NK cell cytotoxicity mediated by 2B4 and NTB-A is dependent on SAP acting downstream of receptor phosphorylation

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Stephan eMeinke

2013-01-01

Full Text Available 2B4 (CD244 and NK-T-B-antigen (NTB-A, CD352 are activating receptors on human NK cells and belong to the family of SLAM-related receptors. Engagement of these receptors leads to phosphorylation of their cytoplasmic tails and recruitment of the adapter proteins SAP and EAT-2. X-linked lymphoproliferative syndrome (XLP is a severe immunodeficiency that results from mutations in the SAP gene. 2B4 and NTB-A-mediated cytotoxicity are abrogated in XLP NK cells. To elucidate the molecular basis for this defect we analyzed early signaling events in SAP knockdown cells. Similar to XLP NK cells, knockdown of SAP in primary human NK cells leads to a reduction of 2B4 and NTB-A-mediated cytotoxicity. We found that early signaling events such as raft recruitment and receptor phosphorylation are not affected by the absence of SAP, indicating the defect in the absence of SAP is downstream of these events. In addition, knockdown of EAT-2 does not impair 2B4 or NTB-A-mediated cytotoxicity. Surprisingly, EAT-2 recruitment to both receptors is abrogated in the absence of SAP, revealing a novel cooperativity between these adapters.

Measuring T cell-mediated cytotoxicity using fluorogenic caspase substrates.

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Chahroudi, A; Silvestri, G; Feinberg, M B

2003-10-01

Cytotoxic T lymphocytes (CTLs) play a major role in the immune response against viruses and other intracellular pathogens. In addition, CTLs are implicated in the control of tumor cells in certain settings. Accurate measures of CTL function are of critical importance to study the pathogenesis of infectious diseases and to evaluate the efficacy of new vaccines and immunotherapies. To this end, we have recently developed a flow cytometry-based CTL (FCC) assay that measures the CTL-induced caspase activation within target cells using cell permeable fluorogenic caspase substrates. This novel assay reliably detects, by flow cytometry or fluorescence/confocal microscopy, antigen-specific CTLs in a wide variety of human and murine systems, and is safer and more informative than the standard 51Cr-release assay. In addition, the flow cytometric CTL (FCC) assay provides an alternative method that is often more sensitive and physiologically informative when compared to previously described FCC assays, as it measures a biological indicator of apoptosis within the target cell. The FCC assay may thus represent a useful tool to further understand the molecular and cellular mechanisms that underlie CTL-mediated killing during tumorigenesis or following infection with viruses or other intracellular pathogens.

Oxaliplatin regulates expression of stress ligands in ovarian cancer cells and modulates their susceptibility to natural killer cell-mediated cytotoxicity.

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Siew, Yin-Yin; Neo, Soek-Ying; Yew, Hui-Chuing; Lim, Shun-Wei; Ng, Yi-Cheng; Lew, Si-Min; Seetoh, Wei-Guang; Seow, See-Voon; Koh, Hwee-Ling

2015-12-01

Selected cytotoxic chemicals can provoke the immune system to recognize and destroy malignant tumors. Most of the studies on immunogenic cell death are focused on the signals that operate on a series of receptors expressed by dendritic cells to induce tumor antigen-specific T-cell responses. Here, we explored the effects of oxaliplatin, an immunogenic cell death inducer, on the induction of stress ligands and promotion of natural killer (NK) cell-mediated cytotoxicity in human ovarian cancer cells. The results indicated that treatment of tumor cells with oxaliplatin induced the production of type I interferons and chemokines and enhanced the expression of major histocompatibility complex class I-related chains (MIC) A/B, UL16-binding protein (ULBP)-3, CD155 and TNF-related apoptosis-inducing ligand (TRAIL)-R1/R2. Furthermore, oxaliplatin but not cisplatin treatment enhanced susceptibility of ovarian cancer cells to NK cell-mediated cytolysis. In addition, activated NK cells completely abrogated the growth of cancer cells that were pretreated with oxaliplatin. However, cancer cells pretreated with the same concentration of oxaliplatin alone were capable of potentiating regrowth over a period of time. These results suggest an advantage in combining oxaliplatin and NK cell-based therapy in the treatment of ovarian cancer. Further investigation on such potential combination therapy is warranted. © The Japanese Society for Immunology. 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Oxidative Mechanisms of Monocyte-Mediated Cytotoxicity

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Weiss, Stephen J.; Lobuglio, Albert F.; Kessler, Howard B.

1980-01-01

Human monocytes stimulated with phorbol myristate acetate were able to rapidly destroy autologous erythrocyte targets. Monocyte-mediated cytotoxicity was related to phorbol myristate acetate concentration and monocyte number. Purified preparations of lymphocytes were incapable of mediating erythrocyte lysis in this system. The ability of phorbol myristate acetate-stimulated monocytes to lyse erythrocyte targets was markedly impaired by catalase or superoxide dismutase but not by heat-inactivated enzymes or albumin. Despite a simultaneous requirement for superoxide anion and hydrogen peroxide in the cytotoxic event, a variety of hydroxyl radical and singlet oxygen scavengers did not effect cytolysis. However, tryptophan significantly inhibited cytotoxicity. The myeloperoxidase inhibitor cyanide enhanced erythrocyte destruction, whereas azide reduced it modestly. The inability of cyanide to reduce cytotoxicity coupled with the protective effect of superoxide dismutase suggests that cytotoxicity is independent of the classic myeloperoxidase system. We conclude that monocytes, stimulated with phorbol myristate acetate, generate superoxide anion and hydrogen peroxide, which together play an integral role in this cytotoxic mechanism.

Enhanced killing of chordoma cells by antibody-dependent cell-mediated cytotoxicity employing the novel anti-PD-L1 antibody avelumab.

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Fujii, Rika; Friedman, Eitan R; Richards, Jacob; Tsang, Kwong Y; Heery, Christopher R; Schlom, Jeffrey; Hodge, James W

2016-06-07

Chordoma, a rare bone tumor derived from the notochord, has been shown to be resistant to conventional therapies. Checkpoint inhibition has shown great promise in immune-mediated therapy of diverse cancers. The anti-PD-L1 mAb avelumab is unique among checkpoint inhibitors in that it is a fully human IgG1 capable of mediating antibody-dependent cell-mediated cytotoxicity (ADCC) of PD-L1-expressing tumor cells. Here, we investigated avelumab as a potential therapy for chordoma. We examined 4 chordoma cell lines, first for expression of PD-L1, and in vitro for ADCC killing using NK cells and avelumab. PD-L1 expression was markedly upregulated by IFN-γ in all 4 chordoma cell lines, which significantly increased sensitivity to ADCC. Brachyury is a transcription factor that is uniformly expressed in chordoma. Clinical trials are ongoing in which chordoma patients are treated with brachyury-specific vaccines. Co-incubating chordoma cells with brachyury-specific CD8+ T cells resulted in significant upregulation of PD-L1 on the tumor cells, mediated by the CD8+ T cells' IFN-γ production, and increased sensitivity of chordoma cells to avelumab-mediated ADCC. Residential cancer stem cell subpopulations of chordoma cells were also killed by avelumab-mediated ADCC to the same degree as non-cancer stem cell populations. These findings suggest that as a monotherapy for chordoma, avelumab may enable endogenous NK cells, while in combination with T-cell immunotherapy, such as a vaccine, avelumab may enhance NK-cell killing of chordoma cells via ADCC.

In vitro bacterial cytotoxicity of CNTs: reactive oxygen species mediate cell damage edges over direct physical puncturing.

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Rajavel, Krishnamoorthy; Gomathi, Rajkumar; Manian, Sellamuthu; Rajendra Kumar, Ramasamy Thangavelu

2014-01-21

Understanding the bacterial cytotoxicity of CNTs is important for a wide variety of applications in the biomedical, environmental, and health sectors. A majority of the earlier reports attributed the bactericidal cytotoxicity of CNTs to bacterial cell membrane damage by direct physical puncturing. Our results reveal that bacterial cell death via bacterial cell membrane damage is induced by reactive oxygen species (ROS) produced from CNTs and is not due to direct physical puncturing by CNTs. To understand the actual mechanism of bacterial killing, we elucidated the bacterial cytotoxicity of SWCNTs and MWCNTs against Gram-negative human pathogenic bacterial species Escherichia coli, Shigella sonnei, Klebsiella pneumoniae, and Pseudomonas aeruginosa and its amelioration upon functionalizing the CNTs with antioxidant tannic acid (TA). Interestingly, the bacterial cells treated with CNTs exhibited severe cell damage under laboratory (ambient) and sunlight irradiation conditions. However, CNTs showed no cytotoxicity to the bacterial cells when incubated in the dark. The quantitative assessments carried out by us made it explicit that CNTs are effective generators of ROS such as (1)O2, O2(•-), and (•)OH in an aqueous medium under both ambient and sunlight-irradiated conditions. Both naked and TA-functionalized CNTs showed negligible ROS production in the dark. Furthermore, strong correlations were obtained between ROS produced by CNTs and the bacterial cell mortality (with the correlation coefficient varying between 0.7618 and 0.9891) for all four tested pathogens. The absence of bactericidal cytotoxicity in both naked and functionalized CNTs in the dark reveals that the presence of ROS is the major factor responsible for the bactericidal action compared to direct physical puncturing. This understanding of the bactericidal activity of the irradiated CNTs, mediated through the generation of ROS, could be interesting for novel applications such as regulated ROS delivery

Au@Pt nanoparticles as catalase mimics to attenuate tumor hypoxia and enhance immune cell-mediated cytotoxicity

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Liang, Hong; Wu, Ying; Ou, Xiang-Yu; Li, Jing-Ying; Li, Juan

2017-11-01

Hypoxic tumor microenvironment (TME) is closely linked to tumor progression, heterogeneity and immune suppression. Therefore, the development of effective methods to overcome hypoxia and substantially enhance the immunotherapy efficacy remains a desirable goal. Herein, we engineered a biocompatible Au core/Pt shell nanoparticles (Au@Pt NPs) to reoxygenate the TME by reacting with endogenous H2O2. Treatment with Au@Pt NPs appeared to improve oxygen in intracellular environments and decrease hypoxia-inducible factor-1α expression. Furthermore, the integration of high catalytic efficiency of Au@Pt NPs with cytokine-induced killer (CIK) cell immunotherapy, could lead to significantly improve the effect of CIK cell-mediated cytotoxicity. These results suggest great potential of Au@Pt NPs for regulation of the hypoxic TME and enhance immune cell mediated anti-tumor immunity.

Ursolic acid-mediated changes in glycolytic pathway promote cytotoxic autophagy and apoptosis in phenotypically different breast cancer cells.

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Lewinska, Anna; Adamczyk-Grochala, Jagoda; Kwasniewicz, Ewa; Deregowska, Anna; Wnuk, Maciej

2017-06-01

Plant-derived pentacyclic triterpenotids with multiple biological activities are considered as promising candidates for cancer therapy and prevention. However, their mechanisms of action are not fully understood. In the present study, we have analyzed the effects of low dose treatment (5-20 µM) of ursolic acid (UA) and betulinic acid (BA) on breast cancer cells of different receptor status, namely MCF-7 (ER + , PR +/- , HER2 - ), MDA-MB-231 (ER - , PR - , HER2 - ) and SK-BR-3 (ER - , PR - , HER2 + ). UA-mediated response was more potent than BA-mediated response. Triterpenotids (5-10 µM) caused G0/G1 cell cycle arrest, an increase in p21 levels and SA-beta-galactosidase staining that was accompanied by oxidative stress and DNA damage. UA (20 µM) also diminished AKT signaling that affected glycolysis as judged by decreased levels of HK2, PKM2, ATP and lactate. UA-induced energy stress activated AMPK that resulted in cytotoxic autophagy and apoptosis. UA-mediated elevation in nitric oxide levels and ATM activation may also account for AMPK activation-mediated cytotoxic response. Moreover, UA-promoted apoptosis was associated with decreased pERK1/2 signals and the depolarization of mitochondrial membrane potential. Taken together, we have shown for the first time that UA at low micromolar range may promote its anticancer action by targeting glycolysis in phenotypically distinct breast cancer cells.

The Impact of HLA Class I-Specific Killer Cell Immunoglobulin-Like Receptors on Antibody-Dependent Natural Killer Cell-Mediated Cytotoxicity and Organ Allograft Rejection.

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Rajalingam, Raja

2016-01-01

Natural killer (NK) cells of the innate immune system are cytotoxic lymphocytes that play an important roles following transplantation of solid organs and hematopoietic stem cells. Recognition of self-human leukocyte antigen (HLA) class I molecules by inhibitory killer cell immunoglobulin-like receptors (KIRs) is involved in the calibration of NK cell effector capacities during the developmental stage, allowing the subsequent recognition and elimination of target cells with decreased expression of self-HLA class I (due to virus infection or tumor transformation) or HLA class I disparities (in the setting of allogeneic transplantation). NK cells expressing an inhibitory KIR-binding self-HLA can be activated when confronted with allografts lacking a ligand for the inhibitory receptor. Following the response of the adaptive immune system, NK cells can further destroy allograft endothelium by antibody-dependent cell-mediated cytotoxicity (ADCC), triggered through cross-linking of the CD16 Fc receptor by donor-specific antibodies bound to allograft. Upon recognizing allogeneic target cells, NK cells also secrete cytokines and chemokines that drive maturation of dendritic cells to promote cellular and humoral adaptive immune responses against the allograft. The cumulative activating and inhibitory signals generated by ligation of the receptors regulates mature NK cell killing of target cells and their production of cytokines and chemokines. This review summarizes the role of NK cells in allograft rejection and proposes mechanistic concepts that indicate a prominent role for KIR-HLA interactions in facilitating NK cells for Fc receptor-mediated ADCC effector function involved in antibody-mediated rejection of solid organ transplants.

The impact of HLA class I-specific killer cell immunoglobulin-like receptors on antibody-dependent natural killer cell-mediated cytotoxicity and organ allograft rejection

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Raja Rajalingam

2016-12-01

Full Text Available Natural killer (NK cells of the innate immune system are cytotoxic lymphocytes that play important roles following transplantation of solid organs and hematopoietic stem cells. Recognition of self HLA class I molecules by inhibitory killer cell immunoglobulin-like receptors (KIR is involved in the calibration of NK cell effector capacities during a developmental stage, allowing the subsequent recognition and elimination of target cells with decreased expression of self HLA class I (due to virus infection or tumor transformation or HLA class I disparities (in the setting of allogeneic transplantation. NK cells expressing an inhibitory KIR binding self HLA can be activated when confronted with allografts lacking a ligand for the inhibitory receptor. Following the response of the adaptive immune system, NK cells can further destroy allograft endothelium by antibody-dependent cell-mediated cytotoxicity (ADCC, triggered through cross-linking of the CD16 Fc receptor by donor-specific antibodies bound to allograft. Upon recognizing allogeneic target cells, NK cells also secrete cytokines and chemokines that drive maturation of dendritic cells to promote cellular and humoral adaptive immune responses against the allograft. The cumulative activating and inhibitory signals generated by ligation of the receptors regulates mature NK cell killing of target cells and their production of cytokines and chemokines. This review summarizes the role of NK cells in allograft rejection and proposes mechanistic concepts that indicate a prominent role for KIR-HLA interactions in facilitating NK cells for Fc receptor-mediated ADCC effector function involved in antibody-mediated rejection of solid organ transplants.

Biochemical studies of immune RNA using a cell-mediated cytotoxicity assay

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Griffin, G.D.; Sellin, H.G.; Novelli, G.D.

1980-01-01

Immune RNA (iRNA), a subcellular macromolecular species usually prepared by phenol extraction of lymphoid tissue, can confer some manifestation(s) of cellular immunity on naive lymphocytes. Experiments were done to develop an assay system to detect activation of lymphocytes by iRNA to become cytotoxic toward tumor cells, and to study certain properties of iRNA using this system. Guinea pigs were immunized with human mammary carcinoma cells and the iRNA, prepared from spleens of animals shown by prior assay to have blood lymphocytes highly cytotoxic against the tumor cells, was assayed by ability of iRNA-activated lymphocytes to lyse /sup 51/Cr-labelled tumor cells. The ability of iRNA to activate lymphocytes to tumor cytotoxicity could only be differentiated from a cytotoxic activation by RNA preparations from unimmunized animals at very low doses of RNA. The most active iRNA preparations were from cytoplasmic subcellular fractions, extracted by a cold phenol procedure, while iRNA isolated by hot phenol methods was no more active than control RNA prepared by the same techniques. Attempts to demonstrate poly(A) sequences in iRNA were inconclusive.

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

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

2012-03-01

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

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

International Nuclear Information System (INIS)

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

2012-01-01

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

Eosinophilia of dystrophin-deficient muscle is promoted by perforin-mediated cytotoxicity by T cell effectors

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Cai, B.; Spencer, M. J.; Nakamura, G.; Tseng-Ong, L.; Tidball, J. G.

2000-01-01

Previous investigations have shown that cytotoxic T lymphocytes (CTLs) contribute to muscle pathology in the dystrophin-null mutant mouse (mdx) model of Duchenne muscular dystrophy through perforin-dependent and perforin-independent mechanisms. We have assessed whether the CTL-mediated pathology includes the promotion of eosinophilia in dystrophic muscle, and thereby provides a secondary mechanism through which CTLs contribute to muscular dystrophy. Quantitative immunohistochemistry confirmed that eosinophilia is a component of the mdx dystrophy. In addition, electron microscopic observations show that eosinophils traverse the basement membrane of mdx muscle fibers and display sites of close apposition of eosinophil and muscle membranes. The close membrane apposition is characterized by impingement of eosinophilic rods of major basic protein into the muscle cell membrane. Transfer of mdx splenocytes and mdx muscle extracts to irradiated C57 mice by intraperitoneal injection resulted in muscle eosinophilia in the recipient mice. Double-mutant mice lacking dystrophin and perforin showed less eosinophilia than was displayed by mdx mice that expressed perforin. Finally, administration of prednisolone, which has been shown previously to reduce the concentration of CTLs in dystrophic muscle, produced a significant reduction in eosinophilia. These findings indicate that eosinophilia is a component of the mdx pathology that is promoted by perforin-dependent cytotoxicity of effector T cells. However, some eosinophilia of mdx muscle is independent of perforin-mediated processes.

Identification of the proteins related to SET-mediated hepatic cytotoxicity of trichloroethylene by proteomic analysis.

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Ren, Xiaohu; Yang, Xifei; Hong, Wen-Xu; Huang, Peiwu; Wang, Yong; Liu, Wei; Ye, Jinbo; Huang, Haiyan; Huang, Xinfeng; Shen, Liming; Yang, Linqing; Zhuang, Zhixiong; Liu, Jianjun

2014-05-16

Trichloroethylene (TCE) is an effective solvent for a variety of organic materials. Since the wide use of TCE as industrial degreasing of metals, adhesive paint and polyvinyl chloride production, TCE has turned into an environmental and occupational toxicant. Exposure to TCE could cause severe hepatotoxicity; however, the toxic mechanisms of TCE remain poorly understood. Recently, we reported that SET protein mediated TCE-induced cytotoxicity in L-02 cells. Here, we further identified the proteins related to SET-mediated hepatic cytotoxicity of TCE using the techniques of DIGE (differential gel electrophoresis) and MALDI-TOF-MS/MS. Among the 20 differential proteins identified, 8 were found to be modulated by SET in TCE-induced cytotoxicity and three of them (cofilin-1, peroxiredoxin-2 and S100-A11) were validated by Western-blot analysis. The functional analysis revealed that most of the identified SET-modulated proteins are apoptosis-associated proteins. These data indicated that these proteins may be involved in SET-mediated hepatic cytotoxicity of TCE in L-02 cells. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Curcumin enhances the mitomycin C-induced cytotoxicity via downregulation of MKK1/2-ERK1/2-mediated Rad51 expression in non-small cell lung cancer cells

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Ko, Jen-Chung; Tsai, Min-Shao; Weng, Shao-Hsing; Kuo, Ya-Hsun; Chiu, Yu-Fan; Lin, Yun-Wei

2011-01-01

Curcumin (diferuloylmethane), a major active component of turmeric (Curcuma longa), has been reported to suppress the proliferation of a wide variety of tumor cells. Rad51 is a key protein in the homologous recombination (HR) pathway of DNA double-strand break repair, and HR represents a novel target for cancer therapy. A high expression of Rad51 has been reported in chemo- or radio-resistant carcinomas. Therefore, in the current study, we will examine whether curcumin could enhance the effects of mitomycin C (MMC), a DNA interstrand cross-linking agent, to induce cytotoxicity by decreasing Rad51 expression. Exposure of two human non-small lung cancer (NSCLC) cell lines (A549 and H1975) to curcumin could suppress MMC-induced MKK1/2-ERK1/2 signal activation and Rad51 protein expression. Enhancement of ERK1/2 activation by constitutively active MKK1/2 (MKK1/2-CA) increased Rad51 protein levels in curcumin and MMC co-treated human lung cells. Moreover, the synergistic cytotoxic effect induced by curcumin combined with MMC was decreased by MKK1-CA-mediated enhancement of ERK1/2 activation by a significant degree. In contrast, MKK1/2 inhibitor, U0126 was shown to augment the cytotoxicity of curcumin and MMC through downregulation of ERK1/2 activation and Rad51 expression. Depletion of endogenous Rad51 expression by siRad51 RNA transfection significantly enhanced MMC and/or curcumin induced cell death and cell growth inhibition. In contrast, an overexpression of Rad51 protected lung cancer cells from synergistic cytotoxic effects induced by curcumin and MMC. We concluded that Rad51 inhibition may be an additional action mechanism for enhancing the chemosensitization of MMC by curcumin in NSCLC. - Highlights: → Curcumin downregulates MKK-ERK-mediated Rad51 expression. → Curcumin enhances mitomycin C-induced cytotoxicity. → Rad51 protects cells from cytotoxic effects induced by curcumin and mitomycin C. → Rad51 inhibition enhances the chemosensitization of

Cell-mediated cytotoxicity for melanome tumor cells: detection by a (3H)proline release assay

International Nuclear Information System (INIS)

Saal, J.G.; Rieber, E.P.; Riethmueller, G.

1976-01-01

An in vitro lymphocyte-mediated cytotoxicity assay using [ 3 H]proline-labelled target cells is described. The assay, modified from an original procedure of Bean et al., assesses the release of [ 3 H]proline by filtering the total culture fluid containing both trypsinised tumor cells and effector cells. Filtration is performed with a semiautomatic harvesting device using low suction pressure and large-diameter glass filters. Pretreatment of filters with whole serum diminishes adsorption of cell-free radioactive material considerably and thus increases the sensitivity of the assay. Nearly 100% of the radioactivity could be recovered with this harvesting device. The technique allowed the detection of cytolytic activities of lymphocytes after 6 h of incubation. Lymphocytes from patients with primary malignant melanoma showed a significantly higher cytolytic reactivity (p > 0.001) than normal donors' lymphocytes against three different melanoma cell lines. In a series of parallel experiments on 36 patients and 18 normal donors, this modification of the [ 3 ]proline test was compared with three different assays: the conventional microcytotoxicity test of Takasugi and Klein, the original [ 3 H]proline microcytotoxicity test of Bean et al., and the viability count of tumor cells. (Auth.)

Sensitive Detection of the Natural Killer Cell-Mediated Cytotoxicity of Anti-CD20 Antibodies and Its Impairment by B-Cell Receptor Pathway Inhibitors

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Floyd Hassenrück

2018-01-01

Full Text Available The antibody-dependent cell-mediated cytotoxicity (ADCC of the anti-CD20 monoclonal antibodies (mAbs rituximab and obinutuzumab against the cell line Raji and isolated CLL cells and its potential impairment by kinase inhibitors (KI was determined via lactate dehydrogenase release or calcein retention, respectively, using genetically modified NK92 cells expressing CD16-176V as effector cells. Compared to peripheral blood mononuclear cells, recombinant effector cell lines showed substantial alloreactivity-related cytotoxicity without addition of mAbs but afforded determination of ADCC with reduced interassay variability. The cytotoxicity owing to alloreactivity was less susceptible to interference by KI than the ADCC of anti-CD20 mAbs, which was markedly diminished by ibrutinib, but not by idelalisib. Compared to rituximab, the ADCC of obinutuzumab against primary CLL cells showed approximately 30% higher efficacy and less interference with KI. Irreversible BTK inhibitors at a clinically relevant concentration of 1 μM only weakly impaired the ADCC of anti-CD20 mAbs, with less influence in combinations with obinutuzumab than with rituximab and by acalabrutinib than by ibrutinib or tirabrutinib. In summary, NK cell line-based assays permitted the sensitive detection of ADCC of therapeutic anti-CD20 mAbs against CLL cells and of the interference of KI with this important killing mechanism.

KHYG-1, a model for the study of enhanced natural killer cell cytotoxicity.

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Suck, Garnet; Branch, Donald R; Smyth, Mark J; Miller, Richard G; Vergidis, Joanna; Fahim, Soad; Keating, Armand

2005-10-01

To compare the cytotoxicity of KHYG-1 with other natural killer (NK)/NK T-cell lines and identify molecules that may be associated with enhanced cytotoxicity, thereby eventually leading to improved NK cell-mediated cancer immunotherapy. NK/NK T-cell lines KHYG-1, NK-92, YT, and SNT-8 were compared with a novel flow cytometric cytotoxicity assay under different culture conditions. Transcription, expression, and phosphorylation studies were performed using polymerase chain reaction sequence-specific primers, reverse transcription polymerase chain reaction, immunoblotting, and flow cytometry. KHYG-1 is a highly cytotoxic cell line, exceeding the cytolytic capacity of the other cell lines against K562. KHYG-1 is also highly cytotoxic against the leukemia cell lines EM2, EM3, and HL60. The novel activation receptor NKp44 and its adaptor, DAP12, NKG2D, and constitutively phosphorylated ERK2 may be associated with the enhanced cytotoxicity of KHYG-1. This cell line most likely mediates cytolysis by granzyme M (but not granzymes A and B) together with perforin, which is constitutively fully cleaved to the 60-kD form, in contrast to the other cell lines. KHYG-1 is a valuable model for the study of enhanced cytotoxicity by NK cells. In addition to the activation of NKp44, KHYG-1 may induce apoptosis of tumor cells by the newly described granzyme M/perforin pathway. Targeted modifications of effector molecules demonstrated in this model could generate NK cells with even greater killing ability that may be particularly attractive for clinical application. Moreover, our demonstration of greater cytotoxicity of KHYG-1 versus NK-92 cells, already in clinical trials, suggests a direct therapeutic role for KHYG-1.

Effect of radiation on cell-mediated cytotoxicity and lymphocyte subpopulations in patients with ovarian carcinoma

International Nuclear Information System (INIS)

Kohorn, E.I.; Mitchell, M.S.; Dwyer, J.M.; Knowlton, A.H.; Klein-Angerer, S.

1978-01-01

Lymphocyte subpopulations and cell-mediated cytotoxicity (CMI) were studied during radiation therapy in 16 patients with ovarian carcinoma. The total lymphocyte count became depressed in all patients. The depression was more marked among T cells, while the proportion of B cells remained unaffected. In patients with Stage I and II ovarian cancer, CMI was depressed significantly by radiotherapy after 7 days of treatment, remained low at 14 days but recovered despite continuation of radiation. This depression of CMI occurred at a delivered dose of 1,000 rads with subsequent recovery. Patients with Stage III ovarian cancer given pelvic and abdominal radiation were found to have no consistent depression of CMI, a finding similar to that in Stage III ovarian carcinoma patients given chemotherapy

Heterogenous populations of cytotoxic cells in the peritoneal cavity of BALB/c mice immunized with allogeneic EL4 leukemia cells

International Nuclear Information System (INIS)

Zighelboim, J.; Bonavida, B.; Fahey, J.L.

1974-01-01

Adherent cells, presumably macrophages, obtained from the peritoneal cavity shortly after rejection of the allogeneic leukemia EL4, produced effective cell-mediated cytotoxicity (CMC) in vitro. These cytotoxic cells were sensitive to anti-macrophage serum and resistant to anti-thymocyte serum and 10,000 roentgen irradiation. In contrast, a second population of specifically cytotoxic cells were nonadherent, sensitive to x-rays and anti-thymocyte serum, but not to anti-macrophage serum. The two cell populations had a cooperative cytotoxic effect in vitro against allogeneic tumor cells

Arctigenin suppresses unfolded protein response and sensitizes glucose deprivation-mediated cytotoxicity of cancer cells.

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Sun, Shengrong; Wang, Xiong; Wang, Changhua; Nawaz, Ahmed; Wei, Wen; Li, Juanjuan; Wang, Lijun; Yu, De-Hua

2011-01-01

The involvement of unfolded protein response (UPR) activation in tumor survival and resistance to chemotherapies suggests a new anticancer strategy targeting UPR pathway. Arctigenin, a natural product, has been recently identified for its antitumor activity with selective toxicity against cancer cells under glucose starvation with unknown mechanism. Here we found that arctigenin specifically blocks the transcriptional induction of two potential anticancer targets, namely glucose-regulated protein-78 (GRP78) and its analog GRP94, under glucose deprivation, but not by tunicamycin. The activation of other UPR pathways, e.g., XBP-1 and ATF4, by glucose deprivation was also suppressed by arctigenin. A further transgene experiment showed that ectopic expression of GRP78 at least partially rescued arctigenin/glucose starvation-mediated cell growth inhibition, suggesting the causal role of UPR suppression in arctigenin-mediated cytotoxicity under glucose starvation. These observations bring a new insight into the mechanism of action of arctigenin and may lead to the design of new anticancer therapeutics. © Georg Thieme Verlag KG Stuttgart · New York.

CEA/CD3-bispecific T cell-engaging (BiTE) antibody-mediated T lymphocyte cytotoxicity maximized by inhibition of both PD1 and PD-L1.

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Osada, Takuya; Patel, Sandip P; Hammond, Scott A; Osada, Koya; Morse, Michael A; Lyerly, H Kim

2015-06-01

Bispecific T cell-engaging (BiTE) antibodies recruit polyclonal cytotoxic T cells (CTL) to tumors. One such antibody is carcinoembryonic antigen (CEA) BiTE that mediates T cell/tumor interaction by simultaneously binding CD3 expressed by T cells and CEA expressed by tumor cells. A widely operative mechanism for mitigating cytotoxic T cell-mediated killing is the interaction of tumor-expressed PD-L1 with T cell-expressed PD-1, which may be partly reversed by PD-1/PD-L1 blockade. We hypothesized that PD-1/PD-L1 blockade during BiTE-mediated T cell killing would enhance CTL function. Here, we determined the effects of PD-1 and PD-L1 blockade during initial T cell-mediated killing of CEA-expressing human tumor cell lines in vitro, as well as subsequent T cell-mediated killing by T lymphocytes that had participated in tumor cell killing. We observed a rapid upregulation of PD-1 expression and diminished cytolytic function of T cells after they had engaged in CEA BiTE-mediated killing of tumors. T cell cytolytic activity in vitro could be maximized by administration of anti-PD-1 or anti-PD-L1 antibodies alone or in combination if applied prior to a round of T cell killing, but T cell inhibition could not be fully reversed by this blockade once the T cells had killed tumor. In conclusion, our findings demonstrate that dual blockade of PD-1 and PD-L1 maximizes T cell killing of tumor directed by CEA BiTE in vitro, is more effective if applied early, and provides a rationale for clinical use.

Rapid kinetics of lysis in human natural cell-mediated cytotoxicity: some implications

International Nuclear Information System (INIS)

Bloom, E.T.; Babbitt, J.T.

1983-01-01

The entire lytic process of natural cell-mediated cytotoxicity against sensitive target cells can occur rapidly, within minutes. This was demonstrated by 51 chromium release and in single-cell assays. At the cellular level, most of the target cell lysis occurred within 15-30 min after binding to effector cells. The enriched natural killer cell subpopulation of lymphocytes obtained by Percoll density gradient centrifugation (containing greater than 70% large granular lymphocytes (LGL)) was the most rapidly lytic population by 51 chromium release. However, in the single-cell assay, the rate of lysis of bound target cells was quite similar for the LGL-enriched effector subpopulation and the higher density subpopulation of effector cells recognized previously. Both the light and dense effector cells contained similar numbers of target binding cells. Therefore, that the light subpopulation effected lysis more rapidly and to a greater extent than the dense subpopulation suggested that the low-density effector cells probably recycled more rapidly than those of higher density. This was corroborated by the finding that when conjugates were formed at 29 degrees C for the single-cell assay, a significant number of dead unconjugated targets could be observed only on the slides made with the LGL-enriched effector cells but not on those made with dense effector cell. Lysis continued to increase in the chromium-release assay probably because of recycling, recruitment, and/or heterogeneity of the effector cells, and/or because of heterogeneity or delayed death of the target cells

DNA polymerase gamma inhibition by vitamin K3 induces mitochondria-mediated cytotoxicity in human cancer cells.

Science.gov (United States)

Sasaki, Ryohei; Suzuki, Yoko; Yonezawa, Yuko; Ota, Yosuke; Okamoto, Yoshiaki; Demizu, Yusuke; Huang, Peng; Yoshida, Hiromi; Sugimura, Kazuro; Mizushina, Yoshiyuki

2008-05-01

Among the vitamin K (VK) compounds, VK3 exhibits distinct cytotoxic activity in cancer cells and is thought to affect redox cycling; however, the underlying mechanisms remain unclear. Here we demonstrate that VK3 selectively inhibits DNA polymerase (pol) gamma, the key enzyme responsible for mitochondrial DNA replication and repair. VK3 at 30 microM inhibited pol gamma by more than 80%, caused impairment of mitochondrial DNA replication and repair, and induced a significant increase in reactive oxygen species (ROS), leading to apoptosis. At a lower concentration (3 microM), VK3 did not cause a significant increase in ROS, but was able to effectively inhibit cell proliferation, which could be reversed by supplementing glycolytic substrates. The cytotoxic action of VK3 was independent of p53 tumor suppressor gene status. Interestingly, VK3 only inhibited pol gamma but did not affect other pol including human pol alpha, pol beta, pol delta, and pol epsilon. VK1 and VK2 exhibited no inhibitory effect on any of the pol tested. These data together suggest that the inhibition of pol gamma by VK3 is relatively specific, and that this compound seems to exert its anticancer activity by two possible mechanisms in a concentration-dependent manner: (1) induction of ROS-mediated cell death at high concentrations; and (2) inhibition of cell proliferation at lower concentrations likely through the suppression of mitochondrial respiratory function. These findings may explain various cytotoxic actions induced by VK3, and may pave the way for the further use of VK3.

Attenuated RANKL-induced cytotoxicity by Portulaca oleracea ethanol extract enhances RANKL-mediated osteoclastogenesis.

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Erkhembaatar, Munkhsoyol; Choi, Eun-Joo; Lee, Hak-Yong; Lee, Choong Hun; Lee, Young-Rae; Kim, Min Seuk

2015-07-14

Portulaca oleracea (PO) has been widely used as traditional medicine because of its pharmacological activities. However, the effects of PO on osteoclasts that modulate bone homeostasis are still elusive. In this study, we examined the effects of PO ethanol extract (POEE) on receptor activator of nuclear factor-κB ligand (RANKL)-mediated Ca(2+) mobilization, nuclear factor of activated T-cell c1 (NFATc1) amplification, tartrate-resistant acid phosphatase-positive (TRAP+) multinucleated cell (MNC) formation, and cytotoxicity. Our results demonstrated that POEE suppressed RANKL-induced Ca(2+) oscillations by inhibition of Ca(2+) release from internal Ca(2+) stores, resulting in reduction of NFATc1 amplification. Notably, POEE attenuated RANKL-mediated cytotoxicity and cleavage of polyadenosine 5'-diphosphate-ribose polymerase (PARP), resulted in enhanced formation of TRAP+ MNCs. These results present in vitro effects of POEE on RANKL-mediated osteoclastogenesis and suggest the possible use of PO in treating bone disorders, such as osteopetrosis.

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

Science.gov (United States)

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

1994-01-01

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

Paucity of natural killer and cytotoxic T cells in human neuromyelitis optica lesions

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Saadoun, Samira; Bridges, Leslie R.; Verkman, A. S.; Papadopoulos, Marios C.

2013-01-01

Neuromyelitis optica is a severe inflammatory demyelinating disease of the central nervous system. Most patients with neuromyelitis optica have circulating immunoglobulin G (IgG) antibodies against the astrocytic water channel protein aquaporin-4 (AQP4), which are pathogenic. Anti-AQP4 IgG-mediated complement-dependent astrocyte toxicity is a key mechanism of central nervous system damage in neuromyelitis optica, but the role of natural killer and cytotoxic T cells is unknown. Our objective was to determine whether natural killer and cytotoxic T cells play a role in human neuromyelitis optica lesions. We immunostained four actively demyelinating lesions, obtained from patients with anti-AQP4 IgG positive neuromyelitis optica, for Granzyme B and Perforin. The inflammatory cells were perivascular neutrophils, eosinophils and macrophages, with only occasional Granzyme B+ or Perforin + cells. Greater than 95% of inflamed vessels in each lesion had no surrounding Granzyme B+ or Perforin + cells. Granzyme B+ or Perforin+ cells were abundant in human spleen (positive control). Although natural killer cells produce central nervous system damage in mice injected with anti-AQP4 IgG, our findings here indicate that natural killer-mediated and T cell-mediated cytotoxicity are probably not involved in central nervous system damage in human neuromyelitis optica. PMID:23108041

Generation of TCR-Expressing Innate Lymphoid-like Helper Cells that Induce Cytotoxic T Cell-Mediated Anti-leukemic Cell Response.

Science.gov (United States)

Ueda, Norihiro; Uemura, Yasushi; Zhang, Rong; Kitayama, Shuichi; Iriguchi, Shoichi; Kawai, Yohei; Yasui, Yutaka; Tatsumi, Minako; Ueda, Tatsuki; Liu, Tian-Yi; Mizoro, Yasutaka; Okada, Chihiro; Watanabe, Akira; Nakanishi, Mahito; Senju, Satoru; Nishimura, Yasuharu; Kuzushima, Kiyotaka; Kiyoi, Hitoshi; Naoe, Tomoki; Kaneko, Shin

2018-06-05

CD4 + T helper (Th) cell activation is essential for inducing cytotoxic T lymphocyte (CTL) responses against malignancy. We reprogrammed a Th clone specific for chronic myelogenous leukemia (CML)-derived b3a2 peptide to pluripotency and re-differentiated the cells into original TCR-expressing T-lineage cells (iPS-T cells) with gene expression patterns resembling those of group 1 innate lymphoid cells. CD4 gene transduction into iPS-T cells enhanced b3a2 peptide-specific responses via b3a2 peptide-specific TCR. iPS-T cells upregulated CD40 ligand (CD40L) expression in response to interleukin-2 and interleukin-15. In the presence of Wilms tumor 1 (WT1) peptide, antigen-specific dendritic cells (DCs) conditioned by CD4-modified CD40L high iPS-T cells stimulated WT1-specific CTL priming, which eliminated WT1 peptide-expressing CML cells in vitro and in vivo. Thus, CD4 modification of CD40L high iPS-T cells generates innate lymphoid helper-like cells inducing bcr-abl-specific TCR signaling that mediates effectiveanti-leukemic CTL responses via DC maturation, showing potential for adjuvant immunotherapy against leukemia. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Analysis of the Effects of Cell Stress and Cytotoxicity on In ...

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Chemical toxicity can arise from disruption of specific biomolecular functions or through more generalized cell stress and cytotoxicity-mediated processes. Here, concentration-dependent responses of 1063 chemicals including pharmaceuticals, natural products, pesticidals, consumer, and industrial chemicals across a diverse battery of 821 in vitro assay endpoints from 7 high-throughput assay technology platforms were analyzed in order to better distinguish between these types of activities. Both cell-based and cell-free assays showed a rapid increase in the frequency of responses at concentrations where cell stress / cytotoxicity responses were observed in cell-based assays. Chemicals that were positive on at least two viability/cytotoxicity assays within the concentration range tested (typically up to 100 M) activated a median of 12% of assay endpoints while those that were not cytotoxic in this concentration range activated 1.3% of the assays endpoints. The results suggest that activity can be broadly divided into: (1) specific biomolecular interactions against one or more targets (e.g., receptors or enzymes) at concentrations below which overt cytotoxicity-associated activity is observed; and (2) activity associated with cell stress or cytotoxicity, which may result from triggering of specific cell stress pathways, chemical reactivity, physico-chemical disruption of proteins or membranes, or broad low-affinity non-covalent interactions. Chemicals showing a g

Trichomonas vaginalis and Tritrichomonas foetus: interaction with fibroblasts and muscle cells - new insights into parasite-mediated host cell cytotoxicity

Directory of Open Access Journals (Sweden)

Ricardo Chaves Vilela

2012-09-01

Full Text Available Trichomonas vaginalis and Tritrichomonas foetus are parasitic, flagellated protists that inhabit the urogenital tract of humans and bovines, respectively. T. vaginalis causes the most prevalent non-viral sexually transmitted disease worldwide and has been associated with an increased risk for human immunodeficiency virus-1 infection in humans. Infections by T. foetus cause significant losses to the beef industry worldwide due to infertility and spontaneous abortion in cows. Several studies have shown a close association between trichomonads and the epithelium of the urogenital tract. However, little is known concerning the interaction of trichomonads with cells from deeper tissues, such as fibroblasts and muscle cells. Published parasite-host cell interaction studies have reported contradictory results regarding the ability of T. foetus and T. vaginalis to interact with and damage cells of different tissues. In this study, parasite-host cell interactions were examined by culturing primary human fibroblasts obtained from abdominal biopsies performed during plastic surgeries with trichomonads. In addition, mouse 3T3 fibroblasts, primary chick embryo myogenic cells and L6 muscle cells were also used as models of target cells. The parasite-host cell cultures were processed for scanning and transmission electron microscopy and were tested for cell viability and cell death. JC-1 staining, which measures mitochondrial membrane potential, was used to determine whether the parasites induced target cell damage. Terminal deoxynucleotidyltransferase-mediated dUTP nick end labelling staining was used as an indicator of chromatin damage. The colorimetric crystal violet assay was performed to ana-lyse the cytotoxicity induced by the parasite. The results showed that T. foetus and T. vaginalis adhered to and were cytotoxic to both fibroblasts and muscle cells, indicating that trichomonas infection of the connective and muscle tissues is likely to occur; such

CD4+ T cell-mediated cytotoxicity is associated with MHC class II expression on malignant CD19+ B cells in diffuse large B cell lymphoma.

Science.gov (United States)

Zhou, Yong; Zha, Jie; Lin, Zhijuan; Fang, Zhihong; Zeng, Hanyan; Zhao, Jintao; Luo, Yiming; Li, Zhifeng; Xu, Bing

2018-01-15

Diffuse large B cell lymphoma (DLBCL) is a common B cell malignancy with approximately 30% of patients present relapsed or refractory disease after first-line therapy. Research of further treatment options is needed. Cytotoxic CD4 + T cells express cytolytic molecules and have potential antitumor function. Here, we showed that the CD19 + cells from DLBCL patients presented significantly reduced expression of MHC II molecules than those from healthy controls. Three years after the first-line treatment, patients that presented relapsed disease had significantly lower MHC II expression on their CD19 + cells than patients who did not show recurrence. Examining cytotoxic CD4 + T cells show that DLBCL patients presented significantly elevated frequencies of granzyme A-, granzyme B-, and/or perforin-expressing cytotoxic CD4 + T cells. Also, frequency of cytotoxic CD4 + T cells in DLBCL patients was positively correlated with the MHC II expression level. Subsequently, the cytotoxic potential of CD4 + T cells against autologous CD19 + cells was investigated. We found that the cytotoxic potential of CD4 + T cells was highest in MHC II-high, intermediate in MHC II-mid, and lowest in MHC II-low patients. The percentage of MHC II-expressing viable CD19 + cells presented a significant reduction after longer incubation with cytotoxic CD4 + T cells, suggesting that cytotoxic CD4 + T cells preferentially eliminated MHC II-expressing CD19 + cells. Blocking MHC II on CD19 + cells significantly reduced the cytolytic capacity of CD4 + T cells. Despite these discoveries, the frequency of cytotoxic CD4 + T cells did not predict the clinical outcome of DLBCL patients. Together, these results demonstrated that cytotoxic CD4 + T cells presented an MHC II-dependent cytotoxic potential against autologous CD19 + cells and could potentially represent a future treatment option for DLBCL. Copyright © 2017 Elsevier Inc. All rights reserved.

Cystatin F as a regulator of immune cell cytotoxicity.

Science.gov (United States)

Kos, Janko; Nanut, Milica Perišić; Prunk, Mateja; Sabotič, Jerica; Dautović, Esmeralda; Jewett, Anahid

2018-05-10

Cysteine cathepsins are lysosomal peptidases involved in the regulation of innate and adaptive immune responses. Among the diverse processes, regulation of granule-dependent cytotoxicity of cytotoxic T-lymphocytes (CTLs) and natural killer (NK) cells during cancer progression has recently gained significant attention. The function of cysteine cathepsins is regulated by endogenous cysteine protease inhibitors-cystatins. Whereas other cystatins are generally cytosolic or extracellular proteins, cystatin F is present in endosomes and lysosomes and is thus able to regulate the activity of its target directly. It is delivered to endosomal/lysosomal vesicles as an inactive, disulphide-linked dimer. Proteolytic cleavage of its N-terminal part leads to the monomer, the only form that is a potent inhibitor of cathepsins C, H and L, involved in the activation of granzymes and perforin. In NK cells and CTLs the levels of active cathepsin C and of granzyme B are dependent on the concentration of monomeric, active cystatin F. In tumour microenvironment, inactive dimeric cystatin F can be secreted from tumour cells or immune cells and further taken up by the cytotoxic cells. Subsequent monomerization and inhibition of cysteine cathepsins within the endosomal/lysosomal vesicles impairs granzyme and perforin activation, and provokes cell anergy. Further, the glycosylation pattern has been shown to be important in controlling secretion of cystatin F from target cells, as well as internalization by cytotoxic cells and trafficking to endosomal/lysosomal vesicles. Cystatin F is therefore an important mediator used by bystander cells to reduce NK and T-cell cytotoxicity.

Concanavalin A-mediated in vitro activation of a secondary cytotoxic T-cell response in virus-primed splenocytes

DEFF Research Database (Denmark)

Thomsen, Allan Randrup; Jensen, B L

1980-01-01

In a recent report it was shown that what appeared to be secondary cytotoxic T cells could be obtained from lymphocytic choriomeningitis virus (LCMV)-primed splenocytes after stimulation in vitro with the non-specific T cell mitogen concanavalin A (Con A). The present experiments attempt to chara......In a recent report it was shown that what appeared to be secondary cytotoxic T cells could be obtained from lymphocytic choriomeningitis virus (LCMV)-primed splenocytes after stimulation in vitro with the non-specific T cell mitogen concanavalin A (Con A). The present experiments attempt...... to characterize further these effector cells and, in particular, to establish whether the Con A-activated cytotoxic effectors are qualitatively different from the secondary cytotoxic T cells induced by restimulation with the homologous antigen. It was found that: (1) in vitro activation with Con A could......, since no evidence was found to indicate a role for other cell types or soluble (cytotoxic or arming) factors; (4) cytotoxicity was specific with regard to both virus and 'self'. By comparison with previous data on LCMV-induced cytotoxic T cells, it is concluded that Con A induces the generation...

Zinc-Dependent Protection of Tobacco and Rice Cells From Aluminum-Induced Superoxide-Mediated Cytotoxicity

Science.gov (United States)

Lin, Cun; Hara, Ayaka; Comparini, Diego; Bouteau, François; Kawano, Tomonori

2015-01-01

Al3+ toxicity in growing plants is considered as one of the major factors limiting the production of crops on acidic soils worldwide. In the last 15 years, it has been proposed that Al3+ toxicity are mediated with distortion of the cellular signaling mechanisms such as calcium signaling pathways, and production of cytotoxic reactive oxygen species (ROS) causing oxidative damages. On the other hand, zinc is normally present in plants at high concentrations and its deficiency is one of the most widespread micronutrient deficiencies in plants. Earlier studies suggested that lack of zinc often results in ROS-mediated oxidative damage to plant cells. Previously, inhibitory action of Zn2+ against lanthanide-induced superoxide generation in tobacco cells have been reported, suggesting that Zn2+ interferes with the cation-induced ROS production via stimulation of NADPH oxidase. In the present study, the effect of Zn2+ on Al3+-induced superoxide generation in the cell suspension cultures of tobacco (Nicotiana tabacum L., cell-line, BY-2) and rice (Oryza sativa L., cv. Nipponbare), was examined. The Zn2+-dependent inhibition of the Al3+-induced oxidative burst was observed in both model cells selected from the monocots and dicots (rice and tobacco), suggesting that this phenomenon (Al3+/Zn2+ interaction) can be preserved in higher plants. Subsequently induced cell death in tobacco cells was analyzed by lethal cell staining with Evans blue. Obtained results indicated that presence of Zn2+ at physiological concentrations can protect the cells by preventing the Al3+-induced superoxide generation and cell death. Furthermore, the regulation of the Ca2+ signaling, i.e., change in the cytosolic Ca2+ ion concentration, and the cross-talks among the elements which participate in the pathway were further explored. PMID:26648960

The anti-lung cancer activity of SEP is mediated by the activation and cytotoxicity of NK cells via TLR2/4 in vivo.

Science.gov (United States)

Ke, Mengyun; Wang, Hui; Zhang, Min; Tian, Yuwei; Wang, Yizhou; Li, Bing; Yu, Jie; Dou, Jie; Xi, Tao; Zhou, Changlin

2014-05-01

Strongylocentrotus nudus egg polysaccharide (SEP) has been reported to display antitumor activity. However, the effects of SEP and its underlying mechanism in the treatment of lung cancer remain unclear, particularly with an immunodeficient mouse model of human non-small cell lung cancer (NSCLC). In the present study, we investigated the anti-lung cancer effects of SEP and its underlying mechanism of action in both Lewis lung cancer (LLC)-bearing C57/BL6J mice and human NSCLC H460-bearing nude mice. Although SEP showed no inhibitory effects on tumor cells in vitro, it markedly stimulated the percentage of CD3-NK1.1(+) cells and natural killer (NK) cell cytotoxicity in the spleens of nude mice and C57/BL6J mice. In LLC-bearing mice, SEP not only inhibited tumor growth but also promoted NK-mediated cytotoxicity, the NK1.1(+) cell population, and IL-2 and IFN-γ secretion. SEP significantly suppressed H460 growth in nude mice, which was abrogated by the selective depletion of NK cells via the intraperitoneal injection of anti-asialo GM-1 antibodies. Furthermore, anti-TLR2/4 antibodies blocked both SEP and NK cell binding and SEP-induced perforin secretion. SEP-induced proliferation and IFN-γ secretion by NK cells in wild type mice were partially impaired in TLR2 or TLR4 knockout mice. These results suggest that SEP-promoted NK cytotoxicity, which was partially mediated via TLR2 and TLR4, was the main contributing factor to lung cancer inhibition in vivo and that SEP may be a potential immunotherapy candidate for the treatment of lung cancer. Copyright © 2014 Elsevier Inc. All rights reserved.

Altered effector function of peripheral cytotoxic cells in COPD

Directory of Open Access Journals (Sweden)

Corne Jonathan M

2009-06-01

Full Text Available Abstract Background There is mounting evidence that perforin and granzymes are important mediators in the lung destruction seen in COPD. We investigated the characteristics of the three main perforin and granzyme containing peripheral cells, namely CD8+ T lymphocytes, natural killer (NK; CD56+CD3- cells and NKT-like (CD56+CD3+ cells. Methods Peripheral blood mononuclear cells (PBMCs were isolated and cell numbers and intracellular granzyme B and perforin were analysed by flow cytometry. Immunomagnetically selected CD8+ T lymphocytes, NK (CD56+CD3- and NKT-like (CD56+CD3+ cells were used in an LDH release assay to determine cytotoxicity and cytotoxic mechanisms were investigated by blocking perforin and granzyme B with relevant antibodies. Results The proportion of peripheral blood NKT-like (CD56+CD3+ cells in smokers with COPD (COPD subjects was significantly lower (0.6% than in healthy smokers (smokers (2.8%, p +CD3- cells from COPD subjects were significantly less cytotoxic than in smokers (16.8% vs 51.9% specific lysis, p +CD3+ cells (16.7% vs 52.4% specific lysis, p +CD3- and NKT-like (CD56+CD3+ cells from smokers and HNS. Conclusion In this study, we show that the relative numbers of peripheral blood NK (CD56+CD3- and NKT-like (CD56+CD3+ cells in COPD subjects are reduced and that their cytotoxic effector function is defective.

Poly(ADP-ribose) polymerase-independent potentiation of nitrosourea cytotoxicity by 3-aminobenzamide in human malignant glioma cells.

Science.gov (United States)

Winter, S; Weller, M

2000-06-16

Poly(ADP-ribose) polymerase is a zinc-finger DNA-binding protein that detects specifically DNA strand breaks generated by genotoxic agents and is thought to be involved in DNA repair. Here, we examined the effects of 3-aminobenzamide, a poly(ADP-ribose) polymerase inhibitor, on the chemosensitivity of human malignant glioma cells. 3-Aminobenzamide selectively potentiated the cytotoxicity of the nitrosoureas, nimustine, carmustine and lomustine in 10 of 12 human malignant glioma cell lines. In contrast, 3-aminobenzamide did not modulate the cytotoxic effects of doxorubicine, teniposide, vincristine, camptothecin or cytarabine. The nitrosoureas did not induce poly(ADP-ribose) polymerase activity in the glioma cells. Ectopic expression of truncated poly(ADP-ribose) polymerase containing the poly(ADP-ribose) polymerase DNA-binding domain, which acts as a dominant-negative mutant, in LN-18 or LN-229 cells did not alter the 3-aminobenzamide effect on nitrosourea-mediated cytotoxicity. Thus, 3-aminobenzamide may target another nicotinamide adenine dinucleotide (NAD)-requiring enzyme, but not poly(ADP-ribose) polymerase, when enhancing nitrosourea cytotoxicity in human malignant glioma cells. Carmustine cytotoxicity was associated with a G2/M arrest. Coexposure to carmustine and 3-aminobenzamide overcame this G2/M arrest in T98G cells, which are sensitized to carmustine by 3-aminobenzamide, but not in U251MG cells, which are refractory to 3-aminobenzamide-mediated sensitization to carmustine. Thus, 3-aminobenzamide-mediated sensitization to carmustine cytotoxicity may result from interference with the stable G2/M arrest response to carmustine in human glioma cells.

The ginsenoside PPD exerts anti-endometriosis effects by suppressing estrogen receptor-mediated inhibition of endometrial stromal cell autophagy and NK cell cytotoxicity.

