dermatitis and psoriasis patients. Taken together, our results highlight Bfl-1 as a major effector in activation-induced human mast cell survival.

  13. Viral DNA Sensors IFI16 and Cyclic GMP-AMP Synthase Possess Distinct Functions in Regulating Viral Gene Expression, Immune Defenses, and Apoptotic Responses during Herpesvirus Infection.

    Science.gov (United States)

    Diner, Benjamin A; Lum, Krystal K; Toettcher, Jared E; Cristea, Ileana M

    2016-11-15

    The human interferon-inducible protein IFI16 is an important antiviral factor that binds nuclear viral DNA and promotes antiviral responses. Here, we define IFI16 dynamics in space and time and its distinct functions from the DNA sensor cyclic dinucleotide GMP-AMP synthase (cGAS). Live-cell imaging reveals a multiphasic IFI16 redistribution, first to viral entry sites at the nuclear periphery and then to nucleoplasmic puncta upon herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV) infections. Optogenetics and live-cell microscopy establish the IFI16 pyrin domain as required for nuclear periphery localization and oligomerization. Furthermore, using proteomics, we define the signature protein interactions of the IFI16 pyrin and HIN200 domains and demonstrate the necessity of pyrin for IFI16 interactions with antiviral proteins PML and cGAS. We probe signaling pathways engaged by IFI16, cGAS, and PML using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated knockouts in primary fibroblasts. While IFI16 induces cytokines, only cGAS activates STING/TBK-1/IRF3 and apoptotic responses upon HSV-1 and HCMV infections. cGAS-dependent apoptosis upon DNA stimulation requires both the enzymatic production of cyclic dinucleotides and STING. We show that IFI16, not cGAS or PML, represses HSV-1 gene expression, reducing virus titers. This indicates that regulation of viral gene expression may function as a greater barrier to viral replication than the induction of antiviral cytokines. Altogether, our findings establish coordinated and distinct antiviral functions for IFI16 and cGAS against herpesviruses. How mammalian cells detect and respond to DNA viruses that replicate in the nucleus is poorly understood. Here, we decipher the distinct functions of two viral DNA sensors, IFI16 and cGAS, during active immune signaling upon infection with two herpesviruses, herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV). We show that IFI16

  14. Anti-apoptotic effects of pan-caspase inhibitor (Z-VAD), SOD or catalase on antimycin A-induced HeLa cell death.

    Science.gov (United States)

    Han, Yong Hwan; Kim, Suhn Hee; Kim, Sung Zoo; Park, Woo Hyun

    2009-01-01

    Antimycin A (AMA) is an inhibitor of the electron transport chain in mitochondria. In this study, we investigated the anti-apoptotic effects of pan-caspase inhibitor (Z-VAD), superoxide dismutase (SOD) or catalase on AMA-induced HeLa cell death in relation to the cell cycle. Treatment with Z-VAD, SOD or catalase rescued some HeLa cells from AMA-induced apoptosis, but did not prevent the growth inhibition of HeLa cells by AMA. DNA flow cytometric analysis indicated that treatment with AMA significantly induced an S-phase arrest of the cell cycle at 72 h. Interestingly, Z-VAD, SOD and catalase intensified S-phase arrest in AMA-treated cells. In conclusion, treatment with Z-VAD, SOD or catalase decreased apoptotic levels in AMA-treated cells, which was associated with the enhancement of the S-phase arrest of the cell cycle in these cells.

  15. Induction of non-apoptotic programmed cell death by oncogenic RAS in human epithelial cells and its suppression by MYC overexpression.

    Science.gov (United States)

    Dendo, Kasumi; Yugawa, Takashi; Nakahara, Tomomi; Ohno, Shin-Ichi; Goshima, Naoki; Arakawa, Hirofumi; Kiyono, Tohru

    2018-02-09

    Oncogenic mutations of RAS genes, found in about 30% of human cancers, are considered to play important roles in cancer development. However, oncogenic RAS can also induce senescence in mouse and human normal fibroblasts. In some cell lines, oncogenic RAS has been reported to induce non-apoptotic programed cell death (PCD). Here, we investigated effects of oncogenic RAS expression in several types of normal human epithelial cells. Oncogenic RAS but not wild-type RAS stimulated macropinocytosis with accumulation of large-phase lucent vacuoles in the cytoplasm, subsequently leading to cell death which was indistinguishable from a recently proposed new type of PCD, methuosis. A RAC1 inhibitor suppressed accumulation of macropinosomes and overexpression of MYC attenuated oncogenic RAS-induced such accumulation, cell cycle arrest and cell death. MYC suppression or rapamycin treatment in some cancer cell lines harbouring oncogenic mutations in RAS genes induced cell death with accumulation of macropinosomes. These results suggest that this type of non-apoptotic PCD is a tumour-suppressing mechanism acting against oncogenic RAS mutations in normal human epithelial cells, which can be overcome by MYC overexpression, raising the possibility that its induction might be a novel approach to treatment of RAS-mutated human cancers. © The Author(s) 2017. Published by Oxford University Press.

  16. Nanosecond pulsed electric fields induce poly(ADP-ribose) formation and non-apoptotic cell death in HeLa S3 cells

    Energy Technology Data Exchange (ETDEWEB)

    Morotomi-Yano, Keiko; Akiyama, Hidenori [Institute of Pulsed Power Science, Kumamoto University, Kumamoto 860-8555 (Japan); Yano, Ken-ichi, E-mail: yanoken@kumamoto-u.ac.jp [Priority Organization for Innovation and Excellence, Kumamoto University, Kumamoto 860-8555 (Japan)

    2013-08-30

    Highlights: •Nanosecond pulsed electric field (nsPEF) is a new and unique means for life sciences. •Apoptosis was induced by nsPEF exposure in Jurkat cells. •No signs of apoptosis were detected in HeLa S3 cells exposed to nsPEFs. •Formation of poly(ADP-ribose) was induced in nsPEF-exposed HeLa S3 cells. •Two distinct modes of cell death were activated by nsPEF in a cell-dependent manner. -- Abstract: Nanosecond pulsed electric fields (nsPEFs) have recently gained attention as effective cancer therapy owing to their potency for cell death induction. Previous studies have shown that apoptosis is a predominant mode of nsPEF-induced cell death in several cell lines, such as Jurkat cells. In this study, we analyzed molecular mechanisms for cell death induced by nsPEFs. When nsPEFs were applied to Jurkat cells, apoptosis was readily induced. Next, we used HeLa S3 cells and analyzed apoptotic events. Contrary to our expectation, nsPEF-exposed HeLa S3 cells exhibited no molecular signs of apoptosis execution. Instead, nsPEFs induced the formation of poly(ADP-ribose) (PAR), a hallmark of necrosis. PAR formation occurred concurrently with a decrease in cell viability, supporting implications of nsPEF-induced PAR formation for cell death. Necrotic PAR formation is known to be catalyzed by poly(ADP-ribose) polymerase-1 (PARP-1), and PARP-1 in apoptotic cells is inactivated by caspase-mediated proteolysis. Consistently, we observed intact and cleaved forms of PARP-1 in nsPEF-exposed and UV-irradiated cells, respectively. Taken together, nsPEFs induce two distinct modes of cell death in a cell type-specific manner, and HeLa S3 cells show PAR-associated non-apoptotic cell death in response to nsPEFs.

  17. Nanosecond pulsed electric fields induce poly(ADP-ribose) formation and non-apoptotic cell death in HeLa S3 cells

    International Nuclear Information System (INIS)

    Morotomi-Yano, Keiko; Akiyama, Hidenori; Yano, Ken-ichi

    2013-01-01

    Highlights: •Nanosecond pulsed electric field (nsPEF) is a new and unique means for life sciences. •Apoptosis was induced by nsPEF exposure in Jurkat cells. •No signs of apoptosis were detected in HeLa S3 cells exposed to nsPEFs. •Formation of poly(ADP-ribose) was induced in nsPEF-exposed HeLa S3 cells. •Two distinct modes of cell death were activated by nsPEF in a cell-dependent manner. -- Abstract: Nanosecond pulsed electric fields (nsPEFs) have recently gained attention as effective cancer therapy owing to their potency for cell death induction. Previous studies have shown that apoptosis is a predominant mode of nsPEF-induced cell death in several cell lines, such as Jurkat cells. In this study, we analyzed molecular mechanisms for cell death induced by nsPEFs. When nsPEFs were applied to Jurkat cells, apoptosis was readily induced. Next, we used HeLa S3 cells and analyzed apoptotic events. Contrary to our expectation, nsPEF-exposed HeLa S3 cells exhibited no molecular signs of apoptosis execution. Instead, nsPEFs induced the formation of poly(ADP-ribose) (PAR), a hallmark of necrosis. PAR formation occurred concurrently with a decrease in cell viability, supporting implications of nsPEF-induced PAR formation for cell death. Necrotic PAR formation is known to be catalyzed by poly(ADP-ribose) polymerase-1 (PARP-1), and PARP-1 in apoptotic cells is inactivated by caspase-mediated proteolysis. Consistently, we observed intact and cleaved forms of PARP-1 in nsPEF-exposed and UV-irradiated cells, respectively. Taken together, nsPEFs induce two distinct modes of cell death in a cell type-specific manner, and HeLa S3 cells show PAR-associated non-apoptotic cell death in response to nsPEFs

  18. A chalcone-related small molecule that induces methuosis, a novel form of non-apoptotic cell death, in glioblastoma cells

    OpenAIRE

    Overmeyer, Jean H; Young, Ashley M; Bhanot, Haymanti; Maltese, William A

    2011-01-01

    Abstract Background Methuosis is a unique form of non-apoptotic cell death triggered by alterations in the trafficking of clathrin-independent endosomes, ultimately leading to extreme vacuolization and rupture of the cell. Results Here we describe a novel chalcone-like molecule, 3-(2-methyl-1H- indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one (MIPP) that induces cell death with the hallmarks of methuosis. MIPP causes rapid accumulation of vacuoles derived from macropinosomes, based on time-lapse mi...

  19. Cytotoxicity and apoptotic effects of tea polyphenol-loaded chitosan nanoparticles on human hepatoma HepG2 cells.

    Science.gov (United States)

    Liang, Jin; Li, Feng; Fang, Yong; Yang, Wenjian; An, Xinxin; Zhao, Liyan; Xin, Zhihong; Cao, Lin; Hu, Qiuhui

    2014-03-01

    Tea polyphenols have strong antioxidant and antitumor activities. However, these health benefits are limited due to their poor in vivo stability and low bioavailability. Chitosan nanoparticles as delivery systems may provide an alternative approach for enhancing bioavailability of poorly absorbed drugs. In this study, tea polyphenol-loaded chitosan nanoparticles have been prepared using two different chitosan biomaterials, and their antitumor effects were evaluated in HepG2 cells, including cell cytotoxicity comparison, cell morphology analysis, cell apoptosis and cell cycle detection. The results indicated that the tea polyphenol-loaded chitosan nanoparticles showed a branch shape and heterogeneous distribution in prepared suspension. MTT assay suggested that tea polyphenol-loaded chitosan nanoparticles could inhibit the proliferation of HepG2 cells, and the cytotoxicity rates were increased gradually and appeared an obvious dose-dependent relationship. Transmission electron microscope images showed that the HepG2 cells treated with tea polyphenol-loaded chitosan nanoparticles exhibited some typical apoptotic features, such as microvilli disappearance, margination of nuclear chromatin, intracytoplasmic vacuoles and the mitochondrial swelling. In addition, the tea polyphenol-loaded chitosan nanoparticles had relatively weak inhibitory effects on HepG2 cancer cells compared with tea polyphenols. Tea polyphenols not only induced cancer cell apoptosis, but also promoted their necrosis. However, tea polyphenol-loaded chitosan nanoparticles exhibited their antitumor effects mainly through inducing cell apoptosis. Our results revealed that the inhibition effects of tea polyphenol-loaded chitosan nanoparticles on tumor cells probably depended on their controlled drug release and effective cell delivery. The chitosan nanoparticles themselves as the delivery carrier showed limited antitumor effects compared with their encapsulated drugs. Copyright © 2013. Published by

  20. Melipona quadrifasciata (Hymenoptera: Apidae) fat body persists through metamorphosis with a few apoptotic cells and an increased autophagy.

    Science.gov (United States)

    Santos, Douglas Elias; Azevedo, Dihego Oliveira; Campos, Lúcio Antônio Oliveira; Zanuncio, José Cola; Serrão, José Eduardo

    2015-03-01

    Fat body, typically comprising trophocytes, provides energy during metamorphosis. The fat body can be renewed once the larval phase is complete or recycled and relocated to form the fat body of the adult insect. This study aims to identify the class of programmed cell death that occurs within the fat body cells during the metamorphosis of the stingless bee Melipona quadrifasciata. Using immunodetection techniques, the fat body of the post-defecating larvae and the white-, pink-, brown-, and black-eyed pupae were tested for cleaved caspase-3 and DNA integrity, followed by ultrastructural analysis and identification of autophagy using RT-PCR for the Atg1 gene. The fat body of M. quadrifasciata showed some apoptotic cells positive for cleaved caspase-3, although without DNA fragmentation. During development, the fat body cells revealed an increased number of mitochondria and free ribosomes, in addition to higher amounts of autophagy Atg1 mRNA, than that of the pupae. The fat body of M. quadrifasciata showed few cells which underwent apoptosis, but there was evidence of increased autophagy at the completion of the larval stage. All together, these data show that some fat body cells persist during metamorphosis in the stingless bee M. quadrifasciata.

  1. Protection of MES23.5 dopaminergic cells by obestatin is mediated by proliferative rather than anti-apoptotic action.

    Science.gov (United States)

    Shen, Xiao-Li; Jia, Feng-Ju; Song, Ning; Xie, Jun-Xia; Jiang, Hong

    2014-02-01

    Obestatin is an endogenous peptide sharing a precursor with ghrelin. This study aims to investigate whether and how obestatin protects MES23.5 dopaminergic cells against 1-methyl-4-phenylpyridinium (MPP(+))-induced neurotoxicity. MES23.5 cells were pretreated with obestatin (10(-13)-10(-6) mol/L) for 20 min prior to incubation with 200 μmol/L MPP(+) for 12 or 24 h, or treated with obestatin alone (10(-13) to 10(-6) mol/L) for 0, 6, 12, and 24 h. The methyl thiazolyl tetrazolium (MTT) assay was used to measure cell viability. Flow cytometry was used to measure the caspase-3 activity and the mitochondrial transmembrane potential. Proliferating cell nuclear antigen (PCNA) protein levels were determined by Western blotting. Obestatin (10(-13) to 10(-7) mol/L) pretreatment blocked or even reversed the MPP(+)-induced reduction of viability in MES23.5 cells, but had no effect on MPP(+)-induced mitochondrial transmembrane potential collapse and caspase-3 activation. When applied alone, obestatin increased viability. Elevated PCNA levels occurred with 10(-7), 10(-9), 10(-11) and 10(-13) mol/L obestatin treatment for 12 h. The results suggest that the protective effects of obestatin against MPP(+) in MES23.5 cells are due to its proliferation-promoting rather than anti-apoptotic effects.

