Anti-cancer Activity of Novel TM4SF5-Targeting Antibodies through TM4SF5 Neutralization and Immune Cell-Mediated Cytotoxicity

PubMed Central

Ahn, Hye-Mi; Ryu, Jihye; Song, Jin Myeong; Lee, Yunhee; Kim, Hye-Jin; Ko, Dongjoon; Choi, Inpyo; Kim, Sang Jick; Lee, Jung Weon; Kim, Semi

2017-01-01

The transmembrane four L6 family member 5 (TM4SF5) protein is a novel molecular target for the prevention and treatment of hepatocellular carcinoma. TM4SF5 is highly expressed in liver, colon, esophageal, and pancreatic cancers and is implicated in tumor progression. Here, we screened monoclonal antibodies that specifically bound to the extracellular loop 2 (EC2) of TM4SF5 from a phage-displayed murine antibody (single-chain variable fragment; scFv) library. We constructed and characterized chimeric antibodies, Ab27 and Ab79, of scFv fused with Fc domain of human IgG1. The affinity (KD) of Ab27 and Ab79 for soluble EC2 was approximately 9.2 nM and 16.9 nM, respectively, as determined by surface plasmon resonance analysis. Ab27 and Ab79 efficiently bound to native TM4SF5 on the cell surface were internalized into the cancer cells, leading to a decrease in cell surface TM4SF5. Ab27 and Ab79 inhibited the proliferation and invasion of TM4SF5-positive liver and colon cancer cells and reduced FAK and c-Src phosphorylation. Ab27 and Ab79 also enhanced anoikis sensitivity and reduced survivin. Ab27 mediated antibody-dependent cell-mediated cytotoxicity in vitro. Ab27 and Ab79 efficiently inhibited tumor growth in a liver cancer xenograft model. These results strongly support the further development of Ab27 as a novel anti-cancer agent in the clinic. PMID:28255353

Localized Irradiation of Cell Membrane by Auger Electrons Is Cytotoxic Through Oxidative Stress-Mediated Nontargeted Effects

PubMed Central

Paillas, Salomé; Ladjohounlou, Riad; Lozza, Catherine; Pichard, Alexandre; Boudousq, Vincent; Jarlier, Marta; Sevestre, Samuel; Le Blay, Marion; Deshayes, Emmanuel; Sosabowski, Jane; Chardès, Thierry; Navarro-Teulon, Isabelle; Mairs, Robert J.

2016-01-01

Abstract Aims: We investigated whether radiation-induced nontargeted effects are involved in the cytotoxic effects of anticell surface monoclonal antibodies labeled with Auger electron emitters, such as iodine 125 (monoclonal antibodies labeled with 125I [125I-mAbs]). Results: We showed that the cytotoxicity of 125I-mAbs targeting the cell membrane of p53+/+ HCT116 colon cancer cells is mainly due to nontargeted effects. Targeted and nontargeted cytotoxicities were inhibited in vitro following lipid raft disruption with Methyl-β-cyclodextrin (MBCD) or filipin or use of radical oxygen species scavengers. 125I-mAb efficacy was associated with acid sphingomyelinase activation and modulated through activation of the AKT, extracellular signal-related kinase ½ (ERK1/2), p38 kinase, c-Jun N-terminal kinase (JNK) signaling pathways, and also of phospholipase C-γ (PLC-γ), proline-rich tyrosine kinase 2 (PYK-2), and paxillin, involved in Ca2+ fluxes. Moreover, the nontargeted response induced by directing 5-[(125)I]iodo-2′-deoxyuridine to the nucleus was comparable to that of 125I-mAb against cell surface receptors. In vivo, we found that the statistical significance of tumor growth delay induced by 125I-mAb was removed after MBCD treatment and observed oxidative DNA damage beyond the expected Auger electron range. These results suggest the involvement of nontargeted effects in vivo also. Innovation: Low-energy Auger electrons, such as those emitted by 125I, have a short tissue range and are usually targeted to the nucleus to maximize their cytotoxicity. In this study, we show that targeting the cancer cell surface with 125I-mAbs produces a lipid raft-mediated nontargeted response that compensates for the inferior efficacy of non-nuclear targeting. Conclusion: Our findings describe the mechanisms involved in the efficacy of 125I-mAbs targeting the cancer cell surface. Antioxid. Redox Signal. 25, 467–484. PMID:27224059

Uninfected Bystander Cells Impact the Measurement of HIV-Specific Antibody-Dependent Cellular Cytotoxicity Responses

PubMed Central

Richard, Jonathan; Prévost, Jérémie; Baxter, Amy E.; Ding, Shilei; Medjahed, Halima; Delgado, Gloria G.; Brassard, Nathalie; Stürzel, Christina M.; Kirchhoff, Frank; Hahn, Beatrice H.; Parsons, Matthew S.; Kaufmann, Daniel E.; Evans, David T.

2018-01-01

ABSTRACT The conformation of the HIV-1 envelope glycoprotein (Env) substantially impacts antibody recognition and antibody-dependent cellular cytotoxicity (ADCC) responses. In the absence of the CD4 receptor at the cell surface, primary Envs sample a “closed” conformation that occludes CD4-induced (CD4i) epitopes. The virus controls CD4 expression through the actions of Nef and Vpu accessory proteins, thus protecting infected cells from ADCC responses. However, gp120 shed from infected cells can bind to CD4 present on uninfected bystander cells, sensitizing them to ADCC mediated by CD4i antibodies (Abs). Therefore, we hypothesized that these bystander cells could impact the interpretation of ADCC measurements. To investigate this, we evaluated the ability of antibodies to CD4i epitopes and broadly neutralizing Abs (bNAbs) to mediate ADCC measured by five ADCC assays commonly used in the field. Our results indicate that the uninfected bystander cells coated with gp120 are efficiently recognized by the CD4i ligands but not the bNabs. Consequently, the uninfected bystander cells substantially affect in vitro measurements made with ADCC assays that fail to identify responses against infected versus uninfected cells. Moreover, using an mRNA flow technique that detects productively infected cells, we found that the vast majority of HIV-1-infected cells in in vitro cultures or ex vivo samples from HIV-1-infected individuals are CD4 negative and therefore do not expose significant levels of CD4i epitopes. Altogether, our results indicate that ADCC assays unable to differentiate responses against infected versus uninfected cells overestimate responses mediated by CD4i ligands. PMID:29559570

PEROXIDASE-MEDIATED MAMMALIAN CELL CYTOTOXICITY

PubMed Central

Edelson, Paul J.; Cohn, Zanvil A.

1973-01-01

Lactoperoxidase, in the presence of hydrogen peroxide and iodide is cytotoxic for human and mouse lymphoid cells, and human erythrocytes. Myeloperoxidase, in amounts equivalent to 1.5 x 106 neutrophils, readily replaces lactoperoxidase, and allows the substitution of the iodide ion by chloride. The myeloperoxidase-mediated reaction is rapid, and highly efficient, leading to 85–90% cell death in 90 min, as measured by 51chromium release and dye exclusion. The mixture of granulocytes. monocytes, and lymphocytes present in an inflammatory exudate, and the intimate cell-to-cell association characteristic of cytotoxic phenomena may provide the in vivo requirements for such a system. PMID:4717124

Modulation of cytotoxic T lymphocyte, natural killer cell, antibody-dependent cellular cytotoxicity, and antibody-dependent complement-mediated cytotoxicity by Vernonia cinerea L. and vernolide-A in BALB/c mice via enhanced production of cytokines IL-2 and IFN-γ.

PubMed

Pratheeshkumar, Poyil; Kuttan, Girija

2012-02-01

Effect of Vernonia cinerea L. and vernolide-A on cell-mediated immune (CMI) response was studied in normal as well as tumor-bearing BALB/c mice. Administration of V. cinerea and vernolide-A significantly enhanced natural killer (NK) cell activity in both normal as well as tumor-bearing animals, and the activity was observed earlier than in tumor-bearing control animals. Antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent complement-mediated cytotoxicity (ACC) were also enhanced significantly in both normal as well as tumor-bearing animals after V. cinerea and vernolide-A administration compared with untreated control tumor-bearing animals. Extract and vernolide-A showed a significant increase in cytotoxic T lymphocyte (CTL) production in both the in vivo and in vitro models. The level of cytokines such as interleukin (IL)-2 and interferon (IFN)-γ were also enhanced by the treatment of V. cinerea and vernolide-A in both normal as well as tumor-bearing animals. This study demonstrated that V. cinerea extract and vernolide-A stimulate the CTL, NK cell, ADCC, and ADCC through enhanced secretion of IL-2 and IFN-γ.

Comparison of Antibody-Dependent Cell-Mediated Cytotoxicity and Virus Neutralization by HIV-1 Env-Specific Monoclonal Antibodies

PubMed Central

von Bredow, Benjamin; Arias, Juan F.; Heyer, Lisa N.; Moldt, Brian; Le, Khoa; Robinson, James E.; Burton, Dennis R.