Science.gov (United States)

Zhang, Bing; Zhou, Wen-Jie; Gu, Chun-Jie; Wu, Ke; Yang, Hui-Li; Mei, Jie; Yu, Jia-Jun; Hou, Xiao-Fan; Sun, Jian-Song; Xu, Feng-Yuan; Li, Da-Jin; Jin, Li-Ping; Li, Ming-Qing

2018-05-14

Endometriosis (EMS) is an estrogen-dependent gynecological disease with a low autophagy level of ectopic endometrial stromal cells (eESCs). Impaired NK cell cytotoxic activity is involved in the clearance obstruction of the ectopic endometrial tissue in the abdominopelvic cavity. Protopanaxadiol (PPD) and protopanaxatriol (PPT) are two metabolites of ginsenosides, which have profound biological functions, such as anti-cancer activities. However, the role and mechanism of ginsenosides and metabolites in endometriosis are completely unknown. Here, we found that the compounds PPD, PPT, ginsenoside-Rg3 (G-Rg3), ginsenoside-Rh2 (G-Rh2), and esculentoside A (EsA) led to significant decreases in the viability of eESCs, particularly PPD (IC50 = 30.64 µM). In vitro and in vivo experiments showed that PPD promoted the expression of progesterone receptor (PR) and downregulated the expression of estrogen receptor α (ERα) in eESCs. Treatment with PPD obviously induced the autophagy of eESCs and reversed the inhibitory effect of estrogen on eESC autophagy. In addition, eESCs pretreated with PPD enhanced the cytotoxic activity of NK cells in response to eESCs. PPD decreased the numbers and suppressed the growth of ectopic lesions in a mouse EMS model. These results suggest that PPD plays a role in anti-EMS activation, possibly by restricting estrogen-mediated autophagy regulation and enhancing the cytotoxicity of NK cells. This result provides a scientific basis for potential therapeutic strategies to treat EMS by PPD or further structural modification.

Cell-mediated immunity to Plasmodium falciparum infection: evidence against the involvement of cytotoxic lymphocytes

DEFF Research Database (Denmark)

Theander, T G; Andersen, B J; Pedersen, B K

1988-01-01

stimulated PBMC from malaria-immune donors by SPag and purified protein derivative (PPD) in culture for 7 days. The PBMC were then co-incubated with P. falciparum for 48 h, and parasitaemia was determined by microscopy. Parasite growth was only significantly impaired after incubation with PBMC stimulated...... by either SPag or PPD in the presence of immune serum. Studies on subpopulations of PBMC indicated that the inhibitory cells resided among the adherent cell fraction. Furthermore we tested PBMC for cytotoxic activity against P. falciparum-infected autologous or heterologous erythrocytes. Experiments were...... done both in the absence and the presence of immune serum. Neither fresh PBMC nor PBMC activated by SPag or PPD for 7 days prior to assay were cytotoxic, indicating that cytotoxic T cells, natural killer (NK) cells, and K cells did not possess cytotoxic activity directed against parasitized...

Immune response to a mammary adenocarcinoma. V. Sera from tumor-bearing rats contain multiple factors blocking cell-mediated cytotoxicity.

Science.gov (United States)

Huber, S A; Lucas, Z J

1978-12-01

Sera from Fischer rats 3 to 13 days after i.p. injection of syngeneic 13762A mammary adenocarcinoma contain three factors specifically blocking cell-mediated cytotoxicity (CMC). The major blocking factor is a 160,000-dalton IgG that combines specifically to cytolytic lymphocytes but not to tumor cells or tumor antigen, and that is not dissociated after treatment with 8 M urea. The other factors have been putatively identified as tumor antigen (less than 70,000 daltons) and as soluble antigen-antibody complexes (greater than 200,000 daltons). Injecting the tumor antigen into tumor-free rats induced spleen cells specifically cytotoxic to the 13762A tumor and provided partial protection to challenge with live tumor cells. Treating soluble antigen-antibody complexes with 8 M urea decreased the size of the blocking activity from greater than 200,000 to less than 70,000 daltons. Although the IgG fraction dissociated from the complex did not block CMC, it did recombine with the tumor antigen fraction to transfer activity to the greater than 200,000-dalton fraction. In contrast, mixing tumor antigen with the IgG fraction that did block CMC did not alter the size of the blocking activities.

Virus-specific HLA-restricted lysis of herpes simplex virus-infected human monocytes and macrophages mediated by cytotoxic T lymphocytes

Energy Technology Data Exchange (ETDEWEB)

Torpey, D.J. III

1987-01-01

Freshly-isolated peripheral blood human monocytes and 5 day in vitro cultured macrophages were infected with herpes simplex virus type 1 (HSV-1), labeled with /sup 51/Cr, and used as target cells in a 12-14 hour cell-mediated cytotoxicity assay. Mononuclear leukocytes (MNL) from HSV-1 non-immune individuals, whether unstimulated or stimulated with HSV-1 antigen, did not mediate significant lysis of either target cell. HSV-immune MNL, both freshly-isolated and cultured for 5 days without antigen, demonstrated only low levels of natural killer (NK) cell-mediate lysis. MNL from HSV-immune individuals incubated for 5 days in vitro with HSV-1 antigen mediated significant virus-specific lysis of both target cells. Mean virus-specific lysis of autologous monocytes was 8.5(/+-/2.0)% compared to a three-fold greater virus-specific lysis of autologous macrophages. Greater than 70% of this lytic activity was mediated by Leu-11-negative, T3-positive cytotoxic T lymphocytes (CTL). Allogeneic target cells lacking a common HLA determinant were not significantly lysed while T8-positive CTL mediated infrequent lysis of target cells sharing a common HLA-A and/or HLA-B determinant. T4-positive lymphocytes were demonstrated to be the predominant cell mediating lysis of autologous target cells and allogeneic target cells sharing both HLA-A and/or HLA-B plus HLA-DR determinants with the CTL; the T4-positive cell was the sole CTL mediator of lysis of allogeneic target cells having a common HLA-DR determinant.

Virus-specific HLA-restricted lysis of herpes simplex virus-infected human monocytes and macrophages mediated by cytotoxic T lymphocytes

International Nuclear Information System (INIS)

Torpey, D.J. III.

1987-01-01

Freshly-isolated peripheral blood human monocytes and 5 day in vitro cultured macrophages were infected with herpes simplex virus type 1 (HSV-1), labeled with 51 Cr, and used as target cells in a 12-14 hour cell-mediated cytotoxicity assay. Mononuclear leukocytes (MNL) from HSV-1 non-immune individuals, whether unstimulated or stimulated with HSV-1 antigen, did not mediate significant lysis of either target cell. HSV-immune MNL, both freshly-isolated and cultured for 5 days without antigen, demonstrated only low levels of natural killer (NK) cell-mediate lysis. MNL from HSV-immune individuals incubated for 5 days in vitro with HSV-1 antigen mediated significant virus-specific lysis of both target cells. Mean virus-specific lysis of autologous monocytes was 8.5(/+-/2.0)% compared to a three-fold greater virus-specific lysis of autologous macrophages. Greater than 70% of this lytic activity was mediated by Leu-11-negative, T3-positive cytotoxic T lymphocytes (CTL). Allogeneic target cells lacking a common HLA determinant were not significantly lysed while T8-positive CTL mediated infrequent lysis of target cells sharing a common HLA-A and/or HLA-B determinant. T4-positive lymphocytes were demonstrated to be the predominant cell mediating lysis of autologous target cells and allogeneic target cells sharing both HLA-A and/or HLA-B plus HLA-DR determinants with the CTL; the T4-positive cell was the sole CTL mediator of lysis of allogeneic target cells having a common HLA-DR determinant

A potential therapy for chordoma via antibody-dependent cell-mediated cytotoxicity employing NK or high-affinity NK cells in combination with cetuximab.

Science.gov (United States)

Fujii, Rika; Schlom, Jeffrey; Hodge, James W

2018-05-01

OBJECTIVE Chordoma is a rare bone tumor derived from the notochord and is resistant to conventional therapies such as chemotherapy, radiotherapy, and targeting therapeutics. Expression of epidermal growth factor receptor (EGFR) in a large proportion of chordoma specimens indicates a potential target for therapeutic intervention. In this study the authors investigated the potential role of the anti-EGFR antibody cetuximab in immunotherapy for chordoma. METHODS Since cetuximab is a monoclonal antibody of the IgG1 isotype, it has the potential to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) employing natural killer (NK) cells as effectors. Polymorphisms in the CD16 allele expressed on NK cells have been shown to influence the degree of ADCC of tumor cells, with the high-affinity valine (V)/V allele being responsible for more lysis than the V/phenylalanine (F) or FF allele. Unfortunately, however, only approximately 10% of the population expresses the VV allele on NK cells. An NK cell line, NK-92, has now been engineered to endogenously express IL-2 and the high-affinity CD16 allele. These irradiated high-affinity (ha)NK cells were analyzed for lysis of chordoma cells with and without cetuximab, and the levels of lysis observed in ADCC were compared with those of NK cells from donors expressing the VV, VF, and FF alleles. RESULTS Here the authors demonstrate for the first time 1) that cetuximab in combination with NK cells can mediate ADCC of chordoma cells; 2) the influence of the NK CD16 polymorphism in cetuximab-mediated ADCC for chordoma cell lysis; 3) that engineered haNK cells-that is, cells transduced to express the CD16 V158 FcγRIIIa receptor-bind cetuximab with similar affinity to normal NK cells expressing the high-affinity VV allele; and 4) that irradiated haNK cells induce ADCC with cetuximab in chordoma cells. CONCLUSIONS These studies provide rationale for the use of cetuximab in combination with irradiated haNK cells for therapy for

Role of T-bet, the master regulator of Th1 cells, in the cytotoxicity of murine CD4+ T cells.

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Eshima, Koji; Misawa, Kana; Ohashi, Chihiro; Iwabuchi, Kazuya

2018-05-01

Although CD4 + T cells are generally regarded as helper T cells, some activated CD4 + T cells have cytotoxic properties. Given that CD4 + cytotoxic T lymphocytes (CTLs) often secrete IFN-γ, CTL activity among CD4 + T cells may be attributable to Th1 cells, where a T-box family molecule, T-bet serves as the "master regulator". However, although the essential contribution of T-bet to expression of IFN-γ has been well-documented, it remains unclear whether T-bet is involved in CD4 + T cell-mediated cytotoxicity. In this study, to investigate the ability of T-bet to confer cytolytic activity on CD4 + T cells, the T-bet gene (Tbx21) was introduced into non-cytocidal CD4 + T cell lines and their cytolytic function analyzed. Up-regulation of FasL (CD178), which provided the transfectant with cytotoxicity, was observed in Tbx21transfected CD4 + T cells but not in untransfected parental cells. In one cell line, T-bet transduction also induced perforin gene (Prf1) expression and Tbx21 transfectants efficiently killed Fas - target cells. Although T-bet was found to repress up-regulation of CD40L (CD154), which controls FasL-mediated cytolysis, the extent of CD40L up-regulation on in vitro-differentiated Th1 cells was similar to that on Th2 cells, suggesting the existence of a compensatory mechanism. These results collectively indicate that T-bet may be involved in the expression of genes, such as FasL and Prf1, which confer cytotoxicity on Th1 cells. © 2018 The Societies and John Wiley & Sons Australia, Ltd.

Cytotoxicity of pyrrolizidine alkaloid in human hepatic parenchymal and sinusoidal endothelial cells: Firm evidence for the reactive metabolites mediated pyrrolizidine alkaloid-induced hepatotoxicity.

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Yang, Mengbi; Ruan, Jianqing; Fu, Peter P; Lin, Ge

2016-01-05

Pyrrolizidine alkaloids (PAs) widely distribute in plants and can cause hepatic sinusoidal obstruction syndrome (HSOS), which typically presents as a primary sinusoidal endothelial cell damage. It is well-recognized that after ingestion, PAs undergo hepatic cytochromes P450 (CYPs)-mediated metabolic activation to generate dehydropyrrolizidine alkaloids (DHPAs), which are hydrolyzed to dehydroretronecine (DHR). DHPAs and DHR are reactive metabolites having same core pyrrole moiety, and can bind proteins to form pyrrole-protein adducts, which are believed as the primary cause for PA-induced HSOS. However, to date, the direct evidences supporting the toxicity of DHPAs and DHR in the liver, in particular in the sinusoidal endothelial cells, are lacking. Using human hepatic sinusoidal endothelial cells (HSEC) and HepG2 (representing hepatic parenchymal cells), cells that lack CYPs activity, this study determined the direct cytotoxicity of dehydromonocrotaline, a representative DHPA, and DHR, but no cytotoxicity of the intact PA (monocrotaline) in both cell lines, confirming that reactive metabolites mediate PA intoxication. Comparing with HepG2, HSEC had significantly lower basal glutathione (GSH) level, and was significantly more susceptible to the reactive metabolites with severer GSH depletion and pyrrole-protein adducts formation. The toxic potency of two reactive metabolites was also compared. DHPA was more reactive than DHR, leading to severer toxicity. In conclusion, our results unambiguously provided the first direct evidence for the critical role of DHPA and DHR in the reactive metabolites-mediated PA-induced hepatotoxicity, which occurs predominantly in HSEC due to severe GSH depletion and the significant formation of pyrrole-protein adducts in HSEC. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Idarubicin induces mTOR-dependent cytotoxic autophagy in leukemic cells

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Ristic, Biljana; Bosnjak, Mihajlo; Arsikin, Katarina; Mircic, Aleksandar; Suzin-Zivkovic, Violeta; Bogdanovic, Andrija; Perovic, Vladimir; Martinovic, Tamara; Kravic-Stevovic, Tamara; Bumbasirevic, Vladimir; Trajkovic, Vladimir; Harhaji-Trajkovic, Ljubica

2014-01-01

We investigated if the antileukemic drug idarubicin induces autophagy, a process of programmed cellular self-digestion, in leukemic cell lines and primary leukemic cells. Transmission electron microscopy and acridine orange staining demonstrated the presence of autophagic vesicles and intracellular acidification, respectively, in idarubicin-treated REH leukemic cell line. Idarubicin increased punctuation/aggregation of microtubule-associated light chain 3B (LC3B), enhanced the conversion of LC3B-I to autophagosome-associated LC3B-II in the presence of proteolysis inhibitors, and promoted the degradation of the selective autophagic target p62, thus indicating the increase in autophagic flux. Idarubicin inhibited the phosphorylation of the main autophagy repressor mammalian target of rapamycin (mTOR) and its downstream target p70S6 kinase. The treatment with the mTOR activator leucine prevented idarubicin-mediated autophagy induction. Idarubicin-induced mTOR repression was associated with the activation of the mTOR inhibitor AMP-activated protein kinase and down-regulation of the mTOR activator Akt. The suppression of autophagy by pharmacological inhibitors or LC3B and beclin-1 genetic knockdown rescued REH cells from idarubicin-mediated oxidative stress, mitochondrial depolarization, caspase activation and apoptotic DNA fragmentation. Idarubicin also caused mTOR inhibition and cytotoxic autophagy in K562 leukemic cell line and leukocytes from chronic myeloid leukemia patients, but not healthy controls. By demonstrating mTOR-dependent cytotoxic autophagy in idarubicin-treated leukemic cells, our results warrant caution when considering combining idarubicin with autophagy inhibitors in leukemia therapy. - Highlights: • Idarubicin induces autophagy in leukemic cell lines and primary leukemic cells. • Idarubicin induces autophagy by inhibiting mTOR in leukemic cells. • mTOR suppression by idarubicin is associated with AMPK activation and Akt blockade. �

Idarubicin induces mTOR-dependent cytotoxic autophagy in leukemic cells

Energy Technology Data Exchange (ETDEWEB)

Ristic, Biljana [Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Dr. Subotica 1, 11000 Belgrade (Serbia); Bosnjak, Mihajlo [Institute of Histology and Embryology, School of Medicine, University of Belgrade, Belgrade (Serbia); Arsikin, Katarina [Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Dr. Subotica 1, 11000 Belgrade (Serbia); Mircic, Aleksandar; Suzin-Zivkovic, Violeta [Institute of Histology and Embryology, School of Medicine, University of Belgrade, Belgrade (Serbia); Bogdanovic, Andrija [Clinic for Hematology, Clinical Centre of Serbia, School of Medicine, University of Belgrade, Belgrade (Serbia); Perovic, Vladimir [Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Dr. Subotica 1, 11000 Belgrade (Serbia); Martinovic, Tamara; Kravic-Stevovic, Tamara; Bumbasirevic, Vladimir [Institute of Histology and Embryology, School of Medicine, University of Belgrade, Belgrade (Serbia); Trajkovic, Vladimir, E-mail: vtrajkovic@med.bg.ac.rs [Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Dr. Subotica 1, 11000 Belgrade (Serbia); Harhaji-Trajkovic, Ljubica, E-mail: buajk@yahoo.com [Institute for Biological Research, University of Belgrade, Belgrade, Despot Stefan Blvd. 142, 11000 Belgrade (Serbia)

2014-08-01

We investigated if the antileukemic drug idarubicin induces autophagy, a process of programmed cellular self-digestion, in leukemic cell lines and primary leukemic cells. Transmission electron microscopy and acridine orange staining demonstrated the presence of autophagic vesicles and intracellular acidification, respectively, in idarubicin-treated REH leukemic cell line. Idarubicin increased punctuation/aggregation of microtubule-associated light chain 3B (LC3B), enhanced the conversion of LC3B-I to autophagosome-associated LC3B-II in the presence of proteolysis inhibitors, and promoted the degradation of the selective autophagic target p62, thus indicating the increase in autophagic flux. Idarubicin inhibited the phosphorylation of the main autophagy repressor mammalian target of rapamycin (mTOR) and its downstream target p70S6 kinase. The treatment with the mTOR activator leucine prevented idarubicin-mediated autophagy induction. Idarubicin-induced mTOR repression was associated with the activation of the mTOR inhibitor AMP-activated protein kinase and down-regulation of the mTOR activator Akt. The suppression of autophagy by pharmacological inhibitors or LC3B and beclin-1 genetic knockdown rescued REH cells from idarubicin-mediated oxidative stress, mitochondrial depolarization, caspase activation and apoptotic DNA fragmentation. Idarubicin also caused mTOR inhibition and cytotoxic autophagy in K562 leukemic cell line and leukocytes from chronic myeloid leukemia patients, but not healthy controls. By demonstrating mTOR-dependent cytotoxic autophagy in idarubicin-treated leukemic cells, our results warrant caution when considering combining idarubicin with autophagy inhibitors in leukemia therapy. - Highlights: • Idarubicin induces autophagy in leukemic cell lines and primary leukemic cells. • Idarubicin induces autophagy by inhibiting mTOR in leukemic cells. • mTOR suppression by idarubicin is associated with AMPK activation and Akt blockade. �

Cancer cell specific cytotoxic gene expression mediated by ARF tumor suppressor promoter constructs

International Nuclear Information System (INIS)

Kurayoshi, Kenta; Ozono, Eiko; Iwanaga, Ritsuko; Bradford, Andrew P.; Komori, Hideyuki; Ohtani, Kiyoshi

2014-01-01

Highlights: • ARF promoter showed higher responsiveness to deregulated E2F activity than the E2F1 promoter. • ARF promoter showed higher cancer cell-specificity than E2F1 promoter to drive gene expression. • HSV-TK driven by ARF promoter showed higher cancer cell-specific cytotoxicity than that driven by E2F1 promoter. - Abstract: In current cancer treatment protocols, such as radiation and chemotherapy, side effects on normal cells are major obstacles to radical therapy. To avoid these side effects, a cancer cell-specific approach is needed. One way to specifically target cancer cells is to utilize a cancer specific promoter to express a cytotoxic gene (suicide gene therapy) or a viral gene required for viral replication (oncolytic virotherapy). For this purpose, the selected promoter should have minimal activity in normal cells to avoid side effects, and high activity in a wide variety of cancers to obtain optimal therapeutic efficacy. In contrast to the AFP, CEA and PSA promoters, which have high activity only in a limited spectrum of tumors, the E2F1 promoter exhibits high activity in wide variety of cancers. This is based on the mechanism of carcinogenesis. Defects in the RB pathway and activation of the transcription factor E2F, the main target of the RB pathway, are observed in almost all cancers. Consequently, the E2F1 promoter, which is mainly regulated by E2F, has high activity in wide variety of cancers. However, E2F is also activated by growth stimulation in normal growing cells, suggesting that the E2F1 promoter may also be highly active in normal growing cells. In contrast, we found that the tumor suppressor ARF promoter is activated by deregulated E2F activity, induced by forced inactivation of pRB, but does not respond to physiological E2F activity induced by growth stimulation. We also found that the deregulated E2F activity, which activates the ARF promoter, is detected only in cancer cell lines. These observations suggest that ARF promoter

Cancer cell specific cytotoxic gene expression mediated by ARF tumor suppressor promoter constructs

Energy Technology Data Exchange (ETDEWEB)

Kurayoshi, Kenta [Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda, Hyogo 669-1337 (Japan); Ozono, Eiko [Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary, University of London, John Vane Science Centre, Charterhouse Square, London EC1M 6BQ (United Kingdom); Iwanaga, Ritsuko; Bradford, Andrew P. [Department of Obstetrics and Gynecology, University of Colorado School of Medicine, Anschutz Medical Campus, 12800 East 19th Avenue, Aurora, CO 80045 (United States); Komori, Hideyuki [Center for Stem Cell Biology, Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109 (United States); Ohtani, Kiyoshi, E-mail: btm88939@kwansei.ac.jp [Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda, Hyogo 669-1337 (Japan)

2014-07-18

Highlights: • ARF promoter showed higher responsiveness to deregulated E2F activity than the E2F1 promoter. • ARF promoter showed higher cancer cell-specificity than E2F1 promoter to drive gene expression. • HSV-TK driven by ARF promoter showed higher cancer cell-specific cytotoxicity than that driven by E2F1 promoter. - Abstract: In current cancer treatment protocols, such as radiation and chemotherapy, side effects on normal cells are major obstacles to radical therapy. To avoid these side effects, a cancer cell-specific approach is needed. One way to specifically target cancer cells is to utilize a cancer specific promoter to express a cytotoxic gene (suicide gene therapy) or a viral gene required for viral replication (oncolytic virotherapy). For this purpose, the selected promoter should have minimal activity in normal cells to avoid side effects, and high activity in a wide variety of cancers to obtain optimal therapeutic efficacy. In contrast to the AFP, CEA and PSA promoters, which have high activity only in a limited spectrum of tumors, the E2F1 promoter exhibits high activity in wide variety of cancers. This is based on the mechanism of carcinogenesis. Defects in the RB pathway and activation of the transcription factor E2F, the main target of the RB pathway, are observed in almost all cancers. Consequently, the E2F1 promoter, which is mainly regulated by E2F, has high activity in wide variety of cancers. However, E2F is also activated by growth stimulation in normal growing cells, suggesting that the E2F1 promoter may also be highly active in normal growing cells. In contrast, we found that the tumor suppressor ARF promoter is activated by deregulated E2F activity, induced by forced inactivation of pRB, but does not respond to physiological E2F activity induced by growth stimulation. We also found that the deregulated E2F activity, which activates the ARF promoter, is detected only in cancer cell lines. These observations suggest that ARF promoter

Protective effects of lichen metabolites evernic and usnic acids against redox impairment-mediated cytotoxicity in central nervous system-like cells.

Science.gov (United States)

Fernández-Moriano, Carlos; Divakar, Pradeep Kumar; Crespo, Ana; Gómez-Serranillos, M Pilar

2017-07-01

Lichens species produce unique secondary metabolites that attract increasing pharmacological interest, including their redox modulatory activities. Current work evaluated for the first time the in vitro cytoprotective properties, based on the antioxidant activities, of the Parmeliaceae lichens Evernia prunastri and Usnea ghattensis and the mechanism of action of their major phenolic constituents: the evernic and usnic acids, respectively. In two models of central nervous system-like cells (U373-MG and SH-SY5Y cell lines), exogenous H 2 O 2 induced oxidative stress-mediated cytotoxicity. We first assessed their radical scavenging capacities (ORAC and DPPH tests) and the phenolic content of the extracts. At the optimal concentrations, pretreatments with evernic acid displayed significant protection against H 2 O 2 -induced cytotoxic damage in both models. It reversed the alterations in oxidative stress markers (including ROS generation, glutathione system and lipid peroxidation levels) and cellular apoptosis (caspase-3 activity). Such effects were in part mediated by a notable enhancement of the expression of intracellular phase-II antioxidant enzymes; a plausible involvement of the Nrf2 cytoprotective pathway is suggested. Usnic acid exerted similar effects, to some extent more moderate. Results suggest that lichen polyketides evernic and usnic acids merit further research as promising antioxidant candidates in the therapy of oxidative stress-related diseases, including the neurodegenerative disorders. Copyright © 2017 Elsevier Ltd. All rights reserved.

Subamolide B Isolated from Medicinal Plant Cinnamomum subavenium Induces Cytotoxicity in Human Cutaneous Squamous Cell Carcinoma Cells through Mitochondrial and CHOP-Dependent Cell Death Pathways

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Shu-Yi Yang

2013-01-01

Full Text Available Subamolide B is a butanolide isolated from Cinnamomum subavenium, a medicinal plant traditionally used to treat various ailments including carcinomatous swelling. We herein reported for the first time that subamolide B potently induced cytotoxicity against diverse human skin cancer cell lines while sparing nonmalignant cells. Mechanistic studies on human cutaneous squamous cell carcinoma (SCC cell line SCC12 highlighted the involvement of apoptosis in subamolide B-induced cytotoxicity, as evidenced by the activation of caspases-8, -9, -4, and -3, the increase in annexin V-positive population, and the partial restoration of cell viability by cotreatment with the pan-caspase inhibitor z-VAD-fmk. Additionally, subamolide B evoked cell death pathways mediated by FasL/Fas, mitochondria, and endoplasmic reticulum (ER stress, as supported by subamolide B-induced FasL upregulation, BCL-2 suppression/cytosolic release of cytochrome c, and UPR activation/CHOP upregulation, respectively. Noteworthy, ectopic expression of c-FLIPL or dominant-negative mutant of FADD failed to impair subamolide B-induced cytotoxicity, whereas BCL-2 overexpression or CHOP depletion greatly rescued subamolide B-stimulated cells. Collectively, these results underscored the central role of mitochondrial and CHOP-mediated cell death pathways in subamolide B-induced cytotoxicity. Our findings further implicate the potential of subamolide B for cutaneous SCC therapy or as a lead compound for developing novel chemotherapeutic agents.

Lactobacillus delbrueckii ssp. bulgaricus B-30892 can inhibit cytotoxic effects and adhesion of pathogenic Clostridium difficile to Caco-2 cells

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Banerjee Pratik

2009-04-01

Full Text Available Abstract Background Probiotic microorganisms are receiving increasing interest for use in the prevention, treatment, or dietary management of certain diseases, including antibiotic-associated diarrhea (AAD. Clostridium difficile is the most common cause of AAD and the resulting C. difficile – mediated infection (CDI, is potentially deadly. C. difficile associated diarrhea (CDAD is manifested by severe inflammation and colitis, mostly due to the release of two exotoxins by C. difficile causing destruction of epithelial cells in the intestine. The aim of this study was to determine the effect of probiotic bacteria Lactobacillus delbrueckii ssp. bulgaricus B-30892 (LDB B-30892 on C. difficile-mediated cytotoxicity using Caco-2 cells as a model. Methods Experiments were carried out to test if the cytotoxicity induced by C. difficile-conditioned-medium on Caco-2 cells can be altered by cell-free supernatant (CFS from LDB B-30892 in different dilutions (1:2 to 1:2048. In a similar experimental setup, comparative evaluations of other probiotic strains were made by contrasting the results from these strains with the results from LDB B-30892, specifically the ability to affect C. difficile induced cytotoxicity on Caco-2 monolayers. Adhesion assays followed by quantitative analysis by Giemsa staining were conducted to test if the CFSs from LDB B-30892 and other probiotic test strains have the capability to alter the adhesion of C. difficile to the Caco-2 monolayer. Experiments were also performed to evaluate if LDB B-30892 or its released components have any bactericidal effect on C. difficile. Results and discussion Co-culturing of LDB B-30892 with C. difficile inhibited the C. difficile-mediated cytotoxicity on Caco-2 cells. When CFS from LDB B-30892-C. difficile co-culture was administered (up to a dilution of 1:16 on Caco-2 monolayer, there were no signs of cytotoxicity. When CFS from separately grown LDB B-30892 was mixed with the cell-free toxin

Uremic Toxins Enhance Statin-Induced Cytotoxicity in Differentiated Human Rhabdomyosarcoma Cells

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Hitoshi Uchiyama

2014-09-01

Full Text Available The risk of myopathy and rhabdomyolysis is considerably increased in statin users with end-stage renal failure (ESRF. Uremic toxins, which accumulate in patients with ESRF, exert cytotoxic effects that are mediated by various mechanisms. Therefore, accumulation of uremic toxins might increase statin-induced cytotoxicity. The purpose of this study was to determine the effect of four uremic toxins—hippuric acid, 3-carboxy-4-methyl-5-propyl-2-furanpropionate, indole-3-acetic acid, and 3-indoxyl sulfate—on statin-induced myopathy. Differentiated rhabdomyosarcoma cells were pre-treated with the uremic toxins for seven days, and then the cells were treated with pravastatin or simvastatin. Cell viability and apoptosis were assessed by viability assays and flow cytometry. Pre-treatment with uremic toxins increased statin- but not cisplatin-induced cytotoxicity (p < 0.05 vs. untreated. In addition, the pre-treatment increased statin-induced apoptosis, which is one of the cytotoxic factors (p < 0.05 vs. untreated. However, mevalonate, farnesol, and geranylgeraniol reversed the effects of uremic toxins and lowered statin-induced cytotoxicity (p < 0.05 vs. untreated. These results demonstrate that uremic toxins enhance statin-induced apoptosis and cytotoxicity. The mechanism underlying this effect might be associated with small G-protein geranylgeranylation. In conclusion, the increased severity of statin-induced rhabdomyolysis in patients with ESRF is likely due to the accumulation of uremic toxins.

Adenosine, but not guanosine, protects vaginal epithelial cells from Trichomonas vaginalis cytotoxicity.

Science.gov (United States)

Menezes, Camila Braz; Frasson, Amanda Piccoli; Meirelles, Lucia Collares; Tasca, Tiana

2017-02-01

Trichomonas vaginalis causes the most common non-viral sexually transmitted disease worldwide. The cytoadherence and cytotoxicity upon the vaginal epithelial cells are crucial for the infection. Extracellular nucleotides are released during cell damage and, along with their nucleosides, can activate purinoceptors. The opposing effects of nucleotides versus nucleosides are regulated by ectonucleotidases. Herein we evaluated the hemolysis and cytolysis induced by T. vaginalis, as well as the extracellular nucleotide hydrolysis along with the effects mediated by nucleotides and nucleosides on cytotoxicity. In addition, the gene expression of purinoceptors in host cells was determined. The hemolysis and cytolysis exerted by all T. vaginalis isolates presented positive Pearson correlation. All T. vaginalis isolates were able to hydrolyze nucleotides, showing higher NTPDase than ecto-5'-nucleotidase activity. The most cytotoxic isolate, TV-LACM6, hydrolyzes ATP, GTP with more efficiency than AMP and GMP. The vaginal epithelial cell line (HMVII) expressed the genes for all subtypes of P1, P2X and P2Y receptors. Finally, when nucleotides and nucleosides were tested, the cytotoxic effect elicited by TV-LACM6 was increased with nucleotides. In contrast, the cytotoxicity was reversed by adenosine in presence of EHNA, but not by guanosine, contributing to the understanding of the purinergic signaling role on T. vaginalis cytotoxicity. Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Cell mediated immunity in patients with osteosarcoma

International Nuclear Information System (INIS)

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

1975-01-01

Because of the difficulty of obtaining suitable material, earlier studies on cell mediated immunity in the radium patients failed to include positive controls. Recently we were fortunate in obtaining samples of lymphocytes from two suitable patients who had had amputations for spontaneous osteosarcoma six months previously. Lymphocytes from both of these patients showed cytotoxicity to cultured cells derived from a human osteogenic sarcoma but not to normal fibroblasts. These results help to validate our test for early detection of osteosarcoma in the radium patients using measurements of cytotoxicity

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

Science.gov (United States)

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

2017-08-01

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

Inhibition of clone formation as an assay for T cell-mediated cytotoxicity: short-term kinetics and comparison with 51Cr release

International Nuclear Information System (INIS)

Lees, R.K.; MacDonald, H.R.; Sinclair, N.R.; University of Western Ontario London

1977-01-01

The short-term kinetics of T cell-mediated cytotoxicity was investigated using a cloning inhibition assay. Murine cytotoxic thymus-derived lymphocytes generated in vitro in mixed leukocyte cultures were incubated for various periods of time at 37degC with allogeneic mastocytoma target cells. The mixtures were then plated in soft agar, and mastocytoma clone formation was assessed after 5-7 days incubation. Using this technique, it was demonstrated that events leading to the loss of cloning ability could be detected after 1-3 min incubation at 37degC, and after 20-30 min, 95% of the clone forming cells had been inactivated. When these results were compared directly with those obtained using the conventional 51 Cr-release assay, it was found that the events leading to loss of cloning ability occurred more rapidly than indicated by the isotope assay. However, a modification of the 51 Cr-release assay involving EDTA addition gave comparable result to the cloning inhibition assay. These results raise the possibility that the events leading to 51 Cr-release of tumor target cells may be related in time to those leading to the loss of cloning ability

Evaluation of a UCMK/dCK fusion enzyme for gemcitabine-mediated cytotoxicity

International Nuclear Information System (INIS)

Johnson, Adam J.; Brown, Melissa N.; Black, Margaret E.

2011-01-01

Highlights: ► Goal was to enhance dFdC cytotoxicity by the creation of a UCMK/dCK fusion enzyme. ► The UCMK/dCK fusion enzyme possesses both native activities. ► The fusion renders cells equally sensitive to dFdC relative to dCK expression alone. ► Dual activities of fusion not sufficient to augment cell dFdC sensitivity in vitro. ► Data may warrant the implementation of UCMK mutagenesis studies. -- Abstract: While gemcitabine (2′-2′-difluoro-2′-deoxycytidine, dFdC) displays wide-ranging antineoplastic activity as a single agent, variable response rates and poor intracellular metabolism often limit its clinical efficacy. In an effort to enhance dFdC cytotoxicity and help normalize response rates, we created a bifunctional fusion enzyme that combines the enzymatic activities of deoxycytidine kinase (dCK) and uridine/cytidine monophosphate kinase (UCMK) in a single polypeptide. Our goal was to evaluate whether the created fusion could induce beneficial, functional changes toward dFdC, expedite dFdC conversion to its active antimetabolites and consequently amplify cell dFdC sensitivity. While kinetic analyses revealed the UCMK/dCK fusion enzyme to possess both native activities, the fusion rendered cells sensitive to the cytotoxic effects of dFdC at the same level as dCK expression alone. These results suggest that increased wild-type UCMK expression does not provide a significant enhancement in dFdC-mediated cytotoxicity and may warrant the implementation of studies aimed at engineering UCMK variants with improved activity toward gemcitabine monophosphate.

Cytotoxicity of synthetic cannabinoids on primary neuronal cells of the forebrain: the involvement of cannabinoid CB1 receptors and apoptotic cell death

International Nuclear Information System (INIS)

Tomiyama, Ken-ichi; Funada, Masahiko

2014-01-01

The abuse of herbal products containing synthetic cannabinoids has become an issue of public concern. The purpose of this paper was to evaluate the acute cytotoxicity of synthetic cannabinoids on mouse brain neuronal cells. Cytotoxicity induced by synthetic cannabinoid (CP-55,940, CP-47,497, CP-47,497-C8, HU-210, JWH-018, JWH-210, AM-2201, and MAM-2201) was examined using forebrain neuronal cultures. These synthetic cannabinoids induced cytotoxicity in the forebrain cultures in a concentration-dependent manner. The cytotoxicity was suppressed by preincubation with the selective CB 1 receptor antagonist AM251, but not with the selective CB 2 receptor antagonist AM630. Furthermore, annexin-V-positive cells were found among the treated forebrain cells. Synthetic cannabinoid treatment induced the activation of caspase-3, and preincubation with a caspase-3 inhibitor significantly suppressed the cytotoxicity. These synthetic cannabinoids induced apoptosis through a caspase-3-dependent mechanism in the forebrain cultures. Our results indicate that the cytotoxicity of synthetic cannabinoids towards primary neuronal cells is mediated by the CB 1 receptor, but not by the CB 2 receptor, and further suggest that caspase cascades may play an important role in the apoptosis induced by these synthetic cannabinoids. In conclusion, excessive synthetic cannabinoid abuse may present a serious acute health concern due to neuronal damage or deficits in the brain. - Highlights: • Synthetic cannabinoids (classical cannabinoids, non-classical cannabinoids, and aminoalkylindole derivatives) induce cytotoxicity in mouse forebrain cultures. • Synthetic cannabinoid-induced cytotoxicity towards forebrain cultures is mediated by the CB 1 receptor, but not by the CB 2 receptor, and involves caspase-dependent apoptosis. • A high concentration of synthetic cannabinoids may be toxic to neuronal cells that express CB 1 receptors

Antibody Fc engineering improves frequency and promotes kinetic boosting of serial killing mediated by NK cells

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Romain, Gabrielle; Senyukov, Vladimir; Rey-Villamizar, Nicolas; Merouane, Amine; Kelton, William; Liadi, Ivan; Mahendra, Ankit; Charab, Wissam; Georgiou, George; Roysam, Badrinath; Lee, Dean A.

2014-01-01

The efficacy of most therapeutic monoclonal antibodies (mAbs) targeting tumor antigens results primarily from their ability to elicit potent cytotoxicity through effector-mediated functions. We have engineered the fragment crystallizable (Fc) region of the immunoglobulin G (IgG) mAb, HuM195, targeting the leukemic antigen CD33, by introducing the triple mutation Ser293Asp/Ala330Leu/Ile332Glu (DLE), and developed Time-lapse Imaging Microscopy in Nanowell Grids to analyze antibody-dependent cell-mediated cytotoxicity kinetics of thousands of individual natural killer (NK) cells and mAb-coated target cells. We demonstrate that the DLE-HuM195 antibody increases both the quality and the quantity of NK cell-mediated antibody-dependent cytotoxicity by endowing more NK cells to participate in cytotoxicity via accrued CD16-mediated signaling and by increasing serial killing of target cells. NK cells encountering targets coated with DLE-HuM195 induce rapid target cell apoptosis by promoting simultaneous conjugates to multiple target cells and induce apoptosis in twice the number of target cells within the same period as the wild-type mAb. Enhanced target killing was also associated with increased frequency of NK cells undergoing apoptosis, but this effect was donor-dependent. Antibody-based therapies targeting tumor antigens will benefit from a better understanding of cell-mediated tumor elimination, and our work opens further opportunities for the therapeutic targeting of CD33 in the treatment of acute myeloid leukemia. PMID:25232058

Lactobacillus delbrueckii ssp. bulgaricus B-30892 can inhibit cytotoxic effects and adhesion of pathogenic Clostridium difficile to Caco-2 cells

Science.gov (United States)

Banerjee, Pratik; Merkel, Glenn J; Bhunia, Arun K

2009-01-01

Background Probiotic microorganisms are receiving increasing interest for use in the prevention, treatment, or dietary management of certain diseases, including antibiotic-associated diarrhea (AAD). Clostridium difficile is the most common cause of AAD and the resulting C. difficile – mediated infection (CDI), is potentially deadly. C. difficile associated diarrhea (CDAD) is manifested by severe inflammation and colitis, mostly due to the release of two exotoxins by C. difficile causing destruction of epithelial cells in the intestine. The aim of this study was to determine the effect of probiotic bacteria Lactobacillus delbrueckii ssp. bulgaricus B-30892 (LDB B-30892) on C. difficile-mediated cytotoxicity using Caco-2 cells as a model. Methods Experiments were carried out to test if the cytotoxicity induced by C. difficile-conditioned-medium on Caco-2 cells can be altered by cell-free supernatant (CFS) from LDB B-30892 in different dilutions (1:2 to 1:2048). In a similar experimental setup, comparative evaluations of other probiotic strains were made by contrasting the results from these strains with the results from LDB B-30892, specifically the ability to affect C. difficile induced cytotoxicity on Caco-2 monolayers. Adhesion assays followed by quantitative analysis by Giemsa staining were conducted to test if the CFSs from LDB B-30892 and other probiotic test strains have the capability to alter the adhesion of C. difficile to the Caco-2 monolayer. Experiments were also performed to evaluate if LDB B-30892 or its released components have any bactericidal effect on C. difficile. Results and discussion Co-culturing of LDB B-30892 with C. difficile inhibited the C. difficile-mediated cytotoxicity on Caco-2 cells. When CFS from LDB B-30892-C. difficile co-culture was administered (up to a dilution of 1:16) on Caco-2 monolayer, there were no signs of cytotoxicity. When CFS from separately grown LDB B-30892 was mixed with the cell-free toxin preparation (CFT) of

A novel multiparametric flow cytometry-based cytotoxicity assay simultaneously immunophenotypes effector cells: Comparisons to a 4 h 51Cr-release assay

OpenAIRE

Kim, GG; Donnenberg, VS; Donnenberg, AD; Gooding, W; Whiteside, TL

2007-01-01

Natural killer (NK) cell- or T cell-mediated cytotoxicity traditionally is measured in 4-16h 51Cr-release assays (CRA). A new four-color flow cytometry-based cytotoxicity assay (FCC) was developed to simultaneously measure NK cell cytotoxicity and NK cell phenotype (CD3−CD16+CD56+). Target cells, K562 or Daudi, were labeled with Cell Tracker Orange (CTO) prior to the addition of effector cells. Following co-incubation, 7 amino-actinomycin D (7-AAD) was added to measure death of target cells. ...

Daratumumab-mediated lysis of primary multiple myeloma cells is enhanced in combination with the human anti-KIR antibody IPH2102 and lenalidomide

DEFF Research Database (Denmark)

Nijhof, I. S.; Lammerts van Bueren, J. J.; van Kessel, B.

2015-01-01

Despite recent treatment improvements, multiple myeloma remains an incurable disease. Since antibody-dependent cell-mediated cytotoxicity is an important effector mechanism of daratumumab, we explored the possibility of improving daratumumab-mediated cell-mediated cytotoxicity by blocking natural...... killer cell inhibitory receptors with the human monoclonal anti-KIR antibody IPH2102, next to activation of natural killer cells with the immune modulatory drug lenalidomide. In 4-hour antibody-dependent cell-mediated cytotoxicity assays, IPH2102 did not induce lysis of multiple myeloma cell lines...... effective treatment strategies can be designed for multiple myeloma by combining daratumumab with agents that independently modulate natural killer cell function....

Cytotoxic T lymphocyte-mediated cytolysis: an example of programmed cell death in the immune system

International Nuclear Information System (INIS)

Duke, R.C.

1985-01-01

Target cells are programmed to die following interaction with cytotoxic T lymphocytes (CTLs). Within minutes of exposure to CTL the target cell's nuclear DNA is fragmented. Target cell lysis, as measured by 51 Cr release, occurs about 60 minutes after induction of DNA fragmentation. DNA fragmentation results from the action of an endonuclease which cleaves DNA in the linker region between nucleosomes. The origin of this nuclease, whether transferred to the target by the CTL or endogenous to the target cell, has not been resolved. DNA fragmentation occurs only when appropriately sensitized CTL are used and is not merely the result of cell death because killing of target cells by extreme deviation from homeostasis, by interruption of energy production, or by lysis with antibody and complement does not induce DNA cleavage. When Triton X-100 is added to target cells which have interacted with CTL, the DNA fragments do not remain in association with the nucleus. This observation suggests that breakdown of overall nuclear structure is induced concomitantly with DNA fragmentation. Morphologically, disruption of nuclear structure and DNA fragmentation are observed as widespread chromatin condensation (apoptosis). Apoptosis is observed in metabolically active target cells and is not a consequence of cell death. A cell whose DNA is extensively fragmented is condemmed to die. Induction of oligonucleosome-sized DNA is also an early event in glucocorticoid-induced thymocyte death and death of T cells upon removal of growth factor. Several similarities exist between these systems and CTL-mediated cytolysis suggesting a final common biochemical pathway for all three types of cell death

Induction of potent NK cell-dependent anti-myeloma cytotoxic T cells in response to combined mapatumumab and bortezomib.

Science.gov (United States)

Neeson, Paul J; Hsu, Andy K; Chen, Yin R; Halse, Heloise M; Loh, Joanna; Cordy, Reece; Fielding, Kate; Davis, Joanne; Noske, Josh; Davenport, Alex J; Lindqvist-Gigg, Camilla A; Humphreys, Robin; Tai, Tsin; Prince, H Miles; Trapani, Joseph A; Smyth, Mark J; Ritchie, David S

2015-09-01

There is increasing evidence that some cancer therapies can promote tumor immunogenicity to boost the endogenous antitumor immune response. In this study, we used the novel combination of agonistic anti-TRAIL-R1 antibody (mapatumumab, Mapa) with low dose bortezomib (LDB) for this purpose. The combination induced profound myeloma cell apoptosis, greatly enhanced the uptake of myeloma cell apoptotic bodies by dendritic cell (DC) and induced anti-myeloma cytotoxicity by both CD8 + T cells and NK cells. Cytotoxic lymphocyte expansion was detected within 24 h of commencing therapy and was maximized when myeloma-pulsed DC were co-treated with low dose bortezomib and mapatumumab (LDB+Mapa) in the presence of NK cells. This study shows that Mapa has two distinct but connected modes of action against multiple myeloma (MM). First, when combined with LDB, Mapa produced powerful myeloma cell apoptosis; secondly, it promoted DC priming and an NK cell-mediated expansion of anti-myeloma cytotoxic lymphocyte (CTL). Overall, this study indicates that Mapa can be used to drive potent anti-MM immune responses.

Cytotoxicity of synthetic cannabinoids on primary neuronal cells of the forebrain: the involvement of cannabinoid CB{sub 1} receptors and apoptotic cell death

Energy Technology Data Exchange (ETDEWEB)

Tomiyama, Ken-ichi; Funada, Masahiko, E-mail: mfunada@ncnp.go.jp

2014-01-01

The abuse of herbal products containing synthetic cannabinoids has become an issue of public concern. The purpose of this paper was to evaluate the acute cytotoxicity of synthetic cannabinoids on mouse brain neuronal cells. Cytotoxicity induced by synthetic cannabinoid (CP-55,940, CP-47,497, CP-47,497-C8, HU-210, JWH-018, JWH-210, AM-2201, and MAM-2201) was examined using forebrain neuronal cultures. These synthetic cannabinoids induced cytotoxicity in the forebrain cultures in a concentration-dependent manner. The cytotoxicity was suppressed by preincubation with the selective CB{sub 1} receptor antagonist AM251, but not with the selective CB{sub 2} receptor antagonist AM630. Furthermore, annexin-V-positive cells were found among the treated forebrain cells. Synthetic cannabinoid treatment induced the activation of caspase-3, and preincubation with a caspase-3 inhibitor significantly suppressed the cytotoxicity. These synthetic cannabinoids induced apoptosis through a caspase-3-dependent mechanism in the forebrain cultures. Our results indicate that the cytotoxicity of synthetic cannabinoids towards primary neuronal cells is mediated by the CB{sub 1} receptor, but not by the CB{sub 2} receptor, and further suggest that caspase cascades may play an important role in the apoptosis induced by these synthetic cannabinoids. In conclusion, excessive synthetic cannabinoid abuse may present a serious acute health concern due to neuronal damage or deficits in the brain. - Highlights: • Synthetic cannabinoids (classical cannabinoids, non-classical cannabinoids, and aminoalkylindole derivatives) induce cytotoxicity in mouse forebrain cultures. • Synthetic cannabinoid-induced cytotoxicity towards forebrain cultures is mediated by the CB{sub 1} receptor, but not by the CB{sub 2} receptor, and involves caspase-dependent apoptosis. • A high concentration of synthetic cannabinoids may be toxic to neuronal cells that express CB{sub 1} receptors.

Cytotoxic activities of amentoflavone against human breast and cervical cancers are mediated by increasing of PTEN expression levels due to peroxisomes proliferate-activated receptor {gamma} activation

Energy Technology Data Exchange (ETDEWEB)

Lee, Eunjung; Shin, Soyoung; Lee, Jeeyoung; Lee, So Jung; Kim, Jinkyoung; Yoon, Doyoung; Kim, Yangmee [Konkuk Univ., Seoul (Korea, Republic of); Woo, Eunrhan [Chosun Univ., Gwangju (Korea, Republic of)

2012-07-15

Human peroxisomes proliferate-activated receptor gamma (hPPAR{gamma}) has been implicated in numerous pathologies, including obesity, diabetes, and cancer. Previously, we verified that amentoflavone is an activator of hPPAR{gamma} and probed the molecular basis of its action. In this study, we investigated the mechanism of action of amentoflavone in cancer cells and demonstrated that amentoflavone showed strong cytotoxicity against MCF-7 and HeLa cancer cell lines. We showed that hPPAR{gamma} expression in MCF-7 and HeLa cells is specifically stimulated by amentoflavone, and suggested that amentoflavone-induced cytotoxic activities are mediated by activation of hPPAR{gamma} in these two cancer cell lines. Moreover, amentoflavone increased PTEN levels in these two cancer cell lines, indicating that the cytotoxic activities of amentoflavone are mediated by increasing of PTEN expression levels due to hPPAR{gamma} activation.

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

The adaptor molecule SAP plays essential roles during invariant NKT cell cytotoxicity and lytic synapse formation.

Science.gov (United States)

Das, Rupali; Bassiri, Hamid; Guan, Peng; Wiener, Susan; Banerjee, Pinaki P; Zhong, Ming-Chao; Veillette, André; Orange, Jordan S; Nichols, Kim E

2013-04-25

The adaptor molecule signaling lymphocytic activation molecule-associated protein (SAP) plays critical roles during invariant natural killer T (iNKT) cell ontogeny. As a result, SAP-deficient humans and mice lack iNKT cells. The strict developmental requirement for SAP has made it difficult to discern its possible involvement in mature iNKT cell functions. By using temporal Cre recombinase-mediated gene deletion to ablate SAP expression after completion of iNKT cell development, we demonstrate that SAP is essential for T-cell receptor (TCR)-induced iNKT cell cytotoxicity against T-cell and B-cell leukemia targets in vitro and iNKT-cell-mediated control of T-cell leukemia growth in vivo. These findings are not restricted to the murine system: silencing RNA-mediated suppression of SAP expression in human iNKT cells also significantly impairs TCR-induced cytolysis. Mechanistic studies reveal that iNKT cell killing requires the tyrosine kinase Fyn, a known SAP-binding protein. Furthermore, SAP expression is required within iNKT cells to facilitate their interaction with T-cell targets and induce reorientation of the microtubule-organizing center to the immunologic synapse (IS). Collectively, these studies highlight a novel and essential role for SAP during iNKT cell cytotoxicity and formation of a functional IS.

Microchip screening platform for single cell assessment of NK cell cytotoxicity

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Karolin eGuldevall

2016-04-01

Full Text Available Here we report a screening platform for assessment of the cytotoxic potential of individual natural killer (NK cells within larger populations. Human primary NK cells were distributed across a silicon-glass microchip containing 32 400 individual microwells loaded with target cells. Through fluorescence screening and automated image analysis the numbers of NK and live or dead target cells in each well could be assessed at different time points after initial mixing. Cytotoxicity was also studied by time-lapse live-cell imaging in microwells quantifying the killing potential of individual NK cells. Although most resting NK cells (≈75% were non-cytotoxic against the leukemia cell line K562, some NK cells were able to kill several (≥3 target cells within the 12 hours long experiment. In addition, the screening approach was adapted to increase the chance to find and evaluate serial killing NK cells. Even if the cytotoxic potential varied between donors it was evident that a small fraction of highly cytotoxic NK cells were responsible for a substantial portion of the killing. We demonstrate multiple assays where our platform can be used to enumerate and characterize cytotoxic cells, such as NK or T cells. This approach could find use in clinical applications, e.g. in the selection of donors for stem cell transplantation or generation of highly specific and cytotoxic cells for adoptive immunotherapy.

Microchip Screening Platform for Single Cell Assessment of NK Cell Cytotoxicity

Science.gov (United States)

Guldevall, Karolin; Brandt, Ludwig; Forslund, Elin; Olofsson, Karl; Frisk, Thomas W.; Olofsson, Per E.; Gustafsson, Karin; Manneberg, Otto; Vanherberghen, Bruno; Brismar, Hjalmar; Kärre, Klas; Uhlin, Michael; Önfelt, Björn

2016-01-01

Here, we report a screening platform for assessment of the cytotoxic potential of individual natural killer (NK) cells within larger populations. Human primary NK cells were distributed across a silicon–glass microchip containing 32,400 individual microwells loaded with target cells. Through fluorescence screening and automated image analysis, the numbers of NK and live or dead target cells in each well could be assessed at different time points after initial mixing. Cytotoxicity was also studied by time-lapse live-cell imaging in microwells quantifying the killing potential of individual NK cells. Although most resting NK cells (≈75%) were non-cytotoxic against the leukemia cell line K562, some NK cells were able to kill several (≥3) target cells within the 12-h long experiment. In addition, the screening approach was adapted to increase the chance to find and evaluate serial killing NK cells. Even if the cytotoxic potential varied between donors, it was evident that a small fraction of highly cytotoxic NK cells were responsible for a substantial portion of the killing. We demonstrate multiple assays where our platform can be used to enumerate and characterize cytotoxic cells, such as NK or T cells. This approach could find use in clinical applications, e.g., in the selection of donors for stem cell transplantation or generation of highly specific and cytotoxic cells for adoptive immunotherapy. PMID:27092139

Differential effects of IL-2 and IL-21 on expansion of the CD4+ CD25+ Foxp3+ T regulatory cells with redundant roles in natural killer cell mediated antibody dependent cellular cytotoxicity in chronic lymphocytic leukemia.

Science.gov (United States)

Gowda, Aruna; Ramanunni, Asha; Cheney, Carolyn; Rozewski, Darlene; Kindsvogel, Wayne; Lehman, Amy; Jarjoura, David; Caligiuri, Michael; Byrd, John C; Muthusamy, Natarajan

2010-01-01

CD4(+) CD25(+) regulatory T cells are expanded in solid and hematological malignancies including chronic lymphocytic leukemia (CLL). Several cytokines and co-stimulatory molecules are required for generation, survival and maintenance of their suppressive effect. We and others have shown direct cytotoxic effect of the novel common gamma chain cytokine interleukin (IL)-21 on primary B cells from CLL patients. Since members of this family of cytokines are known to exhibit their effects on diverse immune cells, we have examined the effects of IL-21 on CLL patient derived regulatory T cell (Treg) induction, expansion and the inhibitory effect on natural killer cells in vitro. We demonstrate here the expression of IL-21 receptor in CD4(+)CD25(High) regulatory cells from CLL patients. In contrast to IL-2, the IL-21 cytokine failed to mediate expansion of regulatory T cells or induced expression of Foxp3 in CD4(+)CD25(Intermediate) or CD4(+)CD25(Dim/-) T cells in whole blood derived from CLL patients. Interestingly, in contrast to their differential effects on expansion of the CD4(+)CD25(+)Foxp3(+)T cells, IL-2 and IL-21 exhibited a redundant role in Treg mediated suppression of NK cell mediated antibody dependent cytotoxicity function. Given the infusion related toxicities and pro-survival effect of IL-2 in CLL, these studies provide a rationale to explore IL-21 as an alternate gamma chain cytokine in CLL therapy.