  2. Effects of highly ripened cheeses on HL-60 human leukemia cells: antiproliferative activity and induction of apoptotic DNA damage.

    Science.gov (United States)

    Yasuda, S; Ohkura, N; Suzuki, K; Yamasaki, M; Nishiyama, K; Kobayashi, H; Hoshi, Y; Kadooka, Y; Igoshi, K

    2010-04-01

    To establish cheese as a dairy product with health benefits, we examined the multifunctional role of cheeses. In this report, we clarify whether different types of commercial cheeses may possess antiproliferative activity using HL-60 human promyelocytic leukemia cell lines as a cancer model. Among 12 cheese extracts tested, 6 (Montagnard, Pont-l'Eveque, Brie, Camembert, Danablue, and Blue) revealed strong growth inhibition activity and induction of DNA fragmentation in HL-60 cells. Based on the quantification of nitrogen contents in different cheese samples, a positive correlation between the ripeness of various cheeses and their antiproliferative activity tested in HL-60 cells was displayed. Four varieties of Blue cheese ripened for 0, 1, 2, or 3 mo demonstrated that the Blue cheese ripened for a long term was capable of causing the strong suppression of the cell growth and the induction of apoptotic DNA damage as well as nucleic morphological change in HL-60 cells. Collectively, these results obtained suggest a potential role of highly ripened cheeses in the prevention of leukemic cell proliferation. Copyright (c) 2010 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  3. 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.

  4. Kaurene diterpene induces apoptosis in U87 human malignant glioblastoma cells by suppression of anti-apoptotic signals and activation of cysteine proteases

    Energy Technology Data Exchange (ETDEWEB)

    Lizarte, F.S. Neto; Tirapelli, D.P.C. [Universidade de São Paulo, Departamento de Cirurgia e Anatomia, Faculdade de Medicina de Ribeirão Preto, Ribeirão Preto, SP (Brazil); Ambrosio, S.R. [Universidade de Franca, Núcleo de Pesquisa em Ciências e Tecnologia, Franca, SP (Brazil); Tirapelli, C.R. [Universidade de São Paulo, Laboratório de Farmacologia, Departamento de Enfermagem Psiquiátrica e Ciências Humanas, Escola de Enfermagem de Ribeirão Preto, Ribeirão Preto, SP (Brazil); Oliveira, F.M. [Universidade de São Paulo, Departamento de Clínica Médica, Faculdade de Medicina de Ribeirão Preto, Ribeirão Preto, SP (Brazil); Novais, P.C. [Universidade de São Paulo, Departamento de Cirurgia e Anatomia, Faculdade de Medicina de Ribeirão Preto, Ribeirão Preto, SP (Brazil); Peria, F.M.; Oliveira, H.F. [Universidade de São Paulo, Departamento de Clínica Médica, Faculdade de Medicina de Ribeirão Preto, Ribeirão Preto, SP (Brazil); Carlotti, C.G. Junior; Tirapelli, L.F. [Universidade de São Paulo, Departamento de Cirurgia e Anatomia, Faculdade de Medicina de Ribeirão Preto, Ribeirão Preto, SP (Brazil)

    2013-01-11

    Gliomas are the most common and malignant primary brain tumors in humans. Studies have shown that classes of kaurene diterpene have anti-tumor activity related to their ability to induce apoptosis. We investigated the response of the human glioblastoma cell line U87 to treatment with ent-kaur-16-en-19-oic acid (kaurenoic acid, KA). We analyzed cell survival and the induction of apoptosis using flow cytometry and annexin V staining. Additionally, the expression of anti-apoptotic (c-FLIP and miR-21) and apoptotic (Fas, caspase-3 and caspase-8) genes was analyzed by relative quantification (real-time PCR) of mRNA levels in U87 cells that were either untreated or treated with KA (30, 50, or 70 µM) for 24, 48, and 72 h. U87 cells treated with KA demonstrated reduced viability, and an increase in annexin V- and annexin V/PI-positive cells was observed. The percentage of apoptotic cells was 9% for control cells, 26% for cells submitted to 48 h of treatment with 50 µM KA, and 31% for cells submitted to 48 h of treatment with 70 µM KA. Similarly, in U87 cells treated with KA for 48 h, we observed an increase in the expression of apoptotic genes (caspase-8, -3) and a decrease in the expression of anti-apoptotic genes (miR-21 and c-FLIP). KA possesses several interesting properties and induces apoptosis through a unique mechanism. Further experiments will be necessary to determine if KA may be used as a lead compound for the development of new chemotherapeutic drugs for the treatment of primary brain tumors.

  5. Kaurene diterpene induces apoptosis in U87 human malignant glioblastoma cells by suppression of anti-apoptotic signals and activation of cysteine proteases

    International Nuclear Information System (INIS)

    Lizarte, F.S. Neto; Tirapelli, D.P.C.; Ambrosio, S.R.; Tirapelli, C.R.; Oliveira, F.M.; Novais, P.C.; Peria, F.M.; Oliveira, H.F.; Carlotti, C.G. Junior; Tirapelli, L.F.

    2013-01-01

    Gliomas are the most common and malignant primary brain tumors in humans. Studies have shown that classes of kaurene diterpene have anti-tumor activity related to their ability to induce apoptosis. We investigated the response of the human glioblastoma cell line U87 to treatment with ent-kaur-16-en-19-oic acid (kaurenoic acid, KA). We analyzed cell survival and the induction of apoptosis using flow cytometry and annexin V staining. Additionally, the expression of anti-apoptotic (c-FLIP and miR-21) and apoptotic (Fas, caspase-3 and caspase-8) genes was analyzed by relative quantification (real-time PCR) of mRNA levels in U87 cells that were either untreated or treated with KA (30, 50, or 70 µM) for 24, 48, and 72 h. U87 cells treated with KA demonstrated reduced viability, and an increase in annexin V- and annexin V/PI-positive cells was observed. The percentage of apoptotic cells was 9% for control cells, 26% for cells submitted to 48 h of treatment with 50 µM KA, and 31% for cells submitted to 48 h of treatment with 70 µM KA. Similarly, in U87 cells treated with KA for 48 h, we observed an increase in the expression of apoptotic genes (caspase-8, -3) and a decrease in the expression of anti-apoptotic genes (miR-21 and c-FLIP). KA possesses several interesting properties and induces apoptosis through a unique mechanism. Further experiments will be necessary to determine if KA may be used as a lead compound for the development of new chemotherapeutic drugs for the treatment of primary brain tumors

  6. Cytoplasmic and nuclear anti-apoptotic roles of αB-crystallin in retinal pigment epithelial cells.

    Directory of Open Access Journals (Sweden)

    Woo Jin Jeong

    Full Text Available In addition to its well-characterized role in the lens, αB-crystallin performs other functions. Methylglyoxal (MGO can alter the function of the basement membrane of retinal pigment epithelial (RPE cells. Thus, if MGO is not efficiently detoxified, it can induce adverse reactions in RPE cells. In this study, we examined the mechanisms underlying the anti-apoptotic activity of αB-crystallin in the human retinal pigment epithelial cell line ARPE-19 following MGO treatment using various assays, including nuclear staining, flow cytometry, DNA electrophoresis, pulse field gel electrophoresis, western blot analysis, confocal microscopy and co-immunoprecipitation assays. To directly assess the role of phosphorylation of αB-crystallin, we used site-directed mutagenesis to convert relevant serine residues to alanine residues. Using these techniques, we demonstrated that MGO induces apoptosis in ARPE-19 cells. Silencing αB-crystallin sensitized ARPE-19 cells to MGO-induced apoptosis, indicating that αB-crystallin protects ARPE-19 cells from MGO-induced apoptosis. Furthermore, we found that αB-crystallin interacts with the caspase subtypes, caspase-2L, -2S, -3, -4, -7, -8, -9 and -12 in untreated control ARPE-19 cells and that MGO treatment caused the dissociation of these caspase subtypes from αB-crystallin; transfection of S19A, S45A or S59A mutants caused the depletion of αB-crystallin from the nuclei of untreated control RPE cells leading to the release of caspase subtypes. Additionally, transfection of these mutants enhanced MGO-induced apoptosis in ARPE-19 cells, indicating that phosphorylation of nuclear αB-crystallin on serine residues 19, 45 and 59 plays a pivotal role in preventing apoptosis in ARPE-19 cells. Taken together, these results suggest that αB-crystallin prevents caspase activation by physically interacting with caspase subtypes in the cytoplasm and nucleus, thereby protecting RPE cells from MGO-induced apoptosis.

  7. Moxifloxacin and ciprofloxacin induces S-phase arrest and augments apoptotic effects of cisplatin in human pancreatic cancer cells via ERK activation

    International Nuclear Information System (INIS)

    Yadav, Vikas; Varshney, Pallavi; Sultana, Sarwat; Yadav, Jyoti; Saini, Neeru

    2015-01-01

    Pancreatic cancer, one of the most dreadful gastrointestinal tract malignancies, with the current chemotherapeutic drugs has posed a major impediment owing to poor prognosis and chemo-resistance thereby suggesting critical need for additional drugs as therapeutics in combating the situation. Fluoroquinolones have shown promising and significant anti-tumor effects on several carcinoma cell lines. Previously, we reported growth inhibitory effects of fourth generation fluoroquinolone Gatifloxacin, while in the current study we have investigated the anti-proliferative and apoptosis-inducing mechanism of older generation fluoroquinolones Moxifloxacin and Ciprofloxacin on the pancreatic cancer cell-lines MIA PaCa-2 and Panc-1. Cytotoxicity was measured by MTT assay. Apoptosis induction was evaluated using annexin assay, cell cycle assay and activation of caspase-3, 8, 9 were measured by western blotting and enzyme activity assay. Herein, we found that both the fluoroquinolones suppressed the proliferation of pancreatic cancer cells by causing S-phase arrest and apoptosis. Blockade in S-phase of cell cycle was associated with decrease in the levels of p27, p21, CDK2, cyclin-A and cyclin-E. Herein we also observed triggering of extrinsic as well as intrinsic mitochondrial apoptotic pathway as suggested by the activation of caspase-8, 9, 3, and Bid respectively. All this was accompanied by downregulation of antiapoptotic protein Bcl-xL and upregulation of proapoptotic protein Bak. Our results strongly suggest the role of extracellular-signal-regulated kinases (ERK1/2), but not p53, p38 and c-JUN N-terminal kinase (JNK) in fluoroquinolone induced growth inhibitory effects in both the cell lines. Additionally, we also found both the fluoroquinolones to augment the apoptotic effects of broad spectrum anticancer drug Cisplatin via ERK. The fact that these fluoroquinolones synergize the effect of cisplatin opens new insight into therapeutic index in treatment of pancreatic

  8. Casticin induced apoptotic cell death and altered associated gene expression in human colon cancer colo 205 cells.

    Science.gov (United States)

    Shang, Hung-Sheng; Liu, Jia-You; Lu, Hsu-Feng; Chiang, Han-Sun; Lin, Chia-Hain; Chen, Ann; Lin, Yuh-Feng; Chung, Jing-Gung

    2017-08-01

    Casticin, a polymethoxyflavone, derived from natural plant Fructus Viticis exhibits biological activities including anti-cancer characteristics. The anti-cancer and alter gene expression of casticin on human colon cancer cells and the underlying mechanisms were investigated. Flow cytometric assay was used to measure viable cell, cell cycle and sub-G1 phase, reactive oxygen species (ROS) and Ca 2+ productions, level of mitochondria membrane potential (ΔΨ m ) and caspase activity. Western blotting assay was used to detect expression of protein level associated with cell death. Casticin induced cell morphological changes, decreased cell viability and induced G2/M phase arrest in colo 205 cells. Casticin increased ROS production but decreased the levels of ΔΨ m , and Ca 2+ , increased caspase-3, -8, and -9 activities. The cDNA microarray indicated that some of the cell cycle associated genes were down-regulated such as cyclin-dependent kinase inhibitor 1A (CDKN1A) (p21, Cip1) and p21 protein (Cdc42/Rac)-activated kinase 3 (PAK3). TNF receptor-associated protein 1 (TRAP1), CREB1 (cAMP responsive element binding protein 1) and cyclin-dependent kinase inhibitor 1B (CDKN1B) (p27, Kip1) genes were increased but matrix metallopeptidase 2 (MMP-2), toll-like receptor 4 (TLR4), PRKAR2B (protein kinase, cAMP-dependent, regulatory, type II, bet), and CaMK4 (calcium/calmodulin-dependent protein kinase IV) genes were inhibited. Results suggest that casticin induced cell apoptosis via the activation of the caspase- and/or mitochondria-dependent signaling cascade, the accumulation of ROS and altered associated gene expressions in colo 205 human colon cancer cells. © 2016 Wiley Periodicals, Inc.

  9. Distinct Dasatinib-Induced Mechanisms of Apoptotic Response and Exosome Release in Imatinib-Resistant Human Chronic Myeloid Leukemia Cells

    Directory of Open Access Journals (Sweden)

    Juan Liu

    2016-04-01

    Full Text Available Although dasatinib is effective in most imatinib mesylate (IMT-resistant chronic myeloid leukemia (CML patients, the underlying mechanism of its effectiveness in eliminating imatinib-resistant cells is only partially understood. This study investigated the effects of dasatinib on signaling mechanisms driving-resistance in imatinib-resistant CML cell line K562 (K562RIMT. Compared with K562 control cells, exsomal release, the phosphoinositide 3-kinase (PI3K/protein kinase B (Akt/ mammalian target of rapamycin (mTOR signaling and autophagic activity were increased significantly in K562RIMT cells and mTOR-independent beclin-1/Vps34 signaling was shown to be involved in exosomal release in these cells. We found that Notch1 activation-mediated reduction of phosphatase and tensin homolog (PTEN was responsible for the increased Akt/mTOR activities in K562RIMT cells and treatment with Notch1 γ-secretase inhibitor prevented activation of Akt/mTOR. In addition, suppression of mTOR activity by rapamycin decreased the level of activity of p70S6K, induced upregulation of p53 and caspase 3, and led to increase of apoptosis in K562RIMT cells. Inhibition of autophagy by spautin-1 or beclin-1 knockdown decreased exosomal release, but did not affect apoptosis in K562RIMT cells. In summary, in K562RIMT cells dasatinib promoted apoptosis through downregulation of Akt/mTOR activities, while preventing exosomal release and inhibiting autophagy by downregulating expression of beclin-1 and Vps34. Our findings reveal distinct dasatinib-induced mechanisms of apoptotic response and exosomal release in imatinib-resistant CML cells.