2016-01-01

ABSTRACT Although antibodies to the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein have been studied extensively for their ability to block viral infectivity, little data are currently available on nonneutralizing functions of these antibodies, such as their ability to eliminate virus-infected cells by antibody-dependent cell-mediated cytotoxicity (ADCC). HIV-1 Env-specific antibodies of diverse specificities, including potent broadly neutralizing and nonneutralizing antibodies, were therefore tested for ADCC against cells infected with a lab-adapted HIV-1 isolate (HIV-1NL4-3), a primary HIV-1 isolate (HIV-1JR-FL), and a simian-human immunodeficiency virus (SHIV) adapted for pathogenic infection of rhesus macaques (SHIVAD8-EO). In accordance with the sensitivity of these viruses to neutralization, HIV-1NL4-3-infected cells were considerably more sensitive to ADCC, both in terms of the number of antibodies and magnitude of responses, than cells infected with HIV-1JR-FL or SHIVAD8-EO. ADCC activity generally correlated with antibody binding to Env on the surfaces of virus-infected cells and with viral neutralization; however, neutralization was not always predictive of ADCC, as instances of ADCC in the absence of detectable neutralization, and vice versa, were observed. These results reveal incomplete overlap in the specificities of antibodies that mediate these antiviral activities and provide insights into the relationship between ADCC and neutralization important for the development of antibody-based vaccines and therapies for combating HIV-1 infection. IMPORTANCE This study provides fundamental insights into the relationship between antibody-dependent cell-mediated cytotoxicity (ADCC) and virus neutralization that may help to guide the development of antibody-based vaccines and immunotherapies for the prevention and treatment of HIV-1 infection. PMID:27122574

Comparison of Antibody-Dependent Cell-Mediated Cytotoxicity and Virus Neutralization by HIV-1 Env-Specific Monoclonal Antibodies.

PubMed

von Bredow, Benjamin; Arias, Juan F; Heyer, Lisa N; Moldt, Brian; Le, Khoa; Robinson, James E; Zolla-Pazner, Susan; Burton, Dennis R; Evans, David T

2016-07-01

Although antibodies to the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein have been studied extensively for their ability to block viral infectivity, little data are currently available on nonneutralizing functions of these antibodies, such as their ability to eliminate virus-infected cells by antibody-dependent cell-mediated cytotoxicity (ADCC). HIV-1 Env-specific antibodies of diverse specificities, including potent broadly neutralizing and nonneutralizing antibodies, were therefore tested for ADCC against cells infected with a lab-adapted HIV-1 isolate (HIV-1NL4-3), a primary HIV-1 isolate (HIV-1JR-FL), and a simian-human immunodeficiency virus (SHIV) adapted for pathogenic infection of rhesus macaques (SHIVAD8-EO). In accordance with the sensitivity of these viruses to neutralization, HIV-1NL4-3-infected cells were considerably more sensitive to ADCC, both in terms of the number of antibodies and magnitude of responses, than cells infected with HIV-1JR-FL or SHIVAD8-EO ADCC activity generally correlated with antibody binding to Env on the surfaces of virus-infected cells and with viral neutralization; however, neutralization was not always predictive of ADCC, as instances of ADCC in the absence of detectable neutralization, and vice versa, were observed. These results reveal incomplete overlap in the specificities of antibodies that mediate these antiviral activities and provide insights into the relationship between ADCC and neutralization important for the development of antibody-based vaccines and therapies for combating HIV-1 infection. This study provides fundamental insights into the relationship between antibody-dependent cell-mediated cytotoxicity (ADCC) and virus neutralization that may help to guide the development of antibody-based vaccines and immunotherapies for the prevention and treatment of HIV-1 infection. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

IgM-mediated opsonization and cytotoxicity in the shark.

PubMed

McKinney, E C; Flajnik, M F

1997-02-01

Two types of cytotoxic reactions have been observed using cells from the nurse shark: spontaneous cytotoxicity mediated by cells of the macrophage lineage and antibody-dependent killing carried out by a different effector cell population. Previous data showed that removal of phagocytic cells using iron particles abolished macrophage-mediated killing, but not antibody-dependent reactions. The current study used single cell assays and showed that the effector of antibody-driven reactions was the neutrophil. Surprisingly, the mechanism of killing was shown to be phagocytosis mediated by both 7S and 19S immunoglobulin M (IgM). Reactions proceeded with as little as 0.01 microg of purified 19S or 7S IgM and were complete within 4-6 h. In contrast, purified immunoglobulin did not adsorb to macrophages and had no effect on target cell binding or cytotoxicity. Pretreatment of cells with cytochalasin D abolished the phagocytic reaction, but not spontaneous cytotoxicity. These data show that antibody-mediated killing results from opsonization and phagocytosis; the mechanism of macrophage killing is currently unknown. In addition, these data show that the shark neutrophil, not the macrophage lineage, carries a receptor for Fc mu.

A targeted complement-dependent strategy to improve the outcome of mAb therapy, and characterization in a murine model of metastatic cancer

PubMed Central

Elvington, Michelle; Huang, Yuxiang; Morgan, B. Paul; Qiao, Fei; van Rooijen, Nico; Atkinson, Carl

2012-01-01

Complement inhibitors expressed on tumor cells provide an evasion mechanism against mAb therapy and may modulate the development of an acquired antitumor immune response. Here we investigate a strategy to amplify mAb-targeted complement activation on a tumor cell, independent of a requirement to target and block complement inhibitor expression or function, which is difficult to achieve in vivo. We constructed a murine fusion protein, CR2Fc, and demonstrated that the protein targets to C3 activation products deposited on a tumor cell by a specific mAb, and amplifies mAb-dependent complement activation and tumor cell lysis in vitro. In syngeneic models of metastatic lymphoma (EL4) and melanoma (B16), CR2Fc significantly enhanced the outcome of mAb therapy. Subsequent studies using the EL4 model with various genetically modified mice and macrophage-depleted mice revealed that CR2Fc enhanced the therapeutic effect of mAb therapy via both macrophage-dependent FcγR-mediated antibody-dependent cellular cytotoxicity, and by direct complement-mediated lysis. Complement activation products can also modulate adaptive immunity, but we found no evidence that either mAb or CR2Fc treatment had any effect on an antitumor humoral or cellular immune response. CR2Fc represents a potential adjuvant treatment to increase the effectiveness of mAb therapy of cancer. PMID:22442351

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.

PubMed

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

An early history of T cell-mediated cytotoxicity.

PubMed

Golstein, Pierre; Griffiths, Gillian M

2018-04-16

After 60 years of intense fundamental research into T cell-mediated cytotoxicity, we have gained a detailed knowledge of the cells involved, specific recognition mechanisms and post-recognition perforin-granzyme-based and FAS-based molecular mechanisms. What could not be anticipated at the outset was how discovery of the mechanisms regulating the activation and function of cytotoxic T cells would lead to new developments in cancer immunotherapy. Given the profound recent interest in therapeutic manipulation of cytotoxic T cell responses, it is an opportune time to look back on the early history of the field. This Timeline describes how the early findings occurred and eventually led to current therapeutic applications.

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

PubMed

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 (<20%). In contrast, the anti-CD20 mAb rituximab depleted >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.

Antibody-Dependent Cellular Cytotoxicity against Reactivated HIV-1-Infected Cells

PubMed Central

Lee, Wen Shi; Richard, Jonathan; Lichtfuss, Marit; Smith, Amos B.; Park, Jongwoo; Courter, Joel R.; Melillo, Bruno N.; Sodroski, Joseph G.; Kaufmann, Daniel E.; Parsons, Matthew S.

2015-01-01

ABSTRACT Lifelong antiretroviral therapy (ART) for HIV-1 does not diminish the established latent reservoir. A possible cure approach is to reactivate the quiescent genome from latency and utilize immune responses to eliminate cells harboring reactivated HIV-1. It is not known whether antibodies within HIV-1-infected individuals can recognize and eliminate cells reactivated from latency through antibody-dependent cellular cytotoxicity (ADCC). We found that reactivation of HIV-1 expression in the latently infected ACH-2 cell line elicited antibody-mediated NK cell activation but did not result in antibody-mediated killing. The lack of CD4 expression on these HIV-1 envelope (Env)-expressing cells likely resulted in poor recognition of CD4-induced antibody epitopes on Env. To examine this further, cultured primary CD4+ T cells from HIV-1+ subjects were used as targets for ADCC. These ex vivo-expanded primary cells were modestly susceptible to ADCC mediated by autologous or heterologous HIV-1+ serum antibodies. Importantly, ADCC mediated against these primary cells could be enhanced following incubation with a CD4-mimetic compound (JP-III-48) that exposes CD4-induced antibody epitopes on Env. Our studies suggest that with sufficient reactivation and expression of appropriate Env epitopes, primary HIV-1-infected cells can be targets for ADCC mediated by autologous serum antibodies and innate effector cells. The results of this study suggest that further investigation into the potential of ADCC to eliminate reactivated latently infected cells is warranted. IMPORTANCE An HIV-1 cure remains elusive due to the persistence of long-lived latently infected cells. An HIV-1 cure strategy, termed “shock and kill,” aims to reactivate HIV-1 expression in latently infected cells and subsequently eliminate the reactivated cells through immune-mediated killing. While recent research efforts have focused on reversing HIV-1 latency, it remains unclear whether preexisting immune

miR-143 or miR-145 overexpression increases cetuximab-mediated antibody-dependent cellular cytotoxicity in human colon cancer cells

PubMed Central

Gomes, Sofia E.; Simões, André E. S.; Pereira, Diane M.; Castro, Rui E.; Rodrigues, Cecília M. P.; Borralho, Pedro M.