Cytotoxicity of lambda-cyhalothrin on the macrophage cell line RAW 264.7.

Science.gov (United States)

Zhang, Quan; Wang, Cui; Sun, Liwei; Li, Ling; Zhao, Meirong

2010-01-01

The wide use and wide-spectrum toxicity of synthetic pyrethroids (SPs) insecticides make them an emerging ecotoxicological concern. Some previous studies showed that SPs possessed cytotoxicity in some immune cells such as human lymphocytes and rat bone marrow. However, the cytotoxicity of SPs to macrophages, which are crucial to innate immunity, has not been explored. In the present report, we investigated a new pyrethroid insecticide, lambda-cyhalothrin (LCT), which may increase the generation of reactive oxygen species (ROS) and DNA damage levels and cause cytotoxicity in RAW 264.7 cells in dose- and time-dependent manners. The results for the first time implicated increased endogenous ROS and DNA damage as co-mediators of LCT-induced cytotoxicity in macrophages. Our results also suggested that macrophages were involved in synthetic pyrethroid-induced adverse immune effects. Considering the ubiquitous environmental presence of SPs, this study provided new information relative to the potential long-term physiological and immunological effects associated with chronic exposure to SPs. Hence, the potential immunotoxicity of SPs should be considered in assessing the safety of these compounds in sensitive environmental compartments.

Cytotoxicity screening of Bangladeshi medicinal plant extracts on pancreatic cancer cells

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Abbasi Atiya

2010-09-01

Full Text Available Abstract Background There has been a long standing interest in the identification of medicinal plants and derived natural products for developing cancer therapeutics. Our study focuses upon pancreatic cancer, due to its high mortality rate, that is attributed in part to the lack of an effective chemotherapeutic agent. Previous reports on the use of medicinal plant extracts either alone or alongside conventional anticancer agents in the treatment of this cancer have shown promising results. This work aims to investigate the therapeutic properties of a library of medicinal plants from Bangladesh. Methods 56 extracts of 44 unique medicinal plants were studied. The extracts were screened for cytotoxicity against the pancreatic adenocarcinoma cell line Panc-1, using a label-free biosensor assay. The top cytotoxic extracts identified in this screen were tested on two additional pancreatic cancer cell lines (Mia-Paca2 and Capan-1 and a fibroblast cell line (Hs68 using an MTT proliferation assay. Finally, one of the most promising extracts was studied using a caspase-3 colorimetric assay to identify induction of apoptosis. Results Crude extracts of Petunia punctata, Alternanthera sessilis, and Amoora chittagonga showed cytotoxicity to three cancer cell lines with IC50 values ranging between 20.3 - 31.4 μg/mL, 13.08 - 34.9 μg/mL, and 42.8 - 49.8 μg/mL, respectively. Furthermore, treatment of Panc-1 cells with Petunia punctata was shown to increase caspase-3 activity, indicating that the observed cytotoxicity was mediated via apoptosis. Only Amoora chittagonga showed low cytotoxicity to fibroblast cells with an IC50 value > 100 μg/mL. Conclusion Based upon the initial screening work reported here, further studies aimed at the identification of active components of these three extracts and the elucidation of their mechanisms as cancer therapeutics are warranted.

Role of Zn doping in oxidative stress mediated cytotoxicity of TiO2 nanoparticles in human breast cancer MCF-7 cells

Science.gov (United States)

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

2016-07-01

We investigated the effect of Zn-doping on structural and optical properties as well as cellular response of TiO2 nanoparticles (NPs) in human breast cancer MCF-7 cells. A library of Zn-doped (1-10 at wt%) TiO2 NPs was prepared. Characterization data indicated that dopant Zn was incorporated into the lattice of host TiO2. The average particle size of TiO2 NPs was decreases (38 to 28 nm) while the band gap energy was increases (3.35 eV-3.85 eV) with increasing the amount of Zn-doping. Cellular data demonstrated that Zn-doped TiO2 NPs induced cytotoxicity (cell viability reduction, membrane damage and cell cycle arrest) and oxidative stress (reactive oxygen species generation & glutathione depletion) in MCF-7 cells and toxic intensity was increases with increasing the concentration of Zn-doping. Molecular data revealed that Zn-doped TiO2 NPs induced the down-regulation of super oxide dismutase gene while the up-regulation of heme oxygenase-1 gene in MCF-7 cells. Cytotoxicity induced by Zn-doped TiO2 NPs was efficiently prevented by N-acetyl-cysteine suggesting that oxidative stress might be the primarily cause of toxicity. In conclusion, our data indicated that Zn-doping decreases the particle size and increases the band gap energy as well the oxidative stress-mediated toxicity of TiO2 NPs in MCF-7 cells.

Functional cell mediated lympholysis I. Description of the assay

International Nuclear Information System (INIS)

Goeken, N.E.; Thompson, J.S.

1981-01-01

The anamnestic response by human bi-directional (BD) mixed lymphocyte cultures (MLC) to restimulation by cells of the original stimulating type is generally strikingly reduced as compared to that of standard one-way cultures. This difference was shown not to be related to a change in kinetics nor was it due to exhaustion of the media or soluble factors since fresh media did not ameliorate the effect nor were supernatants from BD cultures found to be suppressive. The relative inhibition was also not reversed by removal of the allogeneic cells by phenotype specific antiserum. Cytotoxic tests with donor and responder specific antisera revealed that the cells bearing that phenotype were dramatically reduced in BD as compared to one-way cultures. Thus, the diminished secondary response appears to be due to cytotoxic elimination of the responder cells. This allogeneic cytotoxicity is dependent on non-T, phagocytic, adherent cells. The phenomenon is called Functional Cell Mediated Lympholysis (F-CML). (author)

A Developed NK-92MI Cell Line with Siglec-7neg Phenotype Exhibits High and Sustainable Cytotoxicity against Leukemia Cells

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Chin-Han Huang

2018-04-01

Full Text Available Altered sialic acid processing that leads to upregulation of cell surface sialylation is recognized as a key change in malignant tissue glycosylation. This cancer-associated hypersialylation directly impacts the signaling interactions between tumor cells and their surrounding microenvironment, especially the interactions mediated by immune cell surface sialic acid-binding immunoglobulin-like lectins (Siglecs to relay inhibitory signals for cytotoxicity. First, we obtained a Siglec-7neg NK-92MI cell line, NK-92MI-S7N, by separating a group of Siglec-7neg cell population from an eight-month-long-term NK-92MI in vitro culture by fluorescence-activated cell sorting (FACS. The effect of Siglec-7 loss on NK-92MI-S7N cells was characterized by the cell morphology, proliferation, and cytotoxic activity via FACS, MTS assay, cytotoxic assay, and natural killer (NK degranulation assay. We found the expression levels of Siglec-7 in NK-92MI were negatively correlated with NK cytotoxicity against leukemia cells. This NK-92MI-S7N cell not only shared very similar phenotypes with its parental cells but also possessed a high and sustainable killing activity. Furthermore, this Siglec-7neg NK line was unexpectedly capable of eliminating a NK-92MI-resistant leukemia cell, THP-1, through enhancing the effector-target interaction. In this study, a NK cell line with high and sustainable cytotoxicity was established and this cell may provide a potential application in NK-based treatment for leukemia patients.

Cytotoxicity and Genotoxicity of Cypermethrin in Hepatocarcinoma Cells: A Dose- and Time-Dependent Study.

Science.gov (United States)

AlKahtane, Abdullah A; Alarifi, Saud; Al-Qahtani, Ahmed A; Ali, Daoud; Alomar, Suliman Y; Aleissia, Mohammed S; Alkahtani, Saad

2018-01-01

Most of the agricultural workers are potentially exposed to pesticides through different routes. Inhalation exposures may result in numerous diseases that can adversely affect an individual's health and capacity to perform at work. The aim of this study was to determine the cytotoxic potential of cypermethrin pesticide on cultured human hepatocarcinoma (HepG2) cells. The HepG2 cells were exposed to cypermethrin (0, 5, 15, 40 ng/mL) for 24 and 48 hours. We observed that cypermethrin caused cell death of HepG2 cells using 3-(4, 5-dimethylthiozolyl-2)-2,5-diphenyl tetrazolium bromide (MTT) and lactate dehydrogenase tests. Furthermore, cypermethrin reduced HepG2 cells viability in a time and dose dependent basis, that was probably mediated through the induction of reactive oxygen species (ROS) and apoptosis. An increase in ROS generation with a concomitant increase in expression of the proapoptotic protein Bcl-2 and cytochrome c and decrease in the antiapoptosis protein Bax suggested that a mitochondria-mediated pathway was involved in cypermethrin-induced apoptosis. These findings provide insights into the underlying mechanisms involved in cytotoxicity of cypermethrin in HepG2 cells.

Increased Peripheral Blood Pro-Inflammatory/Cytotoxic Lymphocytes in Children with Bronchiectasis.

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G Hodge

Full Text Available Bronchiectasis (BE in children is common in some communities including Indigenous children in Australia. Relatively little is known about the nature of systemic inflammation in these children, especially the contribution of specific pro-inflammatory and cytotoxic lymphocyte subsets: T-cells, natural killer (NK cells and NKT-like cells. We have shown that these cells produce increased cytotoxic (granzyme b and perforin and inflammatory (IFNγ and TNFα mediators in several adult chronic lung diseases and hypothesised that similar changes would be evident in children with BE.Intracellular cytotoxic mediators perforin and granzyme b and pro-inflammatory cytokines were measured in T cell subsets, NKT-like and NK cells from blood and bronchoalveolar samples from 12 children with BE and 10 aged-matched control children using flow cytometry.There was a significant increase in the percentage of CD8+ T cells and T and NKT-like subsets expressing perforin/granzyme and IFNγ and TNFα in blood in BE compared with controls. There was a further increase in the percentage of pro-inflammatory cytotoxic T cells in Indigenous compared with non-Indigenous children. There was no change in any of these mediators in BAL.Childhood bronchiectasis is associated with increased systemic pro-inflammatory/cytotoxic lymphocytes in the peripheral blood. Future studies need to examine the extent to which elevated levels of pro-inflammatory cytotoxic cells predict future co-morbidities.

Cytotoxic effects of local anesthesia through lidocaine/ropivacaine on human melanoma cell lines

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Ding-Kun Kang

Full Text Available Abstract Background: Local anesthetics (LAs are generally considered as safe, but cytotoxicity has been reported for several local anesthetics used in humans, which is not well investigated. In the present study, the cytotoxicity of lidocaine, ropivacaine and the combination of lidocaine and ropivacaine were evaluated on human melanoma cell lines. Melphalan, a nitrogen mustard alkylating agent, was used as a control agent for comparison of cytotoxic activity. Methods: Melanoma cell lines, A375 and Hs294T, were exposed to 1 h to different concentrations of above agents. Cell-viability after exposure was determined by flow cytometry. Results: Investigated LAs showed detrimental cytotoxicity on studied melanoma cell lines in time- (p < 0.001, concentration- (p < 0.001, and agent dependant. In both A375 and Hs294T cell lines, minimum cell viability rates were found after 72 h of exposure to these agents. Lidocaine 2% caused a reduction of vital cells to 10% ± 2% and 14% ± 2% in A375 and Hs294T, respectively after 72 h of exposure. Ropivacaine 0.75% after 72 h reduced viable cells to 15% ± 3% and 25% ± 3% in A375 and Hs294T, respectively. Minimum cell viability after 72 h exposure to the combination was 10% ± 2% and 18% ± 2% in A375 and Hs294T, respectively. Minimum cell viability after 72 h exposure to melphalan was 8% ± 1% and 12% ± 2%, in A375 and Hs294T, respectively. Conclusion: LAs have cytotoxic activity on human melanoma cell lines in a time-, concentration- and agent-dependant manner. Apoptosis in the cell lines was mediated through activity of caspases-3 and caspases-8.

Immune-mediated bone marrow failure syndromes of progenitor and stem cells: molecular analysis of cytotoxic T cell clones.

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Ramon Tiu

2007-03-01

Full Text Available The unique structure of the T cell receptor (TCR enables molecular identification of individual T cell clones and provides an unique opportunity for the design of molecular diagnostic tests based on the structure of the rearranged TCR chain e.g., using the TCR CDR3 region. Initially, clonal T cell malignancies, including T cell large granular lymphocyte leukemia (T-LGL, mucosis fungoides and peripheral T cell lymphoma were targets for the TCR-based analytic assays such as detection of clonality by T-gamma rearrangement using y-chain-specific PCR or Southern Blotting. Study of these disorders facilitated further analytic concepts and application of rational methods of TCR analysis to investigations of polyclonal T cell-mediated diseases. In hematology, such conditions include graft versus host disease (GvHD and immune-mediated bone marrow failure syndromes. In aplastic anemia (AA, myelodysplastic syndrome (MDS or paroxysmal nocturnal hemoglobinuria (PNH, cytotoxic T cell responses may be directed against certain antigens located on stem or more lineage-restricted progenitor cells in single lineage cytopenias. The nature of the antigenic targets driving polyclonal CTL responses remains unclear. Novel methods of TCR repertoire analysis, include VB flow cytometry, peptide-specific tetramer staining, in vitro stimulation assays and TCR CDR3-specific PCR. Such PCR assay can be either VB family-specific or multiplexed for all VB families. Amplified products can be characterized and quantitated to facilitate detection of the most immunodominant clonotypes. Such clonotypes may serve as markers for the global polyclonal T cell response. Identification of these clonotypes can be performed in blood and tissue biopsy material by various methods. Once immunodominant clonotypes corresponding to pathogenic CTL clones are identified they can serve as surrogate markers for the activity of the pathophysiologic process or even indicate the presence of specific

Chaperone protein HYPK interacts with the first 17 amino acid region of Huntingtin and modulates mutant HTT-mediated aggregation and cytotoxicity

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Choudhury, Kamalika Roy [Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, 1/AF Bidhannagar, Kolkata 700064 (India); Centre for Neuroscience, Indian Institute of Science, Bangalore 560012 (India); Bhattacharyya, Nitai P., E-mail: nitai_sinp@yahoo.com [Biomedical Genomics Centre, PG Polyclinic Building, 5, Suburbun Hospital Road, Kolkata 700020 (India)

2015-01-02

Highlights: • HYPK reduces mutant HTT-mediated aggregate formation and cytotoxicity. • Interaction of HYPK with HTT requires N-terminal 17 amino acid of HTT (HTT-N17). • Deletion of HTT-N17 leads to SDS-soluble, smaller, nuclear aggregates. • These smaller aggregates do not associate with HYPK and are more cytotoxic. • Maybe, interaction of HYPK with amphipathic HTT-N17 block HTT aggregate formation. - Abstract: Huntington’s disease is a polyglutamine expansion disorder, characterized by mutant HTT-mediated aggregate formation and cytotoxicity. Many reports suggests roles of N-terminal 17 amino acid domain of HTT (HTT-N17) towards subcellular localization, aggregate formation and subsequent pathogenicity induced by N-terminal HTT harboring polyQ stretch in pathogenic range. HYPK is a HTT-interacting chaperone which can reduce N-terminal mutant HTT-mediated aggregate formation and cytotoxicity in neuronal cell lines. However, how HYPK interacts with N-terminal fragment of HTT remained unknown. Here we report that specific interaction of HYPK with HTT-N17 is crucial for the chaperone activity of HYPK. Deletion of HTT-N17 leads to formation of tinier, SDS-soluble nuclear aggregates formed by N-terminal mutant HTT. The increased cytotoxicity imparted by these tiny aggregates might be contributed due to loss of interaction with HYPK.

Oxidative stress-mediated cytotoxicity of zirconia nanoparticles on PC12 and N2a cells

Energy Technology Data Exchange (ETDEWEB)

Asadpour, Elham [Shiraz University of Medical Sciences, Anesthesiology and Critical Care Research Center (Iran, Islamic Republic of); Sadeghnia, Hamid R. [Mashhad University of Medical Sciences, Department of Pharmacology, Faculty of Medicine (Iran, Islamic Republic of); Ghorbani, Ahmad [Mashhad University of Medical Sciences, Pharmacological Research Center of Medicinal Plants (Iran, Islamic Republic of); Sedaghat, Mehran, E-mail: m-sedaghat81@yahoo.com [Mashhad University of Medical Sciences, Department of Neurosurgery (Iran, Islamic Republic of); Boroushaki, Mohammad T., E-mail: boroushakimt@mums.ac.ir [Mashhad University of Medical Sciences, Department of Pharmacology, Faculty of Medicine (Iran, Islamic Republic of)

2016-01-15

In recent years, there is a growing interest in the application of nanoparticles like zirconium dioxide (zirconia <100 nm), for many purposes. Since a comprehensive study on the toxic effects of zirconia has not been done, we decided to investigate the effects of zirconia nanoparticles on cultured PC12 and N2a cells. In this study, cytotoxic effect of different concentrations of zirconia nanoparticles at three different time intervals were evaluated using MTT and ROS (reactive oxygen species) assays. Also, Lipid peroxidation, glutathione (GSH) content changes, and DNA damage were measured. Zirconia nanoparticles caused a significant reduction in cell viability and GSH content of the cells, and induce a significant increase in intracellular ROS and MDA content of PC12 and N2a cells. Moreover, it increases the percentage of DNA tail of treated cells as compared with control group. Zirconia nanoparticles have cytotoxic and genotoxic effects in PC12 and N2a cells in a time and concentration-dependent manner in concentration more than 31 µg/mL.

Mind Bomb Regulates Cell Death during TNF Signaling by Suppressing RIPK1’s Cytotoxic Potential

Directory of Open Access Journals (Sweden)

Rebecca Feltham

2018-04-01

Full Text Available Summary: Tumor necrosis factor (TNF is an inflammatory cytokine that can signal cell survival or cell death. The mechanisms that switch between these distinct outcomes remain poorly defined. Here, we show that the E3 ubiquitin ligase Mind Bomb-2 (MIB2 regulates TNF-induced cell death by inactivating RIPK1 via inhibitory ubiquitylation. Although depletion of MIB2 has little effect on NF-κB activation, it sensitizes cells to RIPK1- and caspase-8-dependent cell death. We find that MIB2 represses the cytotoxic potential of RIPK1 by ubiquitylating lysine residues in the C-terminal portion of RIPK1. Our data suggest that ubiquitin conjugation of RIPK1 interferes with RIPK1 oligomerization and RIPK1-FADD association. Disruption of MIB2-mediated ubiquitylation, either by mutation of MIB2’s E3 activity or RIPK1’s ubiquitin-acceptor lysines, sensitizes cells to RIPK1-mediated cell death. Together, our findings demonstrate that Mind Bomb E3 ubiquitin ligases can function as additional checkpoint of cytokine-induced cell death, selectively protecting cells from the cytotoxic effects of TNF. : Feltham et al. show that MIB2 directly ubiquitylates RIPK1 upon TNF stimulation, suppressing the cytotoxic potential of RIPK1 and acting as a checkpoint within the TNF signaling pathway. Keywords: MIB2, RIPK1, TNF, cell death, caspase-8, IAPs, ubiquitin

Effect of radiotherapy on lymphocyte cytotoxicity in vitro

Energy Technology Data Exchange (ETDEWEB)

Wasserman, J; Melen, B [Central Microbiological Laboratory, Stockholm County Council (Sweden); Blomgren, H; Glas, U; Perlmann, P

1975-11-01

The cytotoxic functions of highly purified blood lymphocytes from patients with breast cancer were studied before and after radiotherapy. Addition of PHA or of rabbit antibodies to target cells (chicken erythrocytes) were chosen as two means of inducing lymphocyte cytotoxicity in vitro. The proportion of T and non-T lymphocytes was determined by means of E and EAC rosette tests. The antibody-induced cytotoxicity of lymphocytes decreased following radiotherapy while that mediated by PHA remained unchanged. There was some reduction in the percentage of EAC rosette-forming cells. These results, as well as earlier observations, suggest that the decrease in the peripheral blood of the proportion of lymphocytes with receptors for activated complement is responsible for changes in the antibody-mediated lymphocyte cytotoxicity.

Potential genotoxic and cytotoxicity of emamectin benzoate in human normal liver cells.

Science.gov (United States)

Zhang, Zhijie; Zhao, Xinyu; Qin, Xiaosong

2017-10-10

Pesticide residue inducing cancer-related health problems draw people more attention recently. Emamectin benzoate (EMB) has been widely used in agriculture around the world based on its specificity targets. Although potential risk and the molecular mechanism of EMB toxicity to human liver has not been well-characterized. Unlike well-reported toxicity upon central nervous system, potential genotoxic and cytotoxicity of EMB in human liver cell was ignored and very limited. In this study, we identify genotoxicity and cytotoxicity of EMB to human normal liver cells (QSG7701 cell line) in vitro . We demonstrate that EMB inhibited the viability of QSG7701 cells and induced the DNA damage. Established assays of cytotoxicity were performed to characterize the mechanism of EMB toxicity on QSG7701 cells. Typical chromatin condensation and DNA fragmentation indicated the apoptosis of QSG7701 cells induced by EMB. And the intracellular biochemical results demonstrated that EMB-enhanced apoptosis of QSG7701 cells concurrent with generated ROS, a loss of mitochondrial membrane potential, the cytochrome-c release, up regulate the Bax/Bcl-2 and the activation of caspase-9/-3. Our results of EMB induces the death of QSG7701 cells maybe via mitochondrial-mediated intrinsic apoptotic pathways would contribute to promote the awareness of EMB as an extensive used pesticide to human being effects and reveal the underlying mechanisms of potential genotoxic.

Cell-mediated immunity in herpes simplex virus-infected mice: functional analysis of lymph node cells during periods of acute and latent infection, with reference to cytotoxic and memory cells.

Science.gov (United States)

Nash, A A; Quartey-Papafio, R; Wildy, P

1980-08-01

The functional characteristics of lymphoid cells were investigated during acute and latent infection of mice with herpes simplex virus (HSV). Cytotoxic T cells were found in the draining lymph node (DLN) 4 days p.i. and had reached maximum activity between 6 and 9 days. After the 12th day and during the period of latent infection (> 20 days) no cytotoxic cell activity was observed. Cytotoxic activity could only be detected when the lymphoid cells had been cultured for a period of 3 days. In general, the cell killing was specific for syngeneic infected target cells, although some killing of uninfected targets was observed. In contrast to the cytotoxic response, DLN cells responding to HSV in a proliferation assay were detected towards the end of the acute phase and at lease up to 9 months thereafter. The significance of these observations for the pathogenesis of HSV is discussed.

Flow cytometric assay detecting cytotoxicity against human endogenous retrovirus antigens expressed on cultured multiple sclerosis cells

DEFF Research Database (Denmark)

Møller-Larsen, A; Brudek, T; Petersen, T

2013-01-01

on their surface. Polyclonal antibodies against defined peptides in the Env- and Gag-regions of the HERVs were raised in rabbits and used in antibody-dependent cell-mediated cytotoxicity (ADCC) -assays. Rituximab® (Roche), a chimeric monoclonal antibody against CD20 expressed primarily on B cells, was used...

TRX-ASK1-JNK signaling regulation of cell density-dependent cytotoxicity in cigarette smoke-exposed human bronchial epithelial cells.

Science.gov (United States)

Lee, Yong Chan; Chuang, Chun-Yu; Lee, Pak-Kei; Lee, Jin-Soo; Harper, Richart W; Buckpitt, Alan B; Wu, Reen; Oslund, Karen

2008-05-01

Cigarette smoke is a major environmental air pollutant that injures airway epithelium and incites subsequent diseases including chronic obstructive pulmonary disease. The lesion that smoke induces in airway epithelium is still incompletely understood. Using a LIVE/DEAD cytotoxicity assay, we observed that subconfluent cultures of bronchial epithelial cells derived from both human and monkey airway tissues and an immortalized normal human bronchial epithelial cell line (HBE1) were more susceptible to injury by cigarette smoke extract (CSE) and by direct cigarette smoke exposure than cells in confluent cultures. Scraping confluent cultures also caused an enhanced cell injury predominately in the leading edge of the scraped confluent cultures by CSE. Cellular ATP levels in both subconfluent and confluent cultures were drastically reduced after CSE exposure. In contrast, GSH levels were significantly reduced only in subconfluent cultures exposed to smoke and not in confluent cultures. Western blot analysis demonstrated ERK activation in both confluent and subconfluent cultures after CSE. However, activation of apoptosis signal-regulating kinase 1 (ASK1), JNK, and p38 were demonstrated only in subconfluent cultures and not in confluent cultures after CSE. Using short interfering RNA (siRNA) to JNK1 and JNK2 and a JNK inhibitor, we attenuated CSE-mediated cell death in subconfluent cultures but not with an inhibitor of the p38 pathway. Using the tetracycline (Tet)-on inducible approach, overexpression of thioredoxin (TRX) attenuated CSE-mediated cell death and JNK activation in subconfluent cultures. These results suggest that the TRX-ASK1-JNK pathway may play a critical role in mediating cell density-dependent CSE cytotoxicity.

T cell-mediated hepatitis in mice infected with lymphocytic choriomeningitis virus. Liver cell destruction by H-2 class I-restricted virus-specific cytotoxic T cells as a physiological correlate of the 51Cr-release assay

International Nuclear Information System (INIS)

Zinkernagel, R.M.; Haenseler, E.; Leist, T.; Cerny, A.; Hengartner, H.; Althage, A.

1986-01-01

A model for immunologically T cell-mediated hepatitis was established in mice infected with lymphocytic choriomeningitis virus (LCMV). The severity of hepatitis was monitored histologically and by determination of changes in serum levels of the enzymes alanine aminotransferase (ALT), aspartate aminotransferase (AST), glutamate dehydrogenase (GLDH), and alkaline phosphatase (AP). Kinetics of histological disease manifestations, increases of liver enzyme levels in the serum, and cytotoxic T cell activities in livers and spleens all correlated and were dependent upon several parameters: LCMV-isolate; LCMV-WE caused extensive hepatitis, LCMV-Armstrong virtually none. Virus dose. Route of infection; i.v. or i.p. infection caused hepatitis, whereas infection into the footpad did not. The general genetic background of the murine host; of the strains tested, Swiss mice and A-strain mice were more susceptible than C57BL or CBA mice; BALB/c and DBA/2 mice were least susceptible. The degree of immunocompetence of the murine host; T cell deficient nu/nu mice never developed hepatitis, whereas nu/+ or +/+ mice always did. B cell-depleted anti-IgM-treated mice developed immune-mediated hepatitis comparably or even more extensively than control mice. Local cytotoxic T cell activity; mononuclear cells isolated from livers during the period of overt hepatitis were two to five times more active than equal numbers of spleen cells. Adoptive transfer of nylon wool-nonadherent anti-Thy-1.2 and anti-Lyt-2 plus C-sensitive, anti-L3T4 plus C-resistant lymphocytes into irradiated mice preinfected with LCMV-WE caused a rapid time- and dose-dependent linear increase of serum enzyme levels. This increase was caused by adoptive transfer of lymphocytes if immune cell donors and recipient mice shared class I, but not when they shared class II histocompatibility antigens

Role of Proteus mirabilis MR/P fimbriae and flagella in adhesion, cytotoxicity and genotoxicity induction in T24 and Vero cells.

Science.gov (United States)

Scavone, Paola; Villar, Silvia; Umpiérrez, Ana; Zunino, Pablo

2015-06-01

Proteus mirabilis is frequently associated with complicated urinary tract infections (UTI). It is proposed that several virulence factors are associated with P. mirabilis uropathogenicity. The aim of this work was to elucidate genotoxic and cytotoxic effects mediated by MR/P fimbriae and flagella in eukaryotic cells in vitro. Two cell lines (kidney- and bladder-derived) were infected with a clinical wild-type P. mirabilis strain and an MR/P and a flagellar mutant. We evaluated adhesion, genotoxicity and cytotoxicity by microscopy, comet assay and triple staining technique, respectively. Mutant strains displayed lower adhesion rates than the P. mirabilis wild-type strain and were significantly less effective to induce genotoxic and cytotoxic effects compared to the wild type. We report for the first time that P. mirabilis MR/P fimbriae and flagella mediate genotoxic and cytotoxic effects on eukaryotic cells, at least in in vitro conditions. These results could contribute to design new strategies for the control of UTI. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Chemically dispersed oil is cytotoxic and genotoxic to sperm whale skin cells.

Science.gov (United States)

Wise, Catherine F; Wise, James T F; Wise, Sandra S; Wise, John Pierce

2018-06-01

Two major oil crises in United States history, the 1989 Exxon-Valdez oil spill in Alaska and the 2010 Deepwater Horizon Oil Rig explosion in the Gulf of Mexico, drew attention to the need for toxicological experiments on oil and chemically dispersed oil. We are still learning the effects these spills had on wildlife. However, little data is known about the toxicity of these substances in marine mammals. The objective of this study is to determine the toxicity of Alaskan oil, as well as chemically dispersed oil. Oil experiments were performed using the water accommodated fraction of Alaskan oil (WAF) and the chemically enhanced water accommodated fraction of Alaskan oil (CEWAF). The Alaskan WAF is not cytotoxic to sperm whale skin cells though it did induce chromosome damage; S9-mediated metabolism did not affect the cytotoxicity of WAF but did increase the levels of chromosome damage. Alaskan CEWAF is more cytotoxic and genotoxic than the WAF; S9 mediated metabolism increased both cytotoxicity and genotoxicity of CEWAF. Analysis of the PAH content of Alaskan WAF and CEWAF revealed a forty-fold increase in the total levels of PAHs in CEWAF compared to WAF. These findings show that chemically dispersed oil leads to higher levels of PAH exposure which are more toxic and likely to lead to longer and more persistent health effects. Copyright © 2017 Elsevier Inc. All rights reserved.

Metabolic and physiologic studies of nonimmune lymphoid cells cytotoxic for fibroblastic cells in vitro

International Nuclear Information System (INIS)

Mayhew, E.; Bennett, M.

1974-01-01

An in vitro reaction between mouse lymphoid cells and target fibroblastic cells in wells of microtest plates, which appears to simulate the in vivo rejection of hemopoietic allografts, has been analyzed for metabolic and physiologic requirements. Protein synthesis was required for only the first few hours of culture. Inhibition of RNA synthesis and alteration of cell surface charge with various agents were without obvious effects. Metabolic slowing at 4 0 C or deviation of the pH of the culture medium suppressed the reaction. Thymus cells, which are not cytotoxic in this system, significantly but not completely inhibited the cytotoxicity of lymph node cells. Antiserum directed against target cells specifically protected them from the cytotoxic lymphoid cells in the absence of complement. Precursors of cytotoxic lymphoid cells were radiosensitive, unlike the cytotoxic cells themselves. BALB/c anti-C57BL/6 spleen cell serum and 89 Sr both are able to prevent rejection of marrow allografts in vivo. Lymphoid cells incubated with this antiserum plus complement lost much of their cytotoxicity but were still effective at high ratios of aggressor to target cells. Lymphoid cells of mice treated with 89 Sr were effectively cytotoxic but lost practically all of their cytotoxicity after incubation with the antiserum plus complement. Thus, it appears that this reaction detects two different cytotoxic lymphoid cells, either of which can function in vitro. Both cell types may need to cooperate in vivo during marrow allograft rejections

Cytotoxic activity of vitamins K1, K2 and K3 against human oral tumor cell lines.

Science.gov (United States)

Okayasu, H; Ishihara, M; Satoh, K; Sakagami, H

2001-01-01

Vitamin K1, K2 and K3 were compared for their cytotoxic activity, radical generation and O2- scavenging activity. Among these compounds, vitamin K3 showed the highest cytotoxic activity against human oral tumor cell lines (HSC-2, HSG), human promyelocytic leukemic cell line (HL-60) and human gingival fibroblast (HGF). Vitamin K3 induced internucleosomal DNA fragmentation in HL-60 cells, but not in HSC-2 or HSG cells. The cytotoxic activity of vitamins K2 and K1 was one and two orders lower, respectively, than K3. Vitamin K2, but not vitamin K3, showed tumor-specific cytotoxic action. ESR spectroscopy showed that only vitamin K3 produced radical(s) under alkaline condition and most potently enhanced the radical intensity of sodium ascorbate and scavenged O2- (generated by hypoxanthine-xanthine oxidase reaction system); vitamin K2 was much less active whereas vitamin K1 was inactive. These data suggest that the cytotoxic activity of vitamin K3 is generated by radical-mediated oxidation mechanism and that this vitamin has two opposing actions (that is, antioxidant and prooxidant), depending on the experimental conditions.

Semi-automated limit-dilution assay and clonal expansion of all T-cell precursors of cytotoxic lymphocytes

Energy Technology Data Exchange (ETDEWEB)

Wilson, A.; Chen, W.F.; Scollay, R.; Shortman, K. (Walter and Eliza Hall Inst. of Medical Research, Parkville (Australia))

1982-08-13

A limit-dilution microculture system is described, where almost all precursor T cells of the cytotoxic lineage (CTL-p) develop into extended clones of cytotoxic T cells (CTL), which are then detected with a new radio-autographic /sup 111/In-release assay. The principle is to polyclonally activate all T cells with concanavalin A, to expand the resultant clones over an 8-9 day period in cultures saturated with growth factors, then to detect all clones with cytotoxic function by phytohaemagglutinin mediated lysis of P815 tumour cells. The key variables for obtaining high cloning efficiency are the use of flat-bottomed 96-well culture trays, the use of appropriately irradiated spleen filler cells, and the inclusion of a T-cell growth factor supplement. Cultures are set up at input levels of around one T cell per well. Forty percent of T cells then form CTL clones readily detected by the cytotoxic assay. The lytic activity of the average clone is equivalent to 3000 CTL, but clone size appears to be much larger. The precursor cells are predominantly if not entirely from the Lyt 2/sup +/ T-cell subclass and almost all cells of this subclass form cytolytic clones. Analysis of the frequency of positive cultures shows a good fit to the expected Poisson distribution, with no evidence of the CTL-p frequency estimates being distorted by helper or suppressor effects.

Semi-automated limit-dilution assay and clonal expansion of all T-cell precursors of cytotoxic lymphocytes

International Nuclear Information System (INIS)

Wilson, A.; Chen, W.-F.; Scollay, R.; Shortman, K.

1982-01-01

A limit-dilution microculture system is described, where almost all precursor T cells of the cytotoxic lineage (CTL-p) develop into extended clones of cytotoxic T cells (CTL), which are then detected with a new radio-autographic 111 In-release assay. The principle is to polyclonally activate all T cells with concanavalin A, to expand the resultant clones over an 8-9 day period in cultures saturated with growth factors, then to detect all clones with cytotoxic function by phytohaemagglutinin mediated lysis of P815 tumour cells. The key variables for obtaining high cloning efficiency are the use of flat-bottomed 96-well culture trays, the use of appropriately irradiated spleen filler cells, and the inclusion of a T-cell growth factor supplement. Cultures are set up at input levels of around one T cell per well. Forty percent of T cells then form CTL clones readily detected by the cytotoxic assay. The lytic activity of the average clone is equivalent to 3000 CTL, but clone size appears to be much larger. The precursor cells are predominantly if not entirely from the Lyt 2 + T-cell subclass and almost all cells of this subclass form cytolytic clones. Analysis of the frequency of positive cultures shows a good fit to the expected Poisson distribution, with no evidence of the CTL-p frequency estimates being distorted by helper or suppressor effects. (Auth.)

Copper doping enhanced the oxidative stress-mediated cytotoxicity of TiO2 nanoparticles in A549 cells.

Science.gov (United States)

Ahmad, J; Siddiqui, M A; Akhtar, M J; Alhadlaq, H A; Alshamsan, A; Khan, S T; Wahab, R; Al-Khedhairy, A A; Al-Salim, A; Musarrat, J; Saquib, Q; Fareed, M; Ahamed, M

2018-05-01

Physicochemical properties of titanium dioxide nanoparticles (TiO 2 NPs) can be tuned by doping with metals or nonmetals. Copper (Cu) doping improved the photocatalytic behavior of TiO 2 NPs that can be applied in various fields such as environmental remediation and nanomedicine. However, interaction of Cu-doped TiO 2 NPs with human cells is scarce. This study was designed to explore the role of Cu doping in cytotoxic response of TiO 2 NPs in human lung epithelial (A549) cells. Characterization data demonstrated the presence of both TiO 2 and Cu in Cu-doped TiO 2 NPs with high-quality lattice fringes without any distortion. The size of Cu-doped TiO 2 NPs (24 nm) was lower than pure TiO 2 NPs (30 nm). Biological results showed that both pure and Cu-doped TiO 2 NPs induced cytotoxicity and oxidative stress in a dose-dependent manner. Low mitochondrial membrane potential and higher caspase-3 enzyme (apoptotic markers) activity were also observed in A549 cells exposed to pure and Cu-doped TiO 2 NPs. We further observed that cytotoxicity caused by Cu-doped TiO 2 NPs was higher than pure TiO 2 NPs. Moreover, antioxidant N-acetyl cysteine effectively prevented the reactive oxygen species generation, glutathione depletion, and cell viability reduction caused by Cu-doped TiO 2 NPs. This is the first report showing that Cu-doped TiO 2 NPs induced cytotoxicity and oxidative stress in A549 cells. This study warranted further research to explore the role of Cu doping in toxicity mechanisms of TiO 2 NPs.

Tamoxifen enhances erlotinib-induced cytotoxicity through down-regulating AKT-mediated thymidine phosphorylase expression in human non-small-cell lung cancer cells.

Science.gov (United States)

Ko, Jen-Chung; Chiu, Hsien-Chun; Syu, Jhan-Jhang; Jian, Yi-Jun; Chen, Chien-Yu; Jian, Yun-Ting; Huang, Yi-Jhen; Wo, Ting-Yu; Lin, Yun-Wei

2014-03-01

Tamoxifen is a triphenylethylene nonsteroidal estrogen receptor (ER) antagonist used worldwide as an adjuvant hormone therapeutic agent in the treatment of breast cancer. However, the molecular mechanism of tamoxifen-induced cytotoxicity in non-small cell lung cancer (NSCLC) cells has not been identified. Thymidine phosphorylase (TP) is an enzyme of the pyrimidine salvage pathway which is upregulated in cancers. In this study, tamoxifen treatment inhibited cell survival in two NSCLC cells, H520 and H1975. Treatment with tamoxifen decreased TP mRNA and protein levels through AKT inactivation. Furthermore, expression of constitutively active AKT (AKT-CA) vectors significantly rescued the decreased TP protein and mRNA levels in tamoxifen-treated NSCLC cells. In contrast, combination treatment with PI3K inhibitors (LY294002 or wortmannin) and tamoxifen further decreased the TP expression and cell viability of NSCLC cells. Knocking down TP expression by transfection with small interfering RNA of TP enhanced the cytotoxicity and cell growth inhibition of tamoxifen. Erlotinib (Tarceva, OSI-774), an orally available small molecular inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase, is approved for clinical treatment of NSCLC. Compared to a single agent alone, tamoxifen combined with erlotinib resulted in cytotoxicity and cell growth inhibition synergistically in NSCLC cells, accompanied with reduced activation of phospho-AKT and phospho-ERK1/2, and reduced TP protein levels. These findings may have implications for the rational design of future drug regimens incorporating tamoxifen and erlotinib for the treatment of NSCLC. Copyright © 2014 Elsevier Inc. All rights reserved.

Trastuzumab mediates antibody-dependent cell-mediated cytotoxicity and phagocytosis to the same extent in both adjuvant and metastatic HER2/neu breast cancer patients.

Science.gov (United States)

Petricevic, Branka; Laengle, Johannes; Singer, Josef; Sachet, Monika; Fazekas, Judit; Steger, Guenther; Bartsch, Rupert; Jensen-Jarolim, Erika; Bergmann, Michael

2013-12-12

Monoclonal antibodies (mAb), such as trastuzumab are a valuable addition to breast cancer therapy. Data obtained from neoadjuvant settings revealed that antibody-dependent cell-mediated cytotoxicity (ADCC) is a major mechanism of action for the mAb trastuzumab. Conflicting results still call into question whether disease progression, prolonged treatment or concomitant chemotherapy influences ADCC and related immunological phenomena. We analyzed the activity of ADCC and antibody-dependent cell-mediated phagocytosis (ADCP) of peripheral blood mononuclear cells (PBMCs) from human epidermal growth factor receptor 2 (HER2/neu) positive breast cancer patients receiving trastuzumab therapy either in an adjuvant (n = 13) or metastatic (n = 15) setting as well as from trastuzumab treatment-naive (t-naive) HER2/neu negative patients (n = 15). PBMCs from healthy volunteers (n = 24) were used as controls. ADCC and ADCP activity was correlated with the expression of antibody binding Fc-gamma receptor (FcγR)I (CD64), FcγRII (CD32) and FcγRIII (CD16) on CD14+ (monocytes) and CD56+ (NK) cells, as well as the expression of CD107a+ (LAMP-1) on CD56+ cells and the total amount of CD4+CD25+FOXP3+ (Treg) cells. In metastatic patients, markers were correlated with progression-free survival (PFS). ADCC activity was significantly down regulated in metastatic, adjuvant and t-naive patient cohorts as compared to healthy controls. Reduced ADCC activity was inversely correlated with the expression of CD107a on CD56+ cells in adjuvant patients. ADCC and ADCP activity of the patient cohorts were similar, regardless of treatment duration or additional chemotherapy. PFS in metastatic patients inversely correlated with the number of peripheral Treg cells. The reduction of ADCC in patients as compared to healthy controls calls for adjuvant strategies, such as immune-enhancing agents, to improve the activity of trastuzumab. However, efficacy of trastuzumab-specific ADCC and ADCP appears not to

High-Throughput Flow Cytometric Method for the Simultaneous Measurement of CAR-T Cell Characterization and Cytotoxicity against Solid Tumor Cell Lines.

Science.gov (United States)

Martinez, Emily M; Klebanoff, Samuel D; Secrest, Stephanie; Romain, Gabrielle; Haile, Samuel T; Emtage, Peter C R; Gilbert, Amy E

2018-04-01

High-throughput flow cytometry is an attractive platform for the analysis of adoptive cellular therapies such as chimeric antigen receptor T cell therapy (CAR-T) because it allows for the concurrent measurement of T cell-dependent cellular cytotoxicity (TDCC) and the functional characterization of engineered T cells with respect to percentage of CAR transduction, T cell phenotype, and measurement of T cell function such as activation in a single assay. The use of adherent tumor cell lines can be challenging in these flow-based assays. Here, we present the development of a high-throughput flow-based assay to measure TDCC for a CAR-T construct co-cultured with multiple adherent tumor cell lines. We describe optimal assay conditions (such as adherent cell dissociation techniques to minimize impact on cell viability) that result in robust cytotoxicity assays. In addition, we report on the concurrent use of T cell transduction and activation antibody panels (CD25) that provide further dissection of engineered T cell function. In conclusion, we present the development of a high-throughput flow cytometry method allowing for in vitro interrogation of solid tumor, targeting CAR-T cell-mediated cytotoxicity, CAR transduction, and engineered T cell characterization in a single assay.

Sex steroids do not affect shigatoxin cytotoxicity on human renal tubular or glomerular cells

Directory of Open Access Journals (Sweden)

Kohan Donald E

2002-08-01

Full Text Available Abstract Background The greater susceptibility of children to renal injury in post-diarrheal hemolytic-uremic syndrome (HUS may be related, at least in part, to heightened renal cell sensitivity to the cytotoxic effect of Shiga toxin (Stx, the putative mediator of kidney damage in HUS. We hypothesized that sexual maturation, which coincides with a falling incidence of HUS, may induce a relatively Stx-resistant state in the renal cells. Methods Cultured human glomerular endothelial (HGEN, human glomerular visceral epithelial (HGEC and human proximal tubule (HPT cells were exposed to Stx-1 after pre-incubation with progesterone, β-estradiol or testosterone followed by determination of cytotoxicity. Results Under basal conditions, Stx-1 potently and dose-dependently killed HPT and HGEC, but had relatively little effect on HGEN. Pre-incubation for 1, 2 or 7 days with physiologic or pharmacologic concentrations of progesterone, β-estradiol or testosterone had no effect on Stx-1 cytotoxicity dose-response on any cell type. In addition, no steroid altered Gb3 expression (Stx receptor by any cell type at any time point. Conclusion These data do not support the notion that hormonal changes associated with puberty induce an Stx-resistant state within kidney cells.

Antigen-specific cytotoxic T cell and antigen-specific proliferating T cell clones can be induced to cytolytic activity by monoclonal antibodies against T3

NARCIS (Netherlands)

Spits, H.; Yssel, H.; Leeuwenberg, J.; de Vries, J. E.

1985-01-01

T3 is a human differentiation antigen expressed exclusively on mature T cells. In this study it is shown that anti-T3 monoclonal antibodies, in addition to their capacity to induce T cells to proliferate, are able to induce antigen-specific cytotoxic T lymphocyte clones to mediate antigen

Cell-mediated cytotoxicity in rainbow trout, Oncorhynchus mykiss, infected with viral haemorrhagic septicaemia virus

DEFF Research Database (Denmark)

Utke, K.; Bergmann, S.; Lorenzen, Niels

2007-01-01

classical MHC class I locus Onmy-UBA is identical in the rainbow trout clone C25 and in the permanent rainbow trout cell line RTG-2. This enabled us to develop an assay to measure antiviral cytotoxicity in rainbow trout using a system of MHC class I-matched effector and target cells. Peripheral blood...... leucocytes (PBL) isolated from low dose viral haemorrhagic septicaemia virus (VHSV)-infected rainbow trout killed MHC class I-matched and later also xenogeneic MHC class I-mismatched VHSV-infected cells. When compared to PBL from uninfected control fish PBL from infected fish showed a higher transcriptional...

Clinical Cancer Therapy by NK Cells via Antibody-Dependent Cell-Mediated Cytotoxicity

Directory of Open Access Journals (Sweden)

Kory L. Alderson

2011-01-01

Full Text Available Natural killer (NK cells are powerful effector cells that can be directed to eliminate tumor cells through tumor-targeted monoclonal antibodies (mAbs. Some tumor-targeted mAbs have been successfully applied in the clinic and are included in the standard of care for certain malignancies. Strategies to augment the antitumor response by NK cells have led to an increased understanding of how to improve their effector responses. Next-generation reagents, such as molecularly modified mAbs and mAb-cytokine fusion proteins (immunocytokines, ICs designed to augment NK-mediated killing, are showing promise in preclinical and some clinical settings. Continued research into the antitumor effects induced by NK cells and tumor-targeted mAbs suggests that additional intrinsic and extrinsic factors may influence the antitumor response. Therefore more research is needed that focuses on evaluating which NK cell and tumor criteria are best predictive of a clinical response and which combination immunotherapy regimens to pursue for distinct clinical settings.

Xyloside-primed Chondroitin Sulfate/Dermatan Sulfate from Breast Carcinoma Cells with a Defined Disaccharide Composition Has Cytotoxic Effects in Vitro.

Science.gov (United States)

Persson, Andrea; Tykesson, Emil; Westergren-Thorsson, Gunilla; Malmström, Anders; Ellervik, Ulf; Mani, Katrin

2016-07-08

We previously reported that the xyloside 2-(6-hydroxynaphthyl) β-d-xylopyranoside (XylNapOH), in contrast to 2-naphthyl β-d-xylopyranoside (XylNap), specifically reduces tumor growth both in vitro and in vivo Although there are indications that this could be mediated by the xyloside-primed glycosaminoglycans (GAGs) and that these differ in composition depending on xyloside and cell type, detailed knowledge regarding a structure-function relationship is lacking. In this study we isolated XylNapOH- and XylNap-primed GAGs from a breast carcinoma cell line, HCC70, and a breast fibroblast cell line, CCD-1095Sk, and demonstrated that both XylNapOH- and XylNap-primed chondroitin sulfate/dermatan sulfate GAGs derived from HCC70 cells had a cytotoxic effect on HCC70 cells and CCD-1095Sk cells. The cytotoxic effect appeared to be mediated by induction of apoptosis and was inhibited in a concentration-dependent manner by the XylNap-primed heparan sulfate GAGs. In contrast, neither the chondroitin sulfate/dermatan sulfate nor the heparan sulfate derived from CCD-1095Sk cells primed on XylNapOH or XylNap had any effect on the growth of HCC70 cells or CCD-105Sk cells. These observations were related to the disaccharide composition of the XylNapOH- and XylNap-primed GAGs, which differed between the two cell lines but was similar when the GAGs were derived from the same cell line. To our knowledge this is the first report on cytotoxic effects mediated by chondroitin sulfate/dermatan sulfate. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Glycan elongation beyond the mucin associated Tn antigen protects tumor cells from immune-mediated killing.

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Caroline B Madsen

Full Text Available Membrane bound mucins are up-regulated and aberrantly glycosylated during malignant transformation in many cancer cells. This results in a negatively charged glycoprotein coat which may protect cancer cells from immune surveillance. However, only limited data have so far demonstrated the critical steps in glycan elongation that make aberrantly glycosylated mucins affect the interaction between cancer cells and cytotoxic effector cells of the immune system. Tn (GalNAc-Ser/Thr, STn (NeuAcα2-6GalNAc-Ser/Thr, T (Galβ1-3GalNAc-Ser/Thr, and ST (NeuAcα2-6Galβ1-3GalNAc-Ser/Thr antigens are recognized as cancer associated truncated glycans, and are expressed in many adenocarcinomas, e.g. breast- and pancreatic cancer cells. To investigate the role of the cancer associated glycan truncations in immune-mediated killing we created glyco-engineered breast- and pancreatic cancer cells expressing only the shortest possible mucin-like glycans (Tn and STn. Glyco-engineering was performed by zinc finger nuclease (ZFN knockout (KO of the Core 1 enzyme chaperone COSMC, thereby preventing glycan elongation beyond the initial GalNAc residue in O-linked glycans. We find that COSMC KO in the breast and pancreatic cancer cell lines T47D and Capan-1 increases sensitivity to both NK cell mediated antibody-dependent cellular-cytotoxicity (ADCC and cytotoxic T lymphocyte (CTL-mediated killing. In addition, we investigated the association between total cell surface expression of MUC1/MUC16 and NK or CTL mediated killing, and observed an inverse correlation between MUC16/MUC1 expression and the sensitivity to ADCC and CTL-mediated killing. Together, these data suggest that up-regulation of membrane bound mucins protects cells from immune mediated killing, and that particular glycosylation steps, as demonstrated for glycan elongation beyond Tn and STn, can be important for fine tuning of the immune escape mechanisms in cancer cells.

Cellular Adjuvant Properties, Direct Cytotoxicity of Re-differentiated Vα24 Invariant NKT-like Cells from Human Induced Pluripotent Stem Cells

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Shuichi Kitayama

2016-02-01

Full Text Available Vα24 invariant natural killer T (iNKT cells are a subset of T lymphocytes implicated in the regulation of broad immune responses. They recognize lipid antigens presented by CD1d on antigen-presenting cells and induce both innate and adaptive immune responses, which enhance effective immunity against cancer. Conversely, reduced iNKT cell numbers and function have been observed in many patients with cancer. To recover these numbers, we reprogrammed human iNKT cells to pluripotency and then re-differentiated them into regenerated iNKT cells in vitro through an IL-7/IL-15-based optimized cytokine combination. The re-differentiated iNKT cells showed proliferation and IFN-γ production in response to α-galactosylceramide, induced dendritic cell maturation and downstream activation of both cytotoxic T lymphocytes and NK cells, and exhibited NKG2D- and DNAM-1-mediated NK cell-like cytotoxicity against cancer cell lines. The immunological features of re-differentiated iNKT cells and their unlimited availability from induced pluripotent stem cells offer a potentially effective immunotherapy against cancer.

Ochratoxin A: induction of (oxidative) DNA damage, cytotoxicity and apoptosis in mammalian cell lines and primary cells

International Nuclear Information System (INIS)

Kamp, Hennicke G.; Eisenbrand, Gerhard; Schlatter, Josef; Wuerth, Kirsten; Janzowski, Christine

2005-01-01

Ochratoxin A (OTA) is a nephrotoxic/-carcinogenic mycotoxin, produced by several Aspergillus- and Penicillium-strains. Humans are exposed to OTA via food contamination, a causal relationship of OTA to human endemic Balkan nephropathy is still under debate. Since DNA-adducts of OTA or its metabolites could not be identified unambiguously, its carcinogenic effectiveness might be related to secondary effects, such as oxidative cell damage or cell proliferation. In this study, OTA mediated induction of (oxidative) DNA damage, cytotoxicity (necrosis, growth inhibition, apoptosis) and modulation of glutathione were investigated in cell lines (V79, CV-1) and primary rat kidney cells. After 24 h incubation, viability of V79 cells was strongly decreased by OTA concentrations >2.5 μmol/L, whereas CV-1 cells were clearly less sensitive. Strong growth inhibition occurred in both cell lines (IC 50 ∼2 μmol/L). Apoptosis, detected with an immunochemical test and with flow cytometry, was induced by >1 μmol/L OTA. Oxidative DNA damage, detected by comet assay after additional treatment with repair enzymes, was induced in all cell systems already at five-fold lower concentrations. Glutathione in CV-1 cells was depleted after 1 h incubation (>100 μmol/L). In contrast, an increase was measured after 24 h incubation (>0.5 μmol/L). In conclusion, OTA induces oxidative DNA damage at low, not yet cytotoxic concentrations. Oxidative DNA damage might initiate cell transformation eventually in connection with proliferative response following cytotoxic cell death. Both events might represent pivotal factors in the chain of cellular events leading into nephro-carcinogenicity of OTA

In vitro analysis of cytotoxic T cell recruitment mediated by the DC-derived chemokine CCL17

OpenAIRE

sprotocols

2015-01-01

Dendritic cell (DC) licensing in cross-priming requires physical interaction of several rare immune cells, i.e. cytotoxic T cells (CTL), and cross-presenting DCs. Here we describe a novel in vitro method of analyzing chemokine effects on complex recruitment events in a multi-cellular system. To study CTL recruitment towards CCL17-producing DCs, we established a co-culture system of murine splenic DCs with polyclonal splenic CTL from donor mice, which enables visualization of cell motility and...

Cytotoxicity and Apoptotic Activity of Ficus pseudopalma Blanco ...