  10. Antiproliferative and Pro-Apoptotic Effect of Novel Nitro-Substituted Hydroxynaphthanilides on Human Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Tereza Kauerova

    2016-07-01

    Full Text Available Ring-substituted hydroxynaphthanilides are considered as cyclic analogues of salicylanilides, compounds possessing a wide range of pharmacological activities, including promising anticancer properties. The aim of this study was to evaluate the potential anticancer effect of novel nitro-substituted hydroxynaphthanilides with a special focus on structure-activity relationships. The antiproliferative effect was assessed by Water Soluble Tetrazolium Salts-1 (WST-1 assay, and cytotoxicity was evaluated via dye exclusion test. Flow cytometry was used for cell cycle analysis and detection of apoptosis using Annexin V-FITC/PI assay. Protein expression was estimated by Western blotting. Our data indicate that the potential to cause the antiproliferative effect increases with the shift of the nitro substituent from the ortho- to the para-position. The most potent compounds, 3-hydroxy-N-(3-nitrophenylnaphthalene-2-carboxamide (2, and 2-hydroxy-N-(4-nitrophenyl-naphthalene-1-carboxamide (6 showed antiproliferative activity against THP-1 and MCF-7 cancer cells without affecting the proliferation of 3T3-L1 non-tumour cells. Compounds 2 and 6 induced the accumulation of THP-1 and MCF-7 cells in G1 phase associated with the downregulation of cyclin E1 protein levels, while the levels of cyclin B1 were not affected. Moreover, compound 2 was found to exert the pro-apoptotic effect on the THP-1 cells. These results suggest that hydroxynaphthanilides might represent a potential model structure for the development of novel anticancer agents.

  11. Pro-apoptotic activity of new analog of anthracyclines--WP 631 in advanced ovarian cancer cell line.

    Science.gov (United States)

    Gajek, Arkadiusz; Denel, Marta; Bukowska, Barbara; Rogalska, Aneta; Marczak, Agnieszka

    2014-03-01

    In this work we investigated the mode of cell death induced by WP 631, a novel anthracycline antibiotic, in the ovarian cancer cell line (OV-90) derived from the malignant ascites of a patient diagnosed with advanced disease. The effects were compared with those of doxorubicin (DOX), a first generation anthracycline. The ability of WP 631 to induce apoptosis and necrosis was examined by double staining with Annexin V and propidium iodide, measurements of the level of intracellular calcium ions and cytochrome c, PARP cleavage. We also investigated the possible involvement of the caspases activation, DNA degradation (comet assay) and intracellular reactive oxygen species (ROS) production in the development of the apoptotic events and their significance for drug efficiency. The results obtained clearly demonstrate that antiproliferative capacity of WP 631 in tested cell line was a few times greater than that of DOX. Furthermore, ovarian cancer cells treated with WP 631 showed a higher mean level of basal DNA damage in comparison to DOX. In conclusion, WP 631 is able to induce caspase - dependent apoptosis in human ovarian cancer cells. Obtained results suggested that WP 631 may be a candidate for further evaluation as chemotherapeutic agents for human cancers. Copyright © 2013 Elsevier Ltd. All rights reserved.

  12. Pro-apoptotic effect of a Mycoplasma hyopneumoniae putative type I signal peptidase on PK(15) swine cells.

    Science.gov (United States)

    Paes, Jéssica A; Virginio, Veridiana G; Cancela, Martín; Leal, Fernanda M A; Borges, Thiago J; Jaeger, Natália; Bonorino, Cristina; Schrank, Irene S; Ferreira, Henrique B

    2017-03-01

    Mycoplasma hyopneumoniae is an economically significant swine pathogen that causes porcine enzootic pneumonia (PEP). Important processes for swine infection by M. hyopneumoniae depend on cell surface proteins, many of which are secreted by secretion pathways not completely elucidated so far. A putative type I signal peptidase (SPase I), a possible component of a putative Sec-dependent pathway, was annotated as a product of the sipS gene in the pathogenic M. hyopneumoniae 7448 genome. This M. hyopneumoniae putative SPase I (MhSPase I) displays only 14% and 23% of sequence identity/similarity to Escherichia coli bona fide SPase I, and, in complementation assays performed with a conditional E. coli SPase I mutant, only a partial restoration of growth was achieved with the heterologous expression of a recombinant MhSPase I (rMhSPase I). Considering the putative surface location of MhSPase I and its previously demonstrated capacity to induce a strong humoral response, we then assessed its potential to elicit a cellular and possible immunomodulatory response. In assays for immunogenicity assessment, rMhSPase I unexpectedly showed a cytotoxic effect on murine splenocytes. This cytotoxic effect was further confirmed using the swine epithelial PK(15) cell line in MTT and annexin V-flow cytometry assays, which showed that rMhSPase I induces apoptosis in a dose dependent-way. It was also demonstrated that this pro-apoptotic effect of rMhSPase I involves activation of a caspase-3 cascade. The potential relevance of the rMhSPase I pro-apoptotic effect for M. hyopneumoniae-host interactions in the context of PEP is discussed. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Systems analysis of apoptotic priming in ovarian cancer identifies vulnerabilities and predictors of drug response.

    Science.gov (United States)

    Zervantonakis, Ioannis K; Iavarone, Claudia; Chen, Hsing-Yu; Selfors, Laura M; Palakurthi, Sangeetha; Liu, Joyce F; Drapkin, Ronny; Matulonis, Ursula; Leverson, Joel D; Sampath, Deepak; Mills, Gordon B; Brugge, Joan S

    2017-08-28

    The lack of effective chemotherapies for high-grade serous ovarian cancers (HGS-OvCa) has motivated a search for alternative treatment strategies. Here, we present an unbiased systems-approach to interrogate a panel of 14 well-annotated HGS-OvCa patient-derived xenografts for sensitivity to PI3K and PI3K/mTOR inhibitors and uncover cell death vulnerabilities. Proteomic analysis reveals that PI3K/mTOR inhibition in HGS-OvCa patient-derived xenografts induces both pro-apoptotic and anti-apoptotic signaling responses that limit cell killing, but also primes cells for inhibitors of anti-apoptotic proteins. In-depth quantitative analysis of BCL-2 family proteins and other apoptotic regulators, together with computational modeling and selective anti-apoptotic protein inhibitors, uncovers new mechanistic details about apoptotic regulators that are predictive of drug sensitivity (BIM, caspase-3, BCL-X L ) and resistance (MCL-1, XIAP). Our systems-approach presents a strategy for systematic analysis of the mechanisms that limit effective tumor cell killing and the identification of apoptotic vulnerabilities to overcome drug resistance in ovarian and other cancers.High-grade serous ovarian cancers (HGS-OvCa) frequently develop chemotherapy resistance. Here, the authors through a systematic analysis of proteomic and drug response data of 14 HGS-OvCa PDXs demonstrate that targeting apoptosis regulators can improve response of these tumors to inhibitors of the PI3K/mTOR pathway.

  14. Catalase inhibition in diabetic rats potentiates DNA damage and apoptotic cell death setting the stage for cardiomyopathy.

    Science.gov (United States)

    Ivanović-Matić, Svetlana; Bogojević, Desanka; Martinović, Vesna; Petrović, Anja; Jovanović-Stojanov, Sofija; Poznanović, Goran; Grigorov, Ilijana

    2014-12-01

    Diabetes is a risk factor for cardiovascular disease that has a multifactorial etiology, with oxidative stress as an important component. Our previous observation of a significant diabetes-related increase in rat cardiac catalase (CAT) activity suggested that CAT could play a major role in delaying the development of diabetic cardiomyopathy. Thus, in the present work, we examined the effects of the daily administration of the CAT inhibitor, 3-amino-1,2,4-triazole (1 mg/g), on the hearts of streptozotocin (STZ)-induced diabetic rats. Administration of CAT inhibitor was started from the 15th day after the last STZ treatment (40 mg/kg/5 days), and maintained until the end of the 4th or 6th weeks of diabetes. Compared to untreated diabetic rats, at the end of the observation period, CAT inhibition lowered the induced level of cardiac CAT activity to the basal level and decreased CAT protein expression, mediated through a decline in the nuclear factor erythroid-derived 2-like 2 /nuclear factor-kappa B p65 (Nrf2/NF-κB p65) subunit ratio. The perturbed antioxidant defenses resulting from CAT inhibition promoted increased H₂O₂production (P < 0.05) and lipid peroxidation (P < 0.05). Generated cytotoxic stimuli increased DNA damage (P < 0.05) and activated pro-apoptotic events, observed as a decrease (P < 0.05) in the ratio of the apoptosis regulator proteins Bcl-2/Bax, increased (P < 0.05) presence of the poly(ADP-ribose) polymerase-1 (PARP-1) 85 kDa apoptotic fragment and cytoplasmic levels of cytochrome C. These findings confirm an important function of CAT in the suppression of events leading to diabetes-promoted cardiac dysfunction and cardiomyopathy.

  15. Exposure to apoptotic activated CD4+ T cells induces maturation and APOBEC3G-mediated inhibition of HIV-1 infection in dendritic cells.

    Directory of Open Access Journals (Sweden)

    Venkatramanan Mohanram

    Full Text Available Dendritic cells (DCs are activated by signaling via pathogen-specific receptors or exposure to inflammatory mediators. Here we show that co-culturing DCs with apoptotic HIV-infected activated CD4(+ T cells (ApoInf or apoptotic uninfected activated CD4(+ T cells (ApoAct induced expression of co-stimulatory molecules and cytokine release. In addition, we measured a reduced HIV infection rate in DCs after co-culture with ApoAct. A prerequisite for reduced HIV infection in DCs was activation of CD4(+ T cells before apoptosis induction. DCs exposed to ApoAct or ApoInf secreted MIP-1α, MIP-1β, MCP-1, and TNF-α; this effect was retained in the presence of exogenous HIV. The ApoAct-mediated induction of co-stimulatory CD86 molecules and reduction of HIV infection in DCs were partially abrogated after blocking TNF-α using monoclonal antibodies. APOBEC3G expression in DCs was increased in co-cultures of DCs and ApoAct but not by apoptotic resting CD4(+ T cells (ApoRest. Silencing of APOBEC3G in DC abrogated the HIV inhibitory effect mediated by ApoAct. Sequence analyses of an env region revealed significant induction of G-to-A hypermutations in the context of GG or GA dinucleotides in DNA isolated from DCs exposed to HIV and ApoAct. Thus, ApoAct-mediated DC maturation resulted in induction of APOBEC3G that was important for inhibition of HIV-infection in DCs. These findings underscore the complexity of differential DC responses evoked upon interaction with resting as compared with activated dying cells during HIV infection.

  16. Activated cathepsin L is associated with the switch from autophagy to apoptotic death of SH-SY5Y cells exposed to 6-hydroxydopamine

    Energy Technology Data Exchange (ETDEWEB)

    Li, Lingyun, E-mail: lingyunlee@126.com [Department of Pharmacology, College of Pharmaceutical Sciences, Soochow University, Suzhou 215123 (China); Experimental Center, The Second Affiliated Hospital of Soochow University, Suzhou 215004 (China); Gao, Luyan [Experimental Center, The Second Affiliated Hospital of Soochow University, Suzhou 215004 (China); Song, Yunzhen; Qin, Zheng-Hong [Department of Pharmacology, College of Pharmaceutical Sciences, Soochow University, Suzhou 215123 (China); Liang, Zhongqin, E-mail: liangzhongqin@suda.edu.cn [Department of Pharmacology, College of Pharmaceutical Sciences, Soochow University, Suzhou 215123 (China)

    2016-02-12

    Autophagy and apoptosis are common responses to pathological damage in the process of Parkinson's disease (PD), and lysosome dysfunction may contribute to the etiology of PD's neurodegenerative process. In this study, we demonstrated that the neurotoxin 6-hydroxydopamine (6-OHDA) increased autophagy in SH-SY5Y cells, as determined by detection of the lysosome marker lysosomal-associated membrane protein1, the autophagy protein light chain 3 (LC3)-II and the autophagy substrate P62 protein. Meanwhile, autophagy repression with 3-methyladenine accelerated the activation of caspase-3 and PARP and aggravated the cell apoptotic death induced by 6-OHDA. Furthermore, we found that 6-OHDA treatment resulted in a transient increase in the intracellular and nuclear expression of cathepsin L (CTSL). The CTSL inhibitor, Z-FY-CHO, could promote autophagy, decrease accumulation of P62, and block activation of caspase-3 and PARP. Taken together, these results suggest that activation of autophagy may primarily be a protective process in SH-SY5Y cell death induced by 6-OHDA, and the nuclear translocation of CTSL could enhance the cell apoptotic cascade via disturbing autophagy-apoptotic systems in SH-SY5Y cells. Our findings highlight the potential role of CTSL in the cross talk between autophagy and apoptosis, which might be considered a therapeutic strategy for treatment of pathologic conditions associated with neurodegeneration. - Highlights: • Inhibition of autophagy aggravated the cell apoptotic death in SH-SY5Y cells. • Activation of cathepsin L impaired the autophagy pathway. • Activation of cathepsin L enhanced the cell apoptotic cascade. • Cathepsin L involves in the cross talk between autophagy and apoptosis.