2016-01-01

miR-143 and miR-145 are downregulated in colon cancer. Here, we tested the effect of restoring these miRNAs on sensitization to cetuximab in mutant KRAS (HCT116 and SW480) and wild-type KRAS (SW48) colon cancer cells. We evaluated cetuximab-mediated antibody-dependent cellular cytotoxicity (ADCC) and the modulation of signaling pathways involved in immune effector cell-mediated elimination of cancer cells. Stable miR-143 or miR-145 overexpression increased cell sensitivity to cetuximab, resulting in a significant increase of cetuximab-mediated ADCC independently of KRAS status. Importantly, HCT116 cells overexpressing these miRNAs triggered apoptosis in result of cetuximab-mediated ADCC, effected by peripheral blood mononuclear cells (p < 0.01). This was associated with increased apoptosis and caspase-3/7 activity, and reduced Bcl-2 protein expression (p < 0.01). In addition, caspase inhibition abrogated cetuximab-mediated ADCC in HCT116 cells overexpressing either miR-143 or miR-145 (p < 0.01). Furthermore, Bcl-2 silencing led to high level of cetuximab-mediated ADCC, compared to control siRNA (p < 0.05). Importantly, granzyme B inhibition, abrogated cetuximab-mediated ADCC, reducing caspase-3/7 activity (p < 0.01). Collectively, our data suggests that re-introduction of miR-143 or miR-145 may provide a new approach for development of therapeutic strategies to re-sensitize colon cancer cells to cetuximab by stimulating cetuximab-dependent ADCC to induce cell death. PMID:26824186

Horizontal transfer of miR-106a/b from cisplatin resistant hepatocarcinoma cells can alter the sensitivity of cervical cancer cells to cisplatin.

PubMed

Raji, Grace R; Sruthi, T V; Edatt, Lincy; Haritha, K; Sharath Shankar, S; Sameer Kumar, V B

2017-10-01

Recent studies indicate that horizontal transfer of genetic material can act as a communication tool between heterogenous populations of tumour cells, thus altering the chemosensitivity of tumour cells. The present study was designed to check whether the horizontal transfer of miRNAs released by cisplatin resistant (Cp-r) Hepatocarcinoma cells can alter the sensitivity of cervical cancer cells. For this exosomes secreted by cisplatin resistant and cisplatin sensitive HepG2 cells (EXres and EXsen) were isolated and characterised. Cytotoxicity analysis showed that EXres can make Hela cells resistant to cisplatin. Analysis of miR-106a/b levels in EXres and EXsen showed that their levels vary. Mechanistic studies showed that miR-106a/b play an important role in EXsen and EXres mediated change in chemosensitivity of Hela cells to cisplatin. Further SIRT1 was identified as a major target of miR-106a/b using in silico tools and this was proved by experimentation. Also the effect of miR-106a/b in chemosensitivity was seen to be dependent on regulation of SIRT1 by miR-106a/b. In brief, this study brings into light, the SIRT1 dependent mechanism of miR-106a/b mediated regulation of chemosensitivity upon the horizontal transfer from one cell type to another. Copyright © 2017 Elsevier Inc. All rights reserved.

Anti-ErbB-2 mAb therapy requires type I and II interferons and synergizes with anti-PD-1 or anti-CD137 mAb therapy.

PubMed

Stagg, John; Loi, Sherene; Divisekera, Upulie; Ngiow, Shin Foong; Duret, Helene; Yagita, Hideo; Teng, Michele W; Smyth, Mark J

2011-04-26

Trastuzumab, a monoclonal antibody targeting human epidermal growth factor receptor-2 (HER2/ErbB-2), has become the mainstay of treatment for HER2-positive breast cancer. Nevertheless, its exact mechanism of action has not been fully elucidated. Although several studies suggest that Fc receptor-expressing immune cells are involved in trastuzumab therapy, the relative contribution of lymphocyte-mediated cellular cytotoxicity and antitumor cytokines remains unknown. We report here that anti-ErbB-2 mAb therapy is dependent on the release of type I and type II IFNs but is independent of perforin or FasL. Our study thus challenges the notion that classical antibody-dependent, lymphocyte-mediated cellular cytotoxicity is important for trastuzumab. We demonstrate that anti-ErbB-2 mAb therapy of experimental tumors derived from MMTV-ErbB-2 transgenic mice triggers MyD88-dependent signaling and primes IFN-γ-producing CD8+ T cells. Adoptive cell transfer of purified T cell subsets confirmed the essential role of IFN-γ-producing CD8+ T cells. Notably, anti-ErbB-2 mAb therapy was independent of IL-1R or IL-17Ra signaling. Finally, we investigated whether immunostimulatory approaches with antibodies against programmed death-1 (PD-1) or 41BB (CD137) could be used to capitalize on the immune-mediated effects of trastuzumab. We demonstrate that anti-PD-1 or anti-CD137 mAb can significantly improve the therapeutic activity of anti-ErbB-2 mAb in immunocompetent mice.

Human IgG2 antibodies against epidermal growth factor receptor effectively trigger antibody-dependent cellular cytotoxicity but, in contrast to IgG1, only by cells of myeloid lineage.

PubMed

Schneider-Merck, Tanja; Lammerts van Bueren, Jeroen J; Berger, Sven; Rossen, Kai; van Berkel, Patrick H C; Derer, Stefanie; Beyer, Thomas; Lohse, Stefan; Bleeker, Wim K; Peipp, Matthias; Parren, Paul W H I; van de Winkel, Jan G J; Valerius, Thomas; Dechant, Michael

2010-01-01

Ab-dependent cellular cytotoxicity (ADCC) is usually considered an important mechanism of action for immunotherapy with human IgG1 but not IgG2 Abs. The epidermal growth factor receptor (EGF-R) Ab panitumumab represents the only human IgG2 Ab approved for immunotherapy and inhibition of EGF-R signaling has been described as its principal mechanism of action. In this study, we investigated effector mechanisms of panitumumab compared with zalutumumab, an EGF-R Ab of the human IgG1 isotype. Notably, panitumumab was as effective as zalutumumab in recruiting ADCC by myeloid effector cells (i.e., neutrophils and monocytes) in contrast to NK cell-mediated ADCC, which was only induced by the IgG1 Ab. Neutrophil-mediated tumor cell killing could be stimulated by myeloid growth factors and was triggered via FcgammaRIIa. Panitumumab-mediated ADCC was significantly affected by the functional FcgammaRIIa-R131H polymorphism and was induced more effectively by neutrophils from FcgammaRIIa-131H homozygous donors than from -131R individuals. This polymorphism did not affect neutrophil ADCC induced by the IgG1 Ab zalutumumab. The in vivo activity of both Abs was assessed in two animal models: a high-dose model, in which signaling inhibition is a dominant mechanism of action, and a low-dose model, in which effector cell recruitment plays a prominent role. Zalutumumab was more effective than panitumumab in the high-dose model, reflecting its stronger ability to induce EGF-R downmodulation and growth inhibition. In the low-dose model, zalutumumab and panitumumab similarly prevented tumor growth. Thus, our results identify myeloid cell-mediated ADCC as a potent and additional mechanism of action for EGF-R-directed immunotherapy.

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

PubMed

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

PubMed Central

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. PMID:28066408

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

PubMed

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.

Effect of Aerva lanata on cell-mediated immune responses and cytotoxic T-lymphocyte generation in normal and tumor-bearing mice.

PubMed

Siveen, K S; Kuttan, Girija

2012-01-01

Cell-mediated immunity offers protection against virus-infected cells and tumor cells, involves activation of natural killer (NK) cells, production of antigen-specific cytotoxic T-lymphocytes, and release of various cytokines in response to an antigen. Administration of an ethanolic extract of Aerva lanata was found to stimulate cell-mediated immunological responses in normal and tumor-bearing BALB/c mice. A significant enhancement in NK cell activity in both normal and tumor-bearing hosts was observed after administration of A. lanata. Antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent complement-mediated cytotoxicity (ACC) were significantly enhanced as well in both sets of treated hosts. In addition, in vivo production of IL-2 and IFNg were each significantly enhanced by extract treatment. The stimulatory effect of A. lanata on cytotoxic T-lymphocyte (CTL) production was determined by Winn's neutralization assay using CTL-sensitive EL4 thymoma cells. A. lanata treatment caused a significant increase in CTL production in both in vivo and in vitro models, in each case as indicated by a significant increase in the life-spans of tumor-injected mice. Taken together, all of these results in the murine model indicate that administration of an ethanolic extract of A. lanata could enhance the cell-mediated anti-tumor response.