African Journals Online (AJOL)

Blanco Leaf Extracts Against Human Prostate Cancer Cell. Lines ... Keywords: Ficus pseudopalma, Cytotoxicity, Apopotic, human prostate PRST2 cancer cell, Lupeol,. Quercetin. ..... apoptosis through Fas-receptor mediated pathway in a ...

Dichlorophen and Dichlorovos mediated genotoxic and cytotoxic assessment on root meristem cells of Allium cepa

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Sibhghatulla Shaikh

2012-06-01

Full Text Available Plants are direct recipients of agro – toxics and therefore important materials for assessing environmental chemicals for genotoxicity. The meristematic mitotic cell of Allium cepa is an efficient cytogenetic material for chromosome aberration assay on environmental pollutants. Onion root tips were grown on moistened filter paper in petri dish at room temperature. Germinated root tips were then exposed to three concentrations of each pesticide for 24 h. About 1 – 2 mm length of root tip was cut, fixed in cornoy’s fixative, hydrolyzed in warm 1 N HCL, stained with acetocarmine and squashed on glass slide. About 3000 cells were scored and classified into interphase and normal or aberrant division stage. Cytotoxicity was determined by comparing the mitotic index (MI of treated cells with that of the negative control. The MI of cells treated with Dichlorophen and Dichlorovos at one or more concentration was half or less than that of control are said to be cytotoxic. Genotoxicity was measured by comparing the number of cells/1000 in aberrant division stages at each dose with the negative control using Mann – Whitney U test. Both Dichlorophen and Dichlorovos are genotoxic at higher concentrations i.e. 0.001%, 0.002% and 0.028%, 0.056% inducing chromosome fragment, chromosome lagging and bridges, stick chromosome and multipolar anaphase.

Minocycline enhances mitomycin C-induced cytotoxicity through down-regulating ERK1/2-mediated Rad51 expression in human non-small cell lung cancer cells.

Science.gov (United States)

Ko, Jen-Chung; Wang, Tai-Jing; Chang, Po-Yuan; Syu, Jhan-Jhang; Chen, Jyh-Cheng; Chen, Chien-Yu; Jian, Yun-Ting; Jian, Yi-Jun; Zheng, Hao-Yu; Chen, Wen-Ching; Lin, Yun-Wei

2015-10-01

Minocycline is a semisynthetic tetracycline derivative; it has anti-inflammatory and anti-cancer effects distinct from its antimicrobial function. However, the molecular mechanism of minocycline-induced cytotoxicity in non-small cell lung cancer (NSCLC) cells has not been identified. Rad51 plays a central role in homologous recombination and high levels of Rad51 expression are observed in chemo- or radioresistant carcinomas. Our previous studies have shown that the MKK1/2-ERK1/2 signal pathway maintains the expression of Rad51 in NSCLC cells. In this study, minocycline treatment inhibited cell viability and proliferation of two NSCLC cells, A549 and H1975. Treatment with minocycline decreased Rad51 mRNA and protein levels through MKK1/2-ERK1/2 inactivation. Furthermore, expression of constitutively active MKK1 (MKK1-CA) vectors significantly rescued the decreased Rad51 protein and mRNA levels in minocycline-treated NSCLC cells. However, combined treatment with MKK1/2 inhibitor U0126 and minocycline further decreased the Rad51 expression and cell viability of NSCLC cells. Knocking down Rad51 expression by transfection with small interfering RNA of Rad51 enhanced the cytotoxicity and cell growth inhibition of minocycline. Mitomycin C (MMC) is typically used as a first or second line regimen to treat NSCLC. Compared to a single agent alone, MMC combined with minocycline resulted in cytotoxicity and cell growth inhibition synergistically in NSCLC cells, accompanied with reduced activation of phospho-ERK1/2, and reduced Rad51 protein levels. Overexpression of MKK1-CA or Flag-tagged Rad51 could reverse the minocycline and MMC-induced synergistic cytotoxicity. These findings may have implications for the rational design of future drug regimens incorporating minocycline and MMC for the treatment of NSCLC. Copyright © 2015 Elsevier Inc. All rights reserved.

Autophagy plays a critical role in ChLym-1-induced cytotoxicity of non-hodgkin's lymphoma cells.

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Jiajun Fan

Full Text Available Autophagy is a critical mechanism in both cancer therapy resistance and tumor suppression. Monoclonal antibodies have been documented to kill tumor cells via apoptosis, antibody-dependent cellular cytotoxicity (ADCC and complement-dependent cytotoxicity (CDC. In this study, we report for the first time that chLym-1, a chimeric anti-human HLA-DR monoclonal antibody, induces autophagy in Raji Non-Hodgkin's Lymphoma (NHL cells. Interestingly, inhibition of autophagy by pharmacological inhibitors (3-methyladenine and NH4Cl or genetic approaches (siRNA targeting Atg5 suppresses chLym-1-induced growth inhibition, apoptosis, ADCC and CDC in Raji cells, while induction of autophagy could accelerate cytotoxic effects of chLym-1 on Raji cells. Furthermore, chLym-1-induced autophagy can mediate apoptosis through Caspase 9 activation, demonstrating the tumor-suppressing role of autophagy in antilymphoma effects of chLym-1. Moreover, chLym-1 can activate several upstream signaling pathways of autophagy including Akt/mTOR and extracellular signal-regulated kinase 1/2 (Erk1/2. These results elucidate the critical role of autophagy in cytotoxicity of chLym-1 antibody and suggest a potential therapeutic strategy of NHL therapy by monoclonal antibody chLym-1 in combination with autophagy inducer.

Δ8-Tetrahydrocannabinol induces cytotoxicity in macrophage J774-1 cells: Involvement of cannabinoid receptor 2 and p38 MAPK

International Nuclear Information System (INIS)

Yamaori, Satoshi; Ishii, Hirosuke; Chiba, Kenzo; Yamamoto, Ikuo; Watanabe, Kazuhito

2013-01-01

Tetrahydrocannabinol (THC), a psychoactive component of marijuana, is known to exert cytotoxicity in immune cells. In the present study, we examined the cytotoxicity of Δ 8 -THC in mouse macrophage J774-1 cells and a possible involvement of cannabinoid receptors and stress-responsive mitogen-activated protein kinases (MAPKs) in the cytotoxic process. J774-1 cells were treated with Δ 8 -THC (0–20 μM) for up to 6 h. As measured by the MTT and LDH assays, Δ 8 -THC induced cell death of J774-1 cells in a concentration- and/or exposure time-dependent manner. Δ 8 -THC-induced cell damage was associated with vacuole formation, cell swelling, chromatin condensation, and nuclear fragmentation. The cytotoxic effect of Δ 8 -THC was significantly prevented by a caspase-1 inhibitor Ac-YVAD-cmk but not a caspase-3 inhibitor z-DEVD-fmk. The pretreatment with SR144528, a CB 2 receptor-selective antagonist, effectively suppressed Δ 8 -THC-induced cytotoxicity in J774-1 cells, which exclusively expressed CB 2 receptors as indicated by real-time polymerase chain reaction analysis. In contrast, AM251, a CB 1 receptor-selective antagonist, did not affect the cytotoxicity. Pertussis toxin and α-tocopherol significantly attenuated Δ 8 -THC-induced cytotoxicity suggesting that G i/o protein coupling signal transduction and oxidative stress are responsible for the cytotoxicity. Δ 8 -THC stimulated the phosphorylation of p38 MAPK and c-Jun N-terminal kinase (JNK) in J774-1 cells, which were effectively antagonized by the pretreatment with SR144528. In addition, SB203580, a p38 MARK inhibitor, significantly attenuated the cytotoxic effect of Δ 8 -THC, whereas SP600125, a JNK inhibitor, significantly enhanced the cytotoxicity. These results suggest that the cytotoxicity of Δ 8 -THC to J774-1 cells is exerted mediated through the CB 2 receptor followed by the activation of p38 MAPK

CD3+CD4negCD8neg (double negative) T lymphocytes and NKT cells as the main cytotoxic-related-CD107a+ cells in lesions of cutaneous leishmaniasis caused by Leishmania (Viannia) braziliensis.

Science.gov (United States)

Ferraz, Raquel; Cunha, Clarissa F; Pimentel, Maria Inês F; Lyra, Marcelo R; Pereira-Da-Silva, Tatiana; Schubach, Armando O; Da-Cruz, Alda Maria; Bertho, Alvaro Luiz

2017-05-03

Cutaneous leishmaniasis (CL) is caused by Leishmania (Viannia) braziliensis, which infects dermal macrophages and dendritic cells, causing an intense immune-mediated-tissue inflammation and a skin ulcer with elevated borders that can heal spontaneously or after antimonial therapy. The resolution of lesions depends on an adaptive immune response, and cytotoxic cells seem to have a fundamental role in this process. The aim of this study is to better understand the role of cytotoxicity mediated mechanisms that occur during the immune response in the CL lesion milieu, considering distinct cytotoxic-related CD107a + cells, such as CD8 + , CD4 + , CD4 neg CD8 neg (double-negative, DN) and CD4 + CD8 + (double-positive, DP) T lymphocytes, as well as NK and NKT cells. Lesion derived cells were assessed for T cell subpopulations and NK cells, as well as CD107a expression by flow cytometry. In addition, cytometric bead array (CBA) was used to quantify cytokines and granzyme B concentrations in supernatants from macerated lesions. Flow cytometry analyses revealed that NKT cells are the major CD107a-expressing cell population committed to cytotoxicity in CL lesion, although we also observed high frequencies of CD4 + and DN T cells expressing CD107a. Analysing the pool of CD107a + -cell populations, we found a higher distribution of DN T cells (44%), followed by approximately 25% of NKT cells. Interestingly, NK and CD8 + T cells represented only 3 and 4% of the total-CD107a + -cell pool, respectively. The cytotoxicity activity that occurs in the lesion milieu of CL patients seems to be dominated by DN T and NKT cells. These findings suggest the need for a reevaluation of the role of classical-cytotoxic NK and CD8 + T cells in the pathogenesis of CL, implicating an important role for other T cell subpopulations.

A cell-microelectronic sensing technique for profiling cytotoxicity of chemicals

International Nuclear Information System (INIS)

Boyd, Jessica M.; Huang, Li; Xie Li; Moe, Birget; Gabos, Stephan; Li Xingfang

2008-01-01

A cell-microelectronic sensing technique is developed for profiling chemical cytotoxicity and is used to study different cytotoxic effects of the same class chemicals using nitrosamines as examples. This technique uses three human cell lines (T24 bladder, HepG2 liver, and A549 lung carcinoma cells) and Chinese hamster ovary (CHO-K1) cells in parallel as the living components of the sensors of a real-time cell electronic sensing (RT-CES) method for dynamic monitoring of chemical toxicity. The RT-CES technique measures changes in the impedance of individual microelectronic wells that is correlated linearly with changes in cell numbers during t log phase of cell growth, thus allowing determination of cytotoxicity. Four nitrosamines, N-nitrosodimethylamine (NDMA), N-nitrosodiphenylamine (NDPhA), N-nitrosopiperidine (NPip), and N-nitrosopyrrolidine (NPyr), were examined and unique cytotoxicity profiles were detected for each nitrosamine. In vitro cytotoxicity values (IC 50 ) for NDPhA (ranging from 0.6 to 1.9 mM) were significantly lower than the IC 50 values for the well-known carcinogen NDMA (15-95 mM) in all four cell lines. T24 cells were the most sensitive to nitrosamine exposure among the four cell lines tested (T24 > CHO > A549 > HepG2), suggesting that T24 may serve as a new sensitive model for cytotoxicity screening. Cell staining results confirmed that administration of the IC 50 concentration from the RT-CES experiments inhibited cell growth by 50% compared to the controls, indicating that the RT-CES method provides reliable measures of IC 50 . Staining and cell-cycle analysis confirmed that NDPhA caused cell-cycle arrest at the G0/G1 phase, whereas NDMA did not disrupt the cell cycle but induced cell death, thus explaining the different cytotoxicity profiles detected by the RT-CES method. The parallel cytotoxicity profiling of nitrosamines on the four cell lines by the RT-CES method led to the discovery of the unique cytotoxicity of NDPhA causing cell

A cell-microelectronic sensing technique for profiling cytotoxicity of chemicals

Energy Technology Data Exchange (ETDEWEB)

Boyd, Jessica M [Division of Analytical and Environmental Toxicology, Department of Laboratory Medicine and Pathology, Faculty of Medicine and Dentistry, 10-102 Clinical Sciences Building, Edmonton, Alberta, T6G 2G3 (Canada); Huang, Li [Environmental Health Sciences, Department of Public Health Sciences, School of Public Health, University of Alberta, 10-102 Clinical Sciences Building, Edmonton, Alberta, T6G 2G3 (Canada); Li, Xie; Moe, Birget [Division of Analytical and Environmental Toxicology, Department of Laboratory Medicine and Pathology, Faculty of Medicine and Dentistry, 10-102 Clinical Sciences Building, Edmonton, Alberta, T6G 2G3 (Canada); Gabos, Stephan [Public Health Surveillance and Environmental Health, Alberta Health and Wellness, 10025 Jasper Avenue, Box 1360, Edmonton, Alberta, T5J 2N3 (Canada); Xingfang, Li [Division of Analytical and Environmental Toxicology, Department of Laboratory Medicine and Pathology, Faculty of Medicine and Dentistry, 10-102 Clinical Sciences Building, Edmonton, Alberta, T6G 2G3 (Canada); Environmental Health Sciences, Department of Public Health Sciences, School of Public Health, University of Alberta, 10-102 Clinical Sciences Building, Edmonton, Alberta, T6G 2G3 (Canada)], E-mail: xingfang.li@ualberta.ca

2008-05-12

A cell-microelectronic sensing technique is developed for profiling chemical cytotoxicity and is used to study different cytotoxic effects of the same class chemicals using nitrosamines as examples. This technique uses three human cell lines (T24 bladder, HepG2 liver, and A549 lung carcinoma cells) and Chinese hamster ovary (CHO-K1) cells in parallel as the living components of the sensors of a real-time cell electronic sensing (RT-CES) method for dynamic monitoring of chemical toxicity. The RT-CES technique measures changes in the impedance of individual microelectronic wells that is correlated linearly with changes in cell numbers during t log phase of cell growth, thus allowing determination of cytotoxicity. Four nitrosamines, N-nitrosodimethylamine (NDMA), N-nitrosodiphenylamine (NDPhA), N-nitrosopiperidine (NPip), and N-nitrosopyrrolidine (NPyr), were examined and unique cytotoxicity profiles were detected for each nitrosamine. In vitro cytotoxicity values (IC{sub 50}) for NDPhA (ranging from 0.6 to 1.9 mM) were significantly lower than the IC{sub 50} values for the well-known carcinogen NDMA (15-95 mM) in all four cell lines. T24 cells were the most sensitive to nitrosamine exposure among the four cell lines tested (T24 > CHO > A549 > HepG2), suggesting that T24 may serve as a new sensitive model for cytotoxicity screening. Cell staining results confirmed that administration of the IC{sub 50} concentration from the RT-CES experiments inhibited cell growth by 50% compared to the controls, indicating that the RT-CES method provides reliable measures of IC{sub 50}. Staining and cell-cycle analysis confirmed that NDPhA caused cell-cycle arrest at the G0/G1 phase, whereas NDMA did not disrupt the cell cycle but induced cell death, thus explaining the different cytotoxicity profiles detected by the RT-CES method. The parallel cytotoxicity profiling of nitrosamines on the four cell lines by the RT-CES method led to the discovery of the unique cytotoxicity of NDPh

Ontogeny of human natural killer (NK) cells: fetal NK cells mediate cytolytic function and express cytoplasmic CD3 epsilon,delta proteins

NARCIS (Netherlands)

Phillips, J. H.; Hori, T.; Nagler, A.; Bhat, N.; Spits, H.; Lanier, L. L.

1992-01-01

Natural killer (NK) cells have been defined as CD3 epsilon-, CD16+ and/or CD56+ lymphocytes that mediate major histocompatibility complex (MHC)-unrestricted cytotoxicity against certain tumors and virus-infected cells. Unlike T lymphocytes, NK cells do not rearrange or productively express T cell

Analysis of the Effects of Cell Stress and Cytotoxicity on In Vitro Assay Activity Across a Diverse Chemical and Assay Space

Data.gov (United States)

U.S. Environmental Protection Agency — Chemical toxicity can arise from disruption of specific biomolecular functions or through more generalized cell stress and cytotoxicity-mediated processes. Here,...

Exploiting cannabinoid-induced cytotoxic autophagy to drive melanoma cell death.

Science.gov (United States)

Armstrong, Jane L; Hill, David S; McKee, Christopher S; Hernandez-Tiedra, Sonia; Lorente, Mar; Lopez-Valero, Israel; Eleni Anagnostou, Maria; Babatunde, Fiyinfoluwa; Corazzari, Marco; Redfern, Christopher P F; Velasco, Guillermo; Lovat, Penny E

2015-06-01

Although the global incidence of cutaneous melanoma is increasing, survival rates for patients with metastatic disease remain viability, and activation of apoptosis, whereas cotreatment with chloroquine or knockdown of Atg7, but not Beclin-1 or Ambra1, prevented THC-induced autophagy and cell death in vitro. Administration of Sativex-like (a laboratory preparation comprising equal amounts of THC and cannabidiol (CBD)) to mice bearing BRAF wild-type melanoma xenografts substantially inhibited melanoma viability, proliferation, and tumor growth paralleled by an increase in autophagy and apoptosis compared with standard single-agent temozolomide. Collectively, our findings suggest that THC activates noncanonical autophagy-mediated apoptosis of melanoma cells, suggesting that cytotoxic autophagy induction with Sativex warrants clinical evaluation for metastatic disease.

Dendritic cells decreased the concomitant expanded Tregs and Tregs related IL-35 in cytokine-induced killer cells and increased their cytotoxicity against leukemia cells.

Directory of Open Access Journals (Sweden)

Ying Pan

Full Text Available Regulatory T cells (Tregs are potent immunosuppressive cells and essential for inducing immune tolerance. Recent studies have reported that Tregs and Tregs related cytokines can inhibit the antitumor activity of cytokine-induced killer (CIK cells, but dendritic cells co-cultured CIK (DC-CIK cells can be used for induction of a specific immune response by blocking of Tregs and TGF-β, IL-10. As a novel identified cytokine, IL-35 is specially produced by Tregs and plays an essential role in immune regulation. However, it remains unknown whether IL-35 roles in tumor immunotherapy mediated by CIK and DC-CIK cells. In this study, we cultured CIK and DC-CIK cells from the same healthy adult samples, and investigated their phenotype, proliferation, cytotoxic activity against leukemia cell lines K562 and NB4 by FCM and CCK-8, measured IL-35, TGF-β and IL-10 protein by ELISA, detected Foxp3, IL-35 and IL-35 receptor mRNA by Real-time PCR, respectively. We found Tregs and IL-35 concomitantly expanded by a time-dependent way during the generation of CIK cells, but DC significantly down-regulated the expression of them and simultaneously up-regulated the proliferation ability as well as cytotoxic activity of CIK cells against leukemia cell lines. Therefore, our data suggested that DC decreased concomitant expanded Tregs and Tregs related IL-35 in CIK cells and might contribute to improve their cytotoxicity against leukemia cells in vitro.

Cytotoxic Effects of Fascaplysin against Small Cell Lung Cancer Cell Lines

Science.gov (United States)

Hamilton, Gerhard

2014-01-01

Fascaplysin, the natural product of a marine sponge, exhibits anticancer activity against a broad range of tumor cells, presumably through interaction with DNA, and/or as a highly selective cyclin-dependent kinase 4 (CDK4) inhibitor. In this study, cytotoxic activity of fascaplysin against a panel of small cell lung cancer (SCLC) cell lines and putative synergism with chemotherapeutics was investigated. SCLC responds to first-line chemotherapy with platinum-based drugs/etoposide, but relapses early with topotecan remaining as the single approved therapeutic agent. Fascaplysin was found to show high cytotoxicity against SCLC cells and to induce cell cycle arrest in G1/0 at lower and S-phase at higher concentrations, respectively. The compound generated reactive oxygen species (ROS) and induced apoptotic cell death in the chemoresistant NCI-H417 SCLC cell line. Furthermore, fascaplysin revealed marked synergism with the topoisomerase I-directed camptothecin and 10-hydroxy-camptothecin. The Poly(ADP-ribose)-Polymerase 1 (PARP1) inhibitor BYK 204165 antagonized the cytotoxic activity of fascaplysin, pointing to the involvement of DNA repair in response to the anticancer activity of the drug. In conclusion, fascaplysin seems to be suitable for treatment of SCLC, based on high cytotoxic activity through multiple routes of action, affecting topoisomerase I, integrity of DNA and generation of ROS. PMID:24608973

Mac-1 (CD11b/CD18) is essential for Fc receptor-mediated neutrophil cytotoxicity and immunologic synapse formation.

Science.gov (United States)

van Spriel, A B; Leusen, J H; van Egmond, M; Dijkman, H B; Assmann, K J; Mayadas, T N; van de Winkel, J G

2001-04-15

Receptors for human immunoglobulin (Ig)G and IgA initiate potent cytolysis of antibody (Ab)-coated targets by polymorphonuclear leukocytes (PMNs). Mac-1 (complement receptor type 3, CD11b/CD18) has previously been implicated in receptor cooperation with Fc receptors (FcRs). The role of Mac-1 in FcR-mediated lysis of tumor cells was characterized by studying normal human PMNs, Mac-1-deficient mouse PMNs, and mouse PMNs transgenic for human FcR. All PMNs efficiently phagocytosed Ab-coated particles. However, antibody-dependent cellular cytotoxicity (ADCC) was abrogated in Mac-1(-/-) PMNs and in human PMNs blocked with anti-Mac-1 monoclonal Ab (mAb). Mac-1(-/-) PMNs were unable to spread on Ab-opsonized target cells and other Ab-coated surfaces. Confocal laser scanning and electron microscopy revealed a striking difference in immunologic synapse formation between Mac-1(-/-) and wild-type PMNs. Also, respiratory burst activity could be measured outside membrane-enclosed compartments by using Mac-1(-/-) PMNs bound to Ab-coated tumor cells, in contrast to wild-type PMNs. In summary, these data document an absolute requirement of Mac-1 for FcR-mediated PMN cytotoxicity toward tumor targets. Mac-1(-/-) PMNs exhibit defective spreading on Ab-coated targets, impaired formation of immunologic synapses, and absent tumor cytolysis.

Treatment of a solid tumor using engineered drug-resistant immunocompetent cells and cytotoxic chemotherapy.

Science.gov (United States)

Dasgupta, Anindya; Shields, Jordan E; Spencer, H Trent

2012-07-01

Multimodal therapy approaches, such as combining chemotherapy agents with cellular immunotherapy, suffers from potential drug-mediated toxicity to immune effector cells. Overcoming such toxic effects of anticancer cellular products is a potential critical barrier to the development of combined therapeutic approaches. We are evaluating an anticancer strategy that focuses on overcoming such a barrier by genetically engineering drug-resistant variants of immunocompetent cells, thereby allowing for the coadministration of cellular therapy with cytotoxic chemotherapy, a method we refer to as drug-resistant immunotherapy (DRI). The strategy relies on the use of cDNA sequences that confer drug resistance and recombinant lentiviral vectors to transfer nucleic acid sequences into immunocompetent cells. In the present study, we evaluated a DRI-based strategy that incorporates the immunocompetent cell line NK-92, which has intrinsic antitumor properties, genetically engineered to be resistant to both temozolomide and trimetrexate. These immune effector cells efficiently lysed neuroblastoma cell lines, which we show are also sensitive to both chemotherapy agents. The antitumor efficacy of the DRI strategy was demonstrated in vivo, whereby neuroblastoma-bearing NOD/SCID/γ-chain knockout (NSG) mice treated with dual drug-resistant NK-92 cell therapy followed by dual cytotoxic chemotherapy showed tumor regression and significantly enhanced survival compared with animals receiving either nonengineered cell-based therapy and chemotherapy, immunotherapy alone, or chemotherapy alone. These data show there is a benefit to using drug-resistant cellular therapy when combined with cytotoxic chemotherapy approaches.

4-methoxychalcone enhances cisplatin-induced oxidative stress and cytotoxicity by inhibiting the Nrf2/ARE-mediated defense mechanism in A549 lung cancer cells.

Science.gov (United States)

Lim, Juhee; Lee, Sung Ho; Cho, Sera; Lee, Ik-Soo; Kang, Bok Yun; Choi, Hyun Jin

2013-10-01

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key transcriptional regulator for the protection of cells against oxidative and xenobiotic stresses. Recent studies have demonstrated that high constitutive expression of Nrf2 is observed in many types of cancer cells showing resistance to anti-cancer drugs, suggesting that the suppression of overexpressed Nrf2 could be an attractive therapeutic strategy to overcome cancer drug resistance. In the present study, we aimed to find small molecule compounds that enhance the sensitivity of tumor cells to cisplatin induced cytotoxicity by suppressing Nrf2-mediated defense mechanism. A549 lung cancer cells were shown to be more resistant to the anti-cancer drug cisplatin than HEK293 cells, with higher Nrf2 signaling activity; constitutively high amounts of Nrf2-downstream target proteins were observed in A549 cells. Among the three chalcone derivatives 4-methoxy-chalcone (4-MC), hesperidin methylchalcone, and neohesperidin dihydrochalcone, 4-MC was found to suppress transcriptional activity of Nrf2 in A549 cells but to activate it in HEK293 cells. 4-MC was also shown to down-regulate expression of Nrf2 and the downstream phase II detoxifying enzyme NQO1 in A549 cells. The PI3K/Akt pathway was found to be involved in the 4-MC-induced inhibition of Nrf2/ARE activity in A549 cells. This inhibition of Nrf2 signaling results in the accelerated generation of reactive oxygen species and exacerbation of cytotoxicity in cisplatin-treated A549 cells. Taken together, these results suggest that the small molecule compound 4-MC could be used to enhance the sensitivity of tumor cells to the therapeutic effect of cisplatin through the regulation of Nrf2/ARE signaling.

L-Arginine Increases Cytotoxicity in Irradiated Ehrlich Carcinoma Cell Line: Possible Potential Role of Nitric Oxide

International Nuclear Information System (INIS)

Noaman, E.

2008-01-01

Cancer cells possess nitric oxide syntheses (NOS) which metabolize L-Arginine (L-Arg) for producing nitric oxide (NO) The present study investigates the relations between NO and ionizing radiation in the Ehrlich ascites carcinoma (EAC) cell line. NOS activity was stimulated by exposure of cells to L-Arg just after irradiation. L-Arg (5 m M) supply led to an increase in ionizing radiation induced cytotoxicity (% of viability 18± 3 %) whereas, neither L-Arg itself nor ionizing irradiation caused cell death at the doses used in this study. Also, cells were treated either with L-Thio citrulline (L-Thio), an irreversible inhibitor of NOS or with exogenous superoxide dismutase (SOD) and catalase. L-Thio and SOD prevented L-Arg mediated deleterious effects on Irradiated cells, whereas catalase was ineffective. Intracellular antioxidant enzyme activity was also determined. Ionizing radiation + L-Arg stress altered the activity of catalase (66 % decrease) and glutathione peroxidase (83 % decrease). Our findings demonstrated that L-Arg induces increase the radiation-mediated deleterious effects in Ehrlich ascites carcinoma cells cytotoxicity and that the ratio NO/ O 2 plays a key role in these processes. NO could participate the deleterious effect of irradiation, in conjugation with others reactive oxygen species (ROS) produced during the oxidation of intracellular components by ionizing radiation (dose 6 Gy)

Mechanisms of CDDO-imidazolide-mediated cytoprotection against acrolein-induced neurocytotoxicity in SH-SY5Y cells and primary human astrocytes.

Science.gov (United States)

Speen, Adam; Jones, Colton; Patel, Ruby; Shah, Halley; Nallasamy, Palanisamy; Brooke, Elizabeth A S; Zhu, Hong; Li, Y Robert; Jia, Zhenquan

2015-10-01

Acrolein is a ubiquitous unsaturated aldehyde has been implicated in the pathogenesis of various neurological disorders. However, limited study has been conducted into potential therapeutic protection and underlying mechanism against acrolein-induced cytotoxicity via upregulation of cellular aldehyde-detoxification defenses. In this study we have utilized RA-differentiated human SH-SY5Y cells and primary human astrocytes to investigate the induction of glutathione (GSH) by the synthetic triterpenoid 2-cyano-3,12-dixooleana-1,9-dien-28-imidazolide (CDDO-Im) and the protective effects CDDO-Im-mediated antioxidant defenses on acrolein toxicity. Acrolein exposure to RA-differentiated SH-SY5Y cells resulted in a significant time dependent depletion of cellular GSH preceding a reduction in cell viability and LDH release. Further, we demonstrated the predominance of cellular GSH in protection against acrolein-induced cytotoxicity. Buthionine sulfoximine (BSO) at 25μM dramatically depleted GSH and significantly potentiated acrolein-induced cytotoxicity. Pretreatment of the cells with 100nM CDDO-Im afforded a dramatic protection against acrolein-induced cytotoxicity. Pretreatment of BSO and CDDO was found to prevent the CDDO-Im-mediated GSH induction and partially reversed the cytoprotective effects of CDDO-Im against acrolein cytotoxicity. Overall, this study represents for the first time the CDDO-Im mediated upregulation of GSH is a predominant mechanism against acrolein-induced neurotoxicity. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Hepatoprotective potential of Lavandula coronopifolia extracts against ethanol induced oxidative stress-mediated cytotoxicity in HepG2 cells.

Science.gov (United States)

Farshori, Nida Nayyar; Al-Sheddi, Ebtsam S; Al-Oqail, Mai M; Hassan, Wafaa H B; Al-Khedhairy, Abdulaziz A; Musarrat, Javed; Siddiqui, Maqsood A

2015-08-01

The present investigations were carried out to study the protective potential of four extracts (namely petroleum ether extract (LCR), chloroform extract (LCM), ethyl acetate extract (LCE), and alcoholic extract (LCL)) of Lavandula coronopifolia on oxidative stress-mediated cell death induced by ethanol, a known hepatotoxin in human hapatocellular carcinoma (HepG2) cells. Cells were pretreated with LCR, LCM, LCE, and LCL extracts (10-50 μg/ml) of L. coronopifolia for 24 h and then ethanol was added and incubated further for 24 h. After the exposure, cell viability using (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and neutral red uptake assays and morphological changes in HepG2 cells were studied. Pretreatment with various extracts of L. coronpifolia was found to be significantly effective in countering the cytotoxic responses of ethanol. Antioxidant properties of these L. coronopifolia extracts against reactive oxygen species (ROS) generation, lipid peroxidation (LPO), and glutathione (GSH) levels induced by ethanol were investigated. Results show that pretreatment with these extracts for 24 h significantly inhibited ROS generation and LPO induced and increased the GSH levels reduced by ethanol. The data from the study suggests that LCR, LCM, LCE, and LCL extracts of L. coronopifolia showed hepatoprotective activity against ethanol-induced damage in HepG2 cells. However, a comparative study revealed that the LCE extract was found to be the most effective and LCL the least effective. The hepatoprotective effects observed in the study could be associated with the antioxidant properties of these extracts of L. coronopifolia. © The Author(s) 2013.

Cytotoxicity of anthraquinones from the roots of Pentas schimperi towards multi-factorial drug-resistant cancer cells.

Science.gov (United States)

Kuete, Victor; Donfack, Arno R Nanfack; Mbaveng, Armelle T; Zeino, Maen; Tane, Pierre; Efferth, Thomas

2015-08-01

Multidrug resistance in cancer represents a major problem in chemotherapy. The present study was designed to assess the cytotoxicity of anthraquinones from Pentas schimperi, namely damnacanthal (1), damnacanthol (2), 3-hydroxy-2-hydroxymethyl anthraquinone (3) and schimperiquinone B (4) against nine drug-sensitive and multidrug resistant (MDR) cancer cell lines. The resazurin reduction assay was used to evaluate the cytotoxicity of the above compounds, whilst caspase-Glo assay was used to detect the activation of caspases enzymes by compounds 1 and 2. Cell cycle, mitochondrial membrane potential (MMP) and levels of reactive oxygen species were all analyzed via flow cytometry. Anthraquinones 1 and 2 displayed cytotoxic effects with IC50 values below 81 μM on all the nine tested cancer cell lines whilst 3 and 4 displayed selective activities. The recorded IC50 values for compounds 1 and 2 ranged from 3.12 μM and 12.18 μM (towards leukemia CCRF-CEM cells) and from 30.32 μM and 80.11 μM (towards gliobastoma U87MG.ΔEGFR cells) respectively, and from 0.20 μM (against CCRF-CEM cells) to 195.12 μM (against CEM/ADR5000 cells) for doxorubicin. Compounds 1 and 2 induced apoptosis in CCRF-CEM leukemia cells, mediated by the disruption of the MMP and increase in ROS production. Anthraquinones from Pentas schimperi and mostly 1 and 2 are potential cytotoxic natural products that deserve more investigations to develop novel antineoplastic drugs against multifactorial drug resistant cancers.

Interleukin-2 activation of cytotoxic cells in postmastectomy seroma.

Science.gov (United States)

Gercel-Taylor, C; Hoffman, J P; Taylor, D D; Owens, K J; Eisenberg, B L

1996-02-15

Lymphocytes were isolated from breast seroma fluids and used to study the mechanism of activation of cytotoxic lymphocytes and possible role of immunological potentiation following surgery in breast cancer patients. Single or serial samples were obtained from patients who had undergone mastectomy or lumpectomy with axillary node dissection. Lymphocytes were activated with rIL-2 (interleukin-2) and their cytotoxic activity was studied against Daudi and K562 cells and against a breast tumor line (SKBr-3). All of the patients (21/21) responded to IL-2 stimulation by significant activation of cytotoxic activity. The unstimulated cytotoxic activity of these cells against NK targets was low with less than 10% specific release in cytotoxicity assays. In simultaneous experiments, autologous seroma fluid was included during activation of lymphocytes to study possible regulatory molecules that may be present. In 17/21 patients, the presence of their seroma fluid, during the activation period, enhanced or did not effect the cytotoxic potential of their lymphocytes; inhibition was observed when seroma fluids from 4/21 patients were included. Analysis of the cytotoxic population derived from combined IL-2 and seroma treatments indicates the presence of cells with increased expression of CD56, and CD2, as well as in some cases CD16 expression. Cytotoxic lymphocytes derived from IL-2 and seroma treatments appeared to be more effective killers. Modulation of CD2 expression with seroma alone appeared to result in the generation of this highly cytotoxic population. This study demonstrates the role of CD2 expression in the effectiveness of LAK cell killing and also potential benefit of an immunotherapeutic approach to the postoperative treatment of carcinoma of the breast.

Cytotoxic Effects of Fascaplysin against Small Cell Lung Cancer Cell Lines

Directory of Open Access Journals (Sweden)

Gerhard Hamilton

2014-03-01

Full Text Available Fascaplysin, the natural product of a marine sponge, exhibits anticancer activity against a broad range of tumor cells, presumably through interaction with DNA, and/or as a highly selective cyclin-dependent kinase 4 (CDK4 inhibitor. In this study, cytotoxic activity of fascaplysin against a panel of small cell lung cancer (SCLC cell lines and putative synergism with chemotherapeutics was investigated. SCLC responds to first-line chemotherapy with platinum-based drugs/etoposide, but relapses early with topotecan remaining as the single approved therapeutic agent. Fascaplysin was found to show high cytotoxicity against SCLC cells and to induce cell cycle arrest in G1/0 at lower and S-phase at higher concentrations, respectively. The compound generated reactive oxygen species (ROS and induced apoptotic cell death in the chemoresistant NCI-H417 SCLC cell line. Furthermore, fascaplysin revealed marked synergism with the topoisomerase I-directed camptothecin and 10-hydroxy-camptothecin. The Poly(ADP-ribose-Polymerase 1 (PARP1 inhibitor BYK 204165 antagonized the cytotoxic activity of fascaplysin, pointing to the involvement of DNA repair in response to the anticancer activity of the drug. In conclusion, fascaplysin seems to be suitable for treatment of SCLC, based on high cytotoxic activity through multiple routes of action, affecting topoisomerase I, integrity of DNA and generation of ROS.

ELISPOT Assay for Monitoring Cytotoxic T Lymphocytes (CTL Activity in Cancer Vaccine Clinical Trials

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Thomas J. Sayers

2012-05-01

Full Text Available The profiling and monitoring of immune responses are key elements in the evaluation of the efficacy and development of new biotherapies, and a number of assays have been introduced for analyzing various immune parameters before, during, and after immunotherapy. The choice of immune assays for a given clinical trial depends on the known or suggested immunomodulating mechanisms associated with the tested therapeutic modality. Cell-mediated cytotoxicity represents a key mechanism in the immune response to various pathogens and tumors. Therefore, the selection of monitoring methods for the appropriate assessment of cell-mediated cytotoxicity is thought to be crucial. Assays that can detect both cytotoxic T lymphocytes (CTL frequency and function, such as the IFN-γ enzyme-linked immunospot assay (ELISPOT have gained increasing popularity for monitoring clinical trials and in basic research. Results from various clinical trials, including peptide and whole tumor cell vaccination and cytokine treatment, have shown the suitability of the IFN-γ ELISPOT assay for monitoring T cell responses. However, the Granzyme B ELISPOT assay and Perforin ELISPOT assay may represent a more direct analysis of cell-mediated cytotoxicity as compared to the IFN-γ ELISPOT, since Granzyme B and perforin are the key mediators of target cell death via the granule-mediated pathway. In this review we analyze our own data and the data reported by others with regard to the application of various modifications of ELISPOT assays for monitoring CTL activity in clinical vaccine trials.

Oncolytic Group B Adenovirus Enadenotucirev Mediates Non-apoptotic Cell Death with Membrane Disruption and Release of Inflammatory Mediators

Directory of Open Access Journals (Sweden)

Arthur Dyer

2017-03-01

Full Text Available Enadenotucirev (EnAd is a chimeric group B adenovirus isolated by bioselection from a library of adenovirus serotypes. It replicates selectively in and kills a diverse range of carcinoma cells, shows effective anticancer activity in preclinical systems, and is currently undergoing phase I/II clinical trials. EnAd kills cells more quickly than type 5 adenovirus, and speed of cytotoxicity is dose dependent. The EnAd death pathway does not involve p53, is predominantly caspase independent, and appears to involve a rapid fall in cellular ATP. Infected cells show early loss of membrane integrity; increased exposure of calreticulin; extracellular release of ATP, HSP70, and HMGB1; and influx of calcium. The virus also causes an obvious single membrane blister reminiscent of ischemic cell death by oncosis. In human tumor biopsies maintained in ex vivo culture, EnAd mediated release of pro-inflammatory mediators such as TNF-α, IL-6, and HMGB1. In accordance with this, EnAd-infected tumor cells showed potent stimulation of dendritic cells and CD4+ T cells in a mixed tumor-leukocyte reaction in vitro. Whereas many viruses have evolved for efficient propagation with minimal inflammation, bioselection of EnAd for rapid killing has yielded a virus with a short life cycle that combines potent cytotoxicity with a proinflammatory mechanism of cell death.

The new numerology of immunity mediated by virus-specific CD8(+) T cells.

Science.gov (United States)

Doherty, P C

1998-08-01

Our understanding of virus-specific CD8(+) T cell responses is currently being revolutionized by peptide-based assay systems that allow flow cytometric analysis of effector and memory cytotoxic T lymphocyte populations. These techniques are, for the first time, putting the analysis of T-cell-mediated immunity on a quantitative basis.

Conventional CD4+ T cells present bacterial antigens to induce cytotoxic and memory CD8+ T cell responses.

Science.gov (United States)

Cruz-Adalia, Aránzazu; Ramirez-Santiago, Guillermo; Osuna-Pérez, Jesús; Torres-Torresano, Mónica; Zorita, Virgina; Martínez-Riaño, Ana; Boccasavia, Viola; Borroto, Aldo; Martínez Del Hoyo, Gloria; González-Granado, José María; Alarcón, Balbino; Sánchez-Madrid, Francisco; Veiga, Esteban

2017-11-17

Bacterial phagocytosis and antigen cross-presentation to activate CD8 + T cells are principal functions of professional antigen presenting cells. However, conventional CD4 + T cells also capture and kill bacteria from infected dendritic cells in a process termed transphagocytosis (also known as transinfection). Here, we show that transphagocytic T cells present bacterial antigens to naive CD8 + T cells, which proliferate and become cytotoxic in response. CD4 + T-cell-mediated antigen presentation also occurs in vivo in the course of infection, and induces the generation of central memory CD8 + T cells with low PD-1 expression. Moreover, transphagocytic CD4 + T cells induce protective anti-tumour immune responses by priming CD8 + T cells, highlighting the potential of CD4 + T cells as a tool for cancer immunotherapy.

In vitro cytotoxicity and genotoxicity studies of gold nanoparticles-mediated photo-thermal therapy versus 5-fluorouracil

Energy Technology Data Exchange (ETDEWEB)

Gomaa, Iman E., E-mail: iman.gomaa@guc.edu.eg; Abdel Gaber, Sara A. [German University in Cairo (GUC), Faculty of Pharmacy and Biotechnology (Egypt); Bhatt, Samarth; Liehr, Thomas [Friedrich Schiller University, Jena University Hospital, Institute of Human Genetics (Germany); Glei, Michael [Friedrich Schiller University, Faculty of Biology and Pharmacy, Institute of Nutrition (Germany); El-Tayeb, Tarek A. [Cairo University, The National Institute for Laser Enhanced Sciences (NILES) (Egypt); Abdel-Kader, Mahmoud H. [German University in Cairo (GUC), Faculty of Pharmacy and Biotechnology (Egypt)

2015-02-15

This study evaluates tumour cell-killing efficacy of metallic gold nanoparticles (AuNPs)-mediated photo-thermal therapy (PTT) in comparison to 5-fluorouracil (5-FU) as a standard chemotherapeutic drug. It also focuses on the possible genetic abnormalities of both drugs in normal blood lymphocytes. Both 5-FU and light-activated spherical AuNPs of 15± nm diameter were used to target MCF-7 breast cancer cell line. Alkaline comet assay, standard karyotyping and multiplex fluorescent in situ hybridization were applied in order to investigate the respective possible genotoxic and mutagenic side effects that might result from the application of each therapeutic modality. Results showed that the LC25 of AuNPs-mediated PTT was achieved at a concentration of 100 µM for 12-h incubation and exposure to light energy of 50 J/cm{sup 2}, while the same cytotoxic effect was obtained by incubating the MCF-7 cells with the same concentration of the chemotherapeutic drug 5-FU for 24 h. On the other hand, AuNPs showed insignificant genotoxic effect of DNA damage represented by 4.6 % in comparison to 18.58 % exerted by 5-FU. The chromosomal studies resulted in normal karyotypes for cells treated with AuNPs-mediated PTT, while those treated with 5-FU showed several types of numerical as well as structural chromosomal aberrations. In conclusion, compared to 5-FU, light-activated AuNPs-mediated PTT provides considerable efficacy in breast cancer cells killing with no genetic side effects under the proposed experimental conditions.

In vitro cytotoxicity and genotoxicity studies of gold nanoparticles-mediated photo-thermal therapy versus 5-fluorouracil

International Nuclear Information System (INIS)

Gomaa, Iman E.; Abdel Gaber, Sara A.; Bhatt, Samarth; Liehr, Thomas; Glei, Michael; El-Tayeb, Tarek A.; Abdel-Kader, Mahmoud H.

2015-01-01

This study evaluates tumour cell-killing efficacy of metallic gold nanoparticles (AuNPs)-mediated photo-thermal therapy (PTT) in comparison to 5-fluorouracil (5-FU) as a standard chemotherapeutic drug. It also focuses on the possible genetic abnormalities of both drugs in normal blood lymphocytes. Both 5-FU and light-activated spherical AuNPs of 15± nm diameter were used to target MCF-7 breast cancer cell line. Alkaline comet assay, standard karyotyping and multiplex fluorescent in situ hybridization were applied in order to investigate the respective possible genotoxic and mutagenic side effects that might result from the application of each therapeutic modality. Results showed that the LC25 of AuNPs-mediated PTT was achieved at a concentration of 100 µM for 12-h incubation and exposure to light energy of 50 J/cm 2 , while the same cytotoxic effect was obtained by incubating the MCF-7 cells with the same concentration of the chemotherapeutic drug 5-FU for 24 h. On the other hand, AuNPs showed insignificant genotoxic effect of DNA damage represented by 4.6 % in comparison to 18.58 % exerted by 5-FU. The chromosomal studies resulted in normal karyotypes for cells treated with AuNPs-mediated PTT, while those treated with 5-FU showed several types of numerical as well as structural chromosomal aberrations. In conclusion, compared to 5-FU, light-activated AuNPs-mediated PTT provides considerable efficacy in breast cancer cells killing with no genetic side effects under the proposed experimental conditions

induced acute cytotoxicity in human cervical epithelial carcinoma cells

African Journals Online (AJOL)

Molecular basis of arsenite (As +3 )-induced acute cytotoxicity in human cervical epithelial carcinoma cells. ... Libyan Journal of Medicine ... Methods: After performing cytotoxic assays on a human epithelial carcinoma cell line, expression analysis was done by quantitative polymerase chain reaction, western blotting, and ...

miR-146a down-regulation alleviates H2O2-induced cytotoxicity of PC12 cells by regulating MCL1/JAK/STAT pathway : miR-146a down-regulation relieves H2O2-induced PC12 cells cytotoxicity by MCL1/JAK/STAT.

Science.gov (United States)

Yang, Xuecheng; Mao, Xin; Ding, Xuemei; Guan, Fengju; Jia, Yuefeng; Luo, Lei; Li, Bin; Tan, Hailin; Cao, Caixia

2018-02-26

Oxidative stress and miRNAs have been confirmed to play an important role in neurological diseases. The study aimed to explore the underlying effect and mechanisms of miR-146a in H 2 O 2 -induced injury of PC12 cells. Here, PC12 cells were stimulated with 200 μM of H 2 O 2 to construct oxidative injury model. Cell injury was evaluated on the basis of the changes in cell viability, migration, invasion, apoptosis, and DNA damage. Results revealed that miR-146a expression was up-regulated in H 2 O 2 -induced PC12 cells. Functional analysis showed that down-regulation of miR-146a alleviated H 2 O 2 -induced cytotoxicity in PC12 cells. Dual-luciferase reporter and western blot assay verified that MCL1 was a direct target gene of miR-146a. Moreover, anti-miR-146a-mediated suppression on cell cytotoxicity was abated following MCL1 knockdown in H 2 O 2 -induced PC12 cells. Furthermore, MCL1 activated JAK/STAT signaling pathway and MCL1 overexpression attenuated H 2 O 2 -induced cytotoxicity in PC12 cells by JAK/STAT signaling pathway. In conclusion, this study suggested that suppression of miR-146a abated H 2 O 2 -induced cytotoxicity in PC12 cells via regulating MCL1/JAK/STAT pathway.

Cell-mediated immune responses in rainbow trout after DNA immunization against the viral hemorrhagic septicemia virus

DEFF Research Database (Denmark)

Utke, Katrin; Kock, Holger; Schuetze, Heike

2008-01-01

injection site rather than to injection sites of heterologous vaccines, suggesting the antigen specificity of homing. By demonstrating CMC responses to distinct viral proteins and homing in rainbow trout, these results substantially contribute to the understanding of the teleost immune system.......To identify viral proteins that induce cell-mediated cytotoxicity (CMC) against viral hemorrhagic septicemia virus (VHSV)-infected cells, rainbow trout were immunized with DNA vectors encoding the glycoprotein G or the nucleocapsid protein N of VHSV. The G protein was a more potent trigger...... of cytotoxic cells than the N protein. Peripheral blood leukocytes (PBL) isolated from trout immunized against the G protein killed both VHSV-infected MHC class I matched (RTG-2) and VHSV-infected xenogeneic (EPC) target cells, suggesting the involvement of both cytotoxic T lymphocytes (CTL) and NK cells...

Cytotoxic human CD4(+) T cells

NARCIS (Netherlands)

van de Berg, Pablo J.; van Leeuwen, Ester M.; ten Berge, Ineke J.; van Lier, Rene

2008-01-01

The induction of adaptive immune responses critically depends on helper signals provided by CD4(+) T cells. These signals not only license antigen presenting cells (APC) to activate naïve CD8(+) T cells leading to the formation of vast numbers of cytotoxic T lymphocytes but also support the

Rapamycin Synergizes with Cisplatin in Antiendometrial Cancer Activation by Improving IL-27–Stimulated Cytotoxicity of NK Cells

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Wen-Jie Zhou

2018-01-01

Full Text Available Natural killer (NK cell function is critical for controlling initial tumor growth and determining chemosensitivity of the tumor. A synergistic relationship between rapamycin and cisplatin in uterine endometrial cancer (UEC in vitro has been reported, but the mechanism and the combined therapeutic strategy for endometrial cancer (EC are still unknown. We found a positive correlation between the level of IL-27 and the differentiated stage of UEC. The increase of IL-27 in uterine endometrial cancer cell (UECC lines (Ishikawa, RL95-2 and KLE led to a high cytotoxic activity of NK cells to UECC in the co-culture system. Exposure with rapamycin enhanced the cytotoxicity of NK cells by upregulating the expression of IL-27 in UECC and IL-27 receptors (IL-27Rs: WSX-1 and gp130 on NK cells and further restricted the growth of UEC in Ishikawa-xenografted nude mice. In addition, treatment with rapamycin resulted in an increased autophagy level of UECC, and IL-27 enhanced this ability of rapamycin. Cisplatin-mediated NK cells' cytotoxic activity and anti-UEC activation were independent of IL-27; however, the combination of rapamycin and cisplatin led to a higher cytotoxic activity of NK cells, smaller UEC volume and longer survival rate in vivo. These results suggest that rapamycin and cisplatin synergistically activate the cytotoxicity of NK cells and inhibit the progression of UEC in both an IL-27–dependent and –independent manner. This provides a scientific basis for potential rapamycin-cisplatin combined therapeutic strategies targeted to UEC, especially for the patients with low differentiated stage or abnormally low level of IL-27.

Salinomycin enhances cisplatin-induced cytotoxicity in human lung cancer cells via down-regulation of AKT-dependent thymidylate synthase expression.

Science.gov (United States)

Ko, Jen-Chung; Zheng, Hao-Yu; Chen, Wen-Ching; Peng, Yi-Shuan; Wu, Chia-Hung; Wei, Chia-Li; Chen, Jyh-Cheng; Lin, Yun-Wei

2016-12-15

Salinomycin, a polyether antibiotic, acts as a highly selective potassium ionophore and has anticancer activity on various cancer cell lines. Cisplatin has been proved as chemotherapy drug for advanced human non-small cell lung cancer (NSCLC). Thymidylate synthase (TS) is a key enzyme in the pyrimidine salvage pathway, and increased expression of TS is thought to be associated with resistance to cisplatin. In this study, we showed that salinomycin (0.5-2μg/mL) treatment down-regulating of TS expression in an AKT inactivation manner in two NSCLC cell lines, human lung adenocarcinoma A549 and squamous cell carcinoma H1703 cells. Knockdown of TS using small interfering RNA (siRNA) or inhibiting AKT activity with PI3K inhibitor LY294002 enhanced the cytotoxicity and cell growth inhibition of salinomycin. A combination of cisplatin and salinomycin resulted in synergistic enhancement of cytotoxicity and cell growth inhibition in NSCLC cells, accompanied with reduced activation of phospho-AKT, and TS expression. Overexpression of a constitutive active AKT (AKT-CA) expression vector reversed the salinomycin and cisplatin-induced synergistic cytotoxicity. In contrast, pretreatment with LY294002 further decreased the cell viability in salinomycin and cisplatin cotreated cells. Our findings suggested that the down-regulation of AKT-mediated TS expression by salinomycin enhanced the cisplatin-induced cytotoxicity in NSCLC cells. These results may provide a rationale to combine salinomycin with cisplatin for lung cancer treatment. Copyright © 2016 Elsevier Inc. All rights reserved.

Role of Calcium and Mitochondria in MeHg-Mediated Cytotoxicity

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Daniel Roos

2012-01-01

Full Text Available Methylmercury (MeHg mediated cytotoxicity is associated with loss of intracellular calcium (Ca2+ homeostasis. The imbalance in Ca2+ physiology is believed to be associated with dysregulation of Ca2+ intracellular stores and/or increased permeability of the biomembranes to this ion. In this paper we summarize the contribution of glutamate dyshomeostasis in intracellular Ca2+ overload and highlight the mitochondrial dysfunctions induced by MeHg via Ca2+ overload. Mitochondrial disturbances elicited by Ca2+ may involve several molecular events (i.e., alterations in the activity of the mitochondrial electron transport chain complexes, mitochondrial proton gradient dissipation, mitochondrial permeability transition pore (MPTP opening, thiol depletion, failure of energy metabolism, reactive oxygen species overproduction that could culminate in cell death. Here we will focus on the role of oxidative stress in these phenomena. Additionally, possible antioxidant therapies that could be effective in the treatment of MeHg intoxication are briefly discussed.

Antibody-dependent cellular cytotoxicity and cytokine/chemokine secretion by KHYG-1 cells stably expressing FcγRIIIA.

Science.gov (United States)

Kobayashi, Eiji; Motoi, Sotaro; Sugiura, Masahito; Kajikawa, Masunori; Kojima, Shuji; Kohroki, Junya; Masuho, Yasuhiko

2014-09-01

Antibody-dependent cellular cytotoxicity (ADCC) mediated by natural killer (NK) cells is a major mechanism of tumor therapy with antibodies. NK cells not only manifest cytotoxicity but also secrete a variety of cytokines/chemokines that regulate immune responses. Using a retroviral vector, in this study we established a KHYG-1 cell line that stably expresses FcγRIIIA (CD16A). The KHYG-1/FcγRIIIA cells exerted potent antibody concentration-dependent ADCC, whereas parental KHYG-1 cells did not. In contrast, without antibody, the natural killer activity of KHYG-1/FcγRIIIA cells was less potent than that of parental KHYG-1 cells. During the course of ADCC, KHYG-1/FcγRIIIA cells secreted IFN-γ and MIP-1α dependent upon antibody concentration, but parental KHYG-1 cells did not. These results suggest that KHYG-1/FcγRIIIA cells would be useful in studies to elucidate the function of NK cells and the mechanism of ADCC. Copyright © 2014 Elsevier B.V. All rights reserved.