  17. Apoptotic effect of a novel kefir product, PFT, on multidrug-resistant myeloid leukemia cells via a hole-piercing mechanism

    Science.gov (United States)

    GHONEUM, MAMDOOH; GIMZEWSKI, JAMES

    2014-01-01

    We examined the apoptotic effect of a novel Probiotics Fermentation Technology (PFT) kefir grain product; PFT is a natural mixture composed primarily of Lactobacillus kefiri P-IF, a specific strain of L. kefiri with unique growth characteristics. The aim of this study was to examine the apoptotic effect of PFT on human multidrug-resistant (MDR) myeloid leukemia (HL60/AR) cells in vitro and explore the mechanistic approach underlying its effect. HL60/AR cells were cultured with PFT (0.6–5.0 mg/ml) for 3 days. The apoptotic effect of PFT was assessed through examination of percent apoptosis, caspase 3 activation, Bcl-2 expression levels and changes in mitochondrial membrane potential (MMP). PFT induced apoptosis in HL60/AR cells in a dose-dependent manner which was maximal at 67.5% for 5 mg/ml. Induction of apoptosis was associated with activation of caspase 3, decreased expression of Bcl-2 and decreased polarization of MMP. In addition, PFT showed a unique characteristic of piercing holes in HL60/AR cells, as indicated by AFM studies. This hole induction may be responsible for the apoptotic effect on cancer cells. These results suggest that PFT may act as a potential therapy for the treatment of MDR leukemia. PMID:24430613

  18. Mitochondrial fission proteins regulate programmed cell death in yeast.

    Science.gov (United States)

    Fannjiang, Yihru; Cheng, Wen-Chih; Lee, Sarah J; Qi, Bing; Pevsner, Jonathan; McCaffery, J Michael; Hill, R Blake; Basañez, Gorka; Hardwick, J Marie

    2004-11-15

    The possibility that single-cell organisms undergo programmed cell death has been questioned in part because they lack several key components of the mammalian cell death machinery. However, yeast encode a homolog of human Drp1, a mitochondrial fission protein that was shown previously to promote mammalian cell death and the excessive mitochondrial fragmentation characteristic of apoptotic mammalian cells. In support of a primordial origin of programmed cell death involving mitochondria, we found that the Saccharomyces cerevisiae homolog of human Drp1, Dnm1, promotes mitochondrial fragmentation/degradation and cell death following treatment with several death stimuli. Two Dnm1-interacting factors also regulate yeast cell death. The WD40 repeat protein Mdv1/Net2 promotes cell death, consistent with its role in mitochondrial fission. In contrast to its fission function in healthy cells, Fis1 unexpectedly inhibits Dnm1-mediated mitochondrial fission and cysteine protease-dependent cell death in yeast. Furthermore, the ability of yeast Fis1 to inhibit mitochondrial fission and cell death can be functionally replaced by human Bcl-2 and Bcl-xL. Together, these findings indicate that yeast and mammalian cells have a conserved programmed death pathway regulated by a common molecular component, Drp1/Dnm1, that is inhibited by a Bcl-2-like function.

  19. Neurotoxic, cytotoxic, apoptotic and antiproliferative effects of some marine algae extracts on the NA2B cell line.

    Science.gov (United States)

    Kurt, O; Özdal-Kurt, F; Akçora, C M; Özkut, M; Tuğlu, M I

    2018-02-01

    Oxidative stress contributes to cancer pathologies and to apoptosis. Marine algae exhibit cytotoxic, antiproliferative and apoptotic effects; their metabolites have been used to treat many types of cancer. We investigated in culture extracts of Petalonia fascia, Jania longifurca and Halimeda tuna to determine their effects on mouse neuroblastoma cell line, NA2B. NA2B cells were treated with algae extracts, and the survival and proliferation of NA2B cells were assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The effects of algae extracts on oxidative stress in NA2B cells also were investigated using nitric oxide synthase (NOS) immunocytochemistry and apoptosis was assessed using terminal deoxynucleotidyl transferase dUTP nick end labeling. We observed significant neurite inhibition with moderate damage by the neurotoxicity-screening test (NST) at IC 50 dilutions of the extracts. MTT demonstrated that J. longifurca extracts were more toxic than P. fascia and H. tuna extracts. We found an increase of endothelial and inducible NOS immunostaining for oxidative stress and TUNEL analysis revealed increased apoptosis after application of extract. Our findings suggest that the algae we tested may have potential use for treatment of cancer.

  20. Substance P reduces apoptotic cell death possibly by modulating the immune response at the early stage after spinal cord injury.

    Science.gov (United States)

    Jiang, Mei Hua; Lim, Ji Eun; Chi, Guang Fan; Ahn, Woosung; Zhang, Mingzi; Chung, Eunkyung; Son, Youngsook

    2013-10-23

    Previously, we have reported that substance P (SP) enhanced functional recovery from spinal cord injury (SCI) possibly by the anti-inflammatory modulation associated with the induction of M2-type macrophages at the injured lesion. In this study, we explored the cytokine expression profiles and apoptotic cell death in the lesion site of the SCI after an immediate intravenous injection of SP. SP injection increased the levels of interleukin-4 (IL-4), IL-6, and IL-10 at day 1 after the SCI approximately by 2-, 9-, and 10-folds when compared with the control SCI, respectively. On the basis of double immunofluorescence staining with IL-10 and CD11b, activated macrophages or microglia expressing IL-10 appeared in the margin of the lesion site at day 1 only after the SP injection. This SP-mediated alteration in the lesion microenvironment was shown to be associated with the lower cell death of neuronal cells at day 1 and oligodendrocytes at day 5 by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, which was also accompanied by a decrease in caspase-3 activation. These findings suggest that SP may reduce the inflammation-induced secondary cell death, possibly through immune modulation at an early stage after the SCI.

  1. p70S6 kinase signals cell survival as well as growth, inactivating the pro-apoptotic molecule BAD

    DEFF Research Database (Denmark)

    Harada, H; Andersen, Jens S.; Mann, M

    2001-01-01

    Cytokines often deliver simultaneous, yet distinct, cell growth and cell survival signals. The 70-kDa ribosomal protein S6 kinase (p70S6K) is known to regulate cell growth by inducing protein synthesis components. We purified membrane-based p70S6K as a kinase responsible for site-specific phospho...

  2. The possible FAT1-mediated apoptotic pathways in porcine cumulus cells

    NARCIS (Netherlands)

    Wu, Xinhui; Fu, Yao; Liu, Chang; Chai, Menglong; Chen, Chengzhen; Dai, Lisheng; Gao, Yan; Jiang, Hao; Zhang, Jiabao

    Porcine cumulus cells are localized around oocytes and act as a specific type of granulosa that plays essential roles in the development and maturation of oocytes, the development and atresia of follicles, and the development of embryos. Studies of FAT1 have demonstrated its functions in cell-cell

  3. Comparative studies of cytotoxic and apoptotic properties of different extracts and the essential oil of Lavandula angustifolia on malignant and normal cells.

    Science.gov (United States)

    Tayarani-Najaran, Zahra; Amiri, Atefeh; Karimi, Gholamreza; Emami, Seyed Ahmad; Asili, Javad; Mousavi, Seyed Hadi

    2014-01-01

    Lavender (Lavandula angustifolia Mill.) is a bush-like shrub from Lamiaceae. The herb has been used in alternative medicine for several centuries. In this study, the cytotoxicity and the mechanisms of cell death induced by 3 different extracts of aerial parts and the essential oil of L. angustifolia were compared in normal and cancerous human cells. Malignant (HeLa and MCF-7 cell lines) and nonmalignant (human fibroblasts) cells were incubated with different concentrations of the plant extracts. Cell viability was quantified by MTS assay. Apoptotic cells were determined using propidium iodide staining of DNA fragmentation by flow cytometry (sub-G1 peak). The molecules as apoptotic signal translation, including Bax and cleaved PARP, were identified by Western blot. Ethanol and n-hexane extracts and essential oil exhibited significant cytotoxicity to malignant cells but marginal cytotoxicity to human fibroblasts in vitro and induced a sub-G1 peak in flow cytometry histogram of treated cells compared to the control. Western blot analysis demonstrated that EtOH and n-hexane extracts upregulated Bax expression, also it induced cleavage of PARP in HeLa cells compared to the control. In conclusion, L. angustifolia has cytotoxic and apoptotic effects in HeLa and MCF-7 cell lines, and apoptosis is proposed as the possible mechanism of action.

  4. Histone deacetylase inhibitors strongly sensitise neuroblastoma cells to TRAIL-induced apoptosis by a caspases-dependent increase of the pro- to anti-apoptotic proteins ratio

    International Nuclear Information System (INIS)

    Mühlethaler-Mottet, Annick; Flahaut, Marjorie; Bourloud, Katia Balmas; Auderset, Katya; Meier, Roland; Joseph, Jean-Marc; Gross, Nicole

    2006-01-01

    Neuroblastoma (NB) is the second most common solid childhood tumour, an aggressive disease for which new therapeutic strategies are strongly needed. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in most tumour cells, but not in normal tissues and therefore represents a valuable candidate in apoptosis-inducing therapies. Caspase-8 is silenced in a subset of highly malignant NB cells, which results in full TRAIL resistance. In addition, despite constitutive caspase-8 expression, or its possible restoration by different strategies, NB cells remain weakly sensitive to TRAIL indicating a need to develop strategies to sensitise NB cells to TRAIL. Histone deacetylase inhibitors (HDACIs) are a new class of anti-cancer agent inducing apoptosis or cell cycle arrest in tumour cells with very low toxicity toward normal cells. Although HDACIs were recently shown to increase death induced by TRAIL in weakly TRAIL-sensitive tumour cells, the precise involved sensitisation mechanisms have not been fully identified. NB cell lines were treated with various doses of HDACIs and TRAIL, then cytotoxicity was analysed by MTS/PMS proliferation assays, apoptosis was measured by the Propidium staining method, caspases activity by colorimetric protease assays, and (in)activation of apoptotic proteins by immunoblotting. Sub-toxic doses of HDACIs strongly sensitised caspase-8 positive NB cell lines to TRAIL induced apoptosis in a caspases dependent manner. Combined treatments increased the activation of caspases and Bid, and the inactivation of the anti-apoptotic proteins XIAP, Bcl-x, RIP, and survivin, thereby increasing the pro- to anti-apoptotic protein ratio. It also enhanced the activation of the mitochondrial pathway. Interestingly, the kinetics of caspases activation and inactivation of anti-apoptotic proteins is accelerated by combined treatment with TRAIL and HDACIs compared to TRAIL alone. In contrast, cell surface expression of TRAIL

  5. Zinc ferrite nanoparticles activate IL-1b, NFKB1, CCL21 and NOS2 signaling to induce mitochondrial dependent intrinsic apoptotic pathway in WISH cells

    Energy Technology Data Exchange (ETDEWEB)

    Saquib, Quaiser; Al-Khedhairy, Abdulaziz A.; Ahmad, Javed; Siddiqui, Maqsood A.; Dwivedi, Sourabh; Khan, Shams T. [Department of Zoology, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451 (Saudi Arabia); Chair for DNA Research, Department of Zoology, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451 (Saudi Arabia); Musarrat, Javed, E-mail: musarratj1@yahoo.com [Chair for DNA Research, Department of Zoology, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451 (Saudi Arabia); Department of Agricultural Microbiology, Faculty of Agricultural Sciences, Aligarh Muslim University, Aligarh 202002, U.P. (India)

    2013-12-01

    The present study has demonstrated the translocation of zinc ferrite nanoparticles (ZnFe{sub 2}O{sub 4}-NPs) into the cytoplasm of human amnion epithelial (WISH) cells, and the ensuing cytotoxicity and genetic damage. The results suggested that in situ NPs induced oxidative stress, alterations in cellular membrane and DNA strand breaks. The [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) and neutral red uptake (NRU) cytotoxicity assays indicated 64.48 ± 1.6% and 50.73 ± 2.1% reduction in cell viability with 100 μg/ml of ZnFe{sub 2}O{sub 4}-NPs exposure. The treated WISH cells exhibited 1.2-fold higher ROS level with 0.9-fold decline in membrane potential (ΔΨm) and 7.4-fold higher DNA damage after 48 h of ZnFe{sub 2}O{sub 4}-NPs treatment. Real-time PCR (qPCR) analysis of p53, CASP 3 (caspase-3), and bax genes revealed 5.3, 1.6, and 14.9-fold upregulation, and 0.18-fold down regulation of bcl 2 gene vis-à-vis untreated control. RT{sup 2} Profiler™ PCR array data elucidated differential up-regulation of mRNA transcripts of IL-1b, NFKB1, NOS2 and CCL21 genes in the range of 1.5 to 3.7-folds. The flow cytometry based cell cycle analysis suggested the transfer of 15.2 ± 2.1% (p < 0.01) population of ZnFe{sub 2}O{sub 4}-NPs (100 μg/ml) treated cells into apoptotic phase through intrinsic pathway. Over all, the data revealed the potential of ZnFe{sub 2}O{sub 4}-NPs to induce cellular and genetic toxicity in cells of placental origin. Thus, the significant ROS production, reduction in ΔΨm, DNA damage, and activation of genes linked to inflammation, oxidative stress, proliferation, DNA damage and repair could serve as the predictive toxicity and stress markers for ecotoxicological assessment of ZnFe{sub 2}O{sub 4}-NPs induced cellular and genetic damage. - Highlights: • First report on the molecular toxicity of ZnFe{sub 2}O{sub 4}-NPs in cells of placental origin • WISH cells treated with ZnFe{sub 2}O{sub 4}-NPs exhibited cytoplasmic

  6. Induction of Pro-Apoptotic Endoplasmic Reticulum Stress in Multiple Myeloma Cells by NEO214, Perillyl Alcohol Conjugated to Rolipram.

    Science.gov (United States)

    Chen, Thomas C; Chan, Nymph; Labib, Shirin; Yu, Jiali; Cho, Hee-Yeon; Hofman, Florence M; Schönthal, Axel H

    2018-01-17

    Despite the introduction of new therapies for multiple myeloma (MM), many patients are still dying from this disease and novel treatments are urgently needed. We have designed a novel hybrid molecule, called NEO214, that was generated by covalent conjugation of the natural monoterpene perillyl alcohol (POH), an inducer of endoplasmic reticulum (ER) stress, to rolipram (Rp), an inhibitor of phosphodiesterase-4 (PDE4). Its potential anticancer effects were investigated in a panel of MM cell lines. We found that NEO214 effectively killed MM cells in vitro with a potency that was over an order of magnitude stronger than that of its individual components, either alone or in combination. The cytotoxic mechanism of NEO214 involved severe ER stress and prolonged induction of CCAAT/enhancer-binding protein homologous protein (CHOP), a key pro-apoptotic component of the ER stress response. These effects were prevented by salubrinal, a pharmacologic inhibitor of ER stress, and by CHOP gene knockout. Conversely, combination of NEO214 with bortezomib, a drug in clinical use for patients with MM, resulted in synergistic enhancement of MM cell death. Combination with the adenylate cyclase stimulant forskolin did not enhance NEO214 impact, indicating that cyclic adenosine 3',5'-monophosphate (AMP) pathways might play a lesser role. Our study introduces the novel agent NEO214 as a potent inducer of ER stress with significant anti-MM activity in vitro. It should be further investigated as a potential MM therapy aimed at exploiting this tumor's distinct sensitivity to ER stress.