A novel method for evaluating antibody-dependent cell-mediated cytotoxicity by flowcytometry using cryopreserved human peripheral blood mononuclear cells

PubMed Central

Yamashita, Makiko; Kitano, Shigehisa; Aikawa, Hiroaki; Kuchiba, Aya; Hayashi, Mitsuhiro; Yamamoto, Noboru; Tamura, Kenji; Hamada, Akinobu

2016-01-01

Analyzing the cytotoxic functions of effector cells, such as NK cells against target cancer cells, is thought to be necessary for predicting the clinical efficacy of antibody-dependent cellular cytotoxicity (ADCC) -dependent antibody therapy. The 51Cr release assay has long been the most widely used method for quantification of ADCC activity. However, the reproducibilities of these release assays are not adequate, and they do not allow evaluation of the lysis susceptibilities of distinct cell types within the target cell population. In this study, we established a novel method for evaluating cytotoxicity, which involves the detection and quantification of dead target cells using flowcytometry. CFSE (carboxyfluorescein succinimidyl ester) was used as a dye to specifically stain and thereby label the target cell population, allowing living and dead cells, as well as both target and effector cells, to be quantitatively distinguished. Furthermore, with our new approach, ADCC activity was more reproducibly, sensitively, and specifically detectable, not only in freshly isolated but also in frozen human peripheral blood mononuclear cells (PBMCs), than with the calcein-AM release assay. This assay, validated herein, is expected to become a standard assay for evaluating ADCC activity which will ultimately contribute the clinical development of ADCC dependent-antibody therapies. PMID:26813960

A novel method for evaluating antibody-dependent cell-mediated cytotoxicity by flowcytometry using cryopreserved human peripheral blood mononuclear cells.

PubMed

Yamashita, Makiko; Kitano, Shigehisa; Aikawa, Hiroaki; Kuchiba, Aya; Hayashi, Mitsuhiro; Yamamoto, Noboru; Tamura, Kenji; Hamada, Akinobu

2016-01-27

Analyzing the cytotoxic functions of effector cells, such as NK cells against target cancer cells, is thought to be necessary for predicting the clinical efficacy of antibody-dependent cellular cytotoxicity (ADCC) -dependent antibody therapy. The (51)Cr release assay has long been the most widely used method for quantification of ADCC activity. However, the reproducibilities of these release assays are not adequate, and they do not allow evaluation of the lysis susceptibilities of distinct cell types within the target cell population. In this study, we established a novel method for evaluating cytotoxicity, which involves the detection and quantification of dead target cells using flowcytometry. CFSE (carboxyfluorescein succinimidyl ester) was used as a dye to specifically stain and thereby label the target cell population, allowing living and dead cells, as well as both target and effector cells, to be quantitatively distinguished. Furthermore, with our new approach, ADCC activity was more reproducibly, sensitively, and specifically detectable, not only in freshly isolated but also in frozen human peripheral blood mononuclear cells (PBMCs), than with the calcein-AM release assay. This assay, validated herein, is expected to become a standard assay for evaluating ADCC activity which will ultimately contribute the clinical development of ADCC dependent-antibody therapies.

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

PubMed Central

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

Induction of cell-mediated cytotoxicity by lipoprotein containing histocompatibility antigens.

PubMed Central

Dennert, G

1979-01-01

Lipoprotein was isolated from tumour cells by sonication and ultracentrifugal flotation on KBr gradients. It contained H-2 antigen detectable by antibody binding and induced a primary or secondary cell-mediated cytotoxic response in vitro which was H-2 specific. In a syngeneic model only a secondary cell-mediated response was stimulated and no competitive inhibition of the effector step of cell-mediated lysis could be demonstrated. The implications of these findings are discussed. PMID:521060

Characterization of CD4+ T cell-mediated cytotoxicity in patients with multiple myeloma.

PubMed

Zhang, Xiaole; Gao, Lei; Meng, Kai; Han, Chunting; Li, Qiang; Feng, Zhenjun; Chen, Lei

2018-05-01

Multiple myeloma (MM) is an incurable cancer characterized by the development of malignant plasma cells. The CD8 T cell-mediated cytotoxicity is considered a major player in antitumor immunity, but in MM patients, the CD8 T cells displayed senescence markers and were functionally impaired. To investigate whether cytotoxic CD4 T cells could act as a treatment alternative in MM, we examined the frequency and function of naturally occurring cytotoxic CD4 T cells in MM patients. The cytotoxic CD4 T cells were identified as granzyme-A, granzyme B-, and perforin-expressing CD4 T cells, and their frequencies were significantly upregulated in MM patients when compared with healthy controls. The frequencies of cytotoxic CD4 T cells in MM patients were not associated with the frequencies of cytotoxic CD8 T cells, but were negatively associated with disease severity. Interestingly, the expression levels of inhibitory molecules, including PD-1 and CTLA-4, were significantly lower in cytotoxic CD4 T cells than in cytotoxic CD8 T cells. When co-incubated with autologous CD38 + CD138 + plasma cells, CD4 T cells were capable of eliminating plasma cells with varying degrees of efficacy. In MM patients, the frequency of circulating plasma cells was negatively correlated with the frequency of cytotoxic CD4 T cells. Therefore, CD4 T cell-mediated cytotoxicity existed naturally in MM patients and could potentially act as an option in antitumor therapies. Copyright © 2018 Elsevier Inc. All rights reserved.

RB mutation and RAS overexpression induce resistance to NK cell-mediated cytotoxicity in glioma cells.

PubMed

Orozco-Morales, Mario; Sánchez-García, Francisco Javier; Golán-Cancela, Irene; Hernández-Pedro, Norma; Costoya, Jose A; de la Cruz, Verónica Pérez; Moreno-Jiménez, Sergio; Sotelo, Julio; Pineda, Benjamín

2015-01-01

Several theories aim to explain the malignant transformation of cells, including the mutation of tumor suppressors and proto-oncogenes. Deletion of Rb (a tumor suppressor), overexpression of mutated Ras (a proto-oncogene), or both, are sufficient for in vitro gliomagenesis, and these genetic traits are associated with their proliferative capacity. An emerging hallmark of cancer is the ability of tumor cells to evade the immune system. Whether specific mutations are related with this, remains to be analyzed. To address this issue, three transformed glioma cell lines were obtained (Rb(-/-), Ras(V12), and Rb(-/-)/Ras(V12)) by in vitro retroviral transformation of astrocytes, as previously reported. In addition, Ras(V12) and Rb(-/-)/Ras(V12) transformed cells were injected into SCID mice and after tumor growth two stable glioma cell lines were derived. All these cells were characterized in terms of Rb and Ras gene expression, morphology, proliferative capacity, expression of MHC I, Rae1δ, and Rae1αβγδε, mult1, H60a, H60b, H60c, as ligands for NK cell receptors, and their susceptibility to NK cell-mediated cytotoxicity. Our results show that transformation of astrocytes (Rb loss, Ras overexpression, or both) induced phenotypical and functional changes associated with resistance to NK cell-mediated cytotoxicity. Moreover, the transfer of cell lines of transformed astrocytes into SCID mice increased resistance to NK cell-mediated cytotoxicity, thus suggesting that specific changes in a tumor suppressor (Rb) and a proto-oncogene (Ras) are enough to confer resistance to NK cell-mediated cytotoxicity in glioma cells and therefore provide some insight into the ability of tumor cells to evade immune responses.

Natural Killer Cell Mediated Antibody-Dependent Cellular Cytotoxicity in Tumor Immunotherapy with Therapeutic Antibodies

PubMed Central

Seidel, Ursula J. E.; Schlegel, Patrick; Lang, Peter

2013-01-01

In the last decade several therapeutic antibodies have been Federal Drug Administration (FDA) and European Medicines Agency (EMEA) approved. Although their mechanisms of action in vivo is not fully elucidated, antibody-dependent cellular cytotoxicity (ADCC) mediated by natural killer (NK) cells is presumed to be a key effector function. A substantial role of ADCC has been demonstrated in vitro and in mouse tumor models. However, a direct in vivo effect of ADCC in tumor reactivity in humans remains to be shown. Several studies revealed a predictive value of FcγRIIIa-V158F polymorphism in monoclonal antibody treatment, indicating a potential effect of ADCC on outcome for certain indications. Furthermore, the use of therapeutic antibodies after allogeneic hematopoietic stem cell transplantation is an interesting option. Studying the role of the FcγRIIIa-V158F polymorphism and the influence of Killer-cell Immunoglobuline-like Receptor (KIR) receptor ligand incompatibility on ADCC in this approach may contribute to future transplantation strategies. Despite the success of approved second-generation antibodies in the treatment of several malignancies, efforts are made to further augment ADCC in vivo by antibody engineering. Here, we review currently used therapeutic antibodies for which ADCC has been suggested as effector function. PMID:23543707

Mechanism of arctigenin-mediated specific cytotoxicity against human lung adenocarcinoma cell lines.