Antibody-dependent cellular cytotoxicity (ADCC) activity of a novel anti-PD-L1 antibody avelumab (MSB0010718C) on human tumor cells

OpenAIRE

Boyerinas, Benjamin; Jochems, Caroline; Fantini, Massimo; Heery, Christopher R.; Gulley, James L.; Tsang, Kwong Yok; Schlom, Jeffrey

2015-01-01

Several anti-PD1/PD-L1 monoclonal antibodies (MAb) are currently providing evidence of clinical benefit in subsets of cancer patients. The mode of action of these MAbs is to inhibit PD1 on immune cells interacting with PD-L1 on tumor cells. These MAbs are either designed or engineered to eliminate antibody-dependent cell-mediated cytotoxicity (ADCC), which, however, has been implicated as an important mechanism in several highly effective MAb-mediated cancer therapies. A fully human anti-PD-L...

SIAH1-induced p34SEI-1 polyubiquitination/degradation mediates p53 preferential vitamin C cytotoxicity.

Science.gov (United States)

Lee, Soonduck; Kim, Jinsun; Jung, Samil; Li, Chengping; Yang, Young; Kim, Keun Il; Lim, Jong-Seok; Kim, Yonghwan; Cheon, Choong-Il; Lee, Myeong-Sok

2015-03-01

Vitamin C is considered as an important anticancer therapeutic agent although this view is debatable. In this study, we introduce a physiological mechanism demonstrating how vitamin C exerts anticancer activity that induces cell cycle arrest and apoptosis. Our previous and current data reveal that p53 tumor suppressor is the prerequisite factor for stronger anticancer effects of vitamin C. In addition, vitamin C-mediated cancer cell cytotoxicity appears to be achieved at least partly through the downregulation of the p34SEI-1 oncoprotein. Our previous study showed that p34SEI-1 increases the survival of various types of cancer cells by inhibiting their apoptosis. Present data suggest that vitamin C treatment decreases the p34SEI-1 expression at the protein level and therefore alleviates its anti-apoptotic activity. Of note, SIAH1, E3 ubiquitin ligase, appears to be responsible for the p34SEI-1 polyubiquitination and its subsequent degradation, which is dependent on p53. In summary, vitamin C increases cancer cell death by inducing SIAH1-mediated polyubiquitination/degradation of the p34SEI-1 oncoprotein in a p53-dependent manner.

Autophagy mediates cytotoxicity of human colorectal cancer cells treated with garcinielliptone FC.

Science.gov (United States)

Won, Shen-Jeu; Yen, Cheng-Hsin; Lin, Ting-Yu; Jiang-Shieh, Ya-Fen; Lin, Chun-Nan; Chen, Jyun-Ti; Su, Chun-Li

2018-01-01

The tautomeric pair of garcinielliptone FC (GFC) is a novel tautomeric pair of polyprenyl benzophenonoid isolated from the pericarps of Garcinia subelliptica Merr. (G. subelliptica, Clusiaceae), a tree with abundant sources of polyphenols. Our previous report demonstrated that GFC induced apoptosis on various types of human cancer cell lines including chemoresistant human colorectal cancer HT-29 cells. In the present study, we observed that many autophagy-related genes in GFC-treated HT-29 cells were up- and down-regulated using a cDNA microarray containing oncogenes and kinase genes. GFC-induced autophagy of HT-29 cells was confirmed by observing the formation of acidic vesicular organelles, LC3 puncta, and double-membrane autophagic vesicles using flow cytometry, confocal microscopy, and transmission electron microscopy, respectively. Inhibition of AKT/mTOR/P70S6K signaling as well as formation of Atg5-Atg12 and PI3K/Beclin-1 complexes were observed using Western blot. Administration of autophagy inhibitor (3-methyladenine and shRNA Atg5) and apoptosis inhibitor Z-VAD showed that the GFC-induced autophagy was cytotoxic form and GFC-induced apoptosis enhanced GFC-induced autophagy. Our data suggest the involvement of autophagy and apoptosis in GFC-induced anticancer mechanisms of human colorectal cancer. © 2017 Wiley Periodicals, Inc.

Glycoengineering of therapeutic antibodies enhances monocyte/macrophage-mediated phagocytosis and cytotoxicity.

Science.gov (United States)

Herter, Sylvia; Birk, Martina C; Klein, Christian; Gerdes, Christian; Umana, Pablo; Bacac, Marina

2014-03-01

Therapeutic Abs possess several clinically relevant mechanisms of action including perturbation of tumor cell signaling, activation of complement-dependent cytotoxicity, Ab-dependent cellular cytotoxicity (ADCC), Ab-dependent cellular phagocytosis (ADCP), and induction of adaptive immunity. In view of the important role of phagocytic lineage cells in the mechanism of action of therapeutic Abs, we analyzed FcγR receptor-dependent effector functions of monocytes and macrophages triggered by glycoengineered (GE) Abs (having enhanced FcγRIIIa [CD16a] binding affinity) versus their wild-type (WT) counterparts under different experimental conditions. We first defined the precise FcγR repertoire on classical and nonclassical intermediate monocytes--M1 and M2c macrophage populations. We further show that WT and GE Abs display comparable binding and induce similar effector functions (ADCC and ADCP) in the absence of nonspecific, endogenous IgGs. However, in the presence of these IgGs (i.e., in a situation that more closely mimics physiologic conditions), GE Abs display significantly superior binding and promote stronger monocyte and macrophage activity. These data show that in addition to enhancing CD16a-dependent NK cell cytotoxicity, glycoengineering also enhances monocyte and macrophage phagocytic and cytotoxic activities through enhanced binding to CD16a under conditions that more closely resemble the physiologic setting.

Rapid bioreduction of trivalent aurum using banana stem powder and its cytotoxicity against MCF-7 and HEK-293 cell lines

Energy Technology Data Exchange (ETDEWEB)

Arunkumar, Pichaimani [Bharathidasan University, Cancer Genetics and Nanomedicine Laboratory, Department of Biomedical Science, School of Basic Medical Sciences (India); Vedagiri, Hemamalini [Bharathidasan University, Department of Biotechnology (India); Premkumar, Kumpati, E-mail: pkumpati@hotmail.com [Bharathidasan University, Cancer Genetics and Nanomedicine Laboratory, Department of Biomedical Science, School of Basic Medical Sciences (India)

2013-03-15

Bioreduction of metal ions for the synthesis of nanoparticles of well-defined shape and size has been a great challenge in the field of nanotechnology. In this study, we explored the reduction potential of banana stem powder (BSP) for the synthesis of gold nanoparticles (GNP). The kinetics of GNP synthesis was monitored using UV-Vis spectroscopy. The synthesized GNP was characterized using dynamic light scattering (DLS), transmission electron microscopy, and fourier transform infrared spectroscopy. In addition, the cytotoxic potential of the synthesized GNP was investigated using human breast cancer (MCF-7) and normal human embryonic kidney (HEK-293) cell lines, as evaluated by changes in cell morphology, cell viability (MTT), and metabolic activity. BSP exhibited a strong reduction of Au(III) to Au (0) at room temperature within 5 min of reaction time. The synthesized GNP was found to be spherical with an average diameter of 30 nm by DLS analysis. The cytotoxicity analysis reveals a direct dose-response relationship, indicating that the cytotoxicity increases with increasing concentrations of the GNP. Significant cytotoxicity was observed in cancer cells (MCF-7) compared to normal cells (HEK-293). Also the cellular uptake of GNP was more pronounced in MCF-7 cells than HEK-293 cells as evidenced by zeta potential, implicating the possible reason for differential cytotoxicity. Thus the present study demonstrates the importance of these unique, less time-consuming, and stable BSP-mediated GNP as potential drug delivery vehicles in the application of anticancer therapy.

Aluminum doping tunes band gap energy level as well as oxidative stress-mediated cytotoxicity of ZnO nanoparticles in MCF-7 cells

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

2015-09-01

We investigated whether Aluminum (Al) doping tunes band gap energy level as well as selective cytotoxicity of ZnO nanoparticles in human breast cancer cells (MCF-7). Pure and Al-doped ZnO nanoparticles were prepared by a simple sol-gel method. Characterization study confirmed the formation of single phase of AlxZn1-xO nanocrystals with the size range of 33-55 nm. Al-doping increased the band gap energy of ZnO nanoparticles (from 3.51 eV for pure to 3.87 eV for Al-doped ZnO). Al-doping also enhanced the cytotoxicity and oxidative stress response of ZnO nanoparticles in MCF-7 cells. The IC50 for undoped ZnO nanoparticles was 44 μg/ml while for the Al-doped ZnO counterparts was 31 μg/ml. Up-regulation of apoptotic genes (e.g. p53, bax/bcl2 ratio, caspase-3 & caspase-9) along with loss of mitochondrial membrane potential suggested that Al-doped ZnO nanoparticles induced apoptosis in MCF-7 cells through mitochondrial pathway. Importantly, Al-doping did not change the benign nature of ZnO nanoparticles towards normal cells suggesting that Al-doping improves the selective cytotoxicity of ZnO nanoparticles toward MCF-7 cells without affecting the normal cells. Our results indicated a novel approach through which the inherent selective cytotoxicity of ZnO nanoparticles against cancer cells can be further improved.

Evaluation of mutagenicity and metabolism-mediated cytotoxicity of the naphthoquinone 5-methoxy-3,4-dehydroxanthomegnin from Paepalanthus latipes

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Rodrigo R. Kitagawa

Full Text Available A large number of quinones have been associated with antitumor, antibacterial, antimalarial, and antifungal activities. Results of previous studies of 5-methoxy-3,4-dehydroxanthomegnin, a naphthoquinone isolated from Paepalanthus latipes Silveira, Eriocaulaceae, revealed antitumor, antibacterial, immunomodulatory, and antioxidant activities. In this study, we assessed the mutagenicity and metabolism-mediated cytotoxicity of 5-methoxy-3,4-dehydroxanthomegnin by using the Ames test and a microculture neutral red assay incorporating an S9 fraction (hepatic microsomal fraction and cofactors, respectively. We also evaluated the mutagenic activity in Salmonella typhimurium strains TA100, TA98, TA102, and TA97a, as well as the cytotoxic effect on McCoy cells with and without metabolic activation in both tests. Results indicated that naphthoquinone does not cause mutations by substitution or by addition and deletion of bases in the deoxyribonucleic acid sequence with and without metabolic activation. As previously demonstrated, the in vitro cytotoxicity of 5-methoxy-3,4-dehydroxanthomegnin to McCoy cells showed a significant cytotoxic index (CI50 of 11.9 μg/ml. This index was not altered by addition of the S9 fraction, indicating that the S9 mixture failed to metabolically modify the compound. Our results, allied with more specific biological assays in the future, would contribute to the safe use of 5-methoxy-3,4-dehydroxanthomegnin, compound that has showed in previous studies beneficial properties as a potential anticancer drug.

Protection of HepG2 cells against acrolein toxicity by 2-cyano-3,12-dioxooleana-1,9-dien-28-imidazolide via glutathione-mediated mechanism.

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Shah, Halley; Speen, Adam M; Saunders, Christina; Brooke, Elizabeth A S; Nallasamy, Palanisamy; Zhu, Hong; Li, Y Robert; Jia, Zhenquan

2015-10-01

Acrolein is an environmental toxicant, mainly found in smoke released from incomplete combustion of organic matter. Several studies showed that exposure to acrolein can lead to liver damage. The mechanisms involved in acrolein-induced hepatocellular toxicity, however, are not completely understood. This study examined the cytotoxic mechanisms of acrolein on HepG2 cells. Acrolein at pathophysiological concentrations was shown to cause apoptotic cell death and an increase in levels of protein carbonyl and thiobarbituric acid reactive acid substances. Acrolein also rapidly depleted intracellular glutathione (GSH), GSH-linked glutathione-S-transferases, and aldose reductase, three critical cellular defenses that detoxify reactive aldehydes. Results further showed that depletion of cellular GSH by acrolein preceded the loss of cell viability. To further determine the role of cellular GSH in acrolein-mediated cytotoxicity, buthionine sulfoximine (BSO) was used to inhibit cellular GSH biosynthesis. It was observed that depletion of cellular GSH by BSO led to a marked potentiation of acrolein-mediated cytotoxicity in HepG2 cells. To further assess the contribution of these events to acrolein-induced cytotoxicity, triterpenoid compound 2-cyano-3,12-dioxooleana-1,9-dien-28-imidazolide (CDDO-Im) was used for induction of GSH. Induction of GSH by CDDO-Im afforded cytoprotection against acrolein toxicity in HepG2 cells. Furthermore, BSO significantly inhibited CDDO-Im-mediated induction in cellular GSH levels and also reversed cytoprotective effects of CDDO-Im in HepG2 cells. These results suggest that GSH is a predominant mechanism underlying acrolein-induced cytotoxicity as well as CDDO-Im-mediated cytoprotection. This study may provide understanding on the molecular action of acrolein which may be important to develop novel strategies for the prevention of acrolein-mediated toxicity. © 2014 by the Society for Experimental Biology and Medicine.

A novel multiparametric flow cytometry-based cytotoxicity assay simultaneously immunophenotypes effector cells: comparisons to a 4 h 51Cr-release assay.

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Kim, G G; Donnenberg, V S; Donnenberg, A D; Gooding, W; Whiteside, T L

2007-08-31

Natural killer (NK) cell-or T cell-mediated cytotoxicity traditionally is measured in 4-16 h (51)Cr-release assays (CRA). A new four-color flow cytometry-based cytotoxicity assay (FCC) was developed to simultaneously measure NK cell cytotoxicity and NK cell phenotype (CD3(-)CD16(+)CD56(+)). Target cells, K562 or Daudi, were labeled with Cell Tracker Orange (CTO) prior to the addition of effector cells. Following co-incubation, 7 amino-actinomycin D (7-AAD) was added to measure death of target cells. The phenotype of effectors, viability of targets, the formation of tumor-effector cell conjugates and absolute numbers of all cells were measured based on light scatter (FSC/SSC), double discrimination of the fluorescence peak integral and height, and fluorescence intensity. Kinetic studies (0.5 and 1 to 4 h) at different effector to target (E:T) cell ratios (50, 25, 12, and 6) confirmed that the 3 h incubation was optimal. The FCC assay is more sensitive than the CRA, has a coefficient of variation (CV) 8-13% and reliably measures NK cell-or lymphokine-activated killer (LAK) cell-mediated killing of target cells in normal controls and subjects with cancer. The FCC assay can be used to study a range of phenotypic attributes, in addition to lytic activity of various subsets of effector cells, without radioactive tracers and thus, it is relatively inexpensive. The FCC assay has a potential for providing information about molecular interactions underlying target cell lysis and thus becoming a major tool for studies of disease pathogenesis as well as development of novel immune therapies.

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

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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.

Gangliosides inhibit bee venom melittin cytotoxicity but not phospholipase A2-induced degranulation in mast cells

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Nishikawa, Hirofumi; Kitani, Seiichi

2011-01-01

Sting accident by honeybee causes severe pain, inflammation and allergic reaction through IgE-mediated anaphylaxis. In addition to this hypersensitivity, an anaphylactoid reaction occurs by toxic effects even in a non-allergic person via cytolysis followed by similar clinical manifestations. Auto-injectable epinephrine might be effective for bee stings, but cannot inhibit mast cell lysis and degranulation by venom toxins. We used connective tissue type canine mast cell line (CM-MC) for finding an effective measure that might inhibit bee venom toxicity. We evaluated degranulation and cytotoxicity by measurement of β-hexosaminidase release and MTT assay. Melittin and crude bee venom induced the degranulation and cytotoxicity, which were strongly inhibited by mono-sialoganglioside (G M1 ), di-sialoganglioside (G D1a ) and tri-sialoganglioside (G T1b ). In contrast, honeybee venom-derived phospholipase A 2 induced the net degranulation directly without cytotoxicity, which was not inhibited by G M1 , G D1a and G T1b . For analysis of distribution of Gα q and Gα i protein by western blotting, lipid rafts were isolated by using discontinuous sucrose gradient centrifuge. Melittin disrupted the localization of Gα q and Gα i at lipid raft, but gangliosides stabilized the rafts. As a result from this cell-based study, bee venom-induced anaphylactoid reaction can be explained with melittin cytotoxicity and phospholipase A 2 -induced degranulation. Taken together, gangliosides inhibit the effect of melittin such as degranulation, cytotoxicity and lipid raft disruption but not phospholipase A 2 -induced degranulation in mast cells. Our study shows a potential of gangliosides as a therapeutic tool for anaphylactoid reaction by honeybee sting.

Real-time impedimetric monitoring of Poly(ethylenimine)s-mediated cytotoxicity during gene transfection

DEFF Research Database (Denmark)

Caviglia, Claudia; Carminati, Marco; Heiskanen, Arto

Poly(ethylenimine)s (PEIs) are able to condense DNA and RNA into stable toroidal and globular nanostructures (polyplexes) and are among the most efficient and promising synthetic transfectants, but they induce severe cytotoxicity. The mechanisms of PEI-mediated cytotoxicity have not been fully...... delineated but PEI toxicity appears to predominantly depend on membrane perturbing effects in cellular compartments in which they accumulate. Electrochemical Impedance Spectroscopy (EIS) is used as a non-invasive biophysical approach for the investigation of the electrical properties of biological materials...

Betulinic Acid Exerts Cytotoxic Activity Against Multidrug-Resistant Tumor Cells via Targeting Autocrine Motility Factor Receptor (AMFR

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Mohamed E. M. Saeed

2018-05-01

Full Text Available Betulinic acid (BetA is a naturally occurring pentacyclic triterpene isolated from the outer bark of white-barked birch trees and many other medicinal plants. Here, we studied betulinic acid's cytotoxic activity against drug-resistant tumor cell lines. P-glycoprotein (MDR1/ABCB1 and BCRP (ABCG2 are known ATP-binding cassette (ABC drug transporters that mediating MDR. ABCB5 is a close relative to ABCB1, which also mediates MDR. Constitutive activation of the EGF receptor is tightly linked to the development of chemotherapeutic resistance. BetA inhibited P-gp, BCRP, ABCB5 and mutation activated EGFR overexpressing cells with similar efficacy as their drug-sensitive parental counterparts. Furthermore, the mRNA expressions of ABCB1, BCRP, ABCB5 and EGFR were not related to the 50% inhibition concentrations (IC50 for BetA in a panel of 60 cell lines of the National Cancer Institute (NCI, USA. In addition to well-established MDR mechanisms, we attempted to identify other molecular mechanisms that play a role in mediating BetA's cytotoxic activity. For this reason, we performed COMPARE and hierarchical cluster analyses of the transcriptome-wide microarray-based mRNA expression of the NCI cell lines panel. Various genes significantly correlating to BetA's activity were involved in different biological processes, e.g., cell cycle regulation, microtubule formation, signal transduction, transcriptional regulation, chromatin remodeling, cell adhesion, tumor suppression, ubiquitination and proteasome degradation. Immunoblotting and in silico analyses revealed that the inhibition of AMFR activity might be one of the mechanisms for BetA to overcome MDR phenotypes. In conclusion, BetA may have therapeutic potential for the treatment of refractory tumors.

The Transcription Factor Hobit Identifies Human Cytotoxic CD4(+) T Cells

NARCIS (Netherlands)

Oja, Anna E.; Vieira Braga, Felipe A.; Remmerswaal, Ester B. M.; Kragten, Natasja A. M.; Hertoghs, Kirsten M. L.; Zuo, Jianmin; Moss, Paul A.; van Lier, René A. W.; van Gisbergen, Klaas P. J. M.; Hombrink, Pleun

2017-01-01

The T cell lineage is commonly divided into CD4-expressing helper T cells that polarize immune responses through cytokine secretion and CD8-expressing cytotoxic T cells that eliminate infected target cells by virtue of the release of cytotoxic molecules. Recently, a population of CD4(+) T cells that

Circulating blocking factors of lymphoid-cell cytotoxicity in x-ray-induced rat small-bowel adenocarcinoma

International Nuclear Information System (INIS)

Stevens, R.H.; Brooks, G.P.; Osborne, J.W.

1979-01-01

Circulating blocking factors capable of abrogating cell-mediated immune responses measured by in vitro lymphoid-cell cytotoxicity were identified in the sera of Holtzman outbred rats 6 to 9 months after a single exposure of only the temporarily exteriorized, hypoxic ileum and jejunum to 1700 to 2000 R of X radiation. Such factors were found to exist in the serum of every animal exposed to the ionizing radiation regardless of whether a visibly identifiable small-bowel adenocarcinoma existed or subsequently would develop. Protection of cultured x-ray-induced rat small-bowel cancer cells from destruction by tumor-sensitized lymphoid cells as measured by the release of lactoperoxidase-catalyzed radioiodinated membrane proteins from the tumor target cells was conferred by the action of the blocking factors at both effector and target cell levels. The results of this study demonstrate that exposure of only the rat small intestine to ionizing radiation leads to elaboration of circulating factors identifiable several months postirradiation which will block cell-mediated immune responses directed against cancer cells developing in the exposed tissue

Investigation of internalization and cytotoxicity of 125I-[Tyr3]-octreotide in NCI-H446 cell line

International Nuclear Information System (INIS)

Sun Junjie; Fan Wo; Xu Yujie; Zhang Youjiu; Zhu Ran; Hu Mingjiang

2004-01-01

Objective: To investigate the [Tyr 3 ]-octreotide (TOC) internalizing capacity of NCI-H446 cell line, and the cytotoxicity of 125 I-TOC in NCI-H446 cell line. To assess the therapeutic radiopharmaceutical potentiality of 125 I-TOC for the somatostatin receptor (SSTR) positive tumor. Methods: NCI-H446 cells were incubated together with 125 I-TOC for different periods of time, the amount of internalized 125 I-TOC and the 125 I-TOC bound on the cellular nucleus were detected with γ counter, respectively. The viability of the cells was analyzed by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay at different time points with various doses of 125 I-TOC, free 125 I and TOC. Results: 125 I-TOC was internalized into the nucleus and bound on the nucleus in a time-dependent manner. 125 I-TOC bound on the nucleus increased to the highest level at 24 h, the amount of nucleus bound 125 I-TOC at 24 h was 7 times higher than that at 0.5 h. Cytotoxicity of 125 I-TOC in SSTR positive NCI-H446 cells was also dose- and time-dependent. The supreme effect of cytotoxicity was found at 96 h with 74 kBq 125 I-TOC, the survival ratio of cells was reduced to (44.8 ± 7.2)%. Conclusions: 125 I-TOC can be internalized into SSTR positive cells mediated by SSTR. The NCI-H446 cells can be killed by Auger electron emitting from 125 I-TOC. Effect of cytotoxicity showed dose- and time-dependent

Unraveling the response of plant cells to cytotoxic saponins

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Balestrazzi, Alma; Macovei, Anca; Tava, Aldo; Avato, Pinarosa; Raimondi, Elena

2011-01-01

A wide range of pharmacological properties are ascribed to natural saponins, in addition to their biological activities against herbivores, plant soil-borne pathogens and pests. As for animal cells, the cytotoxicity and the chemopreventive role of saponins are mediated by a complex network of signal transduction pathways which include reactive oxygen species (ROS) and nitric oxide (NO). The involvement of other relevant components of the saponin-related signaling routes, such as the Tumor Necrosis Factor (TNF)α, the interleukin (IL)-6 and the Nuclear Transcription FactorκB (NFκB), has been highlighted in animal cells. By contrast, information concerning the response of plant cells to saponins and the related signal transduction pathways is almost missing. To date, there are only a few common features which link plant and animal cells in their response to saponins, such as the early burst in ROS and NO production and the induction of metallothioneins (MTs), small cysteine-rich, metal-binding proteins. This aspect is discussed in the present paper in view of the recent hypothesis that MTs and NO are part of a novel signal transduction pathway participating in the cell response to oxidative stress. PMID:21673512

Cytotoxicity of extracts of spices to cultured cells.

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Unnikrishnan, M C; Kuttan, R

1988-01-01

The cytotoxicity of the extracts from eight different spices used in the Indian diet was determined using Dalton's lymphoma ascites tumor cells and human lymphocytes in vitro and Chinese Hamster Ovary cells and Vero cells in tissue culture. Alcoholic extracts of the spices were found to be more cytotoxic to these cells than their aqueous extracts. Alcoholic extracts of several spices inhibited cell growth at concentrations of 0.2-1 mg/ml in vitro and 0.12-0.3 mg/ml in tissue culture. Ginger, pippali (native to India; also called dried catkins), pepper, and garlic showed the highest activity followed by asafetida, mustard, and horse-gram (native to India). These extracts also inhibited the thymidine uptake into DNA.

Cyclosporine A and palmitic acid treatment synergistically induce cytotoxicity in HepG2 cells

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Luo, Yi, E-mail: yi.luo@pfizer.com; Rana, Payal; Will, Yvonne

2012-06-01

Immunosuppressant cyclosporine A (CsA) treatment can cause severe side effects. Patients taking immunosuppressant after organ transplantation often display hyperlipidemia and obesity. Elevated levels of free fatty acids have been linked to the etiology of metabolic syndromes, nonalcoholic fatty liver and steatohepatitis. The contribution of free fatty acids to CsA-induced toxicity is not known. In this study we explored the effect of palmitic acid on CsA-induced toxicity in HepG2 cells. CsA by itself at therapeutic exposure levels did not induce detectible cytotoxicity in HepG2 cells. Co-treatment of palmitic acid and CsA resulted in a dose dependent increase in cytotoxicity, suggesting that fatty acid could sensitize cells to CsA-induced cytotoxicity at the therapeutic doses of CsA. A synergized induction of caspase-3/7 activity was also observed, indicating that apoptosis may contribute to the cytotoxicity. We demonstrated that CsA reduced cellular oxygen consumption which was further exacerbated by palmitic acid, implicating that impaired mitochondrial respiration might be an underlying mechanism for the enhanced toxicity. Inhibition of c-Jun N-terminal kinase (JNK) attenuated palmitic acid and CsA induced toxicity, suggesting that JNK activation plays an important role in mediating the enhanced palmitic acid/CsA-induced toxicity. Our data suggest that elevated FFA levels, especially saturated FFA such as palmitic acid, may be predisposing factors for CsA toxicity, and patients with underlying diseases that would elevate free fatty acids may be susceptible to CsA-induced toxicity. Furthermore, hyperlipidemia/obesity resulting from immunosuppressive therapy may aggravate CsA-induced toxicity and worsen the outcome in transplant patients. -- Highlights: ► Palmitic acid and cyclosporine (CsA) synergistically increased cytotoxicity. ► The impairment of mitochondrial functions may contribute to the enhanced toxicity. ► Inhibition of JNK activity attenuated

"Proliferation of cytotoxic and activated T cells during acute Epstein-Barr virus induced Infectious Mononucleosis "

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Mansoori SD

2002-05-01

Full Text Available The immune responses that develop following Epstien-Barr Virus (EBV infection are complex and involve both humoral and to a greater extent cell-mediated immune mechanisms. To evaluate the immune response, flow cytometric analysis of the peripheral blood of six patients during the acute phase of EBV infection was performed. This analysis revealed a significant increase in the percentages and the absolute number of CD8+cytotoxic and activated (HLA-DR+ - T lymphocytes and in some cases with a concomitan decrease in the percentages of B (CD19+ lymphocytes and T helper (CD4+ lymphocytes. These patient invariably had inverted CD4/CD8 ratio. All changes reversed to normal level during the recovery phase of infection. It is therefore concluded that EBV specific cytotoxic and activated T lymphocytes are essential in controlling acute EBV infection presented by the infected B cells.

The kinematics of cytotoxic lymphocytes influence their ability to kill target cells.

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Purnima Bhat

Full Text Available Cytotoxic lymphocytes (CTL have been reported to show a range of motility patterns from rapid long-range tracking to complete arrest, but how and whether these kinematics affect their ability to kill target cells is not known. Many in vitro killing assays utilize cell lines and tumour-derived cells as targets, which may be of limited relevance to the kinetics of CTL-mediated killing of somatic cells. Here, live-cell microscopy is used to examine the interactions of CTL and primary murine skin cells presenting antigens. We developed a qualitative and quantitative killing assay using extended-duration fluorescence time-lapse microscopy coupled with large-volume objective software-based data analysis to obtain population data of cell-to-cell interactions, motility and apoptosis. In vivo and ex vivo activated antigen-specific cytotoxic lymphocytes were added to primary keratinocyte targets in culture with fluorometric detection of caspase-3 activation in targets as an objective determinant of apoptosis. We found that activated CTL achieved contact-dependent apoptosis of non-tumour targets after a period of prolonged attachment - on average 21 hours - which was determined by target cell type, amount of antigen, and activation status of CTL. Activation of CTL even without engagement of the T cell receptor was sufficient to mobilise cells significantly above baseline, while the addition of cognate antigen further enhanced their motility. Highly activated CTL showed markedly increased vector displacement, and velocity, and lead to increased antigen-specific target cell death. These data show that the inherent kinematics of CTL correlate directly with their ability to kill non-tumour cells presenting cognate antigen.

Comprehensive Cross-Clade Characterization of Antibody-Mediated Recognition, Complement-Mediated Lysis, and Cell-Mediated Cytotoxicity of HIV-1 Envelope-Specific Antibodies toward Eradication of the HIV-1 Reservoir.

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Mujib, Shariq; Liu, Jun; Rahman, A K M Nur-Ur; Schwartz, Jordan A; Bonner, Phil; Yue, Feng Yun; Ostrowski, Mario A

2017-08-15

Immunotherapy with passive administration of broadly neutralizing HIV-1 envelope-specific antibodies (bnAbs) in the setting of established infection in vivo has yielded mixed results. The contribution of different antibodies toward the direct elimination of infected cells is poorly understood. In this study, we determined the ability of 12 well-characterized anti-HIV-1 neutralizing antibodies to recognize and eliminate primary CD4 T cells infected with HIV-1 belonging to clades A, B, C, and D, via antibody-dependent complement-mediated lysis (ADCML) and antibody-dependent cell-mediated cytotoxicity (ADCC), in vitro We further tested unique combinations of these antibodies to determine the optimal antibody cocktails to be tested in future clinical trials. We report that antibody binding to infected CD4 T cells is highly variable and correlates with ADCML and ADCC processes. Particularly, antibodies targeting the envelope glycan shield (2G12) and V1/V2 site (PG9, PG16, and PGT145) are best at recognizing HIV-1-infected CD4 T cells. However, only PG9 and PG16 and their combinations with other bnAbs sufficiently induced the elimination of HIV-1-infected CD4 T cells by ADCML, ADCC, or both. Notably, CD4 binding site antibodies VRC01, 3BNC117, and NIH45-46 G54W did not exhibit recognition of infected cells and were unable to induce their killing. Future trials geared toward the development of a cure for HIV/AIDS should incorporate V1/V2 antibodies for maximal clearance of infected cells. With the use of only primary immune cells, we conducted a comprehensive cross-clade physiological analysis to aid the direction of antibodies as therapeutics toward the development of a cure for HIV/AIDS. IMPORTANCE Several antibodies capable of neutralizing the majority of circulating HIV-1 strains have been identified to date and have been shown to prevent infection in animal models. However, the use of combinations of such broadly neutralizing antibodies (bnAbs) for the treatment and

Cell-mediated immunity to herpes simplex in humans: lymphocyte cytotoxicity measured by 51Cr release from infected cells

International Nuclear Information System (INIS)

Russell, A.S.; Percy, J.S.; Kovithavongs, T.

1975-01-01

We assessed cell-mediated immunity to herpes simplex virus type 1 antigen in patients suffering from recurrent cold sores and in a series of healthy controls. Paradoxically, all those subject to recurrent herpetic infections had, without exception, evidence of cell-mediated immunity to herpes antigens. This was demonstrated by lymphocyte transformation and specific 51 Cr release from infected human amnion cells after incubation with peripheral blood mononuclear cells. Where performed, skin tests with herpes antigen were also positive. In addition, serum from these patients specifically sensitized herpes virus-infected cells to killing by nonimmune, control mononuclear cells. These tests were negative in the control patients except in a few cases, and it is suggested that these latter may be the asymptomatic herpes virus carriers previously recognized or that they may have experienced a genital infection. (U.S.)

Sensitization of TNF-induced cytotoxicity in lung cancer cells by concurrent suppression of the NF-κB and Akt pathways

International Nuclear Information System (INIS)

Wang Xia; Chen Wenshu; Lin Yong

2007-01-01

Blockage of either nuclear factor-κB (NF-κB) or Akt sensitizes cancer cells to TNF-induced apoptosis. In this study, we investigated the undetermined effect of concurrent blockage of these two survival pathways on TNF-induced cytotoxicity in lung cancer cells. The results show that Akt contributes to TNF-induced NF-κB activation in lung cancer cells through regulating phosphorylation of the p65/RelA subunit of NF-κB. Although individually blocking IKK or Akt partially suppressed TNF-induced NF-κB activation, concurrent suppression of these pathways completely inhibited TNF-induced NF-κB activation and downstream anti-apoptotic gene expression, and synergistically potentiated TNF-induced cytotoxicity. Moreover, suppression of Akt inhibited the Akt-mediated anti-apoptotic pathway through dephosphorylation of BAD. These results indicate that concurrent suppression of NF-κB and Akt synergistically sensitizes TNF-induced cytotoxicity through blockage of distinct survival pathways downstream of NF-κB and Akt, which may be applied in lung cancer therapy

Lymphocyte-dependent antibody-mediated cytotoxicity in Hashimoto thyroiditis

Science.gov (United States)

Calder, Elizabeth A.; Penhale, W. J.; McLeman, Dena; Barnes, E. W.; Irvine, W. J.

1973-01-01

In the presence of normal human lymphocytes, decomplemented sera from twentynine out of thirty-nine patients with Hashimoto thyroiditis caused significant lysis of thyroglobulin-coated chicken red blood cells, as estimated by the release of 51Cr; the mean% specific 51Cr release being 14·1 ± 1·9 (SEM). Serum from twenty-one control subjects studied concurrently caused no significant lysis of thyroglobulin-coated chicken red blood cells; the mean% specific 51Cr release being −1·6±0·7 (SEM). The degree of cytotoxicity correlated with the titre of thyroglobulin antibodies in the serum, determined by tanned red cell haemagglutination. The active component in the Hashimoto serum was localized in the 19S fraction, was unaffected by pre-absorption with anti-human IgM serum, but was neutralized by pre-absorption with anti-human IgG serum. These findings suggest that the cytotoxic activity of serum from patients with Hashimoto thyroiditis is due to the presence of thyroglobulin antibody of the IgG class in the form of complexes, either alone or with antigen. It is postulated that non-specific lymphocytes may play an important role in the pathogenesis of Hashimoto thyroiditis, being activated by the presence in the gland of thyroglobulin antibody, either alone or in the form of complexes attached to thyroid cells. PMID:4740445

Fludarabine-mediated circumvention of cytarabine resistance is associated with fludarabine triphosphate accumulation in cytarabine-resistant leukemic cells.

Science.gov (United States)

Yamamoto, Shuji; Yamauchi, Takahiro; Kawai, Yasukazu; Takemura, Haruyuki; Kishi, Shinji; Yoshida, Akira; Urasaki, Yoshimasa; Iwasaki, Hiromichi; Ueda, Takanori

2007-02-01

The combination of cytarabine (ara-C) with fludarabine is a common approach to treating resistant acute myeloid leukemia. Success depends on a fludarabine triphosphate (F-ara-ATP)-mediated increase in the active intracellular metabolite of ara-C, ara-C 5'-triphosphate (ara-CTP). Therapy-resistant leukemia may exhibit ara-C resistance, the mechanisms of which might induce cross-resistance to fludarabine with reduced F-ara-ATP formation. The present study evaluated the effect of combining ara-C and fludarabine on ara-C-resistant leukemic cells in vitro. Two variant cell lines (R1 and R2) were 8-fold and 10-fold more ara-C resistant, respectively, than the parental HL-60 cells. Reduced deoxycytidine kinase activity was demonstrated in R1 and R2 cells, and R2 cells also showed an increase in cytosolic 5'-nucleotidase II activity. Compared with HL-60 cells, R1 and R2 cells produced smaller amounts of ara-CTP. Both variants accumulated less F-ara-ATP than HL-60 cells and showed cross-resistance to fludarabine nucleoside (F-ara-A). R2 cells, however, accumulated much smaller amounts of F-ara-ATP and were more F-ara-A resistant than R1 cells. In HL-60 and R1 cells, F-ara-A pretreatment followed by ara-C incubation produced F-ara-ATP concentrations sufficient for augmenting ara-CTP production, thereby enhancing ara-C cytotoxicity. No potentiation was observed in R2 cells. Nucleotidase might preferentially degrade F-ara-A monophosphate over ara-C monophosphate, leading to reduced F-ara-ATP production and thereby compromising the F-ara-A-mediated potentiation of ara-C cytotoxicity in R2 cells. Thus, F-ara-A-mediated enhancement of ara-C cytotoxicity depended on F-ara-ATP accumulation in ara-C-resistant leukemic cells but ultimately was associated with the mechanism of ara-C resistance.

Wld(S reduces paraquat-induced cytotoxicity via SIRT1 in non-neuronal cells by attenuating the depletion of NAD.

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Qiujing Yu

Full Text Available Wld(S is a fusion protein with NAD synthesis activity, and has been reported to protect axonal and synaptic compartments of neurons from various mechanical, genetic and chemical insults. However, whether Wld(S can protect non-neuronal cells against toxic chemicals is largely unknown. Here we found that Wld(S significantly reduced the cytotoxicity of bipyridylium herbicides paraquat and diquat in mouse embryonic fibroblasts, but had no effect on the cytotoxicity induced by chromium (VI, hydrogen peroxide, etoposide, tunicamycin or brefeldin A. Wld(S also slowed down the death of mice induced by intraperitoneal injection of paraquat. Further studies demonstrated that Wld(S markedly attenuated mitochondrial injury including disruption of mitochondrial membrane potential, structural damage and decline of ATP induced by paraquat. Disruption of the NAD synthesis activity of Wld(S by an H112A or F116S point mutation resulted in loss of its protective function against paraquat-induced cell death. Furthermore, Wld(S delayed the decrease of intracellular NAD levels induced by paraquat. Similarly, treatment with NAD or its precursor nicotinamide mononucleotide attenuated paraquat-induced cytotoxicity and decline of ATP and NAD levels. In addition, we showed that SIRT1 was required for both exogenous NAD and Wld(S-mediated cellular protection against paraquat. These findings suggest that NAD and SIRT1 mediate the protective function of Wld(S against the cytotoxicity induced by paraquat, which provides new clues for the mechanisms underlying the protective function of Wld(S in both neuronal and non-neuronal cells, and implies that attenuation of NAD depletion may be effective to alleviate paraquat poisoning.

Cooperation of NAD(P)H:quinone oxidoreductase 1 and UDP-glucuronosyltransferases reduces menadione cytotoxicity in HEK293 cells.

Science.gov (United States)

Nishiyama, Takahito; Izawa, Tadashi; Usami, Mami; Ohnuma, Tomokazu; Ogura, Kenichiro; Hiratsuka, Akira

2010-04-09

Previous studies have shown that NAD(P)H:quinone oxidoreductase 1 (NQO1) plays an important role in the detoxification of menadione (2-methyl-1,4-naphthoquinone, also known as vitamin K3). However, menadiol (2-methyl-1,4-naphthalenediol) formed from menadione by NQO1-mediated reduction continues to be an unstable substance, which undergoes the reformation of menadione with concomitant formation of reactive oxygen species (ROS). Hence, we focused on the roles of phase II enzymes, with particular attention to UDP-glucuronosyltransferases (UGTs), in the detoxification process of menadione. In this study, we established an HEK293 cell line stably expressing NQO1 (HEK293/NQO1) and HEK293/NQO1 cell lines with doxycycline (DOX)-regulated expression of UGT1A6 (HEK293/NQO1/UGT1A6) and UGT1A10 (HEK293/NQO1/UGT1A10), and evaluated the role of NQO1 and UGTs against menadione-induced cytotoxicity. Our results differed from those of previous studies. HEK293/NQO1 was the most sensitive cell line to menadione cytotoxicity among cell lines established in this study. These phenomena were also observed in HEK293/NQO1/UGT1A6 and HEK293/NQO1/UGT1A10 cells in which the expression of UGT was suppressed by DOX treatment. On the contrary, HEK293/NQO1/UGT1A6 and HEK293/NQO1/UGT1A10 cells without DOX treatment were resistant to menadione-induced cytotoxicity. These results demonstrated that NQO1 is not a detoxification enzyme for menadione and that UGT-mediated glucuronidation of menadiol is the most important detoxification process. Copyright 2009 Elsevier Inc. All rights reserved.

Control of CD56 expression and tumor cell cytotoxicity in human Vγ2Vδ2 T cells

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Focaccetti Chiara

2009-09-01

Full Text Available Abstract Background In lymphocyte subsets, expression of CD56 (neural cell adhesion molecule-1 correlates with cytotoxic effector activity. For cells bearing the Vγ2Vδ2 T cell receptor, isoprenoid pyrophosphate stimulation leads to uniform activation and proliferation, but only a fraction of cells express CD56 and display potent cytotoxic activity against tumor cells. Our goal was to show whether CD56 expression was regulated stochastically, similar to conventional activation antigens, or whether CD56 defined a lineage of cells committed to the cytotoxic phenotype. Results Tracking individual cell clones defined by their Vγ2 chain CDR3 region sequences, we found that CD56 was expressed on precursor cytotoxic T cells already present in the population irrespective of their capacity to proliferate after antigen stimulation. Public T cell receptor sequences found in the CD56+ subset from one individual might appear in the CD56- subset of another donor. The commitment of individual clones to CD56+ or CD56- lineages was stable for each donor over a 1 year interval. Conclusion The ability to express CD56 was not predicted by TCR sequence or by the strength of signal received by the TCR. For γδ T cells, cytotoxic effector function is acquired when cytotoxic precursors within the population are stimulated to proliferate and express CD56. Expression of CD56 defines a committed lineage to the cytotoxic phenotype.

PHA-induced cytotoxicity of human lymphocytes against adherent hela-cells

NARCIS (Netherlands)

Huges-Law, G.; de Gast, G. C.; The, T. Hauw

The conditions for a phytohaemagglutinin(PHA)-induced cytotoxicity test of human peripheral blood lymphocytes were investigated. [3H]thymidine prelabelled HeLa cells were used as target cells. Stimulation with 10 μl PHA/ml during 24 h gave the best measure of lymphocyte cytotoxic capacity.

Gangliosides inhibit bee venom melittin cytotoxicity but not phospholipase A(2)-induced degranulation in mast cells.

Science.gov (United States)

Nishikawa, Hirofumi; Kitani, Seiichi

2011-05-01

Sting accident by honeybee causes severe pain, inflammation and allergic reaction through IgE-mediated anaphylaxis. In addition to this hypersensitivity, an anaphylactoid reaction occurs by toxic effects even in a non-allergic person via cytolysis followed by similar clinical manifestations. Auto-injectable epinephrine might be effective for bee stings, but cannot inhibit mast cell lysis and degranulation by venom toxins. We used connective tissue type canine mast cell line (CM-MC) for finding an effective measure that might inhibit bee venom toxicity. We evaluated degranulation and cytotoxicity by measurement of β-hexosaminidase release and MTT assay. Melittin and crude bee venom induced the degranulation and cytotoxicity, which were strongly inhibited by mono-sialoganglioside (G(M1)), di-sialoganglioside (G(D1a)) and tri-sialoganglioside (G(T1b)). In contrast, honeybee venom-derived phospholipase A(2) induced the net degranulation directly without cytotoxicity, which was not inhibited by G(M1), G(D1a) and G(T1b). For analysis of distribution of Gα(q) and Gα(i) protein by western blotting, lipid rafts were isolated by using discontinuous sucrose gradient centrifuge. Melittin disrupted the localization of Gα(q) and Gα(i) at lipid raft, but gangliosides stabilized the rafts. As a result from this cell-based study, bee venom-induced anaphylactoid reaction can be explained with melittin cytotoxicity and phospholipase A(2)-induced degranulation. Taken together, gangliosides inhibit the effect of melittin such as degranulation, cytotoxicity and lipid raft disruption but not phospholipase A(2)-induced degranulation in mast cells. Our study shows a potential of gangliosides as a therapeutic tool for anaphylactoid reaction by honeybee sting. Copyright © 2011 Elsevier Inc. All rights reserved.

Xylopine Induces Oxidative Stress and Causes G2/M Phase Arrest, Triggering Caspase-Mediated Apoptosis by p53-Independent Pathway in HCT116 Cells

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Luciano de Souza Santos

2017-01-01

Full Text Available Xylopine is an aporphine alkaloid that has cytotoxic activity to cancer cells. In this study, the underlying mechanism of xylopine cytotoxicity was assessed in human colon carcinoma HCT116 cells. Xylopine displayed potent cytotoxicity in different cancer cell lines in monolayer cultures and in a 3D model of cancer multicellular spheroids formed from HCT116 cells. Typical morphology of apoptosis, cell cycle arrest in the G2/M phase, increased internucleosomal DNA fragmentation, loss of the mitochondrial transmembrane potential, and increased phosphatidylserine externalization and caspase-3 activation were observed in xylopine-treated HCT116 cells. Moreover, pretreatment with a caspase-3 inhibitor (Z-DEVD-FMK, but not with a p53 inhibitor (cyclic pifithrin-α, reduced xylopine-induced apoptosis, indicating induction of caspase-mediated apoptosis by the p53-independent pathway. Treatment with xylopine also caused an increase in the production of reactive oxygen/nitrogen species (ROS/RNS, including hydrogen peroxide and nitric oxide, but not superoxide anion, and reduced glutathione levels were decreased in xylopine-treated HCT116 cells. Application of the antioxidant N-acetylcysteine reduced the ROS levels and xylopine-induced apoptosis, indicating activation of ROS-mediated apoptosis pathway. In conclusion, xylopine has potent cytotoxicity to different cancer cell lines and is able to induce oxidative stress and G2/M phase arrest, triggering caspase-mediated apoptosis by the p53-independent pathway in HCT116 cells.

Attenuation of everolimus-induced cytotoxicity by a protective autophagic pathway involving ERK activation in renal cell carcinoma cells

Science.gov (United States)

Zeng, Yizhou; Tian, Xiaofang; Wang, Quan; He, Weiyang; Fan, Jing; Gou, Xin

2018-01-01

Aim The mammalian target of rapamycin (mTOR) pathway is a critical target for cancer treatment and the mTOR inhibitor everolimus (RAD001) has been approved for treatment of renal cell carcinoma (RCC). However, the limited efficacy of RAD001 has led to the development of drug resistance. Autophagy is closely related to cell survival and death, which may be activated under RAD001 stimulation. The aim of the present study was to identify the underlying mechanisms of RAD001 resistance in RCC cells through cytoprotective autophagy involving activation of the extracellular signal-regulated kinase (ERK) pathway. Methods and results: RAD001 strongly induced autophagy of RCC cells in a dose- and time-dependent manner, as confirmed by Western blot analysis. Importantly, suppression of autophagy by the pharmacological inhibitor chloroquine effectively enhanced RAD001-induced apoptotic cytotoxicity, as demonstrated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and Western blot analysis, indicating a cytoprotective role for RAD001-induced autophagy. In addition, as was shown by the MTT assay, flow cytometry, and Western blot analysis, RAD001 robustly activated ERK, but not c-Jun N-terminal kinase and p38. Activation of ERK was inhibited by the pharmacological inhibitor selumetinib (AZD6244), which effectively promoted RAD001-induced cell death. Moreover, employing AZD6244 markedly attenuated RAD001-induced autophagy and enhanced RAD001-induced apoptosis, which play a central role in RAD001-induced cell death. Furthermore, RAD001-induced autophagy is regulated by ERK-mediated phosphorylation of Beclin-1 and B-cell lymphoma 2, as confirmed by Western blot analysis. Conclusion These results suggest that RAD001-induced autophagy involves activation of the ERK, which may impair cytotoxicity of RAD001 in RCC cells. Thus, inhibition of the activation of ERK pathway-mediated autophagy may be useful to overcome chemoresistance to RAD001. PMID:29719377

Synthesis of Chromonylthiazolidines and Their Cytotoxicity to Human Cancer Cell Lines

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Hoang Le Tuan Anh

2015-01-01

Full Text Available Nine new chromonylthiazolidine derivatives were successfully semi-synthesized from paeonol. All of the compounds, including starting materials, the intermediate compound and products, were evaluated for their cytotoxic effects toward eight human cancer cell lines. The synthesized chromonylthiazolidines displayed weak cytotoxic effects against the tested cancer cell lines, but selective cytotoxic effects were observed. Compounds 3a and 3b showed the most selective cytotoxic effects against human epidermoid carcinoma (IC50 44.1 ± 3.6 μg/mL and breast cancer (IC50 32.8 ± 1.4 μg/mL cell lines, respectively. The results suggest that chromoylthiazolidines are potential low-cost, and selective anticancer agents.

Targeting Multiple Tumors Using T-Cells Engineered to Express a Natural Cytotoxicity Receptor 2-Based Chimeric Receptor

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Vasyl Eisenberg

2017-09-01

Full Text Available Recent developments in cancer treatment are demonstrating the increasing and powerful potential of immunotherapeutic strategies. In this regard, the adoptive transfer of tumor-specific T-lymphocytes approaches can lead to tumor regression in cancer patients. More recently, the use of T-cells genetically engineered to express cancer-specific receptors such as the anti-CD19 chimeric antigen receptor (CAR continues to show promise for the treatment of hematological malignancies. Still, there is a crucial need to develop efficient CAR-T cell approaches for the treatment of solid tumors. It has been shown that other lymphocytes such as natural killer (NK cells can demonstrate potent antitumor function—nonetheless, their use in immunotherapy is rather limited due to difficulties in expanding these cells to therapeutically relevant numbers and to suppression by endogenous inhibitory mechanisms. Cancer recognition by NK cells is partly mediated by molecules termed natural cytotoxicity receptors (NCRs. In the present study, we hypothesize that it is possible to endow T-cells with an NK recognition pattern, providing them with a mean to recognize tumor cells, in a non-MHC restricted way. To test this, we genetically modified human T-cells with different chimeric receptors based on the human NCR2 molecule and then assessed their antitumor activity in vitro and in vivo. Our results show that expression in primary lymphocytes of an NCR2-derived CAR, termed s4428z, confers T-cells with the ability to specifically recognize heterogeneous tumors and to mediate tumor cytotoxicity in a mouse model. This study demonstrates the benefit of combining tumor recognition capability of NK cells with T cell effectiveness to improve cancer immunotherapy.

Cytotoxicity and apoptosis induced by alfalfa (Medicago sativa) leaf extracts in sensitive and multidrug-resistant tumor cells.

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Gatouillat, Grégory; Magid, Abdulmagid Alabdul; Bertin, Eric; Okiemy-Akeli, Marie-Genevieve; Morjani, Hamid; Lavaud, Catherine; Madoulet, Claudie

2014-01-01

Alfalfa (Medicago sativa) has been used to cure a wide variety of ailments. However, only a few studies have reported its anticancer effects. In this study, extracts were obtained from alfalfa leaves and their cytotoxic effects were assessed on several sensitive and multidrug-resistant tumor cells lines. Using the mouse leukaemia P388 cell line and its doxorubicin-resistant counterpart (P388/DOX), we showed that the inhibition of cell growth induced by alfalfa leaf extracts was mediated through the induction of apoptosis, as evidenced by DNA fragmentation analysis. The execution of programmed cell death was achieved via the activation of caspase-3, leading to PARP cleavage. Fractionation of toluene extract (To-1), the most active extract obtained from crude extract, led to the identification of 3 terpene derivatives and 5 flavonoids. Among them, (-)-medicarpin, (-)-melilotocarpan E, millepurpan, tricin, and chrysoeriol showed cytotoxic effects in P388 as well as P388/DOX cells. These results demonstrate that alfalfa leaf extract may have interesting potential in cancer chemoprevention and therapy.

Role of the Nrf2-heme oxygenase-1 pathway in silver nanoparticle-mediated cytotoxicity

International Nuclear Information System (INIS)

Kang, Su Jin; Ryoo, In-geun; Lee, Young Joon; Kwak, Mi-Kyoung

2012-01-01

Silver nanoparticles (nano-Ag) have been widely used in various commercial products including textiles, electronic appliances and biomedical products. However, there remains insufficient information on the potential risk of nano-Ag to human health and environment. In the current study, we have investigated the role of NF-E2-related factor 2 (Nrf2) transcription factor in nano-Ag-induced cytotoxicity. When Nrf2 expression was blocked using interring RNA expression in ovarian carcinoma cell line, nano-Ag treatment showed a substantial decrease in cell viability with concomitant increases in apoptosis and DNA damage compared to the control cells. Target gene analysis revealed that the expression of heme oxygenase-1 (HO-1) was highly elevated by nano-Ag in nonspecific shRNA expressing cells, while Nrf2 knockdown cells (NRF2i) did not increase HO-1 expression. The role of HO-1 in cytoprotection against nano-Ag was reinforced by results using pharmacological inducer of HO-1: cobalt protoporphyrin-mediated HO-1 activation in the NRF2i cells prevented nano-Ag-mediated cell death. Similarly, pharmacological or genetic inhibition of HO-1 in nonspecific control cells exacerbated nano-Ag toxicity. As the upstream signaling mechanism, nano-Ag required the phosphoinositide 3-kinase (PI3K) and p38MAPK signaling cascades for HO-1 induction. The treatment with either PI3K inhibitor or p38MAPK inhibitor suppressed HO-1 induction and intensified nano-Ag-induced cell death. Taken together, these results suggest that Nrf2-dependent HO-1 up-regulation plays a protective role in nano-Ag-induced DNA damage and consequent cell death. In addition, nano-Ag-mediated HO-1 induction is associated with the PI3K and p38MAPK signaling pathways. -- Highlights: ► Role of Nrf2 signaling in silver nanoparticle toxicity. ► Silver nanoparticle toxicity is increased by Nrf2 blockade. ► Nrf2-dependent HO-1 induction protects cells from silver nanoparticle toxicity. ► PI3K and p38MAPK cascades are

Cytotoxicity of Sambucus ebulus on cancer cell lines and protective ...

African Journals Online (AJOL)

Regarding the traditional utilization of Sambucus ebulus, Iranian native botany and its active ingredients (e.g. ebulitin and ebulin 1), cytotoxicity of ethyl acetate ... cytotoxic agent on liver and colon cancer cells and suggest that vitamins C and E may protect normal cells, when SEE were used in cancer therapy in future.

Reduced LAK cytotoxicity of peripheral blood mononuclear cells in patients with bladder cancer: Decreased LAK cytotoxicity caused by a low incidence of CD56+ and CD57+ mononuclear blood cells

International Nuclear Information System (INIS)

Hermann, G.G.; Petersen, K.R.; Steven, K.; Zeuthen, J.