  7. Autophagy Enhances the Aggressiveness of Human Colorectal Cancer Cells and Their Ability to Adapt to Apoptotic Stimulus

    International Nuclear Information System (INIS)

    Zheng, Hai-yang; Zhang, Xiao-yang; Wang, Xing-fen; Sun, Bao-cun

    2012-01-01

    To investigate LC3B-II and active caspase-3 expression in human colorectal cancer to elucidate the role of autophagy, and to explore the relationship of autophagy with apoptosis in human colorectal cancer. LC3B expression was detected by immunohistochemistry in 53 human colorectal cancer tissues and 20 normal colon tissues. The protein levels of LC3B-II and active caspase-3 were also determined by Western blot analysis in 23 human colorectal cancer tissues and 10 normal colon tissues. LC3B was expressed both in cancer cells and normal epithelial cells. LC3B expression in the peripheral area of cancer tissues was correlated with several clinicopathological factors, including tumor differentiation (P=0.002), growth pattern of the tumor margin (P=0.028), pN (P=0.002), pStage (P=0.032), as well as vessel and nerve plexus invasion (P=0.002). The protein level of LC3B-II in cancer tissue was significantly higher than in normal tissue (P=0.038), but the expression of active forms of procaspase-3 in cancer tissue was lower (P=0.041). There was a statistically significant positive correlation between the expression levels of LC3B-II and the active forms of procaspase-3 (r=0.537, P=0.008). Autophagy has a prosurvival role in human colorectal cancer. Autophagy enhances the aggressiveness of colorectal cancer cells and their ability to adapt to apoptotic stimulus

  8. Pterocarpans phaseollin and neorautenol isolated from Erythrina addisoniae induce apoptotic cell death accompanied by inhibition of ERK phosphorylation

    International Nuclear Information System (INIS)

    Waetjen, W.; Kulawik, A.; Suckow-Schnitker, A.K.; Chovolou, Y.; Rohrig, R.; Ruhl, S.; Kampkoetter, A.; Addae-Kyereme, J.; Wright, C.W.; Passreiter, C.M.

    2007-01-01

    The genus Erythrina (Leguminosae), consisting of over 100 different species, is distributed in tropical regions. In traditional medicine, Erythrina species are used to treat cancer, but little is known about the anticancer mechanisms. From the stem bark of Erythrina addisoniae Hutch. and Dalziel, six prenylated pterocarpans were isolated and analysed for pharmacological activity: While calopocarpin, cristacarpin, orientanol c, and isoneorautenol showed only a weak or moderate toxicity in H4IIE hepatoma cells (EC 50 -value > 25 μM), the toxicity of neorautenol and phaseollin was in the low micromolar range (EC 50 -value: 1 and 1.5 μM, respectively). We further focused on these two substances showing that both increased caspase 3/7 activity and nuclear fragmentation as markers for apoptotic cell death. Neorautenol (10 μM, 2 h), but not phaseollin induced the formation of DNA strand breaks (comet assay). Both substances showed no effect on NF-κB signalling (SEAP assay: basal activity and stimulation with TNF-α), on the other hand both pterocarpans (10 μM, 2 h) decreased the activation of the ERK kinase (p44/p42), an mitogen activated protein kinase which is associated with cell proliferation. We conclude that the pterocarpans phaseollin and neorautenol may be responsible for the anticarcinogenic actions of the plant extract reported in the literature. Further analysis of these substances may lead to new pharmacons to be used in cancer therapy

  9. Hepatic progenitor cell resistance to TGF-β1's proliferative and apoptotic effects

    International Nuclear Information System (INIS)

    Clark, J. Brian; Rice, Lisa; Sadiq, Tim; Brittain, Evan; Song, Lujun; Wang Jian; Gerber, David A.

    2005-01-01

    The success of hepatocellular therapies using stem or progenitor cell populations is dependent upon multiple factors including the donor cell, microenvironment, and etiology of the liver injury. The following experiments investigated the impact of TGF-β1 on a previously described population of hepatic progenitor cells (HPC). The majority of the hepatic progenitor cells were resistant to endogenously produced TGF-β1's proapoptotic and anti-proliferative effects unlike more well-differentiated cellular populations (e.g., mature hepatocytes). Surprisingly, in vitro TGF-β1 supplementation significantly inhibited de novo hepatic progenitor cell colony formation possibly via an indirect mechanism(s). Therefore despite the HPC's direct resistance to supplemental TGF-β1, this cytokine's inhibitory effect on colony formation could have a potential negative impact on the use of these cells as a therapy for patients with liver disease

  10. Expression of apoptotic genes in immature and in vitro matured equine oocytes and cumulus cells.

    Science.gov (United States)

    Leon, P M M; Campos, V F; Kaefer, C; Begnini, K R; McBride, A J A; Dellagostin, O A; Seixas, F K; Deschamps, J C; Collares, T

    2013-08-01

    The gene expression of Bax, Bcl-2, survivin and p53, following in vitro maturation of equine oocytes, was compared in morphologically distinct oocytes and cumulus cells. Cumulus-oocyte complexes (COC) were harvested and divided into two groups: G1 - morphologically healthy cells; and G2 - less viable cells or cells with some degree of atresia. Total RNA was isolated from both immature and in vitro matured COC and real-time reverse transcription polymerase chain reaction (qRT-PCR) was used to quantify gene expression. Our results showed there was significantly higher expression of survivin (P < 0.05) and lower expression of p53 (P < 0.01) in oocytes compared with cumulus cells in G1. No significant difference in gene expression was observed following in vitro maturation or in COC derived from G1 and G2. However, expression of the Bax gene was significantly higher in cumulus cells from G1 (P < 0.02).

  11. Cancer Vaccine Composed of Oligonucleotides Conjugated to Apoptotic Tumor Cells | NCI Technology Transfer Center | TTC

    Science.gov (United States)

    Synthetic oligodeoxynucleotides (ODN) containing unmethylated Cytosine-Guanine (CpG) motifs mimic the immunostimulatory activity of bacterial DNA. CpG ODN directly stimulate B cells and plasmacytoid dendritic cells (pDC), promote the production of T Helper 1 cells (Th1) and pro-inflammatory cytokines, and  trigger the maturation/activation of professional antigen presenting cells. The National Cancer Institute, Laboratory of Experimental Immunology, seeks interested parties to co- develop methods for inducing an immune response to tumors.

  12. The Inhibition of microRNA-128 on IGF-1-Activating mTOR Signaling Involves in Temozolomide-Induced Glioma Cell Apoptotic Death.

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    Peng-Hsu Chen

    Full Text Available Temozolomide (TMZ, an alkylating agent of the imidazotetrazine series, is a first-line chemotherapeutic drug used in the clinical therapy of glioblastoma multiforme, the most common and high-grade primary glioma in adults. Micro (miRNAs, which are small noncoding RNAs, post-transcriptionally regulate gene expressions and are involved in gliomagenesis. However, no studies have reported relationships between TMZ and miRNA gene regulation. We investigated TMZ-mediated miRNA profiles and its molecular mechanisms underlying the induction of glioma cell death. By performing miRNA microarray and bioinformatics analyses, we observed that expression of 248 miRNAs was altered, including five significantly upregulated and 17 significantly downregulated miRNAs, in TMZ-treated U87MG cells. miR-128 expression levels were lower in different glioma cells and strongly associated with poor survival. TMZ treatment significantly upregulated miR-128 expression. TMZ significantly enhanced miR-128-1 promoter activity and transcriptionally regulated miR-128 levels through c-Jun N-terminal kinase 2/c-Jun pathways. The overexpression and knockdown of miR-128 expression significantly affected TMZ-mediated cell viability and apoptosis-related protein expression. Furthermore, the overexpression of miR-128 alone enhanced apoptotic death of glioma cells through caspase-3/9 activation, poly(ADP ribose polymerase degradation, reactive oxygen species generation, mitochondrial membrane potential loss, and non-protective autophagy formation. Finally, we identified that key members in mammalian target of rapamycin (mTOR signaling including mTOR, rapamycin-insensitive companion of mTOR, insulin-like growth factor 1, and PIK3R1, but not PDK1, were direct target genes of miR-128. TMZ inhibited mTOR signaling through miR-128 regulation. These results indicate that miR-128-inhibited mTOR signaling is involved in TMZ-mediated cytotoxicity. Our findings may provide a better understanding

  13. Sodium phenylbutyrate prolongs survival and regulates expression of anti-apoptotic genes in transgenic amyotrophic lateral sclerosis mice.

    Science.gov (United States)

    Ryu, Hoon; Smith, Karen; Camelo, Sandra I; Carreras, Isabel; Lee, Junghee; Iglesias, Antonio H; Dangond, Fernando; Cormier, Kerry A; Cudkowicz, Merit E; Brown, Robert H; Ferrante, Robert J

    2005-06-01

    Multiple molecular defects trigger cell death in amyotrophic lateral sclerosis (ALS). Among these, altered transcriptional activity may perturb many cellular functions, leading to a cascade of secondary pathological effects. We showed that pharmacological treatment, using the histone deacetylase inhibitor sodium phenylbutyrate, significantly extended survival and improved both the clinical and neuropathological phenotypes in G93A transgenic ALS mice. Phenylbutyrate administration ameliorated histone hypoacetylation observed in G93A mice and induced expression of nuclear factor-kappaB (NF-kappaB) p50, the phosphorylated inhibitory subunit of NF-kappaB (pIkappaB) and beta cell lymphoma 2 (bcl-2), but reduced cytochrome c and caspase expression. Curcumin, an NF-kappaB inhibitor, and mutation of the NF-kappaB responsive element in the bcl-2 promoter, blocked butyrate-induced bcl-2 promoter activity. We provide evidence that the pharmacological induction of NF-kappaB-dependent transcription and bcl-2 gene expression is neuroprotective in ALS mice by inhibiting programmed cell death. Phenylbutyrate acts to phosphorylate IkappaB, translocating NF-kappaB p50 to the nucleus, or to directly acetylate NF-kappaB p50. NF-kappaB p50 transactivates bcl-2 gene expression. Up-regulated bcl-2 blocks cytochrome c release and subsequent caspase activation, slowing motor neuron death. These transcriptional and post-translational pathways ultimately promote motor neuron survival and ameliorate disease progression in ALS mice. Phenylbutyrate may therefore provide a novel therapeutic approach for the treatment of patients with ALS.

  14. Identification of Proliferative and Apoptotic Sertoli Cells Using Fluorescence and Confocal Microscopy.

    Science.gov (United States)

    Martínez-Hernández, Jesús; Seco-Rovira, Vicente; Beltrán-Frutos, Ester; Quesada-Cubo, Victor; Ferrer, Concepción; Pastor, Luis Miguel

    2018-01-01

    Sertoli cells, the testicular somatic cells of the seminiferous epithelium, are vital for the survival of the epithelium. They undergo proliferation and apoptosis during fetal, neonatal, and prepubertal development. Apoptosis is increased in certain situations such as exposure to many substances, for example, toxics, or short photoperiod in the non-breeding season of some mammals. Therefore, it has always been considered that Sertoli cells that reach adulthood are quiescent cells, that is to say, nonproliferative, do not die, are terminally differentiated, and whose numbers remain constant. Recently, a degree of both proliferation and apoptosis has been observed in normal adult conditions, suggesting that consideration of this cell as quiescent may be subject to change. All this make it necessary to use histochemical techniques to demonstrate whether Sertoli cells are undergoing proliferation or apoptosis in histological sections and to allow the qualitative and quantitative study of these. In this chapter, we present two double-staining techniques that can be used for identifying Sertoli cells in proliferation or apoptosis by fluorescence microscopy. In both, the Sertoli cells are identified by an immunohistochemistry for vimentin followed by an immunohistochemistry for PCNA or a TUNEL histochemistry.

  15. Lack of a Functional VHL Gene Product Sensitizes Renal Cell Carcinoma Cells to the Apoptotic Effects of the Protein Synthesis Inhibitor Verrucarin A

    Directory of Open Access Journals (Sweden)

    Girma M. Woldemichael

    2012-08-01

    Full Text Available Verrucarin A (VA is a small molecule derived from the fungal plant pathogen Myrothecium verrucaria and was identified as a selective inhibitor of clear cell renal cell carcinoma (CCRCC cell proliferation in a high-throughput screen of a library of naturally occurring small molecules. CCRCC arises as a result of loss-of-function mutations in the von Hippel-Lindau (VHL gene. Here we show that VA inhibits protein translation initiation culminating in apoptosis through the extrinsic signaling pathway. Reintroduction of the VHL gene in CCRCC cells afforded resistance to VA's apoptotic effects. This resistance is mediated in part by the formation of stress granules that entrap signaling molecules that initiate the apoptotic signaling cascade. The VHL gene product was found to be a component of stress granules that develop as result of VA treatment. These findings reveal an important role for the VHL gene product in cytotoxic stress response and have important implications for the rational development of VA-related compounds in chemotherapeutic targeting of CCRCC.

  16. Lack of a functional VHL gene product sensitizes renal cell carcinoma cells to the apoptotic effects of the protein synthesis inhibitor verrucarin A.

    Science.gov (United States)

    Woldemichael, Girma M; Turbyville, Thomas J; Vasselli, James R; Linehan, W Marston; McMahon, James B

    2012-08-01

    Verrucarin A (VA) is a small molecule derived from the fungal plant pathogen Myrothecium verrucaria and was identified as a selective inhibitor of clear cell renal cell carcinoma (CCRCC) cell proliferation in a high-throughput screen of a library of naturally occurring small molecules. CCRCC arises as a result of loss-of-function mutations in the von Hippel-Lindau (VHL) gene. Here we show that VA inhibits protein translation initiation culminating in apoptosis through the extrinsic signaling pathway. Reintroduction of the VHL gene in CCRCC cells afforded resistance to VA's apoptotic effects. This resistance is mediated in part by the formation of stress granules that entrap signaling molecules that initiate the apoptotic signaling cascade. The VHL gene product was found to be a component of stress granules that develop as result of VA treatment. These findings reveal an important role for the VHL gene product in cytotoxic stress response and have important implications for the rational development of VA-related compounds in chemotherapeutic targeting of CCRCC.