PubMed

Susanti, Siti; Iwasaki, Hironori; Inafuku, Masashi; Taira, Naoyuki; Oku, Hirosuke

2013-12-15

The lignan arctigenin (ARG) from the herb Arctium lappa L. possesses anti-cancer activity, however the mechanism of action of ARG has been found to vary among tissues and types of cancer cells. The current study aims to gain insight into the ARG mediated mechanism of action involved in inhibiting proliferation and inducing apoptosis in lung adenocarcinoma cells. This study also delineates the cancer cell specificity of ARG by comparison with its effects on various normal cell lines. ARG selectively arrested the proliferation of cancer cells at the G0/G1 phase through the down-regulation of NPAT protein expression. This down-regulation occurred via the suppression of either cyclin E/CDK2 or cyclin H/CDK7, while apoptosis was induced through the modulation of the Akt-1-related signaling pathway. Furthermore, a GSH synthase inhibitor specifically enhanced the cytotoxicity of ARG against cancer cells, suggesting that the intracellular GSH content was another factor influencing the susceptibility of cancer cells to ARG. These findings suggest that specific cytotoxicity of ARG against lung cancer cells was explained by its selective modulation of the expression of NPAT, which is involved in histone biosynthesis. The cytotoxicity of ARG appeared to be dependent on the intracellular GSH level. Copyright © 2013 Elsevier GmbH. All rights reserved.

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

PubMed Central

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. PMID:29686591

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

PubMed

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.

Elimination of human T cell leukemia virus type-1-infected cells by neutralizing and antibody-dependent cellular cytotoxicity-inducing antibodies against human t cell leukemia virus type-1 envelope gp46.

PubMed

Tanaka, Yuetsu; Takahashi, Yoshiaki; Tanaka, Reiko; Kodama, Akira; Fujii, Hideki; Hasegawa, Atsuhiko; Kannagi, Mari; Ansari, Aftab A; Saito, Mineki

2014-06-01

Human T cell leukemia virus type-1 (HTLV-1) is prevalent worldwide with foci of high prevalence. However, to date no effective vaccine or drug against HTLV-1 infection has been developed. In efforts to define the role of antibodies in the control of HTLV-1 infection, we capitalized on the use of our previously defined anti-gp46 neutralizing monoclonal antibody (mAb) (clone LAT-27) and high titers of human anti-HTLV-1 IgG purified from HAM/TSP patients (HAM-IgG). LAT-27 and HAM-IgG completely blocked syncytium formation and T cell immortalization mediated by HTLV-1 in vitro. The addition of these antibodies to cultures of CD8(+) T cell-depleted peripheral blood mononuclear cells (PBMCs) from HAM/TSP patients at the initiation of culture not only decreased the numbers of Tax-expressing cells and the production of HTLV-1 p24 but also inhibited the spontaneous immortalization of T cells. Coculture of in vitro-HTLV-1-immortalized T cell lines with autologous PBMCs in the presence of LAT-27 or HAM-IgG, but not an F(ab')2 fragment of LAT-27 or nonneutralizing anti-gp46 mAbs, resulted in depletion of HTLV-1-infected cells. A 24-h (51)Cr release assay showed the presence of significant antibody-dependent cellular cytotoxicity (ADCC) activity in LAT-27 and HAM-IgG, but not F(ab')2 of LAT-27, resulting in the depletion of HTLV-1-infected T cells by autologous PBMCs. The depletion of natural killer (NK) cells from the effector PBMCs reduced this ADCC activity. Altogether, the present data demonstrate that the neutralizing and ADCC-inducing activities of anti-HTLV-1 antibodies are capable of reducing infection and eliminating HTLV-1-infected cells in the presence of autologous PBMCs.

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

PubMed

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

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

PubMed

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.

Interleukin-12- and interferon-gamma-mediated natural killer cell activation by Agaricus blazei Murill.

PubMed

Yuminamochi, Eri; Koike, Taisuke; Takeda, Kazuyoshi; Horiuchi, Isao; Okumura, Ko

2007-06-01

Dried fruiting bodies of Agaricus blazei Murill (A. blazei) and its extracts have generally used as complementary and alternative medicines (CAMs). Here, we report that the oral administration of A. blazei augmented cytotoxicity of natural killer (NK) cells in wild-type (WT) C57BL/6, C3H/HeJ, and BALB/c mice. Augmented cytotoxicity was demonstrated by purified NK cells from treated wild-type (WT) and RAG-2-deficient mice, but not from interferon-gamma (IFN-gamma) deficient mice. NK cell activation and IFN-gamma production was also observed in vitro when dendritic cell (DC)-rich splenocytes of WT mice were coincubation with an extract of A. blazei. Both parameters were largely inhibited by neutralizing anti-interleukin-12 (IL-12) monoclonal antibody (mAb) and completely inhibited when anti-IL-12 mAb and anti-IL-18 mAb were used in combination. An aqueous extract of the hemicellulase-digested compound of A. blazei particle; (ABPC) induced IFN-gamma production more effectively, and this was completely inhibited by anti-IL-12 mAb alone. NK cell cytotoxicty was augmented with the same extracts, again in an IL-12 and IFN-gamma-dependent manner. These results clearly demonstrated that A. blazei and ABPC augmented NK cell activation through IL-12-mediated IFN-gamma production.

Interleukin-12- and interferon-γ-mediated natural killer cell activation by Agaricus blazei Murill

PubMed Central

Yuminamochi, Eri; Koike, Taisuke; Takeda, Kazuyoshi; Horiuchi, Isao; Okumura, Ko

2007-01-01

Dried fruiting bodies of Agaricus blazei Murill (A. blazei) and its extracts have generally used as complementary and alternative medicines (CAMs). Here, we report that the oral administration of A. blazei augmented cytotoxicity of natural killer (NK) cells in wild-type (WT) C57BL/6, C3H/HeJ, and BALB/c mice. Augmented cytotoxicity was demonstrated by purified NK cells from treated wild-type (WT) and RAG-2-deficient mice, but not from interferon-γ (IFN-γ) deficient mice. NK cell activation and IFN-γ production was also observed in vitro when dendritic cell (DC)-rich splenocytes of WT mice were coincubation with an extract of A. blazei. Both parameters were largely inhibited by neutralizing anti-interleukin-12 (IL-12) monoclonal antibody (mAb) and completely inhibited when anti-IL-12 mAb and anti-IL-18 mAb were used in combination. An aqueous extract of the hemicellulase-digested compound of A. blazei particle; (ABPC) induced IFN-γ production more effectively, and this was completely inhibited by anti-IL-12 mAb alone. NK cell cytotoxicty was augmented with the same extracts, again in an IL-12 and IFN-γ-dependent manner. These results clearly demonstrated that A. blazei and ABPC augmented NK cell activation through IL-12-mediated IFN-γ production. PMID:17346284

T-dependent activation of resting B cells mediated by concanavalin A.

PubMed

Ratcliffe, M J; Julius, M H

1984-03-01

In cultures containing long-term cultured lines of antigen-specific helper T (Th) cells, normal unprimed B cells and concanavalin A (Con A), induction of B cells to immunoglobulin secretion and DNA synthesis was observed. The plaque-forming cell (PFC) response was large (frequently greater than 75 000 PFC/10(6) input B cells) demonstrating the polyspecific nature of the response. Con A-mediated maturation and induction to DNA synthesis of responding B cells was completely Th cell dependent and inhibited with methyl-alpha-D-mannoside. Both resting and blasted B cells, separated by Percoll density centrifugation, were induced to DNA synthesis and immunoglobulin secretion. Responses were completely unrestricted by the B cell major histocompatibility complex, even at the level of the resting B cell. The polyclonal nature of the response taken together with the Con A-mediated bypassing of T cell specificity and restricting haplotype indicates that this response is analogous to lectin-mediated cytotoxicity.

Time-and Concentration-Dependent Cytotoxicity of Ricin in Human Lung Epithelial Cells

DTIC Science & Technology

2007-07-01

lectin, ricin communis agglutinin, which is not directly cytotoxic but does have an affinity for red blood cells and can lead to agglutination and...Time- and Concentration-Dependent Cytotoxicity of Ricin in Human Lung Epithelial Cells Sharmaine Ramasamy and David Proll Human...Disease Control (CDC) Select Agent List. Using human small airway epithelial cells , this is the first study to investigate the time- and dose-dependent

Antibody-Dependent Cell-Mediated Cytotoxicity Effector-Enhanced EphA2 Agonist Monoclonal Antibody Demonstrates Potent Activity against Human Tumors1

PubMed Central

Bruckheimer, Elizabeth M; Fazenbaker, Christine A; Gallagher, Sandra; Mulgrew, Kathy; Fuhrmann, Stacy; Coffman, Karen T; Walsh, William; Ready, Shannon; Cook, Kim; Damschroder, Melissa; Kinch, Michael; Kiener, Peter A; Woods, Rob; Gao, Changshou; Dall'Acqua, William; Wu, Herren; Coats, Steven

2009-01-01

EphA2 is a receptor tyrosine kinase that has been shown to be overexpressed in a variety of human tumor types. Previous studies demonstrated that agonist monoclonal antibodies targeting EphA2 induced the internalization and degradation of the receptor, thereby abolishing its oncogenic effects. In this study, the in vitro and in vivo antibody-dependent cell-mediated cytotoxicity (ADCC) activity of EphA2 effector-enhanced agonist monoclonal antibodies was evaluated. With tumor cell lines and healthy human peripheral blood monocytes, the EphA2 antibodies demonstrated ∼80% tumor cell killing. In a dose-dependent manner, natural killer (NK) cells were required for the in vitro ADCC activity and became activated as demonstrated by the induction of cell surface expression of CD107a. To assess the role of NK cells on antitumor efficacy in vivo, the EphA2 antibodies were evaluated in xenograft models in severe compromised immunodeficient (SCID) mice (which have functional NK cells and monocytes) and SCID nonobese diabetic (NOD) mice (which largely lack functional NK cells and monocytes). Dosing of EphA2 antibody in the SCID murine tumor model resulted in a 6.2-fold reduction in tumor volume, whereas the SCID/nonobese diabetic model showed a 1.6-fold reduction over the isotype controls. Together, these results demonstrate that the anti-EphA2 monoclonal antibodies may function through at least two mechanisms of action: EphA2 receptor activation and ADCC-mediated activity. These novel EphA2 monoclonal antibodies provide additional means by which host effector mechanisms can be activated for selective destruction of EphA2-expressing tumor cells. PMID:19484140

Mechanisms of lectin and antibody-dependent polymorphonuclear leukocyte-mediated cytolysis.