1990-01-01

The cytotoxicity of unstimulated peripheral blood mononuclear cells (US-PBMC), phytohemagglutinin (PHA)-stimulated PBMC (PS-PBMC) and interleukin-2 (IL-2)-activated PBMC (LAK cells) was assessed in patients with noninvasive and invasive transitional-cell bladder cancer and compared with those determined in healthy controls. The differences in the cytotoxicities were correlated with specific changes in the subsets of peripheral blood mononuclear cells (PBMC). PBMC from 37 patients and 13 healthy controls were tested against the bladder cancer cell line T24 in 51 Cr-release assays. The PBMC subsets were analyzed using monoclonal antibodies against T cells, natural killer (NK) -cells, monocytes, and activation markers. The cytotoxicities of US-PBMC, PS-PBMC, and LAK cells were all significantly lower in the cancer patients than in the controls (P less than 0.05). The percentages of PBMC positive for the NK-cell markers CD56 and CD57 were lowest in the patients and were correlated to the decrease in cytotoxicity. Depletion of CD56+ or CD57+ cells from PBMC prior to or after 2 days stimulation with IL-2 demonstrated that these cells are the major source of LAK-cell cytotoxicity and showed that the reduced ability of bladder cancer patient PBMC to develop LAK-cell cytotoxicity is a result of a low incidence of CD56+ and CD57+ cells in the blood. These findings indicate that IL-2 therapy alone might not be a sufficient therapy of bladder cancer patients

Cytotoxicity and Effects on Cell Viability of Nickel Nanowires

KAUST Repository

Rodriguez, Jose E.

2013-05-01

Recently, magnetic nanoparticles are finding an increased use in biomedical applications and research. Nanobeads are widely used for cell separation, biosensing and cancer therapy, among others. Due to their properties, nanowires (NWs) are gaining ground for similar applications and, as with all biomaterials, their cytotoxicity is an important factor to be considered before conducting biological studies with them. In this work, the cytotoxic effects of nickel NWs (Ni NWs) were investigated in terms of cell viability and damage to the cellular membrane. Ni NWs with an average diameter of 30-34 nm were prepared by electrodeposition in nanoporous alumina templates. The templates were obtained by a two-step anodization process with oxalic acid on an aluminum substrate. Characterization of NWs was done using X-Ray diffraction (XRD) and energy dispersive X-Ray analysis (EDAX), whereas their morphology was observed with scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Cell viability studies were carried out on human colorectal carcinoma cells HCT 116 by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) cell proliferation colorimetric assay, whereas the lactate dehydrogenase (LDH) homogenous membrane fluorimetric assay was used to measure the degree of cell membrane rupture. The density of cell seeding was calculated to obtain a specific cell number and confluency before treatment with NWs. Optical readings of the cell-reduced MTT products were measured at 570 nm, whereas fluorescent LDH membrane leakage was recorded with an excitation wavelength of 525 nm and an emission wavelength of 580 - 640 nm. The effects of NW length, cell exposure time, as well as NW:cell ratio, were evaluated through both cytotoxic assays. The results show that cell viability due to Ni NWs is affected depending on both exposure time and NW number. On the other hand, membrane rupture and leakage was only significant at later exposure times. Both

CD83 Antibody Inhibits Human B Cell Responses to Antigen as well as Dendritic Cell-Mediated CD4 T Cell Responses.

Science.gov (United States)

Wong, Kuan Y; Baron, Rebecca; Seldon, Therese A; Jones, Martina L; Rice, Alison M; Munster, David J

2018-05-15

Anti-CD83 Ab capable of Ab-dependent cellular cytotoxicity can deplete activated CD83 + human dendritic cells, thereby inhibiting CD4 T cell-mediated acute graft-versus-host disease. As CD83 is also expressed on the surface of activated B lymphocytes, we hypothesized that anti-CD83 would also inhibit B cell responses to stimulation. We found that anti-CD83 inhibited total IgM and IgG production in vitro by allostimulated human PBMC. Also, Ag-specific Ab responses to immunization of SCID mice xenografted with human PBMC were inhibited by anti-CD83 treatment. This inhibition occurred without depletion of all human B cells because anti-CD83 lysed activated CD83 + B cells by Ab-dependent cellular cytotoxicity and spared resting (CD83 - ) B cells. In cultured human PBMC, anti-CD83 inhibited tetanus toxoid-stimulated B cell proliferation and concomitant dendritic cell-mediated CD4 T cell proliferation and expression of IFN-γ and IL-17A, with minimal losses of B cells (80% of B cells but had no effect on CD4 T cell proliferation and cytokine expression. By virtue of the ability of anti-CD83 to selectively deplete activated, but not resting, B cells and dendritic cells, with the latter reducing CD4 T cell responses, anti-CD83 may be clinically useful in autoimmunity and transplantation. Advantages might include inhibited expansion of autoantigen- or alloantigen-specific B cells and CD4 T cells, thus preventing further production of pathogenic Abs and inflammatory cytokines while preserving protective memory and regulatory cells. Copyright © 2018 by The American Association of Immunologists, Inc.

Soluble fibrin inhibits monocyte adherence and cytotoxicity against tumor cells: implications for cancer metastasis

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Patel Shonak

2006-08-01

Full Text Available Abstract Background Soluble fibrin (sFn is a marker for disseminated intravascular coagulation and may have prognostic significance, especially in metastasis. However, a role for sFn in the etiology of metastatic cancer growth has not been extensively studied. We have reported that sFn cross-linked platelet binding to tumor cells via the major platelet fibrin receptor αIIbβ3, and tumor cell CD54 (ICAM-1, which is the receptor for two of the leukocyte β2 integrins (αLβ2 and aMβ2. We hypothesized that sFn may also affect leukocyte adherence, recognition, and killing of tumor cells. Furthermore, in a rat experimental metastasis model sFn pre-treatment of tumor cells enhanced metastasis by over 60% compared to untreated cells. Other studies have shown that fibrin(ogen binds to the monocyte integrin αMβ2. This study therefore sought to investigate the effect of sFn on β2 integrin mediated monocyte adherence and killing of tumor cells. Methods The role of sFn in monocyte adherence and cytotoxicity against tumor cells was initially studied using static microplate adherence and cytotoxicity assays, and under physiologically relevant flow conditions in a microscope perfusion incubator system. Blocking studies were performed using monoclonal antibodies specific for β2 integrins and CD54, and specific peptides which inhibit sFn binding to these receptors. Results Enhancement of monocyte/tumor cell adherence was observed when only one cell type was bound to sFn, but profound inhibition was observed when sFn was bound to both monocytes and tumor cells. This effect was also reflected in the pattern of monocyte cytotoxicity. Studies using monoclonal blocking antibodies and specific blocking peptides (which did not affect normal coagulation showed that the predominant mechanism of fibrin inhibition is via its binding to αMβ2 on monocytes, and to CD54 on both leukocytes and tumor cells. Conclusion sFn inhibits monocyte adherence and cytotoxicity of

Effects of retinoic acid-inducible gene-I-like receptors activations and ionizing radiation cotreatment on cytotoxicity against human non-small cell lung cancer in vitro.

Science.gov (United States)

Yoshino, Hironori; Iwabuchi, Miyu; Kazama, Yuka; Furukawa, Maho; Kashiwakura, Ikuo

2018-04-01

Retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) are pattern-recognition receptors that recognize pathogen-associated molecular patterns and induce antiviral immune responses. Recent studies have demonstrated that RLR activation induces antitumor immunity and cytotoxicity against different types of cancer, including lung cancer. However a previous report has demonstrated that ionizing radiation exerts a limited effect on RLR in human monocytic cell-derived macrophages, suggesting that RLR agonists may be used as effective immunostimulants during radiation therapy. However, it is unclear whether ionizing radiation affects the cytotoxicity of RLR agonists against cancer cells. Therefore, in the present study the effects of cotreatment with ionizing radiation and RLR agonists on cytotoxicity against human non-small cell lung cancer cells A549 and H1299 was investigated. Treatment with RLR agonist poly(I:C)/LyoVec™ [poly(I:C)] exerted cytotoxic effects against human non-small cell lung cancer. The cytotoxic effects of poly(I:C) were enhanced by cotreatment with ionizing radiation, and poly(I:C) pretreatment resulted in the radiosensitization of non-small cell lung cancer. Furthermore, cotreatment of A549 and H1299 cells with poly(I:C) and ionizing radiation effectively induced apoptosis in a caspase-dependent manner compared with treatment with poly(I:C) or ionizing radiation alone. These results indicate that RLR agonists and ionizing radiation cotreatment effectively exert cytotoxic effects against human non-small cell lung cancer through caspase-mediated apoptosis.

In vitro evaluation of the cytotoxicity and cellular uptake of CMCht/PAMAM dendrimer nanoparticles by glioblastoma cell models

Energy Technology Data Exchange (ETDEWEB)

Pojo, M., E-mail: martapojo@ecsaude.uminho.pt; Cerqueira, S. R.; Mota, T.; Xavier-Magalhaes, A.; Ribeiro-Samy, S. [University of Minho, Life and Health Sciences Research Institute (ICVS), School of Health Sciences (Portugal); Mano, J. F.; Oliveira, J. M., E-mail: miguel.oliveira@dep.uminho.pt; Reis, R. L. [ICVS/3Bs, PT Government Associated Laboratory (Portugal); Sousa, N.; Costa, B. M.; Salgado, A. J. [University of Minho, Life and Health Sciences Research Institute (ICVS), School of Health Sciences (Portugal)

2013-05-15

Glioblastoma (GBM) is simultaneously the most common and most malignant subtype tumor of the central nervous system. These are particularly dramatic diseases ranking first among all human tumor types for tumor-related average years of life lost and for which curative therapies are not available. Recently, the use of nanoparticles as drug delivery systems (DDS) for tumor treatment has gained particular interest. In an attempt to evaluate the potential of carboxymethylchitosan/poly(amidoamine) (CMCht/PAMAM) dendrimer nanoparticles as a DDS, we aimed to evaluate its cytotoxicity and internalization efficiency in GBM cell models. CMCht/PAMAM-mediated cytotoxicity was evaluated in a GBM cell line (U87MG) and in human immortalized astrocytes (hTERT/E6/E7) by MTS and double-stranded DNA quantification. CMCht/PAMAM internalization was assessed by double fluorescence staining. Both cells lines present similar internalization kinetics when exposed to a high dose (400 {mu}g/mL) of these nanoparticles. However, the internalization rate was higher in tumor GBM cells as compared to immortalized astrocytes when cells were exposed to lower doses (200 {mu}g/mL) of CMCht/PAMAM for short periods (<24 h). After 48 h of exposure, both cell lines present {approx}100% of internalization efficiency for the tested concentrations. Importantly, short-term exposures (1, 6, 12, 24, and 48 h) did not show cytotoxicity, and long-term exposures (7 days) to CMCht/PAMAM induced only low levels of cytotoxicity in both cell lines ({approx}20% of decrease in metabolic activity). The high efficiency and rate of internalization of CMCht/PAMAM we show here suggest that these nanoparticles may be an attractive DDS for brain tumor treatment in the future.

CD4 T cell-mediated protection from lethal influenza: perforin and antibody-mediated mechanisms give a one-two punch.

Science.gov (United States)

Brown, Deborah M; Dilzer, Allison M; Meents, Dana L; Swain, Susan L

2006-09-01

The mechanisms whereby CD4 T cells contribute to the protective response against lethal influenza infection remain poorly characterized. To define the role of CD4 cells in protection against a highly pathogenic strain of influenza, virus-specific TCR transgenic CD4 effectors were generated in vitro and transferred into mice given lethal influenza infection. Primed CD4 effectors conferred protection against lethal infection over a broad range of viral dose. The protection mediated by CD4 effectors did not require IFN-gamma or host T cells, but did result in increased anti-influenza Ab titers compared with untreated controls. Further studies indicated that CD4-mediated protection at high doses of influenza required B cells, and that passive transfer of anti-influenza immune serum was therapeutic in B cell-deficient mice, but only when CD4 effectors were present. Primed CD4 cells also acquired perforin (Pfn)-mediated cytolytic activity during effector generation, suggesting a second mechanism used by CD4 cells to confer protection. Pfn-deficient CD4 effectors were less able to promote survival in intact BALB/c mice and were unable to provide protection in B cell-deficient mice, indicating that Ab-independent protection by CD4 effectors requires Pfn. Therefore, CD4 effectors mediate protection to lethal influenza through at least two mechanisms: Pfn-mediated cytotoxicity early in the response promoted survival independently of Ab production, whereas CD4-driven B cell responses resulted in high titer Abs that neutralized remaining virus.

Aberrantly glycosylated MUC1 is expressed on the surface of breast cancer cells and a target for antibody-dependent cell-mediated cytotoxicity

DEFF Research Database (Denmark)

Lavrsen, Kirstine; Madsen, Caroline B; Rasch, Morten G

2013-01-01

to Tn-MUC1 was investigated using BiaCore. The availability of Tn-MUC1 on the surface of breast cancer cells was evaluated by immunohistochemistry, confocal microscopy, and flow cytometry, followed by in vitro assessment of antibody-dependent cellular cytotoxicity by mAb 5E5. Biacore analysis...

Calcein AM release-based cytotoxic cell assay for fish leucocytes.

Science.gov (United States)

Iwanowicz, Luke R; Densmore, Christine L; Ottinger, Christopher A

2004-02-01

A non-specific cytotoxic cell assay for fish is presented that is based on the release of the activated fluorochrome calcein AM from lysed carp epithelioma papulosum cyprini (EPC) cells. To establish the suitability of treating EPC cells with calcein AM the uptake and spontaneous release of the calcein AM by the EPC cells was evaluated. Incubation of 5 microM calcein AM in culture medium with 1x10(5)EPC cells well(-1)for a minimum of 3 h provided sufficient labelling. Spontaneous release of fluorescence from the labelled EPC cells during 10 h of post labelling incubation ranged from 30 to 39% of the total observed fluorescence. Cytotoxic activity of trout leucocytes was evaluated at three leucocyte to target cell ratios (10:1, 2:1 and 1:1) following incubation (4, 6, 8, and 10 h) with calcein AM-labelled EPC cells at 15 degrees C. In some instances, the monoclonal antibody specific for the NCC surface receptor NCCRP-1 (MAb5C.6) was included in the cultures. The activity of NCC cells was significantly inhibited in the presence of 0.25 microg well(-1)of MAb5C.6 relative to no antibody (Pcytotoxic cell activity of approximately 18% was observed following 8 h of incubation at the 2:1 and 1:1 leucocyte to target cell ratios. Percent cytotoxic cell activity using calcein AM was similar to values reported for rainbow trout leucocytes using the 51Cr-release assay.

Genetic control of cell-mediated lympholysis to trinitrophenyl (TNP)-modified murine syngeneic cells. Expression of Ir gene function at the cytotoxic precursor and helper cell

International Nuclear Information System (INIS)

Fujiwara, H.; Shearer, G.M.

1981-01-01

The present study investigates some of the cellular mechanisms responsible for the defect in cytotoxic T lymphocyte (CTL) response to trinitrophenyl (TNP)-modified H-2/sup b/ self components in C57Bl/6(H-2/sup b/) or (C57BL/6 x C3H/He)(H-2/sup b/ x H-2/sup k/)F 1 mice. C3H/He, C57BL/6, and (C57BL/6 x C3H/He)F 1 mice were immunized to TNP-modified self by skin painting with trinitrochlorobenzene, and their spleen cells were used a) for in vitro secondary sensitization to syngeneic spleen cells conjugated with limiting concentrations of trinitrobenzene sulfonate (TNP-self) or b) as a source of radioresistant helper cells for augmenting the TNP-self CTL response generated by spleen cells from unimmunized C3H/He, C57Bl/6, and F 1 mice. The results indicate that strong or weak in vitro secondary CTL responses could be obtained in the H-2/sup k/ or H-2/sup b/ strain, respectively. This strain-dependent genetic difference was also observed in (H-2/sup b/ x H-2/sup k/)F 1 mice. These results permit the detection of the Ir gene defect in the anti-TNP-H-2/sup b/ self CTL response at both the helper and cytotoxic precursor cell levels

Combined fluorimetric caspase 3/7 assay and bradford protein determination for assessment of polycation-mediated cytotoxicity.

Science.gov (United States)

Larsen, Anna K; Hall, Arnaldur; Lundsgart, Henrik; Moghimi, S Moein

2013-01-01

Cationic polyplexes and lipoplexes are widely used as artificial systems for nucleic acid delivery into the cells, but they can also induce cell death. Mechanistic understanding of cell toxicity and biological side effects of these cationic entities is essential for optimization strategies and design of safe and efficient nucleic acid delivery systems. Numerous methods are presently available to detect and delineate cytotoxicity and cell death-mediated signals in cell cultures. Activation of caspases is part of the classical apoptosis program and increased caspase activity is therefore a well-established hallmark of programmed cell death. Additional methods to monitor cell death-related signals must, however, also be carried out to fully define the type of cell toxicity in play. These may include methods that detect plasma membrane damage, loss of mitochondrial membrane potential, phosphatidylserine exposure, and cell morphological changes (e.g., membrane blebbing, nuclear changes, cytoplasmic swelling, cell rounding). Here we describe a 96-well format protocol for detection of capsase-3/7 activity in cell lysates, based on a fluorescent caspase-3 assay, combined with a method to simultaneously determine relative protein contents in the individual wells.

p53-independent structure-activity relationships of 3-ring mesogenic compounds' activity as cytotoxic effects against human non-small cell lung cancer lines.

Science.gov (United States)

Fukushi, Saori; Yoshino, Hironori; Yoshizawa, Atsushi; Kashiwakura, Ikuo

2016-07-25

We recently demonstrated the cytotoxicity of liquid crystal precursors (hereafter referred to as "mesogenic compounds") in the human non-small cell lung cancer (NSCLC) cell line A549 which carry wild-type p53. p53 mutations are observed in 50 % of NSCLC and contribute to their resistance to chemotherapy. To develop more effective and cancer-specific agents, in this study, we investigated the structure-activity relationships of mesogenic compounds with cytotoxic effects against multiple NSCLC cells. The pharmacological effects of mesogenic compounds were examined in human NSCLC cells (A549, LU99, EBC-1, and H1299) and normal WI-38 human fibroblast. Analyses of the cell cycle, cell-death induction, and capsases expression were performed. The 3-ring compounds possessing terminal alkyl and hydroxyl groups (compounds C1-C5) showed cytotoxicity in NSCLC cells regardless of the p53 status. The compounds C1 and C3, which possess a pyrimidine at the center of the core, induced G2/M arrest, while the compounds without a pyrimidine (C2, C4, and C5) caused G1 arrest; all compounds produced caspase-mediated cell death. These events occurred in a p53-independent manner. Furthermore, it was suggested that compounds induced cell death through p53-independent DNA damage-signaling pathway. Compounds C2, C4, and C5 did not show strong cytotoxicity in WI-38 cells, whereas C1 and C3 did. However, the cytotoxicity of compound C1 against WI-38 cells was improved by modulating the terminal alkyl chain lengths of the compound. We showed the p53-indepdent structure-activity relationships of mesogenic compounds related to the cytotoxic effects. These structure-activity relationships will be helpful in the development of more effective and cancer-specific agents.

Vulnerability of cultured canine lung tumor cells to NK cell-mediated cytolysis

International Nuclear Information System (INIS)

Haley, P.J.; Kohr, J.M.; Kelly, G.; Muggenburg, B.A.; Guilmette, B.A.

1988-01-01

Five cell lines, designated as canine lung epithelial cell (CLEP), derived from radiation induced canine lung tumors and canine thyroid adeno-carcinoma (CTAC) cells were compared for their susceptibility to NK cell-mediated cytolysis using peripheral blood lymphocytes from normal, healthy Beagle dogs as effector cells. Effector cells and chromium 51 radiolabeled target cells were incubated for 16 h at ratios of 12.5:1, 25:1, 50:1, and 100:1. Increasing cytolysis was observed for all cell lines as the effector-to-target-cell ratios increased from 12.5:1 to 100:1. The percent cytotoxicity was significantly less for all lung tumor cell lines as compared to CTAC at the 100:1 ratio. One lung tumor cell line, CLEP-9, had 85% of the lytic vulnerability of the CTAC cell line and significantly greater susceptibility to NK cell-mediated lysis than all of the other lung tumor cell lines. Susceptibility to NK cell cytolysis did not correlate with in vivo malignant behavior of the original tumor. These data suggest that cultured canine lung tumor cells are susceptible to NK cell cytolytic activity in vitro and that at least one of these cell lines (CLEP-9) is a candidate for substitution of the standard canine NK cell target, CTAC, in NK cell assays. The use of lung tumor cells in NK cell assays may provide greater insight into the control of lung tumors by immune mechanisms. (author)

Cytotoxicity Testing: Cell Experiments

Science.gov (United States)

Grünert, Renate; Westendorf, Aron; Buczkowska, Magdalena; Hänsch, Mareike; Grüunert, Sybil; Bednarski, Patrick J.

Screening for new anticancer agents has traditionally been done with in vitro cell culture methods. Even in the genomic era of target-driven drug design, screening for cytotoxic activity is still a standard tool in the search for new anticancer agents, especially if the mode of action of a substance is not yet known. A wide variety of cell culture methods with unique end-points are available for testing the anticancer potential of a substance. Each has its advantages and disadvantages, which must be weighed in the decision to use a particular method. Often several complementary methods are used to gain information on the mode of action of a substance.

5-Aminolevulinic acid-mediated sonosensitization of rat RG2 glioma cells in vitro

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Krzysztof Bilmin

2016-10-01

Full Text Available Sonodynamic therapy (SDT is a promising technique based on the ability of certain substances, called sonosensitizers, to sensitize cancer cells to non-thermal effects of low-energy ultrasound waves, allowing their destruction. Sonosensitization is thought to induce cell death by direct physical effects such as cavitation and acoustical streaming as well as by complementary chemical reactions generating oxygen free radicals. One of the promising sonosensitizers is 5-aminolevulinic acid (ALA which upon selective uptake by cancer cells is metabolized and accumulated as protoporphyrin IX. The objective of the study was to describe ALA-mediated sonodynamic effects in vitro on a rat RG2 glioma cell line. Glioma cells, seeded at the bottom of 96-well plates and incubated with ALA (10 µg/ml for 6 h, were exposed to the sinusoidal US pulses with a resonance frequency of 1 MHz, 1000 µs duration, 0.4 duty-cycle, and average acoustic power varying from 2 W to 6 W. Ultrasound waves were generated by a flat circular piezoelectric transducer with a diameter of 25 mm. Cell viability was determined by MTT assay. Structural cellular changes were visualized with a fluorescence microscope. Signs of cytotoxicity such as a decrease in cell viability, chromatin condensation and apoptosis were found. ALA-mediated SDT evokes cytotoxic effects of low intensity US on rat RG2 glioma cells in vitro . This cell line is indicated for further preclinical assessment of SDT in in vivo conditions.

Silencing of the transcription factor STAT3 sensitizes lung cancer cells to DNA damaging drugs, but not to TNFα- and NK cytotoxicity

Energy Technology Data Exchange (ETDEWEB)

Kulesza, Dorota W. [Laboratory of Transcription Regulation, Department of Cell Biology, The Nencki Institute of Experimental Biology, Warsaw (Poland); Postgraduate School of Molecular Medicine, Warsaw Medical University, Warsaw (Poland); Carré, Thibault; Chouaib, Salem [Unité INSERM U753, Institut de Cancérologie Gustave Roussy, Villejuif Cedex (France); Kaminska, Bozena, E-mail: bozenakk@nencki.gov.pl [Laboratory of Transcription Regulation, Department of Cell Biology, The Nencki Institute of Experimental Biology, Warsaw (Poland)

2013-02-15

Transcription factor STAT3 (Signal Transducers and Activators of Transcription 3) is persistently active in human tumors and may contribute to tumor progression. Inhibition of STAT3 expression/activity could be a good strategy to modulate tumor cell survival and responses to cancer chemotherapeutics or immune cytotoxicity. We silenced STAT3 expression in human A549 lung cancer cells to elucidate its role in cell survival and resistance to chemotherapeutics, TNFα and natural killer (NK)-mediated cytotoxicity. We demonstrate that STAT3 is not essential for basal survival and proliferation of A549 cancer cells. Stable silencing of STAT3 expression sensitized A549 cells to DNA damaging chemotherapeutics doxorubicin and cisplatin in a p53-independent manner. Sensitization to DNA damage-inducing chemotherapeutics could be due to down-regulation of the Bcl-xL expression in STAT3 depleted cells. In contrast, knockdown of STAT3 in cancer cells did not modulate responses to TNFα and NK-mediated cytotoxicity. We found that STAT3 depletion increased the NFκB activity likely providing the compensatory, pro-survival signal. The treatment with TNFα, but not doxorubicin, enhanced this effect. We conclude that STAT3 is not crucial for the control of basal cell proliferation and survival of lung carcinoma cells but modulates susceptibility to DNA damaging chemotherapeutics by regulation of intrinsic pro-survival pathways. - Highlights: ► STAT3 silencing is negligent for basal lung cancer cell viability and proliferation. ► STAT3 depletion sensitizes lung cancer cells to DNA damaging chemotherapeutics. ► STAT3 depletion has no effect on susceptibility to extrinsic apoptosis inducers. ► Increased pro-survival NFκB activity may compensate for STAT3 depletion.

Variation among Staphylococcus aureus membrane vesicle proteomes affects cytotoxicity of host cells.

Science.gov (United States)

Jeon, Hyejin; Oh, Man Hwan; Jun, So Hyun; Kim, Seung Il; Choi, Chi Won; Kwon, Hyo Il; Na, Seok Hyeon; Kim, Yoo Jeong; Nicholas, Asiimwe; Selasi, Gati Noble; Lee, Je Chul

2016-04-01

Staphylococcus aureus secretes membrane-derived vesicles (MVs), which can deliver virulence factors to host cells and induce cytopathology. However, the cytopathology of host cells induced by MVs derived from different S. aureus strains has not yet been characterized. In the present study, the cytotoxic activity of MVs from different S. aureus isolates on host cells was compared and the proteomes of S. aureus MVs were analyzed. The MVs purified from S. aureus M060 isolated from a patient with staphylococcal scalded skin syndrome showed higher cytotoxic activity toward host cells than that shown by MVs from three other clinical S. aureus isolates. S. aureus M060 MVs induced HEp-2 cell apoptosis in a dose-dependent manner, but the cytotoxic activity of MVs was completely abolished by treatment with proteinase K. In a proteomic analysis, the MVs from three S. aureus isolates not only carry 25 common proteins, but also carry ≥60 strain-specific proteins. All S. aureus MVs contained δ-hemolysin (Hld), γ-hemolysin, leukocidin D, and exfoliative toxin C, but exfoliative toxin A (ETA) was specifically identified in S. aureus M060 MVs. ETA was delivered to HEp-2 cells via S. aureus MVs. Both rETA and rHld induced cytotoxicity in HEp-2 cells. In conclusion, MVs from clinical S. aureus isolates differ with respect to cytotoxic activity in host cells, and these differences may result from differences in the MV proteomes. Further proteogenomic analysis or mutagenesis of specific genes is necessary to identify cytotoxic factors in S. aureus MVs. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

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Hironori Iwasaki

2017-01-01

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

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

Science.gov (United States)

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

2015-12-01

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

Evaluation of cell cytotoxic effect on herbal extracts mixtures

International Nuclear Information System (INIS)

Kim, Yong Soo; Gwon, Hui Jeong; Choi, Bo Ram; Lim, Youn Mook; Nho, Young Chang

2009-01-01

Herbal extracts (HE) such as Houttuynia cordata Thunb., Eucommia ulimoides, Plantago asiatica var., Morus alba L., and Ulmus davidiana var., are known to suppress an atopic dermatitis like skin lesions. In this study, to evaluate the cell cytotoxicity effect on L929, HaCaT and HMC-1 cell by the HE, the herbs were extracted with distilled water (at 75 .deg. C) and then the HE mixtures were freeze-dried for 5 days and sterilized with γ-rays. The cytotoxicity was measured by Cell Counting Kit-8 (CCK-8) assay. The result showed that the HE mixtures did not significantly affect cell viability and had no toxicity on the cells. These findings indicate that the HE mixtures can be used as a potential therapeutic agent

Evaluation of cell cytotoxic effect on herbal extracts mixtures

Energy Technology Data Exchange (ETDEWEB)

Kim, Yong Soo; Gwon, Hui Jeong; Choi, Bo Ram; Lim, Youn Mook; Nho, Young Chang [Korea Atomic Energy Research Institute, Jeongeup (Korea, Republic of)

2009-12-15

Herbal extracts (HE) such as Houttuynia cordata Thunb., Eucommia ulimoides, Plantago asiatica var., Morus alba L., and Ulmus davidiana var., are known to suppress an atopic dermatitis like skin lesions. In this study, to evaluate the cell cytotoxicity effect on L929, HaCaT and HMC-1 cell by the HE, the herbs were extracted with distilled water (at 75 .deg. C) and then the HE mixtures were freeze-dried for 5 days and sterilized with {gamma}-rays. The cytotoxicity was measured by Cell Counting Kit-8 (CCK-8) assay. The result showed that the HE mixtures did not significantly affect cell viability and had no toxicity on the cells. These findings indicate that the HE mixtures can be used as a potential therapeutic agent.

Immunomodulatory Effect of Rhaphidophora korthalsii on Natural Killer Cell Cytotoxicity

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Swee Keong Yeap

2012-01-01

Full Text Available The in vivo immunomodulatory effect of ethanolic extracts from leaves of Rhaphidophora korthalsii was determined via immune cell proliferation, T/NK cell phenotyping, and splenocyte cytotoxicity of BALB/c mice after 5 consecutive days of i.p. administration at various concentrations. Splenocyte proliferation index, cytotoxicity, peripheral blood T/NK cell population, and plasma cytokine (IL-2 and IFN-γ in mice were assessed on day 5 and day 15. High concentration of extract (350 μg/mice/day for 5 consecutive days was able to stimulate immune cell proliferation, peripheral blood NK cell population, IL-2, and IFN- γ cytokines, as well as splenocyte cytotoxicity against Yac-1 cell line. Unlike rIL-2 which degraded rapidly, the stimulatory effect from the extract managed to last until day 15. These results suggested the potential of this extract as an alternative immunostimulator, and they encourage further study on guided fractionation and purification to identify the active ingredients that contribute to this in vitro and in vivo immunomodulatory activity.

Cytotoxic Components Against Human Oral Squamous Cell Carcinoma Isolated from Andrographis paniculata.

Science.gov (United States)

Suzuki, Ryuichiro; Matsushima, Yasuaki; Okudaira, Noriyuki; Sakagami, Hiroshi; Shirataki, Yoshiaki

2016-11-01

The 5-year survival rate of patients with oral cancer has remained approximately 50% during the past 30 years, possibly due to the poor tumor selectivity of conventional anticancer drugs. This prompted us to search for new candidates for anticancer drugs that have higher cytotoxicity and tumor selectivity. Dried leaves of Andrographis paniculata were supplied from a market in Shanghai. The methanolic fraction of A. paniculata was further fractionated to identify cytotoxic principles by spectroscopic analysis and comparison with literature values. Viable cell number was determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide method, and tumor specificity was calculated by relative cytotoxicity against oral squamous cell carcinoma cell lines compared to that against normal oral cells. Apoptosis induction was detected by cleaved poly (ADP-ribose) polymerase and caspase-3 on western blot analysis. Major cytotoxicity in the methanol extract of a leaf of A. paniculata was recovered by partitioning with EtOAc, followed by silica gel chromatography. Further purification with reversed-phase high-performance liquid chromatography led to isolation of four known cytotoxic compounds, 14-deoxyandrographolide, andrographolide, neoandrographolide and deoxyandrographiside. Among them, andrographolide had the greatest cytotoxicity and tumor specificity, also inducing caspase-3 activation of HSC-2 oral squamous cell carcinoma cells. The present study identified andrographolide as a major antitumor principle in the methanolic extract of leaves of A. paniculata. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Cytotoxicity and gene expression profiling of polyhexamethylene guanidine hydrochloride in human alveolar A549 cells.

Science.gov (United States)

Jung, Ha-Na; Zerin, Tamanna; Podder, Biswajit; Song, Ho-Yeon; Kim, Yong-Sik

2014-06-01

In Korea, lung disease of children and pregnant women associated with humidifier disinfectant use has become a major concern. A common sterilizer is polyhexamethylene guanidine (PHMG), a member of the guanidine family of antiseptics. This study was done to elucidate the putative cytotoxic effect of PHMG and the PHMG-mediated altered gene expression in human alveolar epithelial A549 cells in vitro. Cell viability analyses revealed the potent cytotoxicity of PHMG, with cell death evident at as low as 5 μg/mL. Death was dose- and time-dependent, and was associated with formation of intracellular reactive oxygen species, and apoptosis significantly, at even 2 μg/mL concentration. The gene expression profile in A549 cells following 24 h exposure to 5 μg/mL of PHMG was investigated using DNA microarray analysis. Changes in gene expression relevant to the progression of cell death included induction of genes related to apoptosis, autophagy, fibrosis, and cell cycle. However, the expressions of genes encoding antioxidant and detoxifying enzymes were down-regulated or not affected. The altered expression of selected genes was confirmed by quantitative reverse transcription-polymerase chain reaction and Western blot analyses. The collective data suggest that PHMG confers cellular toxicity through the generation of intracellular reactive oxygen species and alteration of gene expression. Copyright © 2014 Elsevier Ltd. All rights reserved.

CpG oligodeoxynucleotide induces apoptosis and cell cycle arrest in A20 lymphoma cells via TLR9-mediated pathways.

Science.gov (United States)

Qi, Xu-Feng; Zheng, Li; Kim, Cheol-Su; Lee, Kyu-Jae; Kim, Dong-Heui; Cai, Dong-Qing; Qin, Jun-Wen; Yu, Yan-Hong; Wu, Zheng; Kim, Soo-Ki

2013-07-01

Recent studies have suggested that the anti-cancer activity of CpG-oligodeoxynucleotides (CpG-ODNs) is owing to their immunomodulatory effects in tumor-bearing host. The purpose of this study is to investigate the directly cytotoxic activity of KSK-CpG, a novel CpG-ODN with an alternative CpG motif, against A20 and EL4 lymphoma cells in comparison with previously used murine CpG motif (1826-CpG). To evaluate the potential cytotoxic effects of KSK-CpG on lymphoma cells, cell viability assay, confocal microscopy, flow cytometry, DNA fragmentation, Western blotting, and reverse transcription-polymerase chain reaction (RT-PCR) analysis were used. We found that KSK-CpG induced direct cytotoxicity in A20 lymphoma cells, but not in EL4 lymphoma cells, at least in part via TLR9-mediated pathways. Apoptotic cell death was demonstrated to play an important role in CpG-ODNs-induced cytotoxicity. In addition, both mitochondrial membrane potential decrease and G1-phase arrest were involved in KSK-CpG-induced apoptosis in A20 cells. The activities of apoptotic molecules such as caspase-3, PARP, and Bax were increased, but the activation of p27 Kip1 and ERK were decreased in KSK-CpG-treated A20 cells. Furthermore, autocrine IFN-γ partially contributed to apoptotic cell death in KSK-CpG-treated A20 cells. Collectively, our findings suggest that KSK-CpG induces apoptotic cell death in A20 lymphoma cells at least in part by inducing G1-phase arrest and autocrine IFN-γ via increasing TLR9 expression, without the need for immune system of tumor-bearing host. This new understanding supports the development of TLR9-targeted therapy with CpG-ODN as a direct therapeutic agent for treating B lymphoma. Copyright © 2013 Elsevier Ltd. All rights reserved.

Spontaneous cytotoxic T-Cell reactivity against indoleamine 2,3-dioxygenase-2

DEFF Research Database (Denmark)

Sørensen, Rikke Bæk; Køllgaard, Tania; Andersen, Rikke Sick

2011-01-01

in mouse models of cancer in a nontoxic fashion. Here, we describe the immunogenicity of IDO2 by showing the presence of spontaneous cytotoxic T-cell reactivity against IDO2 in peripheral blood of both healthy donors and cancer patients. Furthermore, we show that these IDO2-specific T cells are cytotoxic...

Cytotoxicity of VEGF121/rGel on vascular endothelial cells resulting in inhibition of angiogenesis is mediated via VEGFR-2

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Hittelman Walter N

2011-08-01

Full Text Available Abstract Background The fusion protein VEGF121/rGel composed of the growth factor VEGF121 and the plant toxin gelonin targets the tumor neovasculature and exerts impressive anti-vascular effects. We have previously shown that VEGF121/rGel is cytotoxic to endothelial cells overexpressing VEGFR-2 but not to endothelial cells overexpressing VEGFR-1. In this study, we examined the basis for the specific toxicity of this construct and assessed its intracellular effects in vitro and in vivo. Methods We investigated the binding, cytotoxicity and internalization profile of VEGF121/rGel on endothelial cells expressing VEGFR-1 or VEGFR-2, identified its effects on angiogenesis models in vitro and ex vivo, and explored its intracellular effects on a number of molecular pathways using microarray analysis. Results Incubation of PAE/VEGFR-2 and PAE/VEGFR-1 cells with 125I-VEGF121/rGel demonstrated binding specificity that was competed with unlabeled VEGF121/rGel but not with unlabeled gelonin. Assessment of the effect of VEGF121/rGel on blocking tube formation in vitro revealed a 100-fold difference in IC50 levels between PAE/VEGFR-2 (1 nM and PAE/VEGFR-1 (100 nM cells. VEGF121/rGel entered PAE/VEGFR-2 cells within one hour of treatment but was not detected in PAE/VEGFR-1 cells up to 24 hours after treatment. In vascularization studies using chicken chorioallantoic membranes, 1 nM VEGF121/rGel completely inhibited bFGF-stimulated neovascular growth. The cytotoxic effects of VEGF121/rGel were not apoptotic since treated cells were TUNEL-negative with no evidence of PARP cleavage or alteration in the protein levels of select apoptotic markers. Microarray analysis of VEGF121/rGel-treated HUVECs revealed the upregulation of a unique "fingerprint" profile of 22 genes that control cell adhesion, apoptosis, transcription regulation, chemotaxis, and inflammatory response. Conclusions Taken together, these data confirm the selectivity of VEGF121/rGel for VEGFR-2

Cytotoxic drug sensitivity testing of tumor cells from patients with ovarian carcinoma using the fluorometric microculture cytotoxicity assay (FMCA).

Science.gov (United States)

Csoka, K; Larsson, R; Tholander, B; Gerdin, E; de la Torre, M; Nygren, P

1994-08-01

The automated fluorometric microculture cytotoxicity assay (FMCA) is based on the measurement of fluorescence generated from cellular hydrolysis of fluorescein diacetate (FDA) to fluorescein by viable cells after a 72-hr culture period in microtiter plates. The FMCA was adopted for chemosensitivity testing of tumor cells from patients with ovarian carcinoma. Thirty-seven samples of solid tumors and malignant effusions were obtained from 35 patients at diagnosis or relapse. Tumor cells from solid samples and effusions were prepared by enzymatic digestion and centrifugation, respectively, followed by Percoll or Ficoll purification. The fluorescence was proportional to the number of cells/well and considerably higher in tumor cells than in contaminating normal cells. The effect of up to 19 cytotoxic drugs was successfully assessed in 70% of the samples and there was a good correlation between drug sensitivity data reported by the FMCA and the DiSC assay performed in parallel. The overall drug sensitivity pattern in vitro corresponded well to the clinical experience. The effect of cisplatin varied considerably between patients and resistance was found also in cases not previously exposed to cytotoxic drugs. The FMCA is a rapid and simple method that seems to report clinically relevant cytotoxic drug sensitivity data in ovarian carcinomas. In the future, this method may contribute to optimizing chemotherapy by assisting in individualized drug selection and new drug development.

Human anti-CAIX antibodies mediate immune cell inhibition of renal cell carcinoma in vitro and in a humanized mouse model in vivo.

Science.gov (United States)

Chang, De-Kuan; Moniz, Raymond J; Xu, Zhongyao; Sun, Jiusong; Signoretti, Sabina; Zhu, Quan; Marasco, Wayne A

2015-06-11

Carbonic anhydrase (CA) IX is a surface-expressed protein that is upregulated by the hypoxia inducible factor (HIF) and represents a prototypic tumor-associated antigen that is overexpressed on renal cell carcinoma (RCC). Therapeutic approaches targeting CAIX have focused on the development of CAIX inhibitors and specific immunotherapies including monoclonal antibodies (mAbs). However, current in vivo mouse models used to characterize the anti-tumor properties of fully human anti-CAIX mAbs have significant limitations since the role of human effector cells in tumor cell killing in vivo is not directly evaluated. The role of human anti-CAIX mAbs on CAIX(+) RCC tumor cell killing by immunocytes or complement was tested in vitro by antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and antibody-dependent cellular phagocytosis (ADCP) as well as on CAIX(+) RCC cellular motility, wound healing, migration and proliferation. The in vivo therapeutic activity mediated by anti-CAIX mAbs was determined by using a novel orthotopic RCC xenograft humanized animal model and analyzed by histology and FACS staining. Our studies demonstrate the capacity of human anti-CAIX mAbs that inhibit CA enzymatic activity to result in immune-mediated killing of RCC, including nature killer (NK) cell-mediated ADCC, CDC, and macrophage-mediated ADCP. The killing activity correlated positively with the level of CAIX expression on RCC tumor cell lines. In addition, Fc engineering of anti-CAIX mAbs was shown to enhance the ADCC activity against RCC. We also demonstrate that these anti-CAIX mAbs inhibit migration of RCC cells in vitro. Finally, through the implementation of a novel orthotopic RCC model utilizing allogeneic human peripheral blood mononuclear cells in NOD/SCID/IL2Rγ(-/-) mice, we show that anti-CAIX mAbs are capable of mediating human immune response in vivo including tumor infiltration of NK cells and activation of T cells, resulting in

Cytotoxicity of cuprous oxide nanoparticles to fish blood cells: hemolysis and internalization

Energy Technology Data Exchange (ETDEWEB)

Chen Liqiang, E-mail: chenlq@ynu.edu.cn; Kang Bin [Yunnan University, Asian International Rivers Center, Yunnan Key Laboratory of International Rivers and Trans-boundary Eco-security (China); Ling Jian [Yunnan University, College of Chemistry and Chemical Engineering (China)

2013-03-15

Cuprous oxide nanoparticles (Cu{sub 2}O NPs) possess unique physical and chemical properties which are employed in a broad variety of applications. However, little is known about the adverse effects of Cu{sub 2}O NPs on organisms. In the current study, in vitro cytotoxicity of Cu{sub 2}O NPs (ca. 60 nm in diameter) to the blood cells of freshwater fish Carassius auratus was evaluated. A concentration-dependent hemolytic activity of Cu{sub 2}O NPs to red blood cells (RBCs) and the phagocytosis of Cu{sub 2}O NPs by leukocytes were revealed. The results showed that dosages of Cu{sub 2}O NPs greater than 40 {mu}g/mL were toxic to blood cells, and could cause serious membrane damage to RBCs. The EC{sub 50} value of Cu{sub 2}O NPs as obtained from RBCs and whole blood exposure was 26 and 63 {mu}g/mL, respectively. The generation of reactive oxygen species and the direct interaction between Cu{sub 2}O NPs and the cell membrane were suggested as the possible mechanism for cytotoxicity, and the intrinsic hemolytic active of Cu{sub 2}O NPs was the main contributor to the toxicity rather than solubilized copper ions. The adsorption of plasma proteins on the surfaces of Cu{sub 2}O NPs led to their aggregation in whole blood, and aggregate formation can significantly alleviate the hemolytic effect and subsequently mediate the phagocytosis of Cu{sub 2}O NPs by leukocytes.

Study on the cytotoxicity of natural killer cells induced by endothelial cells in vitro in the model of xenotransplantation

International Nuclear Information System (INIS)

Huang Haoyue; Shen Zhenya; Liu Hongcheng; Meng Zili; Teng Xiaomei

2004-01-01

Objective: To explore the change of the cytotoxicity of natural killer cells induced by vascular endothelial cells in vitro and the relationship between this change and the variety of cytokine level. Methods: After fixed by paraformaldehyde, vascular endothelial cells from pigs were co-cultured in vitro with natural killer cells from Chinese monkeys at different ratios. The change of the cytotoxicity of natural killer cells occurring after this contact and the content of IFN-γ and TNF-α in the supernatants were detected. Results: The cytotoxicity of natural killer cells improved gradually in accordance with the co-culture ratio after co-cultured with fixed vascular endothelial cells. The secretion of INF-γ and TNF-α also improved gradually. Conclusion: After contact with xeno-target cells, the cytotoxicity of natural killer cells and the secretion of cytokines are related to the ratio of effective cells and target cells

C22:0- and C24:0-dihydroceramides confer mixed cytotoxicity in T-cell acute lymphoblastic leukemia cell lines.

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Michael W Holliday

Full Text Available We previously reported that fenretinide (4-HPR was cytotoxic to acute lymphoblastic leukemia (ALL cell lines in vitro in association with increased levels of de novo synthesized dihydroceramides, the immediate precursors of ceramides. However, the cytotoxic potentials of native dihydroceramides have not been defined. Therefore, we determined the cytotoxic effects of increasing dihydroceramide levels via de novo synthesis in T-cell ALL cell lines and whether such cytotoxicity was dependent on an absolute increase in total dihydroceramide mass versus an increase of certain specific dihydroceramides. A novel method employing supplementation of individual fatty acids, sphinganine, and the dihydroceramide desaturase-1 (DES inhibitor, GT-11, was used to increase de novo dihydroceramide synthesis and absolute levels of specific dihydroceramides and ceramides. Sphingolipidomic analyses of four T-cell ALL cell lines revealed strong positive correlations between cytotoxicity and levels of C22:0-dihydroceramide (ρ = 0.74-0.81, P ≤ 0.04 and C24:0-dihydroceramide (ρ = 0.84-0.90, P ≤ 0.004, but not between total or other individual dihydroceramides, ceramides, or sphingoid bases or phosphorylated derivatives. Selective increase of C22:0- and C24:0-dihydroceramide increased level and flux of autophagy marker, LC3B-II, and increased DNA fragmentation (TUNEL assay in the absence of an increase of reactive oxygen species; pan-caspase inhibition blocked DNA fragmentation but not cell death. C22:0-fatty acid supplemented to 4-HPR treated cells further increased C22:0-dihydroceramide levels (P ≤ 0.001 and cytotoxicity (P ≤ 0.001. These data demonstrate that increases of specific dihydroceramides are cytotoxic to T-cell ALL cells by a caspase-independent, mixed cell death mechanism associated with increased autophagy and suggest that dihydroceramides may contribute to 4-HPR-induced cytotoxicity. The targeted increase of specific acyl chain dihydroceramides

Activating Transcription Factor 3 regulates in part the enhanced tumour cell cytotoxicity of the histone deacetylase inhibitor M344 and cisplatin in combination

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St Germain Carly

2010-09-01

Full Text Available Abstract Background Activating Transcription Factor (ATF 3 is a key regulator of the cellular integrated stress response whose expression has also been correlated with pro-apoptotic activities in tumour cell models. Combination treatments with chemotherapeutic drugs, such as cisplatin, and histone deacetylase (HDAC inhibitors have been demonstrated to enhance tumour cell cytotoxicity. We recently demonstrated a role for ATF3 in regulating cisplatin-induced apoptosis and others have shown that HDAC inhibition can also induce cellular stress. In this study, we evaluated the role of ATF3 in regulating the co-operative cytotoxicity of cisplatin in combination with an HDAC inhibitor. Results The HDAC inhibitor M344 induced ATF3 expression at the protein and mRNA level in a panel of human derived cancer cell lines as determined by Western blot and quantitative RT-PCR analyses. Combination treatment with M344 and cisplatin lead to increased induction of ATF3 compared with cisplatin alone. Utilizing the MTT cell viability assay, M344 treatments also enhanced the cytotoxic effects of cisplatin in these cancer cell lines. The mechanism of ATF3 induction by M344 was found to be independent of MAPKinase pathways and dependent on ATF4, a known regulator of ATF3 expression. ATF4 heterozygote (+/- and knock out (-/- mouse embryonic fibroblast (MEF as well as chromatin immunoprecipitation (ChIP assays were utilized in determining the mechanistic induction of ATF3 by M344. We also demonstrated that ATF3 regulates the enhanced cytotoxicity of M344 in combination with cisplatin as evidenced by attenuation of cytotoxicity in shRNAs targeting ATF3 expressing cells. Conclusion This study identifies the pro-apoptotic factor, ATF3 as a novel target of M344, as well as a mediator of the co-operative effects of cisplatin and M344 induced tumour cell cytotoxicity.

Analysis of protective and cytotoxic immune responses in vivo against metabolically inactivated and untreated cells of a mutagenized tumor line (requirements for tumor immunogenicity)

International Nuclear Information System (INIS)

Wehrmaker, A.; Lehmann, V.; Droege, W.

1986-01-01

The immunogenicity of a mutagenized subline (ESb-D) of the weakly immunogenic T-cell lymphoma L 5178 Y ESb has been characterized. The injection of 10(6) ESb-D cells ip did not establish lethal tumors in untreated DBA/2 mice but established tumors in sublethally irradiated mice. Injection of ESb-D cells into otherwise untreated DBA/2 mice established also a state of protective immunity against the subsequent injection of otherwise lethal doses of ESb tumor cells. Protection was only obtained after injection of intact but not UV-irradiated or mitomycin-C-treated ESb-D cells. A direct T-cell-mediated cytotoxic activity was also demonstrable in the spleen cells of DBA/2 mice after injection of ESb-D cells but not ESb cells. The cytotoxic activity was variant specific for ESb-D target cells, and it was induced only with intact but not UV-irradiated or mitomycin C-treated ESb-D cells. This suggested that the induction of protective and cytotoxic immunity may require the persistence of the antigen or unusually high antigen doses. The in vivo priming for a secondary in vitro cytotoxic response, in contrast, was achieved with intact and also with mitomycin C-treated ESb-D cells but again not with UV-irradiated ESb-D cells. This indicated that the metabolic activity was a minimal requirement for the in vivo immunogenicity of the ESb-D tumor line. The secondary cytotoxic activity was demonstrable on ESb-D and ESb target cells and could be restimulated in vitro about equally well with ESb-D and ESb cells. But the in vivo priming was again only obtained with ESb-D cells and not with ESb cells. These experiments thus demonstrated that the requirements for immunogenicity are more stringent in vivo than in vitro, and more stringent for the induction of direct cytotoxic and protective immunity in vivo than for the in vivo priming for secondary in vitro responses

Human CAR T cells with cell-intrinsic PD-1 checkpoint blockade resist tumor-mediated inhibition

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Cherkassky, Leonid; Morello, Aurore; Villena-Vargas, Jonathan; Feng, Yang; Dimitrov, Dimiter S.; Jones, David R.; Sadelain, Michel; Adusumilli, Prasad S.

2016-01-01

Following immune attack, solid tumors upregulate coinhibitory ligands that bind to inhibitory receptors on T cells. This adaptive resistance compromises the efficacy of chimeric antigen receptor (CAR) T cell therapies, which redirect T cells to solid tumors. Here, we investigated whether programmed death-1–mediated (PD-1–mediated) T cell exhaustion affects mesothelin-targeted CAR T cells and explored cell-intrinsic strategies to overcome inhibition of CAR T cells. Using an orthotopic mouse model of pleural mesothelioma, we determined that relatively high doses of both CD28- and 4-1BB–based second-generation CAR T cells achieved tumor eradication. CAR-mediated CD28 and 4-1BB costimulation resulted in similar levels of T cell persistence in animals treated with low T cell doses; however, PD-1 upregulation within the tumor microenvironment inhibited T cell function. At lower doses, 4-1BB CAR T cells retained their cytotoxic and cytokine secretion functions longer than CD28 CAR T cells. The prolonged function of 4-1BB CAR T cells correlated with improved survival. PD-1/PD-1 ligand [PD-L1] pathway interference, through PD-1 antibody checkpoint blockade, cell-intrinsic PD-1 shRNA blockade, or a PD-1 dominant negative receptor, restored the effector function of CD28 CAR T cells. These findings provide mechanistic insights into human CAR T cell exhaustion in solid tumors and suggest that PD-1/PD-L1 blockade may be an effective strategy for improving the potency of CAR T cell therapies. PMID:27454297

Comparison of mammalian and fish cell line cytotoxicity: impact of endpoint and exposure duration

International Nuclear Information System (INIS)

Guelden, Michael; Moerchel, Sabine; Seibert, Hasso

2005-01-01

Comparisons of acute toxic concentrations of chemicals to fish in vivo and cytotoxic concentrations to fish cell lines in vitro reveal rather good correlations of the toxic potencies in vitro and in vivo, but a clearly lower sensitivity of the fish cells. To examine whether the low sensitivity is specific for fish cells, cytotoxic potencies of reference chemicals from the Multicenter Evaluation of In Vitro Cytotoxicity program (MEIC) reported for the fish cell lines R1 and RTG-2 were compared with those obtained with the mouse Balb/c 3T3 cell line. Cytotoxic potencies (EC 50 values) for MEIC reference chemicals were determined with exponentially growing Balb/c 3T3 cells using three different test protocols. To assess both endpoints, cell proliferation and cell survival, EC 50 values were measured for the decrease in final cell protein after 24 and 72 h of exposure and for the reduction of cell protein increase during 24 h of exposure. EC 50 values obtained with the fish cell lines R1 and RTG-2 using cell survival as endpoint were taken from the MEIC data base. The comparison of cytotoxic potencies shows that, in general, the fish cell lines and the mammalian cell line are almost equally sensitive towards the cytotoxic action of chemicals. The mammalian cell line assay, however, becomes considerably more sensitive, by factors of 3.4-8.5, than the fish cell line assays, if cell growth instead of cell survival is used as endpoint. It is concluded, that cell proliferation might be a better endpoint than cell survival and that mammalian cell lines might be suited to assess fish acute toxicity

Annona muricata leaves have strongest cytotoxic activity against breast cancer cells

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Susi Endrini

2014-12-01

Full Text Available Background Plant-derived herbal compounds have a long history of clinical use, better patient tolerance and acceptance. They are freely available natural compounds that can be safely used to prevent various ailments. Plants have been the basis of traditional medicine throughout the world for thousands of years and are providing mankind with new remedies. The objective of this study was to determine the cytotoxicity of soursop (Anona muricata Linn leaves and pearl grass (Hedyotis corymbosa (L. Lam. on the hormone-dependent human breast carcinoma Michigan Cancer Foundation-7 (MCF-7 cell line. Methods This study used two types of solvents (water and ethanol in the extraction process and two incubation times (24 hours and 48 hours in the MTT assays to analyze the cytotoxic effects of both plants. Results Preliminary results showed that the ethanolic extract of soursop leaves (SE displayed cytotoxic effects against MCF-7 on 24- and 48-hour incubation times with IC50 values of 88.788 ìg/ml and 14.678 mg/ml, respectively. Ethanolic pearl grass extract (PE showed similar results, with IC50 values of 65.011 mg/ml on 24-hour incubation time and 52.329 mg/ml on 48-hour incubation time against MCF-7 cell line. However, the water extract of both plants displayed lower cytotoxic effect against MCF-7 cell line. Conclusion The ethanolic extract of both plants displayed cytotoxic effect against MCF-7. Soursop (Anona muricata Linn leaves have the strongest cytotoxic activity against MCF-7 breast cancer cell line.