  17. Lack of a Functional VHL Gene Product Sensitizes Renal Cell Carcinoma Cells to the Apoptotic Effects of the Protein Synthesis Inhibitor Verrucarin A12

    Science.gov (United States)

    Woldemichael, Girma M; Turbyville, Thomas J; Vasselli, James R; Linehan, W Marston; McMahon, James B

    2012-01-01

    Verrucarin A (VA) is a small molecule derived from the fungal plant pathogen Myrothecium verrucaria and was identified as a selective inhibitor of clear cell renal cell carcinoma (CCRCC) cell proliferation in a high-throughput screen of a library of naturally occurring small molecules. CCRCC arises as a result of loss-of-function mutations in the von Hippel-Lindau (VHL) gene. Here we show that VA inhibits protein translation initiation culminating in apoptosis through the extrinsic signaling pathway. Reintroduction of the VHL gene in CCRCC cells afforded resistance to VA's apoptotic effects. This resistance is mediated in part by the formation of stress granules that entrap signaling molecules that initiate the apoptotic signaling cascade. The VHL gene product was found to be a component of stress granules that develop as result of VA treatment. These findings reveal an important role for the VHL gene product in cytotoxic stress response and have important implications for the rational development of VA-related compounds in chemotherapeutic targeting of CCRCC. PMID:22952429

  18. Regulation of cell wall biosynthesis.

    Science.gov (United States)

    Zhong, Ruiqin; Ye, Zheng-Hua

    2007-12-01

    Plant cell walls differ in their amount and composition among various cell types and even in different microdomains of the wall of a given cell. Plants must have evolved regulatory mechanisms controlling biosynthesis, targeted secretion, and assembly of wall components to achieve the heterogeneity in cell walls. A number of factors, including hormones, the cytoskeleton, glycosylphosphatidylinositol-anchored proteins, phosphoinositides, and sugar nucleotide supply, have been implicated in the regulation of cell wall biosynthesis or deposition. In the past two years, there have been important discoveries in transcriptional regulation of secondary wall biosynthesis. Several transcription factors in the NAC and MYB families have been shown to be the key switches for activation of secondary wall biosynthesis. These studies suggest a transcriptional network comprised of a hierarchy of transcription factors is involved in regulating secondary wall biosynthesis. Further investigation and integration of the regulatory players participating in the making of cell walls will certainly lead to our understanding of how wall amounts and composition are controlled in a given cell type. This may eventually allow custom design of plant cell walls on the basis of our needs.

  19. Regulators of Tfh cell differentiation

    Directory of Open Access Journals (Sweden)

    Gajendra Motiram Jogdand

    2016-11-01

    Full Text Available The follicular helper T (Tfh cells help is critical for activation of B cells, antibody class switching and germinal center formation. The Tfh cells are characterized by the expression of CXCR5, ICOS, PD-1, Bcl-6, and IL-21. They are involved in clearing infections and are adversely linked with autoimmune diseases and also have a role in viral replication as well as clearance. Tfh cells are generated from naïve CD4 T cells with sequential steps involving cytokine signaling (IL-21, IL-6, IL-12, activin A, migration and positioning in the germinal center by CXCR5, surface receptors (ICOS/ICOSL, SAP/SLAM as well as transcription factor (Bcl-6, c-Maf, STAT3 signaling and repressor miR155. On the other hand Tfh generation is negatively regulated at specific steps of Tfh generation by specific cytokine (IL-2, IL-7, surface receptor (PD-1, CTLA-4, transcription factors Blimp-1, STAT5, T-bet, KLF-2 signaling and repressor miR 146a. Interestingly, miR 17-92 and FOXO1 acts as a positive as well as a negative regulator of Tfh differentiation depending on the time of expression and disease specificity. Tfh cells are also generated from the conversion of other effector T cells as exemplified by Th1 cells converting into Tfh during viral infection. The mechanistic details of effector T cells conversion into Tfh are yet to be clear. To manipulate Tfh cells for therapeutic implication and or for effective vaccination strategies, it is important to know positive and negative regulators of Tfh generation. Hence, in this review we have highlighted and interlinked molecular signaling from cytokines, surface receptors, transcription factors, ubiquitin Ligase and miRNA as positive and negative regulators for Tfh differentiation.

  20. Antigen-specific primed cytotoxic T cells eliminate tumour cells in vivo and prevent tumour development, regardless of the presence of anti-apoptotic mutations conferring drug resistance.

    Science.gov (United States)

    Jaime-Sánchez, Paula; Catalán, Elena; Uranga-Murillo, Iratxe; Aguiló, Nacho; Santiago, Llipsy; M Lanuza, Pilar; de Miguel, Diego; A Arias, Maykel; Pardo, Julián

    2018-05-09

    Cytotoxic CD8 + T (Tc) cells are the main executors of transformed and cancer cells during cancer immunotherapy. The latest clinical results evidence a high efficacy of novel immunotherapy agents that modulate Tc cell activity against bad prognosis cancers. However, it has not been determined yet whether the efficacy of these treatments can be affected by selection of tumoural cells with mutations in the cell death machinery, known to promote drug resistance and cancer recurrence. Here, using a model of prophylactic tumour vaccination based on the LCMV-gp33 antigen and the mouse EL4 T lymphoma, we analysed the molecular mechanism employed by Tc cells to eliminate cancer cells in vivo and the impact of mutations in the apoptotic machinery on tumour development. First of all, we found that Tc cells, and perf and gzmB are required to efficiently eliminate EL4.gp33 cells after LCMV immunisation during short-term assays (1-4 h), and to prevent tumour development in the long term. Furthermore, we show that antigen-pulsed chemoresistant EL4 cells overexpressing Bcl-X L or a dominant negative form of caspase-3 are specifically eliminated from the peritoneum of infected animals, as fast as parental EL4 cells. Notably, antigen-specific Tc cells control the tumour growth of the mutated cells, as efficiently as in the case of parental cells. Altogether, expression of the anti-apoptotic mutations does not confer any advantage for tumour cells neither in the short-term survival nor in long-term tumour formation. Although the mechanism involved in the elimination of the apoptosis-resistant tumour cells is not completely elucidated, neither necroptosis nor pyroptosis seem to be involved. Our results provide the first experimental proof that chemoresistant cancer cells with mutations in the main cell death pathways are efficiently eliminated by Ag-specific Tc cells in vivo during immunotherapy and, thus, provide the molecular basis to treat chemoresistant cancer cells with CD8 Tc

  1. Synthesis, characterization and apoptotic activity of quinazolinone Schiff base derivatives toward MCF-7 cells via intrinsic and extrinsic apoptosis pathways

    Science.gov (United States)

    Zahedifard, Maryam; Lafta Faraj, Fadhil; Paydar, Mohammadjavad; Yeng Looi, Chung; Hajrezaei, Maryam; Hasanpourghadi, Mohadeseh; Kamalidehghan, Behnam; Abdul Majid, Nazia; Mohd Ali, Hapipah; Ameen Abdulla, Mahmood

    2015-01-01

    The current study investigated the cytotoxic effect of 3-(5-chloro-2-hydroxybenzylideneamino)-2-(5-chloro-2-hydroxyphenyl)-2,3-dihydroquinazolin-41(H)-one (A) and 3-(5-nitro-2-hydroxybenzylideneamino)-2-(5-nitro-2-hydroxyphenyl)-2,3-dihydroquinazolin-4(1H)-one (B) on MCF-7, MDA-MB-231, MCF-10A and WRL-68 cells. The mechanism involved in apoptosis was assessed to evaluate the possible pathways induced by compound A and B. MTT assay results using A and B showed significant inhibition of MCF-7 cell viability, with IC50 values of 3. 27 ± 0.171 and 4.36 ± 0.219 μg/mL, respectively, after a 72 hour treatment period. Compound A and B did not demonstrate significant cytotoxic effects towards MDA-MB-231, WRL-68 and MCF-10A cells. Acute toxicity tests also revealed an absence of toxic effects on mice. Fluorescent microscopic studies confirmed distinct morphological changes (membrane blebbing and chromosome condensation) corresponding to typical apoptotic features in treated MCF-7 cells. Using Cellomics High Content Screening (HCS), we found that compound A and B could trigger the release of cytochrome c from mitochondria to the cytosol. The release of cytochrome c activated the expression of caspases-9 and then stimulated downstream executioner caspase-3/7. In addition, caspase-8 showed remarkable activity, followed by inhibition of NF-κB activation in A-and B-treated MCF-7 cells. The results indicated that A and B could induce apoptosis via a mechanism that involves either extrinsic or intrinsic pathways. PMID:26108872

  2. Dendritic cells take up and present antigens from viable and apoptotic polymorphonuclear leukocytes.

    Directory of Open Access Journals (Sweden)

    Carlos Alfaro

    Full Text Available Dendritic cells (DC are endowed with the ability to cross-present antigens from other cell types to cognate T cells. DC are poised to meet polymorphonuclear leukocytes (PMNs as a result of being co-attracted by interleukin-8 (IL-8, for instance as produced by tumor cells or infected tissue. Human monocyte-derived and mouse bone marrow-derived DC can readily internalize viable or UV-irradiated PMNs. Such internalization was abrogated at 4°C and partly inhibited by anti-CD18 mAb. In mice, DC which had internalized PMNs containing electroporated ovalbumin (OVA protein, were able to cross-present the antigen to CD8 (OT-1 and CD4 (OT-2 TCR-transgenic T cells. Moreover, in humans, tumor cell debris is internalized by PMNs and the tumor-cell material can be subsequently taken up from the immunomagnetically re-isolated PMNs by DC. Importantly, if human neutrophils had endocytosed bacteria, they were able to trigger the maturation program of the DC. Moreover, when mouse PMNs with E. coli in their interior are co-injected in the foot pad with DC, many DC loaded with fluorescent material from the PMNs reach draining lymph nodes. Using CT26 (H-2(d mouse tumor cells, it was observed that if tumor cells are intracellularly loaded with OVA protein and UV-irradiated, they become phagocytic prey of H-2(d PMNs. If such PMNs, that cannot present antigens to OT-1 T cells, are immunomagnetically re-isolated and phagocytosed by H-2(b DC, such DC productively cross-present OVA antigen determinants to OT-1 T cells. Cross-presentation to adoptively transferred OT-1 lymphocytes at draining lymph nodes also take place when OVA-loaded PMNs (H-2(d are coinjected in the footpad of mice with autologous DC (H-2(b. In summary, our results indicate that antigens phagocytosed by short-lived PMNs can be in turn internalized and productively cross-presented by DC.

  3. Apoptotic Effect of Anti myeloma Polyclonal Antibodies on The Growth of Myeloma Cells

    International Nuclear Information System (INIS)

    Abd El-Ghany, I.Y.; El-Kolaly, M.T.; Moustafa, K.A.; El-Shershaby, H.M.; Sayed, A.A.; Borai, I.H.; El-Lahloby, N.M.

    2013-01-01

    Multiple myeloma (MM) is a malignancy characterized by proliferation of plasma cells. Cancer immunotherapy is a major branch of biological therapy that utilizes living cells and their products. The aim of this study is to produce and evaluate the antiproliferative effect of anti myeloma polyclonal antibodies (with and without labelling with radioactive isotopes) against the growth of myeloma cells. The production of polyclonal antibodies (PAb) was generated by immunizing five healthy female mature white New-Zealand rabbits with myeloma cells (SP2/OR) through primary injection and five booster doses. The preparation of labelled anti myeloma antibodies was carried out using chloramine-T method and it was purified using PD-10 chromatographic column. The results obtained revealed that anti myeloma polyclonal antibodies inhibited proliferation and induced apoptosis of myeloma cell lines in vitro and induced apoptosis after serial intraperitoneal injection of PAb in ascites bearing mice in vivo. The present study suggested that the effect of labelled anti myeloma antibodies on myeloma cells growth inhibition was more effective than that of anti myeloma antibodies without labelling which is due to the cytotoxic effect of ionizing radiation. Apoptosis triggered by PAb was confirmed by flow cytometry, caspase -8 and -9 and β2-microglobulin.

  4. X-linked inhibitor of apoptosis regulates T cell effector function

    DEFF Research Database (Denmark)

    Zehntner, Simone P; Bourbonnière, Lyne; Moore, Craig S

    2007-01-01

    To understand how the balance between pro- and anti-apoptotic signals influences effector function in the immune system, we studied the X-linked inhibitor of apoptosis (XIAP), an endogenous regulator of cellular apoptosis. Real-time PCR showed increased XIAP expression in blood of mice with exper......To understand how the balance between pro- and anti-apoptotic signals influences effector function in the immune system, we studied the X-linked inhibitor of apoptosis (XIAP), an endogenous regulator of cellular apoptosis. Real-time PCR showed increased XIAP expression in blood of mice...... dramatically reduced within the CNS. Flow cytometry showed an 88-93% reduction in T cells. The proportion of TUNEL(+) apoptotic CD4(+) T cells in the CNS was increased from Neurons...... and oligodendrocytes were not affected; neither did apoptosis increase in liver, where XIAP knockdown also occurred. ASO-XIAP increased susceptibility of T cells to activation-induced apoptosis in vitro. Our results identify XIAP as a critical controller of apoptotic susceptibility of effector T cell function...

  5. CREB mediates the insulinotropic and anti-apoptotic effects of GLP-1 signaling in adult mouse β-cells

    Directory of Open Access Journals (Sweden)

    Soona Shin

    2014-11-01

    Conclusions: In sum, our studies using conditional gene deletion put into question current notions about the importance of CREB in regulating β-cell function and mass. However, we reveal an important role for CREB in the β-cell response to GLP-1 receptor signaling, further validating CREB as a therapeutic target for diabetes.