PubMed

Tsunawaki, S; Ikenami, M; Mizuno, D; Yamazaki, M

1983-04-01

The mechanisms of tumor lysis by polymorphonuclear leukocytes (PMNs) were investigated. In antibody-dependent PMN-mediated cytolysis (ADPC), sensitized tumor cells were specifically lysed via Fc receptors on PMNs. On the other hand, lectin-dependent PMN-mediated cytolysis (LDPC) caused nonspecific lysis of several murine tumors after recognition of carbohydrate moieties on the cell membrane of both PMNs and tumor cells. Both ADPC and LDPC depended on glycolysis, and cytotoxicity was mediated by reactive oxygen species; LDPC was dependent on superoxide and ADPC on the myeloperoxidase system. The participation of reactive oxygen species in PMN cytotoxicity was also demonstrated by pharmacological triggering with phorbol myristate acetate. These results indicate that reactive oxygen species have an important role In tumor killing by PMNs and that ADPC and LDPC have partly different cytolytic processes as well as different recognition steps.

Alkaline Ceramidase 2 (ACER2) and Its Product Dihydrosphingosine Mediate the Cytotoxicity of N-(4-Hydroxyphenyl)retinamide in Tumor Cells*

PubMed Central

Mao, Zhehao; Sun, Wei; Xu, Ruijuan; Novgorodov, Sergei; Szulc, Zdzislaw M.; Bielawski, Jacek; Obeid, Lina M.; Mao, Cungui

2010-01-01

Increased generation of dihydrosphingosine (DHS), a bioactive sphingolipid, has been implicated in the cytotoxicity of the synthetic retinoid N-(4-hydroxyphenyl)retinamide (4-HPR) in tumor cells. However, how 4-HPR increases DHS remains unclear. Here we demonstrate that 4-HPR increases the expression of ACER2, which catalyzes the hydrolysis of dihydroceramides to generate DHS, and that ACER2 up-regulation plays a key role in mediating the 4-HPR-induced generation of DHS as well as the cytotoxicity of 4-HPR in tumor cells. Treatment with 4-HPR induced the accumulation of dihydroceramides (DHCs) in tumor cells by inhibiting dihydroceramide desaturase (DES) activity, which catalyzes the conversion of DHCs to ceramides. Treatment with 4-HPR also increased ACER2 expression through a retinoic acid receptor-independent and caspase-dependent manner. Overexpression of ACER2 augmented the 4-HPR-induced generation of DHS as well as 4-HPR cytotoxicity, and 4-HPR-induced death in tumor cells, whereas knocking down ACER2 had the opposite effects. ACER2 overexpression, along with treatment with GT11, another DES inhibitor, markedly increased cellular DHS, leading to tumor cell death, whereas ACER2 overexpression or GT11 treatment alone failed to do so, suggesting that both ACER2 up-regulation and DES inhibition are necessary and sufficient to mediate 4-HPR-induced DHS accumulation, cytotoxicity, and death in tumor cells. Taken together, these results suggest that up-regulation of the ACER2/DHS pathway mediates the cytotoxicity of 4-HPR in tumor cells and that up-regulating or activating ACER2 may improve the anti-cancer activity of 4-HRR and other DHC-inducing agents. PMID:20628055

Hantavirus-infection Confers Resistance to Cytotoxic Lymphocyte-Mediated Apoptosis

PubMed Central

Gupta, Shawon; Braun, Monika; Tischler, Nicole D.; Stoltz, Malin; Sundström, Karin B.; Björkström, Niklas K.; Ljunggren, Hans-Gustaf; Klingström, Jonas

2013-01-01

Hantaviruses cause hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardio-pulmonary syndrome (HCPS; also called hantavirus pulmonary syndrome (HPS)), both human diseases with high case-fatality rates. Endothelial cells are the main targets for hantaviruses. An intriguing observation in patients with HFRS and HCPS is that on one hand the virus infection leads to strong activation of CD8 T cells and NK cells, on the other hand no obvious destruction of infected endothelial cells is observed. Here, we provide an explanation for this dichotomy by showing that hantavirus-infected endothelial cells are protected from cytotoxic lymphocyte-mediated induction of apoptosis. When dissecting potential mechanisms behind this phenomenon, we discovered that the hantavirus nucleocapsid protein inhibits the enzymatic activity of both granzyme B and caspase 3. This provides a tentative explanation for the hantavirus-mediated block of cytotoxic granule-mediated apoptosis-induction, and hence the protection of infected cells from cytotoxic lymphocytes. These findings may explain why infected endothelial cells in hantavirus-infected patients are not destroyed by the strong cytotoxic lymphocyte response. PMID:23555267

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

PubMed

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.

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

PubMed

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.

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

NASA Technical Reports Server (NTRS)

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.

HBV-specific CD4+ cytotoxic T cells in hepatocellular carcinoma are less cytolytic toward tumor cells and suppress CD8+ T cell-mediated antitumor immunity.

PubMed

Meng, Fanzhi; Zhen, Shoumei; Song, Bin

2017-08-01

In East Asia and sub-Saharan Africa, chronic infection is the main cause of the development of hepatocellular carcinoma, an aggressive cancer with low survival rate. Cytotoxic T cell-based immunotherapy is a promising treatment strategy. Here, we investigated the possibility of using HBV-specific CD4 + cytotoxic T cells to eliminate tumor cells. The naturally occurring HBV-specific cytotoxic CD4 + and CD8 + T cells were identified by HBV peptide pool stimulation. We found that in HBV-induced hepatocellular carcinoma patients, the HBV-specific cytotoxic CD4 + T cells and cytotoxic CD8 + T cells were present at similar numbers. But compared to the CD8 + cytotoxic T cells, the CD4 + cytotoxic T cells secreted less cytolytic factors granzyme A (GzmA) and granzyme B (GzmB), and were less effective at eliminating tumor cells. In addition, despite being able to secrete cytolytic factors, CD4 + T cells suppressed the cytotoxicity mediated by CD8 + T cells, even when CD4 + CD25 + regulator T cells were absent. Interestingly, we found that interleukin 10 (IL-10)-secreting Tr1 cells were enriched in the cytotoxic CD4 + T cells. Neutralization of IL-10 abrogated the suppression of CD8 + T cells by CD4 + CD25 - T cells. Neither the frequency nor the absolute number of HBV-specific CD4 + cytotoxic T cells were correlated with the clinical outcome of advanced stage hepatocellular carcinoma patients. Together, this study demonstrated that in HBV-related hepatocellular carcinoma, CD4 + T cell-mediated cytotoxicity was present naturally in the host and had the potential to exert antitumor immunity, but its capacity was limited and was associated with immunoregulatory properties. © 2017 APMIS. Published by John Wiley & Sons Ltd.

Mediation of mouse natural cytotoxic activity by tumour necrosis factor

NASA Astrophysics Data System (ADS)

Ortaldo, John R.; Mason, Llewellyn H.; Mathieson, Bonnie J.; Liang, Shu-Mei; Flick, David A.; Herberman, Ronald B.

1986-06-01

Natural cell-mediated cytotoxic activity in the mouse has been associated with two types of effector cells, the natural killer (NK) cell and the natural cytotoxic (NC) cell, which seem to differ with regard to their patterns of target selectivity, cell surface characteristics and susceptibility to regulatory factors1. During studies on the mechanism of action of cytotoxic molecules, it became evident that WEHI-164, the prototype NC target cell, was highly susceptible to direct lysis by both human and mouse recombinant tumour necrosis factor (TNF). Here we show that NC, but not NK activity mediated by normal splenocytes, is abrogated by rabbit antibodies to recombinant and natural TNF, respectively. Thus, the cell-mediated activity defined as NC is due to release of TNF by normal spleen cells and does not represent a unique natural effector mechanism.

Anti-podocalyxin antibody exerts antitumor effects via antibody-dependent cellular cytotoxicity in mouse xenograft models of oral squamous cell carcinoma.