Annona muricata leaves have strongest cytotoxic activity against breast cancer cells

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Susi Endrini

2015-12-01

Full Text Available BACKGROUND Plant-derived herbal compounds have a long history of clinical use, better patient tolerance and acceptance. They are freely available natural compounds that can be safely used to prevent various ailments. Plants have been the basis of traditional medicine throughout the world for thousands of years and are providing mankind with new remedies. The objective of this study was to determine the cytotoxicity of soursop (Anona muricata Linn leaves and pearl grass (Hedyotis corymbosa (L. Lam. on the hormone-dependent human breast carcinoma Michigan Cancer Foundation-7 (MCF-7 cell line. METHODS This study used two types of solvents (water and ethanol in the extraction process and two incubation times (24 hours and 48 hours in the MTT assays to analyze the cytotoxic effects of both plants. RESULTS Preliminary results showed that the ethanolic extract of soursop leaves (SE displayed cytotoxic effects against MCF-7 on 24- and 48-hour incubation times with IC50 values of 88.788 μg/ml and 14.678 μg/ml, respectively. Ethanolic pearl grass extract (PE showed similar results, with IC50 values of 65.011 μg/ ml on 24-hour incubation time and 52.329 μg/ml on 48-hour incubation time against MCF-7 cell line. However, the water extract of both plants displayed lower cytotoxic effect against MCF-7 cell line. CONCLUSION The ethanolic extract of both plants displayed cytotoxic effect against MCF-7. Soursop (Anona muricata Linn leaves have the strongest cytotoxic activity against MCF-7 breast cancer cell line.

Reduced antibody-dependent cellular cytotoxicity to herpes simplex virus-infected cells of salivary polymorphonuclear leukocytes and inhibition of peripheral blood polymorphonuclear leukocyte cytotoxicity by saliva.

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Ashkenazi, M; Kohl, S

1990-06-15

Blood polymorphonuclear leukocytes (BPMN) have been shown to mediate antibody-dependent cellular cytotoxicity (ADCC) against HSV-infected cells. Although HSV infections are frequently found in the oral cavity, the ADCC capacity of salivary PMN (SPMN) has not been studied, mainly because methods to isolate SPMN were not available. We have recently developed a method to isolate SPMN, and in this study have evaluated their ADCC activity against HSV-infected cells. SPMN were obtained by repeated washings of the oral cavity, and separated from epithelial cells by nylon mesh filtration. ADCC was quantitatively determined by 51Cr release from HSV-infected Chang liver cells. SPMN in the presence of antibody were able to destroy HSV-infected cells, but SPMN were much less effective in mediating ADCC than BPMN (3.4% vs 40.7%, p less than 0.0001). In the presence of antiviral antibody, SPMN were able to adhere to HSV-infected cells, but less so than BPMN (34% vs 67%), and specific antibody-induced adherence was significantly lower in SPMN (p less than 0.04). The spontaneous adherence to HSV-infected cells was higher for SPMN than BPMN. SPMN demonstrated up-regulation of the adhesion glycoprotein CD18, but down-regulation of the FcRIII receptor. Incubation with saliva decreased ADCC capacity of BPMN, up-regulated CD18 expression, and down-regulated FcRIII expression.

Phototoxicity and cytotoxicity of fullerol in human lens epithelial cells

International Nuclear Information System (INIS)

Roberts, Joan E.; Wielgus, Albert R.; Boyes, William K.; Andley, Usha; Chignell, Colin F.

2008-01-01

The water-soluble, hydroxylated fullerene [fullerol, nano-C 60 (OH) 22-26 ] has several clinical applications including use as a drug carrier to bypass the blood ocular barriers. We have assessed fullerol's potential ocular toxicity by measuring its cytotoxicity and phototoxicity induced by UVA and visible light in vitro with human lens epithelial cells (HLE B-3). Accumulation of nano-C 60 (OH) 22-26 in the cells was confirmed spectrophotometrically at 405 nm and cell viability estimated using MTS and LDH assays. Fullerol was cytotoxic to HLE B-3 cells maintained in the dark at concentrations higher than 20 μM. Exposure to either UVA or visible light in the presence of > 5 μM fullerol-induced phototoxic damage. When cells were pretreated with non-toxic antioxidants: 20 μM lutein, 1 mM N-acetyl cysteine, or 1 mM L-ascorbic acid prior to irradiation, only the singlet oxygen quencher-lutein significantly protected against fullerol photodamage. Apoptosis was observed in lens cells treated with fullerol whether or not the cells were irradiated, in the order UVA > visible light > dark. Dynamic light scattering (DLS) showed that in the presence of the endogenous lens protein α-crystallin, large aggregates of fullerol were reduced. In conclusion, fullerol is both cytotoxic and phototoxic to human lens epithelial cells. Although the acute toxicity of water-soluble nano-C 60 (OH) 22-26 is low, these compounds are retained in the body for long periods, raising concern for their chronic toxic effect. Before fullerols are used to deliver drugs to the eye, they should be tested for photo- and cytotoxicity in vivo

TNFα and IFNγ but not perforin are critical for CD8 T cell-mediated protection against pulmonary Yersinia pestis infection.

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Szaba, Frank M; Kummer, Lawrence W; Duso, Debra K; Koroleva, Ekaterina P; Tumanov, Alexei V; Cooper, Andrea M; Bliska, James B; Smiley, Stephen T; Lin, Jr-Shiuan

2014-05-01

Septic pneumonias resulting from bacterial infections of the lung are a leading cause of human death worldwide. Little is known about the capacity of CD8 T cell-mediated immunity to combat these infections and the types of effector functions that may be most effective. Pneumonic plague is an acutely lethal septic pneumonia caused by the Gram-negative bacterium Yersinia pestis. We recently identified a dominant and protective Y. pestis antigen, YopE69-77, recognized by CD8 T cells in C57BL/6 mice. Here, we use gene-deficient mice, Ab-mediated depletion, cell transfers, and bone marrow chimeric mice to investigate the effector functions of YopE69-77-specific CD8 T cells and their relative contributions during pulmonary Y. pestis infection. We demonstrate that YopE69-77-specific CD8 T cells exhibit perforin-dependent cytotoxicity in vivo; however, perforin is dispensable for YopE69-77-mediated protection. In contrast, YopE69-77-mediated protection is severely impaired when production of TNFα and IFNγ by CD8 T cells is simultaneously ablated. Interestingly, TNFα is absolutely required at the time of challenge infection and can be provided by either T cells or non-T cells, whereas IFNγ provided by T cells prior to challenge appears to facilitate the differentiation of optimally protective CD8 T cells. We conclude that cytokine production, not cytotoxicity, is essential for CD8 T cell-mediated control of pulmonary Y. pestis infection and we suggest that assays detecting Ag-specific TNFα production in addition to antibody titers may be useful correlates of vaccine efficacy against plague and other acutely lethal septic bacterial pneumonias.

Glucocorticoid effect on melphalan cytotoxicity, cell-cycle position, cell size, and [3H]uridine incorporation in one of three human melanoma cell lines

International Nuclear Information System (INIS)

Benckhuijsen, C.; Osman, A.M.; Hillebrand, M.J.; Smets, L.A.

1987-01-01

Three human melanoma cell lines of known content of specific glucocorticoid-binding sites were studied for colony formation after a microM dose of glucocorticoid combined with melphalan. In one of the three cell lines, M-5A, subcloned from M-5 (formerly designated RPMI 8322), the effect of combined treatment was markedly increased compared to that of melphalan even if the glucocorticoid was applied for 1 h only, 10 h before the melphalan. Semilogarithmic dose-effect plots for a reduction of final plating efficiency by glucocorticoid were curvilinear, according to a receptor-mediated process. The effects of glucocorticoid, melphalan, and their combination were linearized by bilogarithmic median-effect plotting which allowed the quantitation of a synergism which was more marked in case of glucocorticoid pretreatment, for 1 or 24 h, than on simultaneous exposure. According to sequential DNA per cell cytophotometry, melphalan abolished in M-5A a glucocorticoid-induced arrest in the G1 phase of the cell cycle. The cytotoxic synergism correlated with an apparent stimulation by glucocorticoid of the rate of acid-insoluble incorporation of [ 3 H]uridine and [ 14 C]leucine and an increase in cell size and protein content in M-5A cells but not in the other two cell lines. The way in which glucocorticoids induce an enhanced susceptibility to melphalan is not clear. Our results appear compatible with a hypothesis that chromatin in a transcriptionally activated state is more vulnerable to cytotoxic attack by an alkylating agent than under average conditions

Cisplatin Induces Cytotoxicity through the Mitogen-Activated Protein Kinase Pathways ana Activating Transcription Factor 3

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Carly St. Germain

2010-07-01

Full Text Available The mechanisms underlying the proapoptotic effect of the chemotherapeutic agent, cisplatin, are largely undefined. Understanding the mechanisms regulating cisplatin cytotoxicity may uncover strategies to enhance the efficacy of this important therapeutic agent. This study evaluates the role of activating transcription factor 3 (ATF3 as a mediator of cisplatin-induced cytotoxicity. Cytotoxic doses of cisplatin and carboplatin treatments consistently induced ATF3 expression in five tumor-derived cell lines. Characterization of this induction revealed a p53, BRCA1, and integrated stress response-independent mechanism, all previously implicated in stress-mediated ATF3 induction. Analysis of mitogenactivated protein kinase (MAPK pathway involvement in ATF3 induction by cisplatin revealed a MAPK-dependent mechanism. Cisplatin treatment combined with specific inhibitors to each MAPK pathway (c-Jun N-terminal kinase, extracellularsignal-regulated kinase, and p38 resulted in decreasedATF3 induction at the protein level. MAPK pathway inhibition led to decreased ATF3 messenger RNA expression and reduced cytotoxic effects of cisplatin as measured by the 3-(4,5-dimethylthiazol-2-ylF2,5-diphenyltetrazolium bromide cell viability assay. In A549 lung carcinoma cells, targeting ATF3 with specific small hairpin RNA also attenuated the cytotoxic effects of cisplatin. Similarly, ATF3-/murine embryonic fibroblasts (MEFs were shown to be less sensitive to cisplatin-induced cytotoxicity compared with ATF3+/+ MEFs. This study identifies cisplatin as a MAPK pathway-dependent inducer of ATF3, whose expression influences cisplatin’s cytotoxic effects.

A Mechanism for the Temporal Potentiation of Genipin to the Cytotoxicity of Cisplatin in Colon Cancer Cells.

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Wang, Ruihua; MoYung, K C; Zhao, Y J; Poon, Karen

2016-01-01

To investigate the potentiation effect of Genipin to Cisplatin induced cell senescence in HCT-116 colon cancer cells in vitro. Cell viability was estimated by Propidium iodide and Hoechst 3342, reactive oxygen species (ROS) with DHE, mitochondrial membrane potential (MMP) with JC-1 MMP assay Kit and electron current production with microbial fuel cells (MFC). Genipin inhibited the UCP2 mediated anti-oxidative proton leak significantly promoted the Cisplatin induced ROS and subsequent cell death, which was similar to that of UCP2-siRNA. Cells treated with Cisplatin alone or combined with Genipin, ROS negatively, while MMP positively correlated with cell viability. Cisplatin induced ROS was significantly decreased by detouring electrons to MFC, or increased by Genipin combined treatment. Compensatory effects of UCP2 up-regulation with time against Genipin treatment were suggested. Shorter the Genipin treatment before Cisplatin better promoted the Cisplatin induced ROS and subsequent cell death. The interaction of leaked electron with Cisplatin was important during ROS generation. Inhibition of UCP2-mediated proton leak with Genipin potentiated the cytotoxicity of Cisplatin. Owing to the compensatory effects against Genipin, shorter Genipin treatment before Cisplatin was recommended in order to achieve better potentiation effect.

In vitro cytotoxicity of silver nanoparticles and zinc oxide nanoparticles to human epithelial colorectal adenocarcinoma (Caco-2) cells

International Nuclear Information System (INIS)

Song, Yijuan; Guan, Rongfa; Lyu, Fei; Kang, Tianshu; Wu, Yihang; Chen, Xiaoqiang

2014-01-01

Highlights: • The characterization of Ag NPs and ZnO NPs. • The various morphologies of Caco-2 cells stained with AO/EB. • The viability of Caco-2 cells after Ag NPs and ZnO NPs exposure. • The cytotoxicity of Ag NPs and ZnO NPs on Caco-2 cells by oxidative stress assays. - Abstract: With the increasing applications of silver nanoparticles (Ag NPs) and zinc oxide nanoparticles (ZnO NPs) in foods and cosmetics, the concerns about the potential toxicities to human have been raised. The aims of this study are to observe the cytotoxicity of Ag NPs and ZnO NPs to human epithelial colorectal adenocarcinoma (Caco-2) cells in vitro, and to discover the toxicity mechanism of nanoparticles on Caco-2 cells. Caco-2 cells were exposed to 10, 25, 50, 100, 200 μg/mL of Ag NPs and ZnO NPs (90 nm). AO/EB double staining was used to characterize the morphology of the treated cells. The cell counting kit-8 (CCK-8) assay was used to detect the proliferation of the cells. Reactive oxygen species (ROS), superoxide dismutase (SOD) and glutathione (GSH) assay were used to explore the oxidative damage of Caco-2 cells. The results showed that Ag NPs and ZnO NPs (0–200 μg/mL) had highly significant effect on the Caco-2 cells activity. ZnO NPs exerted higher cytotoxicity than Ag NPs in the same concentration range. ZnO NPs have dose-depended toxicity. The LD 50 of ZnO NPs in Caco-2 cells is 0.431 mg/L. Significant depletion of SOD level, variation in GSH level and release of ROS in cells treated by ZnO NPs were observed, which suggests that cytotoxicity of ZnO NPs in intestine cells might be mediated through cellular oxidative stress. While Caco-2 cells treated with Ag NPs at all experimental concentrations showed no cellular oxidative damage. Moreover, the cells’ antioxidant capacity increased, and reached the highest level when the concentration of Ag NPs was 50 μg/mL. Therefore, it can be concluded that Ag NPs are safer antibacterial material in food packaging materials than

In vitro cytotoxicity of silver nanoparticles and zinc oxide nanoparticles to human epithelial colorectal adenocarcinoma (Caco-2) cells

Energy Technology Data Exchange (ETDEWEB)

Song, Yijuan [Zhejiang Provincial Key Laboratory of Biometrology and Inspection and Quarantine, China Jiliang University, Hangzhou 310018 (China); Guan, Rongfa, E-mail: rongfaguan@163.com [Zhejiang Provincial Key Laboratory of Biometrology and Inspection and Quarantine, China Jiliang University, Hangzhou 310018 (China); Lyu, Fei [Department of Food Science and Technology, Zhejiang University of Technology, Hangzhou 310014 (China); Kang, Tianshu; Wu, Yihang [Zhejiang Provincial Key Laboratory of Biometrology and Inspection and Quarantine, China Jiliang University, Hangzhou 310018 (China); Chen, Xiaoqiang [Hubei University of Technology, Wuhan 430068 (China)

2014-11-15

Highlights: • The characterization of Ag NPs and ZnO NPs. • The various morphologies of Caco-2 cells stained with AO/EB. • The viability of Caco-2 cells after Ag NPs and ZnO NPs exposure. • The cytotoxicity of Ag NPs and ZnO NPs on Caco-2 cells by oxidative stress assays. - Abstract: With the increasing applications of silver nanoparticles (Ag NPs) and zinc oxide nanoparticles (ZnO NPs) in foods and cosmetics, the concerns about the potential toxicities to human have been raised. The aims of this study are to observe the cytotoxicity of Ag NPs and ZnO NPs to human epithelial colorectal adenocarcinoma (Caco-2) cells in vitro, and to discover the toxicity mechanism of nanoparticles on Caco-2 cells. Caco-2 cells were exposed to 10, 25, 50, 100, 200 μg/mL of Ag NPs and ZnO NPs (90 nm). AO/EB double staining was used to characterize the morphology of the treated cells. The cell counting kit-8 (CCK-8) assay was used to detect the proliferation of the cells. Reactive oxygen species (ROS), superoxide dismutase (SOD) and glutathione (GSH) assay were used to explore the oxidative damage of Caco-2 cells. The results showed that Ag NPs and ZnO NPs (0–200 μg/mL) had highly significant effect on the Caco-2 cells activity. ZnO NPs exerted higher cytotoxicity than Ag NPs in the same concentration range. ZnO NPs have dose-depended toxicity. The LD{sub 50} of ZnO NPs in Caco-2 cells is 0.431 mg/L. Significant depletion of SOD level, variation in GSH level and release of ROS in cells treated by ZnO NPs were observed, which suggests that cytotoxicity of ZnO NPs in intestine cells might be mediated through cellular oxidative stress. While Caco-2 cells treated with Ag NPs at all experimental concentrations showed no cellular oxidative damage. Moreover, the cells’ antioxidant capacity increased, and reached the highest level when the concentration of Ag NPs was 50 μg/mL. Therefore, it can be concluded that Ag NPs are safer antibacterial material in food packaging materials

Cytotoxicity of ophthalmic solutions with and without preservatives to human corneal endothelial cells, epithelial cells and conjunctival epithelial cells.

Science.gov (United States)

Ayaki, Masahiko; Yaguchi, Shigeo; Iwasawa, Atsuo; Koide, Ryohei

2008-08-01

The cytotoxicity of a range of commercial ophthalmic solutions in the presence and absence of preservatives was assessed in human corneal endothelial cells (HCECs), corneal epithelia and conjunctival epithelia using in vitro techniques. Cell survival was measured using the WST-1 assay for endothelial cells and the MTT assay for epithelial cells. Commercially available timolol, carteolol, cromoglicate, diclofenac, bromfenac and hyaluronic acid ophthalmic solutions were assessed for cytotoxicity in the presence and absence of preservatives. The preservatives benzalkonium, chlorobutanol and polysorbate were also tested. The survival of cells exposed to test ophthalmic solutions was expressed as a percentage of cell survival in the control solution (distilled water added to media) after 48 h exposure. HCEC survival was 20-30% in ophthalmic solutions diluted 10-fold. The survival of HCEC was significantly greater in all solutions in the absence of preservative than in the presence of preservative. The survival of corneal and conjunctival epithelia was consistent with that of HCECs for all test ophthalmic solutions. The preservatives polysorbate and benzalkonium were highly cytotoxic with cell survival decreasing to 20% at the concentration estimated in commercial ophthalmic solutions. By comparison, the survival of cells exposed to chlorobutanol was 80% or greater. The cytotoxicity of ophthalmic solutions to HCEC, corneal epithelia and conjunctival epithelia decreased in the absence of preservative.

Reproducible fashion of the HSP70B' promoter-induced cytotoxic response on a live cell-based biosensor by cell cycle synchronization.

Science.gov (United States)

Migita, Satoshi; Wada, Ken-Ichi; Taniguchi, Akiyoshi

2010-10-15

Live cell-based sensors potentially provide functional information about the cytotoxic effect of reagents on various signaling cascades. Cells transfected with a reporter vector derived from a cytotoxic response promoter can be used as intelligent cytotoxicity sensors (i.e., sensor cells). We have combined sensor cells and a microfluidic cell culture system that can achieve several laminar flows, resulting in a reliable high-throughput cytotoxicity detection system. These sensor cells can also be applied to single cell arrays. However, it is difficult to detect a cellular response in a single cell array, due to the heterogeneous response of sensor cells. The objective of this study was cell homogenization with cell cycle synchronization to enhance the response of cell-based biosensors. Our previously established stable sensor cells were brought into cell cycle synchronization under serum-starved conditions and we then investigated the cadmium chloride-induced cytotoxic response at the single cell level. The GFP positive rate of synchronized cells was approximately twice as high as that of the control cells, suggesting that cell homogenization is an important step when using cell-based biosensors with microdevices, such as a single cell array. Copyright 2010 Wiley Periodicals, Inc.

Regulation of DC development and DC-mediated T-cell immunity via CISH

OpenAIRE

Miah, Mohammad Alam; Bae, Yong-Soo

2013-01-01

Cytokine inducible SH2-containing protein (CISH) plays a crucial role in type 1 dendritic cell (DC) development as well as in the DC-mediated activation of cytotoxic T lymphocytes (CTLs). CISH expression at late DC developmental stages shuts down the proliferation of DC progenitors by negatively regulating signal transducer and activator of transcription 5 (STAT5) and facilitates the differentiation of DCs into potent stimulators of CTLs.

Regulation of DC development and DC-mediated T-cell immunity via CISH.

Science.gov (United States)

Miah, Mohammad Alam; Bae, Yong-Soo

2013-03-01

Cytokine inducible SH2-containing protein (CISH) plays a crucial role in type 1 dendritic cell (DC) development as well as in the DC-mediated activation of cytotoxic T lymphocytes (CTLs). CISH expression at late DC developmental stages shuts down the proliferation of DC progenitors by negatively regulating signal transducer and activator of transcription 5 (STAT5) and facilitates the differentiation of DCs into potent stimulators of CTLs.

Genipin-induced inhibition of uncoupling protein-2 sensitizes drug-resistant cancer cells to cytotoxic agents.

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Ryan J Mailloux

2010-10-01

Full Text Available Uncoupling protein-2 (UCP2 is known to suppress mitochondrial reactive oxygen species (ROS production and is employed by drug-resistant cancer cells to mitigate oxidative stress. Using the drug-sensitive HL-60 cells and the drug-resistant MX2 subline as model systems, we show that genipin, a UCP2 inhibitor, sensitizes drug-resistant cells to cytotoxic agents. Increased MX2 cell death was observed upon co-treatment with genipin and different doses of menadione, doxorubicin, and epirubicin. DCFH-DA fluorimetry revealed that the increase in MX2 cell death was accompanied by enhanced cellular ROS levels. The drug-induced increase in ROS was linked to genipin-mediated inhibition of mitochondrial proton leak. State 4 and resting cellular respiratory rates were higher in the MX2 cells in comparison to the HL-60 cells, and the increased respiration was readily suppressed by genipin in the MX2 cells. UCP2 accounted for a remarkable 37% of the resting cellular oxygen consumption indicating that the MX2 cells are functionally reliant on this protein. Higher amounts of UCP2 protein were detected in the MX2 versus the HL-60 mitochondria. The observed effects of genipin were absent in the HL-60 cells pointing to the selectivity of this natural product for drug-resistant cells. The specificity of genipin for UCP2 was confirmed using CHO cells stably expressing UCP2 in which genipin induced an ∼22% decrease in state 4 respiration. These effects were absent in empty vector CHO cells expressing no UCP2. Thus, the chemical inhibition of UCP2 with genipin sensitizes multidrug-resistant cancer cells to cytotoxic agents.

Cytotoxicity of the methanol extracts of Elephantopus mollis, Kalanchoe crenata and 4 other Cameroonian medicinal plants towards human carcinoma cells.

Science.gov (United States)

Kuete, Victor; Fokou, Fabrice W; Karaosmanoğlu, Oğuzhan; Beng, Veronique P; Sivas, Hülya

2017-05-25

Cancer still constitutes one of the major health concerns globally, causing serious threats on patients, their families, and the healthcare system. In this study, the cytotoxicity of the methanol extract of Elephantopus mollis whole plant (EMW), Enantia chlorantha bark (ECB), Kalanchoe crenata leaves (KCL), Lophira alata bark (LAB), Millettia macrophylla leaves (MML) and Phragmanthera capitata leaves (PCL) towards five human solid cancer cell lines and normal CRL2120 fibroblasts, was evaluated. Extracts were subjected to qualitative chemical screening of their secondary metabolite contents using standard methods. The cytotoxicity of samples was evaluated using neutral red uptake (NR) assay meanwhile caspase activation was detected by caspase-Glo assay. Flow cytometry was used to analyze the cell cycle distribution and the mitochondrial membrane potential (MMP) whilst spectrophotometry was used to measure the levels of reactive oxygen species (ROS). Phytochemical analysis revealed the presence of polyphenols, triterpenes and sterols in all extracts. The IC 50 values of the best samples ranged from 3.29 μg/mL (towards DLD-1 colorectal adenocarcinoma cells) to 24.38 μg/mL (against small lung cancer A549 cells) for EMW, from 2.33 μg/mL (mesothelioma SPC212 cells) to 28.96 μg/mL (HepG2 hepatocarcinoma) for KCL, and from 0.04 μg/mL (towards SPC212 cells) to 0.55 μg/mL (towards A549 cells) for doxorubicin. EMW induced apoptosis in MCF-7 cells mediated by MMP loss and increased ROS production whilst KCL induced apoptosis via ROS production. This study provides evidences of the cytotoxicity of the tested plant extract and highlights the good activity of Elephantopus mollis and Kalanchoe crenata. They deserve more exploration to develop novel cytotoxic drugs.

Cytotoxicity effect of Zataria multiflora Boiss. on two human colon carcinoma cell lines

Directory of Open Access Journals (Sweden)

F. Sharififar

2017-10-01

Full Text Available Background and objectives: Natural products are one of the major sources for investigations of novel medicines. Zataria multiflora Boiss (ZM has shown pharmacological activities especially in gastrointestinal tract; however, there are limited studies about its cytotoxicity effects. In this study, the effect of Zataria multiflora was examined on two colon cancer cell lines (SW-48 and HT-29. Methods: Hydro-alcoholic extract of ZM and its fractions including chloroform, petroleum ether and methanol extract were prepared by warm maceration method. Different concentrations were prepared and examined on SW-48 and HT-29 cell lines using 2-(4, 5-dimethylthiazol-2-yl 2, 5-diphenyltetrazolium bromide (MTT assay. Results: The results of the present study have shown the cytotoxic effect of some fractions of ZM. The most considerable cytotoxic effect was shown against HT-29 cell line. Also, total ZM extract and the petroleum ether fraction demonstrated cytotoxic effects with IC50 values of 44.22 and 33.42 µg/ml on SW-48 and HT-29 cell lines, respectively. Conclusion: Zataria multiflora was cytotoxic to against colon cancer cell lines HT-29 and SW-48.

Pattern of CD4 T‑lymphocyte Values in Cancer Patients on Cytotoxic ...

African Journals Online (AJOL)

through cell surface receptors and results in replication and or mediation of one ... Keywords: Cancers, CD4 lymphocyte, Cytotoxic chemotherapy, Immunosuppression. Access this ..... of the larynx, ovaries, stomach, and urinary bladder. These.

Correlation of particle properties with cytotoxicity and cellular uptake of hydroxyapatite nanoparticles in human gastric cancer cells

Energy Technology Data Exchange (ETDEWEB)

Cui, Xinhui [State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237 (China); Liang, Tong [Engineering Research Center for Biomedical Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237 (China); Liu, Changsheng [State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237 (China); Engineering Research Center for Biomedical Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237 (China); Yuan, Yuan, E-mail: yyuan@ecust.edu.cn [Engineering Research Center for Biomedical Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237 (China); Qian, Jiangchao, E-mail: jiangchaoqian@ecust.edu.cn [State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237 (China)

2016-10-01

Three types of hydroxyapatite nanoparticles (HAPNs) were synthesized employing a sonochemistry-assisted microwave method by changing microwave power (from 200 to 300 W) or using calcination treatment: L200 (200 W, lyophilization), L300 (300 W, lyophilization) and C200 (200 W, lyophilization & calcination). Their physiochemical properties were characterized and correlated with cytotoxicity to human gastric cancer cells (MGC80-3). The major differences among these HAPN preparations were their size and specific surface area, with the L200 showing a smaller size and higher specific surface area. Although all HAPNs inhibited cell proliferation and induced apoptosis of cancer cells, L200 exhibited the greatest toxicity. All types of HAPNs were internalized through energy-dependent pathways, but the L200 nanoparticles were more efficiently uptaken by MGC80-3 cells. Inhibitor studies with dynasore and methyl-β-cyclodextrin suggested that caveolae-mediated endocytosis and, to a much lesser extent, clathrin-mediated endocytosis, were involved in cellular uptake of the various preparations, whereas the inhibition of endocytosis was more obvious for L200. Using fluorescein isothiocyanate-labeled HAPNs and laser-scanning confocal microscopy, we found that all forms of nanoparticles were present in the cytoplasm, and some L200 HAPNs were even found within nuclei. Treatment with all HAPN preparations led to the increase in the intracellular calcium level with the highest level detected for L200. - Highlights: • Three types of HAPNs (L200, L300 and C200) were synthesized employing a sonochemistry-assisted microwave method. • L200 exhibited the greatest cytotoxicity to human gastric cancer (MGC80-3) cells. • L200 showed a smaller size and higher specific surface area. • The L200 nanoparticles were more efficiently uptaken by MGC80-3 cells through energy-dependent pathways. • L200 caused the most significant increase in the intracellular calcium level.

Natural mineral particles are cytotoxic to rainbow trout gill epithelial cells in vitro.

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Christian Michel

Full Text Available Worldwide increases in fluvial fine sediment are a threat to aquatic animal health. Fluvial fine sediment is always a mixture of particles whose mineralogical composition differs depending on the sediment source and catchment area geology. Nonetheless, whether particle impact in aquatic organisms differs between mineral species remains to be investigated. This study applied an in vitro approach to evaluate cytotoxicity and uptake of four common fluvial mineral particles (quartz, feldspar, mica, and kaolin; concentrations: 10, 50, 250 mg L(-1 in the rainbow trout epithelial gill cell line RTgill-W1. Cells were exposed for 24, 48, 72, and 96 h. Cytotoxicity assays for cell membrane integrity (propidium iodide assay, oxidative stress (H2DCF-DA assay, and metabolic activity (MTT assay were applied. These assays were complemented with cell counts and transmission electron microscopy. Regardless of mineral species, particles ≤ 2 µm in diameter were taken up by the cells, suggesting that particles of all mineral species came into contact and interacted with the cells. Not all particles, however, caused strong cytotoxicity: Among all assays the tectosilicates quartz and feldspar caused sporadic maximum changes of 0.8-1.2-fold compared to controls. In contrast, cytotoxicity of the clay particles was distinctly stronger and even differed between the two particle types: mica induced concentration-dependent increases in free radicals, with consistent 1.6-1.8-fold-changes at the 250 mg L(-1 concentration, and a dilated endoplasmic reticulum. Kaolin caused concentration-dependent increases in cell membrane damage, with consistent 1.3-1.6-fold increases at the 250 mg L(-1 concentration. All effects occurred in the presence or absence of 10% fetal bovine serum. Cell numbers per se were marginally affected. Results indicate that (i. natural mineral particles can be cytotoxic to gill epithelial cells, (ii. their cytotoxic potential differs between mineral

Interferon-β gene transfer induces a strong cytotoxic bystander effect on melanoma cells.

Science.gov (United States)

Rossi, Úrsula A; Gil-Cardeza, María L; Villaverde, Marcela S; Finocchiaro, Liliana M E; Glikin, Gerardo C

2015-05-01

A local gene therapy scheme for the delivery of type I interferons could be an alternative for the treatment of melanoma. We evaluated the cytotoxic effects of interferon-β (IFNβ) gene lipofection on tumor cell lines derived from three human cutaneous and four canine mucosal melanomas. The cytotoxicity of human IFNβ gene lipofection resulted higher or equivalent to that of the corresponding addition of the recombinant protein (rhIFNβ) to human cells. IFNβ gene lipofection was not cytotoxic for only one canine melanoma cell line. When cultured as monolayers, three human and three canine IFNβ-lipofected melanoma cell lines displayed a remarkable bystander effect. As spheroids, the same six cell lines were sensitive to IFNβ gene transfer, two displaying a significant multicell resistance phenotype. The effects of conditioned IFNβ-lipofected canine melanoma cell culture media suggested the release of at least one soluble thermolabile cytotoxic factor that could not be detected in human melanoma cells. By using a secretion signal-free truncated human IFNβ, we showed that its intracellular expression was enough to induce cytotoxicity in two human melanoma cell lines. The lower cytoplasmatic levels of reactive oxygen species detected after intracellular IFNβ expression could be related to the resistance displayed by one human melanoma cell line. As IFNβ gene transfer was effective against most of the assayed melanomas in a way not limited by relatively low lipofection efficiencies, the clinical potential of this approach is strongly supported. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

δ-Aminolevulinic acid cytotoxic effects on human hepatocarcinoma cell lines

Energy Technology Data Exchange (ETDEWEB)

De Siervi, Adriana; Vazquez, Elba S; Rezaval, Carolina; Rossetti, María V; Batlle, Alcira M del [Centro de Investigaciones sobre Porfirinas y Porfirias (CIPYP), Argentine National Research Council (CONICET), Department of Biological Chemistry, FCEN, University of Buenos Aires (Argentina)

2002-01-01

Acute Intermittent Porphyria is a genetic disorder of heme metabolism, characterized by increased levels of porphyrin precursors, δ-aminolevulinic acid (ALA) and porphobilinogen (PBG). ALA has been reported to generate reactive oxygen species and to cause oxidative damage to proteins, subcellular structures and DNA. It is known that oxidative stress can induce apoptosis. The aim of this work was to study the cytotoxic effect of ALA on two hepatocarcinoma cell lines. We have determined the impact of ALA on HEP G2 and HEP 3B hepatocarcinoma cell lines survival as measured by the MTT assay. ALA proved to be cytotoxic in both cell lines however; HEP G2 was more sensitive to ALA than HEP 3B. Addition of hemin or glucose diminished ALA cytotoxicity in HEP G2 cells; instead it was enhanced in HEP 3B cells. Because apoptosis is usually associated with DNA fragmentation, the DNA of ALA treated and untreated cells were analyzed. The characteristic pattern of DNA fragmentation ladders was observed in ALA treated cells. To elucidate the mechanisms of ALA induced apoptosis, we examined its effect on p53 expression. No changes in p53 mRNA levels were observed after exposure of both cell lines to ALA for 24 h. CDK2 and CDK4 protein levels were reduced after ALA treatment at physiological concentrations.

δ-Aminolevulinic acid cytotoxic effects on human hepatocarcinoma cell lines

Directory of Open Access Journals (Sweden)

del Batlle Alcira M

2002-03-01

Full Text Available Abstract Background Acute Intermittent Porphyria is a genetic disorder of heme metabolism, characterized by increased levels of porphyrin precursors, δ-aminolevulinic acid (ALA and porphobilinogen (PBG. ALA has been reported to generate reactive oxygen species and to cause oxidative damage to proteins, subcellular structures and DNA. It is known that oxidative stress can induce apoptosis. The aim of this work was to study the cytotoxic effect of ALA on two hepatocarcinoma cell lines. Results We have determined the impact of ALA on HEP G2 and HEP 3B hepatocarcinoma cell lines survival as measured by the MTT assay. ALA proved to be cytotoxic in both cell lines however; HEP G2 was more sensitive to ALA than HEP 3B. Addition of hemin or glucose diminished ALA cytotoxicity in HEP G2 cells; instead it was enhanced in HEP 3B cells. Because apoptosis is usually associated with DNA fragmentation, the DNA of ALA treated and untreated cells were analyzed. The characteristic pattern of DNA fragmentation ladders was observed in ALA treated cells. To elucidate the mechanisms of ALA induced apoptosis, we examined its effect on p53 expression. No changes in p53 mRNA levels were observed after exposure of both cell lines to ALA for 24 h. CDK2 and CDK4 protein levels were reduced after ALA treatment at physiological concentrations.

δ-Aminolevulinic acid cytotoxic effects on human hepatocarcinoma cell lines

International Nuclear Information System (INIS)

De Siervi, Adriana; Vazquez, Elba S; Rezaval, Carolina; Rossetti, María V; Batlle, Alcira M del

2002-01-01

Acute Intermittent Porphyria is a genetic disorder of heme metabolism, characterized by increased levels of porphyrin precursors, δ-aminolevulinic acid (ALA) and porphobilinogen (PBG). ALA has been reported to generate reactive oxygen species and to cause oxidative damage to proteins, subcellular structures and DNA. It is known that oxidative stress can induce apoptosis. The aim of this work was to study the cytotoxic effect of ALA on two hepatocarcinoma cell lines. We have determined the impact of ALA on HEP G2 and HEP 3B hepatocarcinoma cell lines survival as measured by the MTT assay. ALA proved to be cytotoxic in both cell lines however; HEP G2 was more sensitive to ALA than HEP 3B. Addition of hemin or glucose diminished ALA cytotoxicity in HEP G2 cells; instead it was enhanced in HEP 3B cells. Because apoptosis is usually associated with DNA fragmentation, the DNA of ALA treated and untreated cells were analyzed. The characteristic pattern of DNA fragmentation ladders was observed in ALA treated cells. To elucidate the mechanisms of ALA induced apoptosis, we examined its effect on p53 expression. No changes in p53 mRNA levels were observed after exposure of both cell lines to ALA for 24 h. CDK2 and CDK4 protein levels were reduced after ALA treatment at physiological concentrations

Lactic Acid Bacteria from Kefir Increase Cytotoxicity of Natural Killer Cells to Tumor Cells.

Science.gov (United States)

Yamane, Takuya; Sakamoto, Tatsuji; Nakagaki, Takenori; Nakano, Yoshihisa

2018-03-27

The Japanese fermented beverage, homemade kefir, contains six lactic acid bacteria: Lactococcus. lactis subsp. Lactis , Lactococcus . lactis subsp. Cremoris , Lactococcus. Lactis subsp. Lactis biovar diacetylactis , Lactobacillus plantarum , Leuconostoc meseuteroides subsp. Cremoris and Lactobacillus casei . In this study, we found that a mixture of the six lactic acid bacteria from kefir increased the cytotoxicity of human natural killer KHYG-1 cells to human chronic myelogenous leukemia K562 cells and colorectal tumor HCT116 cells. Furthermore, levels of mRNA expression and secretion of IFN-γ (interferon gamma) increased in KHYG-1 cells that had been treated with the six lactic acid bacteria mixture from kefir. The results suggest that the six lactic acid bacteria mixture from kefir has strong effects on natural immunity and tumor cell cytotoxicity.

Cytotoxicity and accumulation of ergot alkaloids in human primary cells.

Science.gov (United States)

Mulac, Dennis; Humpf, Hans-Ulrich

2011-04-11

Ergot alkaloids are secondary metabolites produced by fungi of the species Claviceps. Toxic effects after consumption of contaminated grains are described since mediaeval times. Of the more than 40 known ergot alkaloids six are found predominantly. These are ergotamine, ergocornine, ergocryptine, ergocristine, ergosine and ergometrine, along with their corresponding isomeric forms (-inine-forms). Toxic effects are known to be induced by an interaction of the ergot alkaloids as neurotransmitters, like dopamine or serotonin. Nevertheless data concerning cytotoxic effects are missing and therefore a screening of the six main ergot alkaloids was performed in human primary cells in order to evaluate the toxic potential. As it is well known that ergot alkaloids isomerize easily the stability was tested in the cell medium. Based on these results factors were calculated to correct the used concentration values to the biologically active lysergic (-ine) form. These factors range from 1.4 for the most stable compound ergometrine to 5.0 for the most unstable ergot alkaloid ergocristine. With these factors, reflecting the instability, several controverse literature data concerning the toxicity could be explained. To evaluate the cytotoxic effects of ergot alkaloids, human cells in primary culture were used. These cells remain unchanged in contrast to cell lines and the data allow a better comparison to the in vivo situation than using immortalized cell lines. To characterize the effects on primary cells, renal proximal tubule epithelial cells (RPTEC) and normal human astrocytes (NHA) were used. The parameters necrosis (LDH-release) and apoptosis (caspase-3-activation, DNA condensation and fragmentation) were distinguished. The results show that depending on the individual structure of the peptide ergot alkaloids the toxic properties change. While ergometrine as a lysergic acid amide did not show any effect, the peptide ergot alkaloids revealed a different toxic potential. Of

Development and validation of TOF-SIMS and CLSM imaging method for cytotoxicity study of ZnO nanoparticles in HaCaT cells

Energy Technology Data Exchange (ETDEWEB)

Lee, Pei-Ling; Chen, Bo-Chia; Gollavelli, Ganesh [Department of Chemistry, National Tsing Hua University, Hsinchu 30013, Taiwan (China); Shen, Sin-Yu [Graduate Institute of Medical Science, Taipei Medical University, Taipei 11031, Taiwan (China); Yin, Yu-Sheng; Lei, Shiu-Ling [Department of Chemistry, National Tsing Hua University, Hsinchu 30013, Taiwan (China); Jhang, Cian-Ling; Lee, Woan-Ruoh [Department of Dermatology, Taipei Medical University, Taipei 11031, Taiwan (China); Ling, Yong-Chien, E-mail: ycling@mx.nthu.edu.tw [Department of Chemistry, National Tsing Hua University, Hsinchu 30013, Taiwan (China); Graduate Institute of Medical Science, Taipei Medical University, Taipei 11031, Taiwan (China)

2014-07-30

Highlights: • Assorted material, chemical, and toxicological analysis methods were used to confirm the shape, size, crystalline structure, and aggregation properties of ZnO NPS as well as their dissolution behavior and effect on HaCaT cell viability. • The developed TOF-SIMS and CLSM imaging method for rapid and sensitive study of ZnO NPs in HaCaT cells was validated by comparative and correlative analyses to aforementioned experimental results. • The imaging results demonstrate spatially-resolved cytotoxicity relationship between intracellular ZnO NPs concentration, {sup 40}Ca/{sup 39}K ratio, phosphocholine fragments, and glutathione fragments. CLSM images reveal the localization of ZnO NPs in cytoplasm and nuclei. • The trend of change in TOF-SIMS spectra and images of ZnO NPs treated HaCaT cells demonstrate the possible mode of actions by ZnO NP involves cell membrane disruption, cytotoxic response, and ROS mediated apoptosis. - Abstract: Zinc oxide nanoparticles (ZnO NPs) exhibit novel physiochemical properties and have found increasing use in sunscreen products and cosmetics. The potential toxicity is of increasing concern due to their close association with human skin. A time-of-flight secondary ion mass spectrometry (TOF-SIMS) and confocal laser scanning microscopy (CLSM) imaging method was developed and validated for rapid and sensitive cytotoxicity study of ZnO NPs using human skin equivalent HaCaT cells as a model system. Assorted material, chemical, and toxicological analysis methods were used to confirm their shape, size, crystalline structure, and aggregation properties as well as dissolution behavior and effect on HaCaT cell viability in the presence of various concentrations of ZnO NPs in aqueous media. Comparative and correlative analyses of aforementioned results with TOF-SIMS and CLSM imaging results exhibit reasonable and acceptable outcome. A marked drop in survival rate was observed with 50 μg/ml ZnO NPs. The CLSM images reveal the

Combined effect of x irradiation and cell-mediated immune reaction

International Nuclear Information System (INIS)

Song, C.W.; Guertin, D.P.

1978-01-01

The combined effect of radiation and cell-mediated immune reaction on tumor cells was investigated in vitro. Mastocytoma P815-X2 cells of DBA mice either were irradiated first and subjected to immune lysis by immune splenic lymphocytes of C57Bl mice, or the tumor cells were subjected to immune reaction first and then irradiated. Cell survival was quantitated by colony formation in soft agar medium. It was observed that cellular immune damage to tumor cells did not influence the response of tumor cells to subsequent radiation. Irradiation of tumor cells first, followed by subjection of the cells to cellular immune reaction, slightly enhanced the death of the tumor cells. It appears that this enhanced death might have resulted from a relative increase in the ratio of the number of cytotoxic immune cells to the number of target tumor cells in the incubation mixture as a consequence of the decrease in the number of viable tumor cells by radiation

Cytotoxic effects of replication-competent adenoviruses on human esophageal carcinoma are enhanced by forced p53 expression

International Nuclear Information System (INIS)

Yang, Shan; Kawamura, Kiyoko; Okamoto, Shinya; Yamauchi, Suguru; Shingyoji, Masato; Sekine, Ikuo; Kobayashi, Hiroshi; Tada, Yuji; Tatsumi, Koichiro; Hiroshima, Kenzo; Shimada, Hideaki; Tagawa, Masatoshi

2015-01-01

Improvement of transduction and augmentation of cytotoxicity are crucial for adenoviruses (Ad)-mediated gene therapy for cancer. Down-regulated expression of type 5 Ad (Ad5) receptors on human tumors hampered Ad-mediated transduction. Furthermore, a role of the p53 pathways in cytotoxicity mediated by replication-competent Ad remained uncharacterized. We constructed replication-competent Ad5 of which the E1 region genes were activated by a transcriptional regulatory region of the midkine or the survivin gene, which is expressed preferentially in human tumors. We also prepared replication-competent Ad5 which were regulated by the same region but had a fiber-knob region derived from serotype 35 (AdF35). We examined the cytotoxicity of these Ad and a possible combinatory use of the replication-competent AdF35 and Ad5 expressing the wild-type p53 gene (Ad5/p53) in esophageal carcinoma cells. Expression levels of molecules involved in cell death, anti-tumor effects in vivo and production of viral progenies were also investigated. Replication-competent AdF35 in general achieved greater cytotoxic effects to esophageal carcinoma cells than the corresponding replication-competent Ad5. Infection with the AdF35 induced cleavages of caspases and increased sub-G1 fractions, but did not activate the autophagy pathway. Transduction with Ad5/p53 in combination with the replication-competent AdF35 further enhanced the cytotoxicity in a synergistic manner. We also demonstrated the combinatory effects in an animal model. Transduction with Ad5/p53 however suppressed production of replication-competent AdF35 progenies, but the combination augmented Ad5/p53-mediated p53 expression levels and the downstream pathways. Combination of replication-competent AdF35 and Ad5/p53 achieved synergistic cytotoxicity due to enhanced p53-mediated apoptotic pathways. The online version of this article (doi:10.1186/s12885-015-1482-8) contains supplementary material, which is available to authorized

An altered gp100 peptide ligand with decreased binding by TCR and CD8alpha dissects T cell cytotoxicity from production of cytokines and activation of NFAT

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Niels eSchaft

2013-09-01

Full Text Available Altered peptide ligands (APLs provide useful tools to study T cell activation and potentially direct immune responses to improve treatment of cancer patients. To better understand and exploit APLs, we studied the relationship between APLs and T cell function in more detail. Here, we tested a broad panel of gp100(280-288 APLs with respect to T cell cytotoxicity, production of cytokines and activation of Nuclear Factor of Activated T cells (NFAT by human T cells gene-engineered with a gp100-HLA-A2-specific TCRalpha/beta. We demonstrated that gp100-specific cytotoxicity, production of cytokines, and activation of NFAT were not affected by APLs with single amino acid substitutions, except for an APL with an amino acid substitution at position 3 (APL A3, which did not elicit any T cell response. A gp100 peptide with a double amino acid mutation (APL S4S6 elicited T cell cytotoxicity and production of IFNgamma, and to a lesser extent TNFalpha, IL-4, and IL-5, but not production of IL-2 and IL-10, or activation of NFAT. Notably, TCR-mediated functions showed decreases in sensitivities for S4S6 versus gp100 wt peptide, which were minor for cytotoxicity but at least a 1000-fold more prominent for the production of cytokines. TCR-engineered T cells did not bind A3-HLA-A2, but did bind S4S6-HLA-A2 although to a lowered extent compared to wt peptide-HLA-A2. Moreover, S4S6-induced T cell function demonstrated an enhanced dependency on CD8alpha. Taken together, most gp100 APLs functioned as agonists, but A3 and S4S6 peptides acted as a null ligand and partial agonist, respectively. Our results further suggest that TCR-mediated cytotoxicity can be dissected from production of cytokines and activation of NFAT, and that the agonist potential of peptide mutants relates to the extent of binding by TCR and CD8alpha. These findings may facilitate the design of APLs to advance the study of T cell activation and their use for therapeutic applications.

Biodiesel exhaust-induced cytotoxicity and proinflammatory mediator production in human airway epithelial cells.

Science.gov (United States)

Mullins, Benjamin J; Kicic, Anthony; Ling, Kak-Ming; Mead-Hunter, Ryan; Larcombe, Alexander N

2016-01-01

Increasing use of biodiesel has prompted research into the potential health effects of biodiesel exhaust exposure. Few studies directly compare the health consequences of mineral diesel, biodiesel, or blend exhaust exposures. Here, we exposed human epithelial cell cultures to diluted exhaust generated by the combustion of Australian ultralow-sulfur-diesel (ULSD), unprocessed canola oil, 100% canola biodiesel (B100), and a blend of 20% canola biodiesel mixed with 80% ULSD. The physicochemical characteristics of the exhaust were assessed and we compared cellular viability, apoptosis, and levels of interleukin (IL)-6, IL-8, and Regulated on Activation, Normal T cell Expressed and Secreted (RANTES) in exposed cultured cells. Different fuel types produced significantly different amounts of exhaust gases and different particle characteristics. All exposures resulted in significant apoptosis and loss of viability when compared with control, with an increasing proportion of biodiesel being correlated with a decrease in viability. In most cases, exposure to exhaust resulted in an increase in mediator production, with the greatest increases most often in response to B100. Exposure to pure canola oil (PCO) exhaust did not increase mediator production, but resulted in a significant decrease in IL-8 and RANTES in some cases. Our results show that canola biodiesel exhaust exposure elicits inflammation and reduces viability of human epithelial cell cultures in vitro when compared with ULSD exhaust exposure. This may be related to an increase in particle surface area and number in B100 exhaust when compared with ULSD exhaust. Exposure to PCO exhaust elicited the greatest loss of cellular viability, but virtually no inflammatory response, likely due to an overall increase in average particle size. © 2014 Wiley Periodicals, Inc.

Analysis of cytotoxic T cell epitopes in relation to cancer

DEFF Research Database (Denmark)

Stranzl, Thomas

The human immune system is a highly adaptable system, defending our bodies against pathogens and tumor cells. Cytotoxic T cells (CTL) are cells of the adaptive immune system, capable of inducing a programmed cell death and thus able to eliminate infected or tumor cells. CTLs discriminate between...

Cytotoxic chemotherapy in the treatment of advanced renal cell carcinoma in the era of targeted therapy.

Science.gov (United States)

Diamond, E; Molina, A M; Carbonaro, M; Akhtar, N H; Giannakakou, P; Tagawa, S T; Nanus, D M

2015-12-01

Renal cell carcinoma (RCC) is a heterogeneous disease with regards to histology, progression, and response to treatment. Cytotoxic chemotherapy has been extensively studied in metastatic RCC (mRCC). Responses in most studies are modest and the mechanisms of resistance remain poorly understood. Targeted therapies have significantly improved outcomes in mRCC; however, most patients eventually relapse and die of their disease. Early clinical data suggest that combinations of chemotherapy and targeted agents are clinically active and are well tolerated. We reviewed the available literature for published clinical trials incorporating traditional chemotherapeutic agents in the treatment of mRCC. These papers were identified through a Medline search and were included if they employed at least one chemotherapeutic agent in the treatment of mRCC. The literature was also reviewed for information regarding mechanisms of chemotherapy resistance. The data regarding the use of cytotoxic chemotherapy in mRCC consist of small, non-randomized phase I and II studies. The major response proportions with single agent chemotherapies are low but combination regimens either with other cytotoxic agents, cytokines, or targeted agents have demonstrated moderate activity. Disparate trial designs and lack of head to head clinical trials make it difficult to compare the efficacy of chemotherapy with that of immunotherapy or targeted agents. Chemotherapy is particularly useful in patients with collecting duct histology and predominantly sarcomatoid differentiation. Chemotherapy resistance may be mediated by overexpression of p-glycoprotein efflux pumps and the dysregulation of the microtubule-hypoxia inducible factor signaling axis. The role of cytotoxic chemotherapy in the treatment for clear cell RCC remains poorly defined. Cytotoxic chemotherapy is considered a standard of care in patients with mRCC with predominantly sarcomatoid differentiation and collecting duct RCC variants (Motzer et al

Effect of citral on the cytotoxicity of doxorubicin in human B-lymphoma cells.

Science.gov (United States)

Dangkong, Darinee; Limpanasithikul, Wacharee

2015-02-01

Doxorubicin is a chemotherapy agent used in non-Hodgkin's lymphoma but side effects limit its use. Citral is a mixture of neral and geranial found in essential oils of lemon grass. We evaluated the activity of citral, doxorubicin, and combination on cytotoxicity, apoptosis, and anti-proliferative effects using human lymphoma Ramos cells. Cells were treated with doxorubicin alone or in combination with citral (10, 20, and 40 μM). Cytotoxic and apoptosis studies were done after 24 and 18 h incubations, respectively. Cytotoxic effects of citral on normal human peripheral blood mononuclear cells (PBMCs) were also investigated for its safety. Changes in the expression of BCL-2 family genes were analyzed by quantitative RT-PCR. Citral had cytotoxicity on cells with an IC50 value of 77.19 ± 4.95 µM. Citral at concentrations of 10, 20, and 40 µM additively increased the cytotoxic and apoptotic effects of doxorubicin, leading to decreased IC50 (µM) of the drug from 2.50 ± 0.01 to 2.16 ± 0.03, 1.90 ± 0.04, and 1.23 ± 0.04, respectively. Enhanced cytotoxicity was not observed in normal human PBMCs. Citral (40 µM) in combination with doxorubicin (1.5 µM) increased the expression of pro-apoptotic protein BAK but significantly decreased the expression of anti-apoptotic protein BCL-XL to 5.26-fold compared with doxorubicin-treated cells. It did not change the anti-proliferative activity of drug. Citral potentiated cytotoxicity of doxorubicin by increasing apoptotic effects. We conclude that citral may have beneficial effects in patients with B cell lymphoma treated with chemotherapy.

MODERATE CYTOTOXICITY OF PROANTHOCYANIDINS TO HUMAN TUMOR-CELL LINES

NARCIS (Netherlands)

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

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

Visfatin Reduces Gap Junction Mediated Cell-to-Cell Communication in Proximal Tubule-Derived Epithelial Cells

Directory of Open Access Journals (Sweden)

Claire E. Hills

2013-11-01

Full Text Available Background/Aims: In the current study we examined if the adipocytokine, visfatin, alters connexin-mediated intercellular communication in proximal tubule-derived epithelial cells. Methods: The effects of visfatin (10-200ng/mL on cell viability and cytotoxicity in HK2-cells were assessed by MTT, crystal violet and lactate dehydrogenase assays. Western blot analysis was used to confirm expression of Cx26, Cx40 and Cx43. The effect of visfatin (10-200ng/mL on TGF-β1 secretion was confirmed by ELISA, and the effects of both TGF-β1 (2-10ng/mL and visfatin (10-200ng/mL on connexin expression were assessed by western blot. Functional intercellular communication was determined using transfer of Lucifer Yellow and paired-whole cell patch clamp electrophysiology. Results: In low glucose (5mM, visfatin (10-200ng/mL did not affect membrane integrity, cytotoxicity or cell viability at 48hrs, but did evoke a concentration-dependent reduction in Cx26 and Cx43 expression. The expression of Cx40 was unaffected. At 48hrs, visfatin (10-200ng/mL increased the secretion of TGF-β1 and the visfatin-evoked changes in connexin expression were mimicked by exogenous application of the pro-fibrotic cytokine (2-10ng/ml. Visfatin reduced dye transfer between coupled cells and decreased functional conductance, with levels falling by 63% as compared to control. Although input resistance was increased following visfatin treatment by 166%, the change was not significant as compared to control. The effects of visfatin on Cx-expression and cell-coupling were blocked in the presence of a TGF-β1 specific neutralizing antibody. Conclusions: The adipocytokine visfatin selectively evoked a non-toxic reduction in connexin expression in HK2-cells. The loss in gap-junction associated proteins was mirrored by a loss in functional conductance between coupled cells. Visfatin increased TGF-β secretion and the pattern of change for connexins expression was mimicked by exogenous

Cytotoxic activity of methanol extracts from Basidiomycete mushrooms on murine cancer cell lines.