  6. Human decidual stromal cells secrete soluble pro-apoptotic factors during decidualization in a cAMP-dependent manner.

    Science.gov (United States)

    Leno-Durán, E; Ruiz-Magaña, M J; Muñoz-Fernández, R; Requena, F; Olivares, E G; Ruiz-Ruiz, C

    2014-10-10

    Is there a relationship between decidualization and apoptosis of decidual stromal cells (DSC)? Decidualization triggers the secretion of soluble factors that induce apoptosis in DSC. The differentiation and apoptosis of DSC during decidualization of the receptive decidua are crucial processes for the controlled invasion of trophoblasts in normal pregnancy. Most DSC regress in a time-dependent manner, and their removal is important to provide space for the embryo to grow. However, the mechanism that controls DSC death is poorly understood. The apoptotic response of DSC was analyzed after exposure to different exogenous agents and during decidualization. The apoptotic potential of decidualized DSC supernatants and prolactin (PRL) was also evaluated. DSC lines were established from samples of decidua from first trimester pregnancies. Apoptosis was assayed by flow cytometry. PRL production, as a marker of decidualization, was determined by enzyme-linked immunosorbent assay. DSCs were resistant to a variety of apoptosis-inducing substances. Nevertheless, DSC underwent apoptosis during decidualization in culture, with cAMP being essential for both apoptosis and differentiation. In addition, culture supernatants from decidualized DSC induced apoptosis in undifferentiated DSC, although paradoxically these supernatants decreased the spontaneous apoptosis of decidual lymphocytes. Exogenously added PRL did not induce apoptosis in DSC and an antibody that neutralized the PRL receptor did not decrease the apoptosis induced by supernatants. Further studies are needed to examine the involvement of other soluble factors secreted by decidualized DSC in the induction of apoptosis. The present results indicate that apoptosis of DSC occurs in parallel to differentiation, in response to decidualization signals, with soluble factors secreted by decidualized DSC being responsible for triggering cell death. These studies are relevant in the understanding of how the regression of decidua

  7. Cytotoxicity and Apoptotic Effects of Polyphenols from Sugar Beet Molasses on Colon Carcinoma Cells in Vitro

    Directory of Open Access Journals (Sweden)

    Mingshun Chen

    2016-06-01

    Full Text Available Three polyphenols were isolated and purified from sugar beet molasses by ultrasonic-aid extraction and various chromatographic techniques, and their structures were elucidated by spectral analysis. Cytotoxicity and the molecular mechanism were measured by methyl thiazolyl tetrazolium (MTT assay, flow cytometry, caspase-3 activity assay and Western blot assay. The results showed that gallic acid, cyanidin-3-O-glucoside chloride and epicatechin have cytotoxicity to the human colon, hepatocellular and breast cancer cells. Cyanidin-3-O-glucoside chloride showed its cytotoxicity against various tumor cell lines, particularly against colon cancer Caco-2 cells with half maximal inhibitory concentration (IC50 value of 23.21 ± 0.14 μg/mL in vitro. Cyanidin-3-O-glucoside chloride may be a potential candidate for the treatment of colon cancer. In the mechanism study, cyanidin-3-O-glucoside chloride increased the ratio of cell cycle at G0/G1 phase and reduced cyclin D1 expression on Caco-2 cells. Cyanidin-3-O-glucoside chloride decreased mutant p21 expression, and increased the ratio of Bax/Bcl-2 and the activation of caspase-3 to induce apoptosis.

  8. Circadian rhythms in the incidence of apoptotic cells and number of clonogenic cells in intestinal crypts after radiation using normal and reversed light conditions

    International Nuclear Information System (INIS)

    Ijiri, K.; Potten, C.S.

    1988-01-01

    Variations in the number of radiation-induced morphologically dead or dying cells (apoptotic cells) in the crypts in the small intestine of the mouse have been studied throughout a 24-h period under a normal light regimen. A clear circadian rhythm was displayed in the apoptotic incidence 3 or 6 h after irradiation for each gamma-ray dose studied (range 0.14-9.0 Gy). The most prominent circadian rhythm was obtained after 0.5 Gy. Peak time of day for inducing apoptosis was 06.00-09.00 h, and the trough occurred at 18.00-21.00 h. Some mice were also transferred to a reversed light cycle, and irradiated on different days after transfer. Apoptosis induced by 0.5 Gy or 9.0 Gy, or number of surviving crypts (microcolonies) after 11.0 Gy or 13.0 Gy was examined. The transition point for reversal of circadian rhythm in apoptosis (after 0.5 Gy) occurred 7 days after transfer and the rhythm was reversed by 14 days. The rhythm for crypt survival (i.e. for clonogenic cell radiosensitivity) was disturbed on 1 day and transition point for reversal occurred 3 days after transfer. The rhythm became reversed by 7 days. (author)

  9. Peroxynitrite decomposition catalyst prevents apoptotic cell death in a human astrocytoma cell line incubated with supernatants of HIV-infected macrophages

    Directory of Open Access Journals (Sweden)

    Perno Carlo

    2002-09-01

    Full Text Available Abstract Background Oxidative stress has shown to contribute in the mechanisms underlying apoptotic cell death occuring in AIDS-dementia complex. Here we investigated the role of peroxynitrite in apoptosis occurring in astroglial cells incubated with supernatants of HIV-infected human primary macrophages (M/M. Results Flow cytometric analysis (FACS of human cultured astrocytes shortly incubated with HIV-1-infected M/M supernatants showed apoptotic cell death, an effect accompanied by pronounced staining for nitrotyrosine (footprint of peroxynitrite and by abnormal formation of malondialdehyde (MDA. Pretreatment of astrocytes with the peroxynitrite decomposition catalyst FeTMPS antagonized HIV-related astrocytic apoptosis, MDA formation and nitrotyrosine staining. Conclusions Taken together, our results suggest that inibition of peroxynitrite leads to protection against peroxidative stress accompanying HIV-related apoptosis of astrocytes. Overall results support the role of peroxynitrite in HIV-related programmed death of astrocytes and suggest the use of peroxynitrite decomposition catalyst to counteract HIV-1-related neurological disorders.

  10. Antiproliferative and apoptotic effects of butyrolactone lignans from Arctium lappa on leukemic cells.

    Science.gov (United States)

    Matsumoto, T; Hosono-Nishiyama, K; Yamada, H

    2006-02-01

    In the course of screening for pharmacologically active substances from extracts of crude drugs used traditionally in Sino-Japanese herbal medicines, it was found that the 70 % ethanol extract from the fruits of Arctium lappa L. (Compositae) showed potent antiproliferative activity against B cell hybridoma cell, MH60. By bioassay-guided purification, a new lignan, (+)-7,8-didehydroarctigenin, together with the known lignans (-)-arctigenin and (-)-matairesinol were isolated as the active ingredients from an aqueous ethanolic extract of the fruits of A. lappa. Of these active compounds, (-)-arctigenin showed the most potent antiproliferative activity against MH60 cells (IC (50) : 1.0 microM), and the activity was suggested to be due to apoptosis.

  11. Antiproliferative and apoptotic effect of Morus nigra extract on human prostate cancer cells

    Directory of Open Access Journals (Sweden)

    Ibrahim Turan

    2017-02-01

    Full Text Available Background: Morus nigra L. belongs to the family Moraceae and is frequently used in traditional medicine. Numerous studies have investigated the antiproliferative effects of various extracts of different Morus species, but studies involving the in vitro cytotoxic effect of M. nigra extract are very limited. The purpose of this study was to evaluate the phenolic composition and antioxidant activity of dimethyl sulfoxide extract of M. nigra (DEM and to investigate, for the first time, the probable cytotoxic effect in human prostate adenocarcinoma (PC-3 cells together with the mechanism involved. Methods: Total polyphenolic contents (TPC, ferric reducing antioxidant power (FRAP and phenolic compounds of DEM were evaluated using spectrophotometric procedures and HPLC. The cytotoxic effect of DEM on PC-3 cells was revealed using the MTT assay. Mechanisms involved in the cytotoxic effect of DEM on PC-3 cells were then investigated in terms of apoptosis, mitochondrial membrane potential and cell cycle using flow cytometry, while caspase activity was investigated using luminometric analysis. Results: TPC and FRAP values were 20.7 ± 0.3 mg gallic acid equivalents and 48.8 ± 1.6 mg trolox equivalents per g sample, respectively. Ascorbic acid and chlorogenic acid were the major phenolic compounds detected at HPLC analysis. DEM arrested the cell cycle of PC-3 cells at the G1 phase, induced apoptosis via increased caspase activity and reduced mitochondrial membrane potential. Conclusions: Our results indicate that M. nigra may be a novel candidate for the development of new natural product based therapeutic agents against prostate cancer.

  12. Endocytosis of Albumin by Podocytes Elicits an Inflammatory Response and Induces Apoptotic Cell Death

    Science.gov (United States)

    Okamura, Kayo; Dummer, Patrick; Kopp, Jeffrey; Qiu, Liru; Levi, Moshe; Faubel, Sarah; Blaine, Judith

    2013-01-01

    The presence of albuminuria is strongly associated with progression of chronic kidney disease. While albuminuria has been shown to injure renal proximal tubular cells, the effects of albumin on podocytes have been less well studied. We have addressed the hypothesis that exposure of podocytes to albumin initiates an injury response. We studied transformed human-urine derived podocytes-like epithelial cells (HUPECS, or podocytes). Upon differentiation, these cells retain certain characteristics of differentiated podocytes, including expression of synaptopodin, CD2AP, and nestin. We exposed podocytes to recombinant human albumin, which lacks lipids and proteins that bind serum albumin; this reagent allowed a direct examination of the effects of albumin. Podocytes endocytosed fluoresceinated albumin and this process was inhibited at 4°C, suggesting an energy-dependent process. Exposure to albumin at concentrations of 5 and 10 mg/ml was associated with increased cell death in a dose-dependent manner. The mechanism of cell death may involve apoptosis, as caspase 3/7 were activated and the pan-caspase inhibitor z-VAD reduced cell death. Albumin exposure also increased nuclear factor (NF)-κB activation and increased transcription and release of interleukin (IL-) 1β, tumor necrosis factor (TNF), and IL-6. We extended these findings to an in vivo model. Glomeruli isolated from mice with nephrotic syndrome also had increased expression of IL-1β and TNF RNA. These data suggest that while podocyte injury begets albuminuria, albumin in the glomerular ultrafiltrate may also beget podocyte injury. Thus, an additional mechanism by which anti-proteinuric therapies are beneficial in the treatment of glomerular diseases may be a reduction in injury to the podocyte by albumin. PMID:23382978

  13. On the use of an appropriate TdT-mediated dUTP-biotin nick end labeling assay to identify apoptotic cells.

    OpenAIRE

    Lebon, C.; Rodriguez, G.V.; Zaoui, I.E.; Jaadane, I.; Behar-Cohen, F.; Torriglia, A.

    2015-01-01

    Apoptosis is an essential cellular mechanism involved in many processes such as embryogenesis, metamorphosis, and tissue homeostasis. DNA fragmentation is one of the key markers of this form of cell death. DNA fragmentation is executed by endogenous endonucleases such as caspase-activated DNase (CAD) in caspase-dependent apoptosis. The TUNEL (TdT-mediated dUTP-biotin nick end labeling) technique is the most widely used method to identify apoptotic cells in a tissue or culture and to assess dr...

  14. Quantification of apoptotic DNA fragmentation in a transformed uterine epithelial cell line, HRE-H9, using capillary electrophoresis with laser-induced fluorescence detector (CE-LIF).

    Science.gov (United States)

    Fiscus, R R; Leung, C P; Yuen, J P; Chan, H C

    2001-01-01

    Apoptotic cell death of uterine epithelial cells is thought to play an important role in the onset of menstruation and the successful implantation of an embryo during early pregnancy. Abnormal apoptosis in these cells can result in dysmenorrhoea and infertility. In addition, decreased rate of epithelial apoptosis likely contributes to endometriosis. A key step in the onset of apoptosis in these cells is cleavage of the genomic DNA between nucleosomes, resulting in polynucleosomal-sized fragments of DNA. The conventional technique for assessing apoptotic DNA fragmentation uses agarose (slab) gel electrophoresis (i.e. DNA laddering). However, recent technological advances in the use of capillary electrophoresis (CE), particularly the introduction of the laser-induced fluorescence detector (LIF), has made it possible to perform DNA laddering with improved automation and much greater sensitivity. In the present study, we have further developed the CE-LIF technique by using a DNA standard curve to quantify accurately the amount of DNA in the apoptotic DNA fragments and have applied this new quantitative technique to study apoptosis in a transformed uterine epithelial cell line, the HRE-H9 cells. Apoptosis was induced in the HRE-H9 cells by serum deprivation for 5, 7 and 24 h, resulting in increased DNA fragmentation of 2.2-, 3.1- and 6.2-fold, respectively, above the 0 h or plus-serum controls. This ultrasensitive CE-LIF technique provides a novel method for accurately measuring the actions of pro- or anti-apoptotic agents or conditions on uterine epithelial cell lines. Copyright 2001 Academic Press.

  15. Nitric oxide synthase expression and apoptotic cell death in brains of AIDS and AIDS dementia patients

    NARCIS (Netherlands)

    Vincent, V. A.; de Groot, C. J.; Lucassen, P. J.; Portegies, P.; Troost, D.; Tilders, F. J.; van Dam, A. M.

    1999-01-01

    To determine the occurrence and cellular localization of inducible nitric oxide synthase (iNOS), NOS activity and its association with cell death in brains of AIDS and AIDS dementia complex (ADC) patients. Post-mortem cerebral cortex tissue of eight AIDS patients, eight ADC patients and eight

  16. Biological Evaluation of Dipyrromethanes in Cancer Cell Lines: Antiproliferative and Pro-apoptotic Properties

    Czech Academy of Sciences Publication Activity Database

    Jorda, Radek; Lopes, S. M.M.; Řezníčková, Eva; Kryštof, Vladimír; Pinho e Melo, T. M.V.D.

    2017-01-01

    Roč. 12, č. 9 (2017), s. 701-711 ISSN 1860-7179 R&D Projects: GA MŠk(CZ) LO1204 Institutional support: RVO:61389030 Keywords : anticancer agents * apoptosis * cell cycle * cytotoxicity * dipyrromethanes Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Oncology Impact factor: 3.225, year: 2016

  17. Identification and apoptotic potential of T-2 toxin metabolites in human cells

    NARCIS (Netherlands)

    Weidner, M.; Welsch, T.; Hübner, F.; Schwerdt, G.; Gekle, M.; Humpf, H.U.