PubMed

Itai, Shunsuke; Ohishi, Tomokazu; Kaneko, Mika K; Yamada, Shinji; Abe, Shinji; Nakamura, Takuro; Yanaka, Miyuki; Chang, Yao-Wen; Ohba, Shun-Ichi; Nishioka, Yasuhiko; Kawada, Manabu; Harada, Hiroyuki; Kato, Yukinari

2018-04-27

Podocalyxin (PODXL) overexpression is associated with progression, metastasis, and poor outcomes in cancers. We recently produced the novel anti-PODXL monoclonal antibody (mAb) PcMab-47 (IgG 1 , kappa). Herein, we engineered PcMab-47 into 47-mG 2a , a mouse IgG 2a -type mAb, to add antibody-dependent cellular cytotoxicity (ADCC). We further developed 47-mG 2a -f, a core fucose-deficient type of 47-mG 2a to augment its ADCC. Immunohistochemical analysis of oral cancer tissues using PcMab-47 and 47-mG 2a revealed that the latter stained oral squamous cell carcinoma (OSCC) cells in a cytoplasmic pattern at a much lower concentration. PcMab-47 and 47-mG 2a detected PODXL in 163/201 (81.1%) and in 197/201 (98.0%) OSCC samples, respectively. 47-mG 2a -f also detected PODXL in OSCCs at a similar frequency as 47-mG 2a . In vitro analysis revealed that both 47-mG 2a and 47-mG 2a -f exhibited strong complement-dependent cytotoxicity (CDC) against CHO/hPODXL cells. In contrast, 47-mG 2a -f exhibited much stronger ADCC than 47-mG 2a against OSCC cells, indicating that ADCC and CDC of those anti-PODXL mAbs depend on target cells. In vivo analysis revealed that both 47-mG 2a and 47-mG 2a -f exerted antitumor activity in CHO/hPODXL xenograft models at a dose of 100 μg or 500 μg/mouse/week administered twice. 47-mG 2a -f, but not 47-mG 2a , exerted antitumor activity in SAS and HSC-2 xenograft models at a dose of 100 μg/mouse/week administered three times. Although both 47-mG 2a and 47-mG 2a -f exerted antitumor activity in HSC-2 xenograft models at a dose of 500 μg/mouse/week administered twice, 47-mG 2a -f also showed higher antitumor activity than 47-mG 2a . These results suggested that a core fucose-deficient anti-PODXL mAb could be useful for antibody-based therapy against PODXL-expressing OSCCs.

Anti-podocalyxin antibody exerts antitumor effects via antibody-dependent cellular cytotoxicity in mouse xenograft models of oral squamous cell carcinoma

PubMed Central

Itai, Shunsuke; Ohishi, Tomokazu; Kaneko, Mika K.; Yamada, Shinji; Abe, Shinji; Nakamura, Takuro; Yanaka, Miyuki; Chang, Yao-Wen; Ohba, Shun-Ichi; Nishioka, Yasuhiko; Kawada, Manabu; Harada, Hiroyuki; Kato, Yukinari

2018-01-01

Podocalyxin (PODXL) overexpression is associated with progression, metastasis, and poor outcomes in cancers. We recently produced the novel anti-PODXL monoclonal antibody (mAb) PcMab-47 (IgG1, kappa). Herein, we engineered PcMab-47 into 47-mG2a, a mouse IgG2a-type mAb, to add antibody-dependent cellular cytotoxicity (ADCC). We further developed 47-mG2a-f, a core fucose-deficient type of 47-mG2a to augment its ADCC. Immunohistochemical analysis of oral cancer tissues using PcMab-47 and 47-mG2a revealed that the latter stained oral squamous cell carcinoma (OSCC) cells in a cytoplasmic pattern at a much lower concentration. PcMab-47 and 47-mG2a detected PODXL in 163/201 (81.1%) and in 197/201 (98.0%) OSCC samples, respectively. 47-mG2a-f also detected PODXL in OSCCs at a similar frequency as 47-mG2a. In vitro analysis revealed that both 47-mG2a and 47-mG2a-f exhibited strong complement-dependent cytotoxicity (CDC) against CHO/hPODXL cells. In contrast, 47-mG2a-f exhibited much stronger ADCC than 47-mG2a against OSCC cells, indicating that ADCC and CDC of those anti-PODXL mAbs depend on target cells. In vivo analysis revealed that both 47-mG2a and 47-mG2a-f exerted antitumor activity in CHO/hPODXL xenograft models at a dose of 100 μg or 500 μg/mouse/week administered twice. 47-mG2a-f, but not 47-mG2a, exerted antitumor activity in SAS and HSC-2 xenograft models at a dose of 100 μg/mouse/week administered three times. Although both 47-mG2a and 47-mG2a-f exerted antitumor activity in HSC-2 xenograft models at a dose of 500 μg/mouse/week administered twice, 47-mG2a-f also showed higher antitumor activity than 47-mG2a. These results suggested that a core fucose-deficient anti-PODXL mAb could be useful for antibody-based therapy against PODXL-expressing OSCCs. PMID:29854293

Distinct regulatory functions of SLP-76 and MIST in NK cell cytotoxicity and IFN-gamma production.

PubMed

Hidano, Shinya; Sasanuma, Hiroki; Ohshima, Keiko; Seino, Ken-ichiro; Kumar, Lalit; Hayashi, Katsuhiko; Hikida, Masaki; Kurosaki, Tomohiro; Taniguchi, Masaru; Geha, Raif S; Kitamura, Daisuke; Goitsuka, Ryo

2008-03-01

Activation of NK cells is triggered by multiple receptors. We demonstrate here that SLP-76 is required for CD16- and NKG2D-mediated NK cell cytotoxicity, while MIST negatively regulates these responses in an SLP-76-dependent manner. Exceptionally, MIST acts as a positive regulator of cytotoxicity against YAC-1 cells, although SLP-76 plays a more key role. SLP-76 acts as a dominant positive regulator for both NKG2D-mediated and YAC-1 cell-triggered IFN-gamma production. Although NKG2D-mediated IFN-gamma production depends on phospholipase C (PLC) gamma 2, YAC-1 cell-triggered IFN-gamma production is PLC gamma 2- and Syk/ZAP-70 independent and nuclear factor-kappa B mediated. SLP-76 is required for this process in the presence of MIST but is dispensable in the absence of MIST. Thus, YAC-1 cell-triggered NKG2D-independent IFN-gamma production appears to be regulated by SLP-76-dependent and -independent pathways, in which the latter is negatively regulated by MIST. Taken together, these results suggest that SLP-76 and MIST distinctly but interactively regulate NK cell cytotoxicity and IFN-gamma production.

Abrogation of TNF-mediated cytotoxicity by space flight involves protein kinase C

NASA Technical Reports Server (NTRS)

Woods, K. M.; Chapes, S. K.; Spooner, B. S. (Principal Investigator)

1994-01-01

Experiments conducted on STS-50 indicated that space flight significantly inhibited tumor necrosis factor (TNF)-mediated killing of LM929 cells compared to ground controls. In ground-based studies, activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate (PMA) also inhibited TNF-mediated killing of LM929 cells. Therefore, we used PKC inhibitors to determine if the inhibitory effects of spaceflight on TNF-mediated cytotoxicity involved the activation of PKC. In experiments conducted onboard space shuttle mission STS-54, we saw that in the presence of the protein kinase C inhibitors H7 and H8, TNF-mediated cytotoxicity was restored to levels of those observed in the ground controls. Subsequent experiments done during the STS-57 mission tested the dose response of two protein kinase inhibitors, H7 and HA1004. We again saw that killing was restored in a dose-dependent manner, with inhibitor concentrations known to inhibit PKC being most effective. These data suggest that space flight ameliorates the action of TNF by affecting PKC in target cells.

Obinutuzumab induces superior B-cell cytotoxicity to rituximab in rheumatoid arthritis and systemic lupus erythematosus patient samples.

PubMed

Reddy, Venkat; Klein, Christian; Isenberg, David A; Glennie, Martin J; Cambridge, Geraldine; Cragg, Mark S; Leandro, Maria J

2017-07-01

A proportion of RA and SLE patients treated with standard doses of rituximab (RTX) display inefficient B cell deletion and poor clinical responses that can be augmented by delivering higher doses, indicating that standard-dose RTX is a sub-optimal therapy in these patients. This study aimed to investigate whether better responses could be achieved with mechanistically different anti-CD20 mAbs. We compared RTX with obinutuzumab (OBZ), a new-generation, glycoengineered type II anti-CD20 mAb, in a series of in vitro assays measuring B cell cytotoxicity in RA and SLE patient samples. We found that OBZ was at least 2-fold more efficient than RTX at inducing B-cell cytotoxicity in in vitro whole blood assays. Dissecting this difference, we found that RTX elicited more potent complement-dependent cellular cytotoxicity than OBZ. In contrast, OBZ was more effective at evoking Fc gamma receptor-mediated effector mechanisms, including activation of NK cells and neutrophils, probably due to stronger interaction with Fc gamma receptors and the ability of OBZ to remain at the cell surface following CD20 engagement, whereas RTX became internalized. OBZ was also more efficient at inducing direct cell death. This was true for all CD19 + B cells as a whole and in naïve (IgD + CD27 - ) and switched (IgD - CD27 + ) memory B cells specifically, a higher frequency of which is associated with poor clinical response after RTX. Taken together, these data provide a mechanistic basis for resistance to rituximab-induced B-cell depletion, and for considering obinutuzumab as an alternative B-cell depleting agent in RA and SLE. © The Author 2017. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Enhancement of antibody-dependent cellular cytotoxicity of cetuximab by a chimeric protein encompassing interleukin-15.