Science.gov (United States)

Tomasi, S; Lohézic-Le Dévéhat, F; Sauleau, P; Bézivin, C; Boustie, J

2004-04-01

Crude methanol extracts of 58 mushroom species were screened for their cytotoxic activities against two murine cancer cell lines, L1210 and 3LL, using the tetrazolium assay. A majority of extracts (74%) exhibited IC50 > 100 microg/ml against both cell lines. A most marked activity against one of the cell lines was noted for nine species (14% of the tested species). While Amanitales and Russulales tested were not found active, Polyporales and Boletales gave better results. Four species exhibited a significant cytotoxic activity (IC50 Suillus granulatus, S. luteus). The last one had never been investigated for its cytotoxic compounds before.

Mentha arvensis (Linn.-mediated green silver nanoparticles trigger caspase 9-dependent cell death in MCF7 and MDA-MB-231 cells

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Banerjee PP

2017-04-01

exhibited significant cytotoxicity toward breast cancer cells (MCF7 and MDA-MB-231, which were at par with that of CSNPs. Cell cycle analyses of MCF7 cells revealed a significant increase in sub-G1 cell population, indicating cytotoxicity of GSNPs. On the other hand, human peripheral blood lymphocytes showed significantly less cytotoxicity compared with MCF7 and MDA-MB-231 cells when treated with the same dose. Expression patterns of proteins suggested that GSNPs triggered caspase 9-dependent cell death in both cell lines. The Ames test showed that GSNPs were nonmutagenic in nature. Conclusion: GSNPs synthesized using Mentha arvensis may be considered as a promising anticancer agent in breast cancer therapy. They are less toxic and nonmutagenic and mediate caspase 9-dependent apoptosis in MCF7 and MDA-MB-231 cells. Keywords: nanoparticles, EDX, TEM, breast cancer cells, anticancer, nonmutagenic 

The cytotoxicity and genotoxicity of soluble and particulate cobalt in human lung fibroblast cells

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Smith, Leah J.; Holmes, Amie L. [Wise Laboratory of Environmental and Genetic Toxicology, University of Southern Maine, 96 Falmouth St., P.O. Box 9300, Portland, ME 04101-9300 (United States); Maine Center for Environmental Toxicology and Health, University of Southern Maine, 96 Falmouth St., P.O. Box 9300, Portland, ME 04101-9300 (United States); Department of Applied Medical Science, University of Southern Maine, 96 Falmouth St., P.O. Box 9300, Portland, ME 04101-9300 (United States); Kandpal, Sanjeev Kumar; Mason, Michael D. [Department of Chemical and Biological Engineering, University of Maine, Orono, ME (United States); Zheng, Tongzhang [Department of Environmental Health Sciences, Yale School of Public Health, New Haven, CT (United States); Wise, John Pierce, E-mail: John.Wise@usm.maine.edu [Wise Laboratory of Environmental and Genetic Toxicology, University of Southern Maine, 96 Falmouth St., P.O. Box 9300, Portland, ME 04101-9300 (United States); Maine Center for Environmental Toxicology and Health, University of Southern Maine, 96 Falmouth St., P.O. Box 9300, Portland, ME 04101-9300 (United States); Department of Applied Medical Science, University of Southern Maine, 96 Falmouth St., P.O. Box 9300, Portland, ME 04101-9300 (United States)

2014-08-01

Cobalt exposure is increasing as cobalt demand rises worldwide due to its use in enhancing rechargeable battery efficiency, super-alloys, and magnetic products. Cobalt is considered a possible human carcinogen with the lung being a primary target. However, few studies have considered cobalt-induced toxicity in human lung cells. Therefore, in this study, we sought to determine the cytotoxicity and genotoxicity of particulate and soluble cobalt in human lung cells. Cobalt oxide and cobalt chloride were used as representative particulate and soluble cobalt compounds, respectively. Exposure to both particulate and soluble cobalt induced a concentration-dependent increase in cytotoxicity, genotoxicity, and intracellular cobalt ion levels. Based on intracellular cobalt ion levels, we found that soluble cobalt was more cytotoxic than particulate cobalt while particulate and soluble cobalt induced similar levels of genotoxicity. However, soluble cobalt induced cell cycle arrest indicated by the lack of metaphases at much lower intracellular cobalt concentrations compared to cobalt oxide. Accordingly, we investigated the role of particle internalization in cobalt oxide-induced toxicity and found that particle-cell contact was necessary to induce cytotoxicity and genotoxicity after cobalt exposure. These data indicate that cobalt compounds are cytotoxic and genotoxic to human lung fibroblasts, and solubility plays a key role in cobalt-induced lung toxicity. - Highlights: • Particulate and soluble cobalt are cytotoxic and genotoxic to human lung cells. • Soluble cobalt induces more cytotoxicity compared to particulate cobalt. • Soluble and particulate cobalt induce similar levels of genotoxicity. • Particle-cell contact is required for particulate cobalt-induced toxicity.

Poly (D,L-lactide-co-glycolide nanoparticles: Uptake by epithelial cells and cytotoxicity

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J. H. Hamman

2014-03-01

Full Text Available Nanoparticles as drug delivery systems offer benefits such as protection of the encapsulated drug against degradation, site-specific targeting and prolonged blood circulation times. The aim of this study was to investigate nanoparticle uptake into Caco-2 cell monolayers, their co-localization within the lysosomal compartment and their cytotoxicity in different cell lines. Rhodamine-6G labelled poly(D,L-lactide-co-glycolide (PLGA nanoparticles were prepared by a double emulsion solvent evaporation freeze-drying method. Uptake and co-localisation of PLGA nanoparticles in lysosomes were visualized by confocal laser scanning microscopy. The cytotoxicity of the nanoparticles was evaluated on different mammalian cells lines by means of Trypan blue exclusion and the MTS assay. The PLGA nanoparticles accumulated in the intercellular spaces of Caco-2 cell monolayers, but were also taken up transcellularly into the Caco-2 cells and partially co-localized within the lysosomal compartment indicating involvement of endocytosis during uptake. PLGA nanoparticles did not show cytotoxic effects in all three cell lines. Intact PLGA nanoparticles are therefore capable of moving across epithelial cell membranes partly by means of endocytosis without causing cytotoxic effects.

Cytotoxicity of Portland cement with different radiopacifying agents: a cell death study.

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Gomes Cornélio, Ana Lívia; Salles, Loise Pedrosa; Campos da Paz, Mariana; Cirelli, Joni Augusto; Guerreiro-Tanomaru, Juliane Maria; Tanomaru Filho, Mário

2011-02-01

The aim of this study was to investigate the cytotoxicity of white Portland cement (PC) alone or associated with bismuth oxide (PCBi), zirconium oxide (PCZir), and calcium tungstate (PCCa) in 2 cell lineages. Murine periodontal ligament cells (mPDL) and rat osteosarcoma cells (ROS 17/2.8) were exposed for 24 hours to specific concentrations of fresh PC and PC associations with radiopacifiers. Zinc oxide-eugenol cement and hydrogen peroxide treatment were applied as cytotoxic positive controls. Cell viability after incubation with the cements was assessed by mitochondrial dehydrogenase enzymatic assay. Cell morphology was microscopically analyzed by cresyl violet staining, and the mechanism of cell death was determined by acridine orange/ethidium bromide methodology. All data were analyzed statistically by analysis of variance and Tukey post hoc test (P cement elutes. PC alone was not cytotoxic, even at 100 mg/mL. Microscopic images showed that none of the PC formulations caused damage to any cell lines. Statistical analysis of apoptosis/necrosis data demonstrated that PC and PC plus radiopacifying agents promoted significant necrosis cell death only at 100 mg/mL. The mPDL cells were more sensitive than ROS17/2.8. The results showed that PC associated with bismuth oxide, zirconium oxide, or calcium tungstate is not cytotoxic to mPDL or ROS17/2.8. Zirconium oxide and calcium tungstate might be good alternatives as radiopacifying agents. Copyright © 2011 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Lactic Acid Bacteria from Kefir Increase Cytotoxicity of Natural Killer Cells to Tumor Cells

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Takuya Yamane

2018-03-01

Full Text Available The Japanese fermented beverage, homemade kefir, contains six lactic acid bacteria: Lactococcus. lactis subsp. Lactis, Lactococcus. lactis subsp. Cremoris, Lactococcus. Lactis subsp. Lactis biovar diacetylactis, Lactobacillus plantarum, Leuconostoc meseuteroides subsp. Cremoris and Lactobacillus casei. In this study, we found that a mixture of the six lactic acid bacteria from kefir increased the cytotoxicity of human natural killer KHYG-1 cells to human chronic myelogenous leukemia K562 cells and colorectal tumor HCT116 cells. Furthermore, levels of mRNA expression and secretion of IFN-γ (interferon gamma increased in KHYG-1 cells that had been treated with the six lactic acid bacteria mixture from kefir. The results suggest that the six lactic acid bacteria mixture from kefir has strong effects on natural immunity and tumor cell cytotoxicity.

Pha-induced T-cell cytotoxity. Mechanism and application in haemodialysis and renal transplant patients.

NARCIS (Netherlands)

Huges-Wirawan, Gladys Ratna Widhi Indrati

1978-01-01

This thesis describes a method to measure PHA-incluced cytotoxicity of human lymphocytes (nonspecific T-cell cytotoxicity), using 3H-thymidine prelabelled target cells (HeLa cells). The method has some advantages over the widely used 51Cr-release assay. Its application in two clinical conditions is

Upregulation of adhesion molecules on leukemia targets improves the efficacy of cytotoxic T cells transduced with chimeric anti-CD19 receptor.

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Laurin, David; Marin, Virna; Biagi, Ettore; Pizzitola, Irene; Agostoni, Valentina; Gallot, Géraldine; Vié, Henri; Jacob, Marie Christine; Chaperot, Laurence; Aspord, Caroline; Plumas, Joël

2013-04-01

T lymphocytes engineered to express chimeric antigen receptors (CARs) interact directly with cell surface molecules, bypassing MHC antigen presentation dependence. We generated human anti-CD19ζ CAR cytotoxic T lymphocytes and cytokine-induced killer cells and studied their sensitivity to the expression of adhesion molecules for the killing of primary B-lineage acute lymphoblastic leukemia (B-ALL) targets. Despite a very low basal expression of surface adhesion molecules, B-ALL blasts were lysed by the anti-CD19ζ-CAR transduced effectors as expected. We next investigated the regulatory role of adhesion molecules during CAR-mediated cytolysis. The blocking of these accessory molecules strongly limited the chimeric effector's cytotoxicity. Thereafter, B-ALL cells surface adhesion molecule level expression was induced by IFN-γ or by the combined use of CD40L and IL-4 and the cells were submitted to anti-CD19ζ-CAR transduced effectors lysis. Upregulation of adhesion molecules expression by blasts potentiated their killing. The improved cytotoxicity observed was dependent on target surface expression of adhesion molecules, particularly CD54. Taken together, these results indicate that adhesion molecules, and principally CD54, are involved in the efficiency of recognition by effector chimeric ζ. These observations have potential implications for the design of immunotherapy treatment approaches for hematological malignancies and tumors based on the adoption of CAR effector cells.

Size-dependent cytotoxicity of Fe3O4 nanoparticles induced by biphasic regulation of oxidative stress in different human hepatoma cells

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Xie Y

2016-07-01

Full Text Available Yuexia Xie,1,2,* Dejun Liu,3,* Chenlei Cai,1,* Xiaojing Chen,1 Yan Zhou,1 Liangliang Wu,1 Yongwei Sun,3 Huili Dai,1,2 Xianming Kong,1,2 Peifeng Liu1,2 1Central Laboratory, 2State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, 3Department of Biliary-Pancreatic Surgery, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, People’s Republic of China *These authors contributed equally to this work Abstract: The application of Fe3O4 nanoparticles (NPs has made great progress in the diagnosis of disease and in the drug delivery system for cancer therapy, but the relative mecha­nisms of potential toxicity induced by Fe3O4 have not kept pace with its development in the application, which has hampered its further clinical application. In this article, we used two kinds of human hepatoma cell lines, SK-Hep-1 and Hep3B, to investigate the cytotoxic effects and the involved mechanisms of small Fe3O4 NPs with different diameters (6 nm, 9 nm, and 14 nm. Results showed that the size of NPs effectively influences the cytotoxicity of hepatoma cells: 6 nm Fe3O4 NPs exhibited negligible cytotoxicity and 9 nm Fe3O4 NPs affected cytotoxicity via cellular mitochondrial dysfunction and by inducing necrosis mediated through the mitochondria-dependent intracellular reactive oxygen species generation. Meanwhile, 14 nm Fe3O4 NPs induced cytotoxicity by impairing the integrity of plasma membrane and promoting massive lactate dehydrogenase leakage. These results explain the detailed mechanism of different diameters of small Fe3O4 NPs-induced cytotoxicity. We anticipate that this study will provide different insights into the cytotoxicity mechanism of Fe3O4 NPs, so as to make them safer to use in clinical application. Keywords: hepatoma cells, nanoparticles, cytotoxicity, mechanism, oxidative stress

Enhanced CDC of B cell chronic lymphocytic leukemia cells mediated by rituximab combined with a novel anti-complement factor H antibody.

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Mark T Winkler

Full Text Available Rituximab therapy for B cell chronic lymphocytic leukemia (B-CLL has met with mixed success. Among several factors to which resistance can be attributed is failure to activate complement dependent cytotoxicity (CDC due to protective complement regulatory proteins, including the soluble regulator complement factor H (CFH. We hypothesized that rituximab killing of non-responsive B-CLL cells could be augmented by a novel human monoclonal antibody against CFH. The B cells from 11 patients with B-CLL were tested ex vivo in CDC assays with combinations of CFH monoclonal antibody, rituximab, and a negative control antibody. CDC of rituximab non-responsive malignant B cells from CLL patients could in some cases be augmented by the CFH monoclonal antibody. Antibody-mediated cytotoxicity of cells was dependent upon functional complement. In one case where B-CLL cells were refractory to CDC by the combination of rituximab plus CFH monoclonal antibody, additionally neutralizing the membrane complement regulatory protein CD59 allowed CDC to occur. Inhibiting CDC regulatory proteins such as CFH holds promise for overcoming resistance to rituximab therapy in B-CLL.

Cytotoxicity of titanium dioxide nanoparticles in mouse fibroblast cells.

Science.gov (United States)

Jin, Cheng-Yu; Zhu, Bang-Shang; Wang, Xue-Feng; Lu, Qing-Hua

2008-09-01

Nanotitanium dioxide (TiO2) is an important industrial material that is widely used as an additive in cosmetics, pharmaceuticals, and food colorants. Although the small size of the TiO2 nanoparticle is useful in various applications, the biosafety of this material needs to be evaluated. In this study, mouse fibroblast (L929) cells were used to evaluate the cytotoxicity of different concentrations (3-600 microg/mL) of homogeneous and weakly aggregated TiO2 nanoparticles in aqueous solution. The L929 cells became round and even shrank as the concentration of TiO2 nanoparticles increased. Moreover, TiO2 nanoparticle-treated cells had condensed fragmented chromatin or were directly necrosed, as observed by acridine orange (AO) staining. The transmission electron microscopy (TEM) analysis showed that in cells cultured in a medium containing 300 microg/mL TiO2, the number of lysosomes increased, and some cytoplasmic organelles were damaged. In addition, there was a significant increase in oxidative stress at higher TiO2 nanoparticle concentrations (>60 microg/mL). As the concentration of TiO2 nanoparticles increased in the culture medium, the levels of reactive oxygen species (ROS) and lactate dehydrogenase (LDH) increased, while those of methyl tetrazolium cytotoxicity (MTT), glutathione (GSH), and superoxide dismutase (SOD) decreased. A possible mechanism for the cytotoxicity of TiO2 nanoparticles is also discussed.

Bacterial CpG-DNA activates dendritic cells in vivo: T helper cell-independent cytotoxic T cell responses to soluble proteins.

Science.gov (United States)

Sparwasser, T; Vabulas, R M; Villmow, B; Lipford, G B; Wagner, H

2000-12-01

Receptors for conserved molecular patterns associated with microbial pathogens induce synthesis of co-stimulatory molecules and cytokines in immature dendritic cells (DC), as do antigen-reactive CD4 T helper cells via CD40 signaling. Once activated, antigen-presenting DC may activate CD8 T cell responses in a T helper cell-independent fashion. Using immunostimulatory CpG-oligonucleotides (ODN) mimicking bacterial CpG-DNA, we tested whether CpG-DNA bypasses the need for T helper cells in CTL responses towards proteins by directly activating antigen-presenting DC to transit into professional APC. We describe that immature DC in situ constitutively process soluble proteins and generate CD8 T cell determinants yet CD8 T cell responses remain abortive. Induction of primary antigen-specific CD8 cytotoxic T lymphocyte (CTL)-mediated responses becomes initiated in wild-type as well as T helper cell-deficient mice, provided soluble protein and CpG-ODN are draining into the same lymph node. Specifically we show that CpG-ODN trigger antigen-presenting immature DC within the draining lymph node to acutely up-regulate co-stimulatory molecules and produce IL-12. These results provide new insights for generating in vivo efficient CTL responses to soluble proteins which may influence vaccination strategies.

Fascin-1 knock-down of human glioma cells reduces their microvilli/filopodia while improving their susceptibility to lymphocyte-mediated cytotoxicity

Science.gov (United States)

Hoa, Neil T; Ge, Lisheng; Erickson, Kate L; Kruse, Carol A; Cornforth, Andrew N; Kuznetsov, Yurii; McPherson, Alex; Martini, Filippo; Jadus, Martin R

2015-01-01

Cancer cells derived from Glioblastoma multiforme possess membranous protrusions allowing these cells to infiltrate surrounding tissue, while resisting lymphocyte cytotoxicity. Microvilli and filopodia are supported by actin filaments cross-linked by fascin. Fascin-1 was genetically silenced within human U251 glioma cells; these knock-down glioma cells lost their microvilli/filopodia. The doubling time of these fascin-1 knock-down cells was doubled that of shRNA control U251 cells. Fascin-1 knock-down cells lost their transmigratory ability responding to interleukin-6 or insulin-like growth factor-1. Fascin-1 silenced U251 cells were more easily killed by cytolytic lymphocytes. Fascin-1 knock-down provides unique opportunities to augment glioma immunotherapy by simultaneously targeting several key glioma functions: like cell transmigration, cell division and resisting immune responses. PMID:25901196

Cytotoxicity of TSP in 3D Agarose Gel Cultured Cell.

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Song-I Chun

Full Text Available A reference reagent, 3-(trimethylsilyl propionic-2, 2, 3, 3-d4 acid sodium (TSP, has been used frequently in nuclear magnetic resonance (NMR and magnetic resonance spectroscopy (MRS as an internal reference to identify cell and tissue metabolites, and determine chemical and protein structures. This reference material has been exploited for the quantitative and dynamic analyses of metabolite spectra acquired from cells. The aim of this study was to evaluate the cytotoxicity of TSP on three-dimensionally, agarose gel, cultured cells.A human osteosarcoma cell line (MG-63 was selected, and cells were three dimensionally cultured for two weeks in an agarose gel. The culture system contained a mixture of conventional culture medium and various concentrations (0, 1, 3, 5, 7, 10, 20 30 mM of TSP. A DNA quantification assay was conducted to assess cell proliferation using Quant-iT PicoGreen dsDNA reagent and kit, and cell viability was determined using a LIVE/DEAD Viability/Cytotoxicity kit. Both examinations were performed simultaneously at 1, 3, 7 and 14 days from cell seeding.In this study, the cytotoxicity of TSP in the 3D culture of MG-63 cells was evaluated by quantifying DNA (cell proliferation and cell viability. High concentrations of TSP (from 10 to 30 mM reduced both cell proliferation and viability (to 30% of the control after one week of exposure, but no such effects were found using low concentrations of TSP (0-10 mM.This study shows that low concentrations of TSP in 3D cell culture medium can be used for quantitative NMR or MRS examinations for up to two weeks post exposure.

Serine esterase and hemolytic activity in human cloned cytotoxic T lymphocytes

OpenAIRE

1988-01-01

Target cell lysis by most murine cytotoxic T lymphocytes appears to be mediated by a complement (C9)-like protein called perforin, contained in high-density cytoplasmic granules. These granules also contain high levels of serine esterase activity, which may also play a role in cytolysis. Analysis of 17 cloned human cytotoxic T lymphocytes revealed the presence of serine esterase that is very similar to its murine counterpart in substrate and inhibitor specificities, pH optimum, and molecular ...

Klebsiella pneumoniae triggers a cytotoxic effect on airway epithelial cells

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Llobet-Brossa Enrique

2009-08-01

Full Text Available Abstract Background Klebsiella pneumoniae is a capsulated Gram negative bacterial pathogen and a frequent cause of nosocomial infections. Despite its clinical relevance, little is known about the features of the interaction between K. pneumoniae and lung epithelial cells on a cellular level, neither about the role of capsule polysaccharide, one of its best characterised virulence factors, in this interaction. Results The interaction between Klebsiella pneumoniae and cultured airway epithelial cells was analysed. K. pneumoniae infection triggered cytotoxicity, evident by cell rounding and detachment from the substrate. This effect required the presence of live bacteria and of capsule polysaccharide, since it was observed with isolates expressing different amounts of capsule and/or different serotypes but not with non-capsulated bacteria. Cytotoxicity was analysed by lactate dehydrogenase and formazan measurements, ethidium bromide uptake and analysis of DNA integrity, obtaining consistent and complementary results. Moreover, cytotoxicity of non-capsulated strains was restored by addition of purified capsule during infection. While a non-capsulated strain was avirulent in a mouse infection model, capsulated K. pneumoniae isolates displayed different degrees of virulence. Conclusion Our observations allocate a novel role to K. pneumoniae capsule in promotion of cytotoxicity. Although this effect is likely to be associated with virulence, strains expressing different capsule levels were not equally virulent. This fact suggests the existence of other bacterial requirements for virulence, together with capsule polysaccharide.

Cytotoxicity and genotoxicity of intravitreal adalimumab administration in rabbit retinal cells

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Álcio Coutinho de Paula

2015-04-01

Full Text Available Purpose: To assess the cytotoxicity and genotoxicity of intravitreal adalimumab treatment in an animal experimental model using cytological and molecular techniques. Methods: Eighteen rabbits were randomly assigned to three groups: control, adalimumab treatment, and placebo. Cytotoxicity on retinal cells was evaluated using flow cytometry assays to determine the level of apoptosis and necrosis. Genotoxicity was evaluated by comet assays to assess DNA damage, and quantitative real-time polymerase chain reaction (qPCR was used to evaluate expression of apoptosis-inducing caspases (8 and 3. Results: No cytotoxicity or genotoxicity was observed in any of the two treatment groups (adalimumab and placebo following intravitreal administration compared with the control group. Flow cytometry analysis revealed that more than 90% of the cells were viable, and only a low proportion of retinal cells presented apoptotic (~10% or necrotic (<1% activity across all groups. Molecular damage was also low with a maximum of 6.4% DNA degradation observed in the comet assays. In addition, no increase in gene expression of apoptosis-inducing caspases was observed on retinal cells by qPCR in both the adalimumab and placebo groups compared with the control group. Conclusion: The use of adalimumab resulted in no detectable cytotoxicity or genotoxicity on retinal cells for up to 60 days upon administration. These results therefore indicate that adalimumab may be a safe option for intravitreal application to treat ocular inflammatory diseases in which TNF-α is involved.

Relationship between spermatogonial stem cell survival and testis function after cytotoxic therapy

International Nuclear Information System (INIS)

Meistrich, M.L.

1986-01-01

This review, with substantial bibliography, concludes that the acute effects of radiation and cytotoxic drugs are a result of toxicity to the most sensitive of the germ cells, which in most cases are the differentiating spermatogonia. Long-term sterility or reduction in sperm production depends directly on killing of spermatogonial stem cells. For a variety of cytotoxic agents, the same relationship holds between the stem cell survival index and the prompt recovery level of sperm production (at 56 days), the maximal recovered level of sperm production, and the time required for fertility to return. It also appears that the spermatogonial stem cell is the target for long-term sterility in man following cytotoxic therapy. It is not known whether the delay in recovery is a result of this direct damage to the stem cell or an effect on another target cell in the tissue. Data obtained in both experimental animals and man indicate very little direct changes in the secretory cells and other stromal cells of the testis, and there is no evidence as yet that any alterations in these cells adversely affect sperm production or reproductive performance. (UK)

Tumor-specific CD4+ T cells develop cytotoxic activity and eliminate virus-induced tumor cells in the absence of regulatory T cells.

Science.gov (United States)

Akhmetzyanova, Ilseyar; Zelinskyy, Gennadiy; Schimmer, Simone; Brandau, Sven; Altenhoff, Petra; Sparwasser, Tim; Dittmer, Ulf

2013-02-01

The important role of tumor-specific cytotoxic CD8(+) T cells is well defined in the immune control of the tumors, but the role of effector CD4(+) T cells is poorly understood. In the current research, we have used a murine retrovirus-induced tumor cell line of C57BL/6 mouse origin, namely FBL-3 cells, as a model to study basic mechanisms of immunological control and escape during tumor formation. This study shows that tumor-specific CD4(+) T cells are able to protect against virus-induced tumor cells. We show here that there is an expansion of tumor-specific CD4(+) T cells producing cytokines and cytotoxic molecule granzyme B (GzmB) in the early phase of tumor growth. Importantly, we demonstrate that in vivo depletion of regulatory T cells (Tregs) and CD8(+) T cells in FBL-3-bearing DEREG transgenic mice augments IL-2 and GzmB production by CD4(+) T cells and increases FV-specific CD4(+) T-cell effector and cytotoxic responses leading to the complete tumor regression. Therefore, the capacity to reject tumor acquired by tumor-reactive CD4(+) T cells largely depends on the direct suppressive activity of Tregs. We suggest that a cytotoxic CD4(+) T-cell immune response may be induced to enhance resistance against oncovirus-associated tumors.

Cytotoxic action of Brazilian propolis in vitro on canine osteosarcoma cells.

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Cinegaglia, N C; Bersano, P R O; Búfalo, M C; Sforcin, J M

2013-09-01

Osteosarcoma (OSA) is a primary bone neoplasm frequently diagnosed in dogs. The biology of OSA in pet dogs is identical to that of pediatric patients, and it has been considered an excellent model in vivo to study human OSA. Since the individual response to chemotherapy is unpredictable and considering that propolis is a natural product with several biological properties, this work evaluated the cytotoxic action of propolis on canine OSA cells. The primary cell culture of canine OSA was obtained from the tumor of a dog with OSA. Cell viability was assessed after incubation with propolis, 70% ethanol (propolis solvent), and carboplatin after 6, 24, 48, and 72 h. Cell viability was analyzed by the crystal violet method. Data showed that canine OSA cells were sensitive to propolis in a dose- and time-dependent manner and had a distinct morphology compared to control. Its solvent (70% ethanol) had no effect on cell viability, suggesting that the cytotoxic action was exclusively due to propolis. Our propolis sample exerted a cytotoxic effect on canine OSA cells, and its introduction as a possible therapeutic agent in vivo could be investigated, providing a new contribution to OSA treatment. Copyright © 2012 John Wiley & Sons, Ltd.

Nitric oxide-releasing nanoparticles: synthesis, characterization, and cytotoxicity to tumorigenic cells

Energy Technology Data Exchange (ETDEWEB)

Pelegrino, Milena T. [Universidade Federal de São Paulo, Exact and Earth Sciences Department (Brazil); Silva, Letícia C.; Watashi, Carolina M. [Universidade Federal do ABC, UFABC, Center of Natural and Human Sciences (Brazil); Haddad, Paula S. [Universidade Federal de São Paulo, Exact and Earth Sciences Department (Brazil); Rodrigues, Tiago; Seabra, Amedea B., E-mail: amedea.seabra@ufabc.edu.br [Universidade Federal do ABC, UFABC, Center of Natural and Human Sciences (Brazil)

2017-02-15

Nitric oxide (NO) is involved in several biological processes, including toxicity against tumor cells. The aim of this study was to synthesize, characterize, and evaluate the cytotoxicity of NO-releasing chitosan nanoparticles. A thiol-containing molecule, mercaptosuccinic acid (MSA), was encapsulated (encapsulation efficiency of 99%) in chitosan/sodium tripolyphosphate nanoparticles (CS NPs). The obtained nanoparticles showed an average hydrodynamic size of 108.40 ± 0.96 nm and polydispersity index of 0.26 ± 0.01. MSA-CS NPs were nitrosated leading to S-nitroso-MSA-CS NPs, which act as NO donor. The cytotoxicity of CS NPs, MSA-CS NPs, and S-nitroso-MSA-CS NPs were evaluated in several tumor cells, including human hepatocellular carcinoma (HepG2), mouse melanoma (B16F10), and human chronic myeloid leukemia (K562) cell lines and Lucena-1, a vincristine-resistant K562 cell line. Both CS NPs and MSA-CS NPs did not cause toxic effects in these cells, whereas S-nitroso-MSA-CS NPs caused potent cytotoxic effects in all the tested tumor cell lines. The half-maximal inhibitory concentration values of S-nitroso-MSA-CS NPs were 19.7, 10.5, 22.8, and 27.8 μg·mL{sup −1} for HepG2, B16F10, K562, and Lucena-1 cells, respectively. In contrast, S-nitroso-MSA-CS NPs exhibited lower cytotoxic to non-tumorigenic melanocytes (Melan-A) when compared with melanoma B16F10. Therefore, the results highlight the potential use of NO-releasing CS NPs in antitumor chemotherapy.

Cytotoxicity Effects of Amoora rohituka and chittagonga on Breast and Pancreatic Cancer Cells

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Leo L. Chan

2011-01-01

Full Text Available Chemotherapeutic agents for cancer are highly toxic to healthy tissues and hence alternative medicine avenues are widely researched. Majority of the recent studies on alternative medicine suggested that Amoora rohituka possesses considerable antitumor and antibacterial properties. In this work, rohituka and chittagonga, fractionated with petroleum ether, dichloromethane, and ethanol, were explored for their anticancer potential against two breast cancer (MCF-7 and HTB-126 and three pancreatic cancer (Panc-1, Mia-Paca2, and Capan1. The human foreskin fibroblast, Hs68, was also included. Cytotoxicity of each extract was analyzed using the MTT assay and label-free photonic crystal biosensor assay. A concentration series of each extract was performed on the six cell lines. For MCF-7 cancer cells, the chittagonga (Pet-Ether and CH2Cl2 and rohituka (Pet-Ether extracts induced cytotoxicity; the chittagonga (EtoAC and rohituka (MeOH extracts did not induce cytotoxicity. For HTB126, Panc-1, Mia-Paca2, and Capan-1 cancer cells, only the chittagonga CH2Cl2 extract showed a significant cytotoxic effect. The extracts were not cytotoxic to normal fibroblast Hs68 cells, which may be correlated to the specificity of Amoora extracts in targeting cancerous cells. Based on these results, further examination of the potential anticancer properties Amoora species and the identification of the active ingredients of these extracts is warranted.

Suppression of cytotoxic T lymphocytes by carrageenan-activated macrophage-like cells

International Nuclear Information System (INIS)

Yung, Y.P.; Cudkowicz, G.

1978-01-01

In the presence of 100 μg/ml of carrageenans (CAR), B6D2F 1 responder spleen cells failed to generate antiparent or anti-allogeneic cytotoxic T lymphocytes in vitro, but instead generated suppressor cells. Cultured CAR-treated cells added to mixtures of B6D2F 1 anti-B6 or B6D2F 1 anti-C3H cytotoxic effectors (induced in vitro) and the appropriate 51 Cr-labeled lymphoma targets reduced or abolished cytolysis (measured as 51 Cr release) depending on the ratio of suppressor to effector cells. Cultured spleen cells not exposed to CAR failed to inhibit both types of cytotoxicity. Presuppressor cells were associated with a splenic subpopulation independent of the thymus (i.e., present in spleens of athymic nude mice), were moderately adherent to Sephadex G-10 columns, but were not phagocytic or ''sticky'' to carbonyl iron particles. Activation of such cells by CAR was not prevented by in vitro exposure to 2000 rads of γ-rays before culture, nor facilitated by antigenic stimulation. The matured suppressor cells remained radioresistant and became strongly adherent to Sephadex G-10. The suppressors lacked surface Thy-1 alloantigen detectable by antibody and rabbit complement. Suppressor cell activity was not restricted by the immunologic specificity and major histocompatibility type of effectors

The 3 major types of innate and adaptive cell-mediated effector immunity.

Science.gov (United States)

Annunziato, Francesco; Romagnani, Chiara; Romagnani, Sergio

2015-03-01

The immune system has tailored its effector functions to optimally respond to distinct species of microbes. Based on emerging knowledge on the different effector T-cell and innate lymphoid cell (ILC) lineages, it is clear that the innate and adaptive immune systems converge into 3 major kinds of cell-mediated effector immunity, which we propose to categorize as type 1, type 2, and type 3. Type 1 immunity consists of T-bet(+) IFN-γ-producing group 1 ILCs (ILC1 and natural killer cells), CD8(+) cytotoxic T cells (TC1), and CD4(+) TH1 cells, which protect against intracellular microbes through activation of mononuclear phagocytes. Type 2 immunity consists of GATA-3(+) ILC2s, TC2 cells, and TH2 cells producing IL-4, IL-5, and IL-13, which induce mast cell, basophil, and eosinophil activation, as well as IgE antibody production, thus protecting against helminthes and venoms. Type 3 immunity is mediated by retinoic acid-related orphan receptor γt(+) ILC3s, TC17 cells, and TH17 cells producing IL-17, IL-22, or both, which activate mononuclear phagocytes but also recruit neutrophils and induce epithelial antimicrobial responses, thus protecting against extracellular bacteria and fungi. On the other hand, type 1 and 3 immunity mediate autoimmune diseases, whereas type 2 responses can cause allergic diseases. Copyright © 2014 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Antibody-dependent cellular cytotoxicity (ADCC) activity of a novel anti-PD-L1 antibody avelumab (MSB0010718C) on human tumor cells

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Fantini, Massimo; Heery, Christopher R.; Gulley, James L.; Tsang, Kwong Yok; Schlom, Jeffrey

2015-01-01

Several anti-PD1/PD-L1 monoclonal antibodies (MAb) are currently providing evidence of clinical benefit in subsets of cancer patients. The mode of action of these MAbs is to inhibit PD1 on immune cells interacting with PD-L1 on tumor cells. These MAbs are either designed or engineered to eliminate antibody-dependent cell-mediated cytotoxicity (ADCC), which, however, has been implicated as an important mechanism in several highly effective MAb-mediated cancer therapies. A fully human anti-PD-L1 MAb would potentially be able to block PD-L1/PD1 interactions and also mediate the ADCC lysis of tumor cells. MSB0010718C (designated avelumab) is a fully human IgG1 anti-PD-L1 MAb. The studies reported here demonstrate (a) the ability of avelumab to lyse a range of human tumor cells in the presence of PBMC or NK effectors; (b) IFNγ can enhance tumor cell PD-L1 expression and in some cases enhance ADCC tumor cell lysis; (c) purified NK cells are potent effectors for avelumab; (d) similar levels of avelumab-mediated ADCC lysis of tumor cells are seen using purified NK as effectors from either healthy donors or cancer patients; (e) very low levels of avelumab-mediated lysis are seen using whole PBMCs as targets; this finding complements results seen in analyses of PBMC subsets of patients receiving avelumab; and (f) the addition of IL12 to NK cells greatly enhances avelumab-mediated ADCC. These studies thus provide an additional mode of action for an anti-PD-L1 MAb and support the rationale for further studies to enhance avelumab-mediated ADCC activity. PMID:26014098

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

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

2015-01-01

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

Cytotoxic, genotoxic and cell-cycle disruptive effects of thio-dimethylarsinate in cultured human cells and the role of glutathione

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Ochi, Takafumi [Laboratory of Toxicology, Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa 229-0195 (Japan); Kita, Kayoko; Suzuki, Toshihide [Laboratory of Toxicology, Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa 229-0195 (Japan); Rumpler, Alice; Goessler, Walter; Francesconi, Kevin A [Karl-Franzens University Graz, Institute of Chemistry-Analytical Chemistry, Universitaetsplatz 1, 8010 Graz (Austria)

2008-04-01

Thio-dimethylarsinate (thio-DMA), a recently discovered urine metabolite in humans, was investigated for its cytotoxic, genotoxic and cell-cycle disruptive effects in the cultured human hepatocarcinoma cell line, HepG2, and Syrian hamster embryo cells. In addition, the role of glutathione (GSH) on the cytotoxic effects of thio-DMA was investigated in terms of the effects of GSH depletion and the effects of exogenously added GSH. LC{sub 50} values of arsenicals for cells incubated for 48 h were 0.026 mM for thio-DMA, 0.343 mM for DMA and 3.66 mM for dithio-DMA. Depletion of cell GSH reduced the cytotoxic effects of thio-DMA. The cytotoxic effects of 0.02 mM and 0.05 mM thio-DMA were enhanced markedly when used in combination with 1 to 3 mM GSH, but decreased again when combined with 5 mM GSH. These results suggested that cytotoxic intermediates were generated by the interaction of thio-DMA with GSH, while an excessive amount of GSH suppressed the generation of these intermediates. Flow-cytometry showed that thio-DMA was an inducer of cells with 4N DNA and hypo 2N DNA. The results also demonstrated that cells arrested in the mitotic phase had abnormalities in their spindle organization and centrosome integrity. In addition, cells arrested in mitosis by thio-DMA had chromosome structural aberrations, such as chromatid gaps, chromatid breaks and chromatid exchanges. Moreover, the cytotoxic effects of thio-DMA may in part be associated with an apoptotic mode of cell death that was evaluated by the appearance of nucleosome level DNA fragmentations and an 85-kDa cleavage fragment of poly (ADP-ribose) polymerase. These findings suggest that the presence of thio-DMA in human urine has implications for human health in terms of arsenic metabolism and toxicity.

Cytotoxic effect of the Her-2/Her-1 inhibitor PKI-166 on renal cancer cells expressing the connexin 32 gene.

Science.gov (United States)

Fujimoto, Eriko; Yano, Tomohiro; Sato, Hiromi; Hagiwara, Kiyokazu; Yamasaki, Hiroshi; Shirai, Sumiko; Fukumoto, Keiko; Hagiwara, Hiromi; Negishi, Etsuko; Ueno, Koichi

2005-02-01

We have reported that connexin (Cx) 32 acts as a tumor suppressor gene in renal cancer cells partly due to Her-2 inactivation. Here, we determined if a Her-2/Her-1 inhibitor (PKI-166) can enhance the tumor-suppressive effect of Cx32 in Caki-2 cells from human renal cell carcinoma. The expression of Cx32 in Caki-2 cells was required for PKI-166-induced cytotoxic effect at lower doses. The cyctotoxicity was dependent on the occurrence of apoptosis and partly mediated by Cx32-driven gap junction intercellular communications. These results suggest that PKI-166 further supports the tumor-suppressive effect of the Cx32 gene in renal cancer cells through the induction of apoptosis.

Antibody-Dependent Cellular Cytotoxicity Activity of a Novel Anti-PD-L1 Antibody Avelumab (MSB0010718C) on Human Tumor Cells.

Science.gov (United States)

Boyerinas, Benjamin; Jochems, Caroline; Fantini, Massimo; Heery, Christopher R; Gulley, James L; Tsang, Kwong Yok; Schlom, Jeffrey

2015-10-01

Several anti-PD-1/PD-L1 monoclonal antibodies (mAb) are currently providing evidence of clinical benefit in subsets of cancer patients. The mode of action of these mAbs is to inhibit PD-1 on immune cells interacting with PD-L1 on tumor cells. These mAbs are either designed or engineered to eliminate antibody-dependent cell-mediated cytotoxicity (ADCC), which, however, has been implicated as an important mechanism in several highly effective mAb-mediated cancer therapies. A fully human anti-PD-L1 mAb would potentially be able to block PD-1/PD-L1 interactions and also mediate the ADCC lysis of tumor cells. MSB0010718C (designated avelumab) is a fully human IgG1 anti-PD-L1 mAb. The studies reported here demonstrate (i) the ability of avelumab to lyse a range of human tumor cells in the presence of PBMC or NK effectors; (ii) IFNγ can enhance tumor cell PD-L1 expression and, in some cases, enhance ADCC tumor cell lysis; (iii) purified NK cells are potent effectors for avelumab; (iv) similar levels of avelumab-mediated ADCC lysis of tumor cells are seen using purified NK as effectors from either healthy donors or cancer patients; (v) very low levels of avelumab-mediated lysis are seen using whole PBMCs as targets; this finding complements results seen in analyses of PBMC subsets of patients receiving avelumab; and (vi) the addition of IL12 to NK cells greatly enhances avelumab-mediated ADCC. These studies thus provide an additional mode of action for an anti-PD-L1 mAb and support the rationale for further studies to enhance avelumab-mediated ADCC activity. ©2015 American Association for Cancer Research.

Dual functions of silver nanoparticles in F9 teratocarcinoma stem cells, a suitable model for evaluating cytotoxicity- and differentiation-mediated cancer therapy

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Han JW

2017-10-01

Full Text Available Jae Woong Han, Sangiliyandi Gurunathan, Yun-Jung Choi, Jin-Hoi Kim Department of Stem Cell and Regenerative Biotechnology, Humanized Pig Research Center (SRC, Konkuk University, Seoul, Republic of Korea Background: Silver nanoparticles (AgNPs exhibit strong antibacterial and anticancer activity owing to their large surface-to-volume ratios and crystallographic surface structure. Owing to their various applications, understanding the mechanisms of action, biological interactions, potential toxicity, and beneficial effects of AgNPs is important. Here, we investigated the toxicity and differentiation-inducing effects of AgNPs in teratocarcinoma stem cells. Materials and methods: AgNPs were synthesized and characterized using various analytical techniques such as UV–visible spectroscopy, X-ray diffraction, energy-dispersive X-ray spectroscopy, and transmission electron microscopy. The cellular responses of AgNPs were analyzed by a series of cellular and biochemical assays. Gene and protein expressions were analyzed by reverse transcription-quantitative polymerase chain reaction and western blotting, respectively. Results: The AgNPs showed typical crystalline structures and spherical shapes (average size =20 nm. High concentration of AgNPs induced cytotoxicity in a dose-dependent manner by increasing lactate dehydrogenase leakage and reactive oxygen species. Furthermore, AgNPs caused mitochondrial dysfunction, DNA fragmentation, increased expression of apoptotic genes, and decreased expression of antiapoptotic genes. Lower concentrations of AgNPs induced neuronal differentiation by increasing the expression of differentiation markers and decreasing the expression of stem cell markers. Cisplatin reduced the viability of F9 cells that underwent AgNPs-induced differentiation. Conclusion: The results showed that AgNPs caused differentially regulated cytotoxicity and induced neuronal differentiation of F9 cells in a concentration-dependent manner

FOXO3a mediates the cytotoxic effects of cisplatin in colon cancer cells

OpenAIRE

de Mattos, Silvia Fernández; Villalonga, Priam; Clardy, Jon; Lam, Eric W-F

2008-01-01

Cisplatin is a conventional chemotherapeutic agent that binds covalently to purine DNA bases and mediates cellular apoptosis. A better understanding of the downstream cellular targets of cisplatin will provide information on its mechanism of action and help to understand the mechanism of drug resistance. In this study, we have investigated the effects of cisplatin in a panel of colon carcinoma cell lines and the involvement of the PI3K/FOXO pathway in cisplatin action and resistance. Cisplati...

Nyctanthes arbortristis mediated synthesis of silver nanoparticles: Cytotoxicity assay against THP-1 human leukemia cell lines

Science.gov (United States)

Kumari, Priti; Kumari, Niraj; Jha, Anal K.; Singh, K. P.; Prasad, K.

2018-05-01

Green synthesis, characterizations and applications of nanoparticles have become an important branch of nanotechnology now a day. In this paper, green synthesis of silver nanoparticles (AgNPs) using the aqueous extract of Nyctanthes arbortristis as a reducing and stabilizing agent, has been discussed. Present synthetic method is very handy, cost-effective and reproducible. Formation of AgNPs was characterized by X-ray diffraction, dynamic light scattering, scanning electron microscopy and UV-visible spectroscopy techniques. The phytochemicals responsible for nano-transformation were principally flavonoids, phenols and glycosides present in the leaves. Further, the dose dependent cytotoxicity assay of biosynthesized AgNPs against THP-1 human leukemia cell lines showed the encouraging results.

Cytotoxic effects of air freshener biocides in lung epithelial cells.

Science.gov (United States)

Kwon, Jung-Taek; Lee, Mimi; Seo, Gun-Baek; Kim, Hyun-Mi; Shim, Ilseob; Lee, Doo-Hee; Kim, Taksoo; Seo, Jung Kwan; Kim, Pilje; Choi, Kyunghee

2013-09-01

This study evaluated the cytotoxicity of mixtures of citral (CTR) and either benzisothiazolinone (BIT, Mix-CTR-BIT) or triclosan (TCS, Mix-CTR-TCS) in human A549 lung epithelial cells. We investigated the effects of various mix ratios of these common air freshener ingredients on cell viability, cell proliferation, reactive oxygen species (ROS) generation, and DNA damage. Mix-CTR-BIT and Mix-CTR-TCS significantly decreased the viability of lung epithelial cells and inhibited cell growth in a dose-dependent manner. In addition, both mixtures increased ROS generation, compared to that observed in control cells. In particular, cell viability, growth, and morphology were affected upon increase in the proportion of BIT or TCS in the mixture. However, comet analysis showed that treatment of cells with Mix-CTR-BIT or Mix-CTR-TCS did not increase DNA damage. Taken together, these data suggested that increasing the content of biocides in air fresheners might induce cytotoxicity, and that screening these compounds using lung epithelial cells may contribute to hazard assessment.

The Influences of Cell Type and ZnO Nanoparticle Size on Immune Cell Cytotoxicity and Cytokine Induction

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Thurber Aaron

2009-01-01

Full Text Available Abstract Nanotechnology represents a new and enabling platform that promises to provide a range of innovative technologies for biological applications. ZnO nanoparticles of controlled size were synthesized, and their cytotoxicity toward different human immune cells evaluated. A differential cytotoxic response between human immune cell subsets was observed, with lymphocytes being the most resistant and monocytes being the most susceptible to ZnO nanoparticle-induced toxicity. Significant differences were also observed between previously activated memory lymphocytes and naive lymphocytes, indicating a relationship between cell-cycle potential and nanoparticle susceptibility. Mechanisms of toxicity involve the generation of reactive oxygen species, with monocytes displaying the highest levels, and the degree of cytotoxicity dependent on the extent of nanoparticle interactions with cellular membranes. An inverse relationship between nanoparticle size and cytotoxicity, as well as nanoparticle size and reactive oxygen species production was observed. In addition, ZnO nanoparticles induce the production of the proinflammatory cytokines, IFN-γ, TNF-α, and IL-12, at concentrations below those causing appreciable cell death. Collectively, these results underscore the need for careful evaluation of ZnO nanoparticle effects across a spectrum of relevant cell types when considering their use for potential new nanotechnology-based biological applications.

The novel oral imatinib microemulsions: physical properties, cytotoxicity activities and improved Caco-2 cell permeability.

Science.gov (United States)

Gundogdu, Evren; Karasulu, Hatice Yesim; Koksal, Cinel; Karasulu, Ercüment

2013-01-01

The objective of this study was to formulate imatinib (IM) loaded to water-in-oil (w/o) microemulsions as an alternative formulation for cancer therapy and to evaluate the cytotoxic effect of microemulsions Caco-2 and MCF-7. Moreover, permeability studies were also performed with Caco-2 cells. W/o microemulsion systems were developed by using pseudo-ternary phase diagram. According to cytotoxicity studies, all formulations did not exert a cytotoxic effect on Caco-2 cells. Furthermore, all formulations had a significant cytotoxic effect on MCF-7 cells and the cytotoxic effect of M3IM was significantly more than that of other microemulsions and IM solution (p < 0.05). The permeability studies of IM across Caco-2 cells showed that permeability value from apical to basolateral was higher than permeability value of other formulations. In conclusion, the microemulsion formulations as a drug carrier, especially M3IM formulation, may be used as an effective alternative breast cancer therapy for oral delivery of IM.

In Vitro Cytotoxic Potential of Essential Oils of Eucalyptus benthamii and Its Related Terpenes on Tumor Cell Lines

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Patrícia Mathias Döll-Boscardin

2012-01-01

Full Text Available Eucalyptus L. is traditionally used for many medicinal purposes. In particular, some Eucalyptus species have currently shown cytotoxic properties. Local Brazilian communities have used leaves of E. benthamii as a herbal remedy for various diseases, including cancer. Considering the lack of available data for supporting this cytotoxic effect, the goal of this paper was to study the in vitro cytotoxic potential of the essential oils from young and adult leaves of E. benthamii and some related terpenes (α-pinene, terpinen-4-ol, and γ-terpinene on Jurkat, J774A.1 and HeLa cells lines. Regarding the cytotoxic activity based on MTT assay, the essential oils showed improved results than α-pinene and γ-terpinene, particularly for Jurkat and HeLa cell lines. Terpinen-4-ol revealed a cytotoxic effect against Jurkat cells similar to that observed for volatile oils. The results of LDH activity indicated that cytotoxic activity of samples against Jurkat cells probably involved cell death by apoptosis. The decrease of cell DNA content was demonstrated due to inhibition of Jurkat cells proliferation by samples as a result of cytotoxicity. In general, the essential oils from young and adult leaves of E. benthamii presented cytotoxicity against the investigated tumor cell lines which confirms their antitumor potential.

In Vitro Cytotoxic Potential of Essential Oils of Eucalyptus benthamii and Its Related Terpenes on Tumor Cell Lines

Science.gov (United States)

Döll-Boscardin, Patrícia Mathias; Sartoratto, Adilson; Sales Maia, Beatriz Helena Lameiro de Noronha; Padilha de Paula, Josiane; Nakashima, Tomoe; Farago, Paulo Vitor; Kanunfre, Carla Cristine

2012-01-01

Eucalyptus L. is traditionally used for many medicinal purposes. In particular, some Eucalyptus species have currently shown cytotoxic properties. Local Brazilian communities have used leaves of E. benthamii as a herbal remedy for various diseases, including cancer. Considering the lack of available data for supporting this cytotoxic effect, the goal of this paper was to study the in vitro cytotoxic potential of the essential oils from young and adult leaves of E. benthamii and some related terpenes (α-pinene, terpinen-4-ol, and γ-terpinene) on Jurkat, J774A.1 and HeLa cells lines. Regarding the cytotoxic activity based on MTT assay, the essential oils showed improved results than α-pinene and γ-terpinene, particularly for Jurkat and HeLa cell lines. Terpinen-4-ol revealed a cytotoxic effect against Jurkat cells similar to that observed for volatile oils. The results of LDH activity indicated that cytotoxic activity of samples against Jurkat cells probably involved cell death by apoptosis. The decrease of cell DNA content was demonstrated due to inhibition of Jurkat cells proliferation by samples as a result of cytotoxicity. In general, the essential oils from young and adult leaves of E. benthamii presented cytotoxicity against the investigated tumor cell lines which confirms their antitumor potential. PMID:22645627

The cytotoxicity of a 2-chloroethylnitrosourea analog of sarcosinamide in the SK-MG-1 human glioma cell line as a possible indicator for transport

Energy Technology Data Exchange (ETDEWEB)

Skalski, V.; Feindel, W.; Panasci, L.C. (Lady Davis Institute for Medical Research, Montreal, Quebec (Canada))

1989-07-01

The cytotoxicities of a new sarcosinamide analog of chloroethylnitrosourea (SarCNU) and of BCNU were examined in the glioma cell line SK-MG-1 in the presence or absence of excess concentrations of amino acids and sarcosinamide. The cytotoxicity of SarCNU, but not of BCNU, was significantly reduced in the presence of excess sarcosinamide. The stability of SarCNU was not significantly altered by increasing concentrations of sarcosinamide. In order to investigate the possibility that sarcosinamide inhibits the uptake of SarCNU, the transport of tritiated sarcosinamide was examined in SK-MG-1 cells. The uptake of 3H-sarcosinamide was inhibited by excess, unlabelled sarcosinamide and SarCNU but not by BCNU, glycine or sarcosine. These results suggest the existence of a carrier-mediated transport for sarcosinamide which can accomodate SarCNU in SK-MG-1 cells.

Cysteine Cathepsins as Regulators of the Cytotoxicity of NK and T Cells

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Perišić Nanut, Milica; Sabotič, Jerica; Jewett, Anahid; Kos, Janko

2014-01-01

Cysteine cathepsins are lysosomal peptidases involved at different levels in the processes of the innate and adaptive immune responses. Some, such as cathepsins B, L, and H are expressed constitutively in most immune cells. In cells of innate immunity they play a role in cell adhesion and phagocytosis. Other cysteine cathepsins are expressed more specifically. Cathepsin X promotes dendritic cell maturation, adhesion of macrophages, and migration of T cells. Cathepsin S is implicated in major histocompatibility complex class II antigen presentation, whereas cathepsin C, expressed in cytotoxic T lymphocytes and natural killer (NK) cells, is involved in processing pro-granzymes into proteolytically active forms, which trigger cell death in their target cells. The activity of cysteine cathepsins is controlled by endogenous cystatins, cysteine protease inhibitors. Of these, cystatin F is the only cystatin that is localized in endosomal/lysosomal vesicles. After proteolytic removal of its N-terminal peptide, cystatin F becomes a potent inhibitor of cathepsin C with the potential to regulate pro-granzyme processing and cell cytotoxicity. This review is focused on the role of cysteine cathepsins and their inhibitors in the molecular mechanisms leading to the cytotoxic activity of T lymphocytes and NK cells in order to address new possibilities for regulation of their function in pathological processes. PMID:25520721