    2012-01-01

    The mycotoxin T-2 toxin, produced by various Fusarium species, is a widespread contaminant of grain and grain products. Knowledge about its toxicity and metabolism in the human body is crucial for any risk assessment as T-2 toxin can be detected in processed and unprocessed food samples. Cell

  18. Apoptotic effect of chalcone derivatives of 2-acetylthiophene in human breast cancer cells.

    Science.gov (United States)

    Fogaça, Tatiana B; Martins, Rosiane M; Begnini, Karine R; Carapina, Caroline; Ritter, Marina; de Pereira, Claudio M P; Seixas, Fabiana K; Collares, Tiago

    2017-02-01

    A variety of chalcones have demonstrated cytotoxic activity toward several cancer cell lines. This study aimed to investigate the cytotoxicity of four chalcones derivatives of 2-acetylthiophene in human breast cancer cell lines. MCF-7 and MDA-MB-231 cells were treated with synthesized chalcones and the cytotoxicity was evaluated by tetrazolium dye (MTT), live/dead, and DAPI assays. Chalcones significantly decreased MCF-7 and MDA-MB-231 cells viability in vitro in a dose dependent manner. After 48h treatment, the IC 50 values ranging from 5.52 to 34.23μM. Chalcone 3c displayed the highest cytotoxic activity from all the tested compounds. Cytotoxic effects of compounds were confirmed in the live/dead assay. In addition, DAPI staining revealed that these compounds induce death by apoptosis. The data speculate that chalcone derivatives of 2-acetylthiophene may represent a source of therapeutic agents for human breast cancer. Copyright © 2016 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

  19. Signal transduction of p53-independent apoptotic pathway induced by hexavalent chromium in U937 cells

    International Nuclear Information System (INIS)

    Hayashi, Yoko; Kondo, Takashi; Zhao Qingli; Ogawa Ryohei; Cui Zhengguo; Feril, Loreto B.; Teranishi, Hidetoyo; Kasuya, Minoru

    2004-01-01

    It has been reported that the hexavalent chromium compound (Cr(VI)) can induce both p53-dependent and p53-independent apoptosis. While a considerable amount of information is available on the p53-dependent pathway, only little is known about the p53-independent pathway. To elucidate the p53-independent mechanism, the roles of the Ca 2+ -calpain- and mitochondria-caspase-dependent pathways in apoptosis induced by Cr(VI) were investigated. When human lymphoma U937 cells, p53 mutated cells, were treated with 20 μM Cr(VI) for 24 h, nuclear morphological changes and DNA fragmentation were observed. Production of hydroxyl radicals revealed by electron paramagnetic resonance (EPR)-spin trapping, and increase of intracellular calcium ion concentration monitored by digital imaging were also observed in Cr(VI)-treated cells. An intracellular Ca 2+ chelator, BAPTA-AM, and calpain inhibitors suppressed the Cr(VI)-induced DNA fragmentation. The number of cells showing low mitochondrial membrane potential (MMP), high level of superoxide anion radicals (O 2 - ), and high activity of caspase-3, which are indicators of mitochondria-caspase-dependent pathway, increased significantly in Cr(VI)-treated cells. An antioxidant, N-acetyl-L-cysteine (NAC), decreased DNA fragmentation and inhibited the changes in MMP, O 2 - formation, and activation of caspase-3 induced by Cr(VI). No increase of the expressions of Fas and phosphorylated JNK was observed after Cr(VI) treatment. Cell cycle analysis revealed that the fraction of G2/M phase tended to increase after 24 h of treatment, suggesting that Cr(VI)-induced apoptosis is related to the G2 block. These results indicate that Ca 2+ -calpain- and mitochondria-caspase-dependent pathways play significant roles in the Cr(VI)-induced apoptosis via the G2 block, which are independent of JNK and Fas activation. The inhibition of apoptosis and all its signal transductions by NAC suggests that intracellular reactive oxygen species (ROS) are

  20. Berberine Induces Apoptotic Cell Death via Activation of Caspase-3 and -8 in HL-60 Human Leukemia Cells: Nuclear Localization and Structure-Activity Relationships.

    Science.gov (United States)

    Okubo, Shinya; Uto, Takuhiro; Goto, Aya; Tanaka, Hiroyuki; Nishioku, Tsuyoshi; Yamada, Katsushi; Shoyama, Yukihiro

    2017-01-01

    Berberine (BBR), an isoquinoline alkaloid, is a well-known bioactive compound contained in medicinal plants used in traditional and folk medicines. In this study, we investigated the subcellular localization and the apoptotic mechanisms of BBR were elucidated. First, we confirmed the incorporation of BBR into the cell visually. BBR showed antiproliferative activity and promptly localized to the nucleus from 5[Formula: see text]min to 15[Formula: see text]min after BBR treatment in HL-60 human promyelocytic leukemia cells. Next, we examined the antiproliferative activity of BBR (1) and its biosynthetically related compounds (2-7) in HL-60 cells. BBR exerted strongest antiproliferative activity among 1-7 and the results of structures and activity relation suggested that a methylenedioxyl group in ring A, an [Formula: see text]-alkyl group at C-9 position, and the frame of isoquinoline may be necessary for antiproliferative activity. Moreover, BBR showed the most potent antiproliferative activity in HL-60 cells among human cancer and normal cell lines tested. Next, we examined the effect of BBR on molecular events known as apoptosis induction. In HL-60 cells, BBR induced chromatin condensation and DNA fragmentation, and triggered the activation of PARP, caspase-3 and caspase-8 without the activation of caspase-9. BBR-induced DNA fragmentation was abolished by pretreatment with inhibitors against caspase-3 and caspase-8, but not against caspase-9. ERK and p38 were promptly phosphorylated after 15 min of BBR treatment, and this was correlated with time of localization to the nucleus of BBR. These results demonstrated that BBR translocated into nucleus immediately after treatments and induced apoptotic cell death by activation of caspase-3 and caspase-8.

  1. Role of BK channels in the apoptotic volume decrease in native eel intestinal cells

    DEFF Research Database (Denmark)

    Lionetto, Maria Giulia; Giordano, Maria Elena; Calisi, Antonio

    2010-01-01

    High conductance Ca(+)-activated K(+) channels (BK channels) have previously been demonstrated in the eel intestine. They are specifically activated following a hypotonic stress and sustain Regulatory Volume Decrease (RVD). The aim of the present work was to address the possible role...... enterocytes that BK channels, which are involved in RVD in these cells, plays also a crucial role in the AVD process and in the progression of apoptosis....

  2. Libidibia ferrea presents antiproliferative, apoptotic and antioxidant effects in a colorectal cancer cell line.

    Science.gov (United States)

    Guerra, Andreza Conceição Véras de Aguiar; Soares, Luiz Alberto Lira; Ferreira, Magda Rhayanny Assunção; Araújo, Aurigena Antunes de; Rocha, Hugo Alexandre de Oliveira; Medeiros, Juliana Silva de; Cavalcante, Rômulo Dos Santos; Júnior, Raimundo Fernandes de Araújo

    2017-08-01

    Colorectal cancer is noted for being one of the most frequent of tumors, with expressive morbidity and mortality rates. In new drug discovery, plants stand out as a source capable of yielding safe and high-efficiency products. Well known in Brazilian popular medicine, Libidibia ferrea (Mart. Ex Tul.) L.P. Queiroz var. ferrea (better known as "ironwood" or "jucá"), has been used to treat a wide spectrum of conditions and to prevent cancer. Using methodologies that involved flow cytometry, spectrophotometry and RT-qPCR assays, crude extracts of the fruits of L. ferrea (20T, 40T, 60T and 80T) were evaluated at 24h and/or 48h for: their ability to inhibit cell proliferation; induce apoptosis through Bcl-2, caspase-3 and Apaf-1; their antioxidant activity and effects on important targets related to cell proliferation (EGFR and AKT) in the HT-29 human colorectal cancer lineage. The results revealed high antiproliferative potential as compared to the controls, induction of apoptosis through the intrinsic pathway, and probable tumor inhibition activity under the mediation of important targets in tumorigenesis. In addition, L. ferrea revealed antioxidant, lipid peroxidation and chemoprotective effects in healthy cells. Thus, L. ferrea derivatives have important anticancer effects, and may be considered promising candidate for colorectal cancer therapy. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  3. Synergistic Effect of Garcinol and Curcumin on Antiproliferative and Apoptotic Activity in Pancreatic Cancer Cells

    Directory of Open Access Journals (Sweden)

    Mansi A. Parasramka

    2012-01-01

    Full Text Available Pancreatic cancer (PaCa is a major health concern due to its aggressiveness and early metastasis. Current treatments for PaCa are limited by development of resistance against therapy. As an alternative strategy, we assessed the combinatorial effect of dietary compounds, garcinol and curcumin, on human PaCa cells (BxPC-3 and Panc-1. A significant (<0.05 dose-dependent reduction in cell viability and increase in apoptosis were observed in both cell lines as compared to untreated controls. A combination index (CI value < 1, for a two-way comparison of curcumin and garcinol, suggests synergism. The potency (Dm of the combination of garcinol and curcumin was 2 to 10 fold that of the individual agents. This indicates that curcumin and garcinol in combination exhibit a high level of synergism, with enhanced bioactivity, thereby reducing the required effective dose required for each individually. This combinatorial strategy may hold promise in future development of therapies against PaCa.

  4. Curcuma longa Is Able to Induce Apoptotic Cell Death of Pterygium-Derived Human Keratinocytes.

    Science.gov (United States)

    Sancilio, Silvia; Di Staso, Silvio; Sebastiani, Stefano; Centurione, Lucia; Di Girolamo, Nick; Ciancaglini, Marco; Di Pietro, Roberta

    2017-01-01

    Pterygium is a relatively common eye disease that can display an aggressive clinical behaviour. To evaluate the in vitro effects of Curcuma longa on human pterygium-derived keratinocytes, specimens of pterygium from 20 patients undergoing pterygium surgical excision were collected. Pterygium explants were put into culture and derived keratinocytes were treated with an alcoholic extract of 1.3% Curcuma longa in 0.001% Benzalkonium Chloride for 3, 6, and 24 h. Cultured cells were examined for CAM5.2 (anti-cytokeratin antibody) and CD140 (anti-fibroblast transmembrane glycoprotein antibody) expression between 3th and 16th passage to assess cell homogeneity. TUNEL technique and Annexin-V/PI staining in flow cytometry were used to detect keratinocyte apoptosis. We showed that Curcuma longa exerts a proapoptotic effect on pterygium-derived keratinocytes already after 3 h treatment. Moreover, after 24 h treatment, Curcuma longa induces a significant increase in TUNEL as well as Annexin-V/PI positive cells in comparison to untreated samples. Our study confirms previous observations highlighting the expression, in pterygium keratinocytes, of nuclear VEGF and gives evidence for the first time to the expression of nuclear and cytoplasmic VEGF-R1. All in all, these findings suggest that Curcuma longa could have some therapeutic potential in the treatment and prevention of human pterygium.

  5. Activation of apoptotic pathway in normal, cancer ovarian cells by epothilone B.

    Science.gov (United States)

    Rogalska, Aneta; Szula, Ewa; Gajek, Arkadiusz; Marczak, Agnieszka; Jóźwiak, Zofia

    2013-09-01

    The epothilones, a new class of microtubule-targeting agents, seem to be a very promising alternative to the current strategy of cancer treatment. We have analyzed the aspects of epothilone B (Epo B) on cellular metabolism of tumor (OV-90) and normal (MM 14) ovarian cells. The observed effects were compared with those of paclitaxel (PTX), which is now a standard for the treatment of ovarian cancer. The results provide direct evidence that Epo B is considerably more cytotoxic to human OV-90 ovarian cancer cells than PTX. We have found, that antitumor efficacy of this new drug is related to its apoptosis-inducing ability, which was confirmed during measurements typical markers of the process. Epo B induced changes in morphology of cells, mitochondrial membrane potential and cytochrome c release. Also a slight increase of the intracellular calcium level was observed. Moreover, we have found that ROS production, stimulated by Epo B, is directly involved in the induction of apoptosis via mitochondrial pathway. Copyright © 2013 Elsevier B.V. All rights reserved.

  6. Pro-apoptotic effect of Persea americana var. Hass (avocado) on Jurkat lymphoblastic leukemia cells.

    Science.gov (United States)

    Bonilla-Porras, Angelica R; Salazar-Ospina, Andrea; Jimenez-Del-Rio, Marlene; Pereañez-Jimenez, Andres; Velez-Pardo, Carlos

    2013-11-05

    Abstract Context: Therapy for leukemia has a limited efficacy. There is a need to search for alternative anti-leukemia therapies. Persea americana Mill var. Hass (Lauraceae) is a tropical fruit (avocado) that might be used against cancer. Objective: To investigate whether P. americana induces death in Jurkat lymphoblastic leukemia cells. Materials and methods: Four ethanol extracts (0.1, 0.5, 1, 2 and 5 mg/mL) from avocado fruit (endocarp, whole seed, seed and leaves) were analyzed against Jurkat cells. Hydrogen peroxide generation by oxidation of 2',7'-dichlorodihydrofluorescein diacetate to the fluorescent compound 2',7'-dichlorfluorescein assay, acridine orange/ethidium bromide staining, flow cytometry analysis of annexin-V/7-amino-actinomycin, mitochondrial membrane potential and immunocytochemistry detection of transcription factor p53, caspase-3 and apoptosis-inducing factor (AIF) were evaluated. Results: Endocarp, seed, whole seed, and leaf (0.1 mg/mL) extracts induced significant apoptosis in Jurkat cells (p avocado and its therapeutic action on leukemia.

  7. Curcuma longa Is Able to Induce Apoptotic Cell Death of Pterygium-Derived Human Keratinocytes

    Directory of Open Access Journals (Sweden)

    Silvia Sancilio

    2017-01-01

    Full Text Available Pterygium is a relatively common eye disease that can display an aggressive clinical behaviour. To evaluate the in vitro effects of Curcuma longa on human pterygium-derived keratinocytes, specimens of pterygium from 20 patients undergoing pterygium surgical excision were collected. Pterygium explants were put into culture and derived keratinocytes were treated with an alcoholic extract of 1.3% Curcuma longa in 0.001% Benzalkonium Chloride for 3, 6, and 24 h. Cultured cells were examined for CAM5.2 (anti-cytokeratin antibody and CD140 (anti-fibroblast transmembrane glycoprotein antibody expression between 3th and 16th passage to assess cell homogeneity. TUNEL technique and Annexin-V/PI staining in flow cytometry were used to detect keratinocyte apoptosis. We showed that Curcuma longa exerts a proapoptotic effect on pterygium-derived keratinocytes already after 3 h treatment. Moreover, after 24 h treatment, Curcuma longa induces a significant increase in TUNEL as well as Annexin-V/PI positive cells in comparison to untreated samples. Our study confirms previous observations highlighting the expression, in pterygium keratinocytes, of nuclear VEGF and gives evidence for the first time to the expression of nuclear and cytoplasmic VEGF-R1. All in all, these findings suggest that Curcuma longa could have some therapeutic potential in the treatment and prevention of human pterygium.

  8. Curcumin affects cell survival and cell volume regulation in human renal and intestinal cells

    Science.gov (United States)

    Kössler, Sonja; Nofziger, Charity; Jakab, Martin; Dossena, Silvia; Paulmichl, Markus

    2012-01-01

    Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1E,6E-heptadiene-3,5-dione or diferuloyl methane) is a polyphenol derived from the Curcuma longa plant, commonly known as turmeric. This substance has been used extensively in Ayurvedic medicine for centuries for its anti-oxidant, analgesic, anti-inflammatory and antiseptic activity. More recently curcumin has been found to possess anti-cancer properties linked to its pro-apoptotic and anti-proliferative actions. The underly