PubMed

Ochoa, Maria Carmen; Minute, Luna; López, Ascensión; Pérez-Ruiz, Elisabeth; Gomar, Celia; Vasquez, Marcos; Inoges, Susana; Etxeberria, Iñaki; Rodriguez, Inmaculada; Garasa, Saray; Mayer, Jan-Peter Andreas; Wirtz, Peter; Melero, Ignacio; Berraondo, Pedro

2018-01-01

Enhancement of antibody-dependent cellular cytotoxicity (ADCC) may potentiate the antitumor efficacy of tumor-targeted monoclonal antibodies. Increasing the numbers and antitumor activity of NK cells is a promising strategy to maximize the ADCC of standard-of-care tumor-targeted antibodies. For this purpose, we have preclinically tested a recombinant chimeric protein encompassing the sushi domain of the IL15Rα, IL-15, and apolipoprotein A-I (Sushi-IL15-Apo) as produced in CHO cells. The size-exclusion purified monomeric fraction of this chimeric protein was stable and retained the IL-15 and the sushi domain bioactivity as measured by CTLL-2 and Mo-7e cell proliferation and STAT5 phosphorylation in freshly isolated human NK and CD8 + T cells. On cell cultures, Sushi-IL15-Apo increases NK cell proliferation and survival as well as spontaneous and antibody-mediated cytotoxicity. Scavenger receptor class B type I (SR-B1) is the receptor for ApoA-I and is expressed on the surface of tumor cells. SR-B1 can adsorb the chimeric protein on tumor cells and can transpresent IL-15 to NK and CD8 + T cells. A transient NK-humanized murine model was developed to test the increase of ADCC attained by the chimeric protein in vivo . The EGFR + human colon cancer cell line HT-29 was intraperitoneally inoculated in immune-deficient Rag2 -/- γc -/- mice that were reconstituted with freshly isolated PBMCs and treated with the anti-EGFR mAb cetuximab. The combination of the Sushi-IL15-Apo protein and cetuximab reduced the number of remaining tumor cells in the peritoneal cavity and delayed tumor engraftment in the peritoneum. Furthermore, Sushi-IL15-Apo increased the anti-tumor effect of a murine anti-EGFR mAb in Rag1 -/- mice bearing subcutaneous MC38 colon cancer transfected to express EGFR. Thus, Sushi-IL15-Apo is a potent tool to increase the number and the activation of NK cells to promote the ADCC activity of antibodies targeting tumor antigens.

Clonidine Induces Apoptosis of Human Corneal Epithelial Cells through Death Receptors-Mediated, Mitochondria-Dependent Signaling Pathway.

PubMed

Fan, Dan; Fan, Ting-Jun

2017-03-01

Clonidine, an α2-adrenoreceptor agonist, is an anti-glaucoma drug clinically used in many developing countries, and its abuse might damage the cornea and impair human vision. However, its cytotoxicity and precise mechanisms need to be elucidated. Herein, we investigated the cytotoxicity of clonidine and its underlying mechanisms, using an in vitro model of human corneal epithelial (HCEP) cells and an in vivo model of cat corneas, respectively. HCEP cells were treated with various doses of clonidine for 1-28 h, resulting in abnormal morphology, decline of cell viability and G1 phase arrest in a time- and/or dose-dependent manner. Moreover, clonidine treatment induced elevation of plasma membrane permeability, phosphatidylserine externalization, DNA fragmentation, and apoptotic body formation in HCEP cells. Furthermore, we found that clonidine treatment resulted in activated caspase-2, -3, -8, and -9, disruption of the mitochondrial transmembrane potential, downregulation of Bcl-2, and upregulation of Bad, cytoplasmic cytochrome c and apoptosis inducing factor, suggesting that clonidine-induced apoptosis is triggered through Fas/TNFR1 death receptors and Bcl-2 family proteins-mediated mitochondria-dependent pathways. Finally, our in vivo results displayed that 0.25% clonidine could induce DNA fragmentation of cat corneal epithelial cells. In summary, our findings suggest that clonidine above 1/32 of its clinical therapeutic dosage is cytotoxic to corneal epithelial cells by inducing cell apoptosis both in vitro and in vivo, and its pro-apoptotic effect on HCEP cells is triggered by a Fas/TNFR1 death receptors-mediated, mitochondria-dependent signaling pathway. © The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Dihydroceramide accumulation mediates cytotoxic autophagy of cancer cells via autolysosome destabilization.

PubMed

Hernández-Tiedra, Sonia; Fabriàs, Gemma; Dávila, David; Salanueva, Íñigo J; Casas, Josefina; Montes, L Ruth; Antón, Zuriñe; García-Taboada, Elena; Salazar-Roa, María; Lorente, Mar; Nylandsted, Jesper; Armstrong, Jane; López-Valero, Israel; McKee, Christopher S; Serrano-Puebla, Ana; García-López, Roberto; González-Martínez, José; Abad, José L; Hanada, Kentaro; Boya, Patricia; Goñi, Félix; Guzmán, Manuel; Lovat, Penny; Jäättelä, Marja; Alonso, Alicia; Velasco, Guillermo

2016-11-01

Autophagy is considered primarily a cell survival process, although it can also lead to cell death. However, the factors that dictate the shift between these 2 opposite outcomes remain largely unknown. In this work, we used Δ 9 -tetrahydrocannabinol (THC, the main active component of marijuana, a compound that triggers autophagy-mediated cancer cell death) and nutrient deprivation (an autophagic stimulus that triggers cytoprotective autophagy) to investigate the precise molecular mechanisms responsible for the activation of cytotoxic autophagy in cancer cells. By using a wide array of experimental approaches we show that THC (but not nutrient deprivation) increases the dihydroceramide:ceramide ratio in the endoplasmic reticulum of glioma cells, and this alteration is directed to autophagosomes and autolysosomes to promote lysosomal membrane permeabilization, cathepsin release and the subsequent activation of apoptotic cell death. These findings pave the way to clarify the regulatory mechanisms that determine the selective activation of autophagy-mediated cancer cell death.

A Novel Assay for Antibody-Dependent Cell-Mediated Cytotoxicity against HIV-1- or SIV-Infected Cells Reveals Incomplete Overlap with Antibodies Measured by Neutralization and Binding Assays

PubMed Central

Alpert, Michael D.; Heyer, Lisa N.; Williams, David E. J.; Harvey, Jackson D.; Greenough, Thomas; Allhorn, Maria

2012-01-01

The resistance of human immunodeficiency virus type 1 (HIV-1) to antibody-mediated immunity often prevents the detection of antibodies that neutralize primary isolates of HIV-1. However, conventional assays for antibody functions other than neutralization are suboptimal. Current methods for measuring the killing of virus-infected cells by antibody-dependent cell-mediated cytotoxicity (ADCC) are limited by the number of natural killer (NK) cells obtainable from individual donors, donor-to-donor variation, and the use of nonphysiological targets. We therefore developed an ADCC assay based on NK cell lines that express human or macaque CD16 and a CD4+ T-cell line that expresses luciferase from a Tat-inducible promoter upon HIV-1 or simian immunodeficiency virus (SIV) infection. NK cells and virus-infected targets are mixed in the presence of serial plasma dilutions, and ADCC is measured as the dose-dependent loss of luciferase activity. Using this approach, ADCC titers were measured in plasma samples from HIV-infected human donors and SIV-infected macaques. For the same plasma samples paired with the same test viruses, this assay was approximately 2 orders of magnitude more sensitive than optimized assays for neutralizing antibodies—frequently allowing the measurement of ADCC in the absence of detectable neutralization. Although ADCC correlated with other measures of Env-specific antibodies, neutralizing and gp120 binding titers did not consistently predict ADCC activity. Hence, this assay affords a sensitive method for measuring antibodies capable of directing ADCC against HIV- or SIV-infected cells expressing native conformations of the viral envelope glycoprotein and reveals incomplete overlap of the antibodies that direct ADCC and those measured in neutralization and binding assays. PMID:22933282

Nitric Oxide-Induced Apoptosis of Human Dental Pulp Cells Is Mediated by the Mitochondria-Dependent Pathway

PubMed Central

Park, Min Young; Jeong, Yeon Jin; Kang, Gi Chang; Kim, Mi-Hwa; Kim, Sun Hun; Chung, Hyun-Ju

2014-01-01

Nitric oxide (NO) is recognized as a mediator and regulator of inflammatory responses. NO is produced by nitric oxide synthase (NOS), and NOS is abundantly expressed in the human dental pulp cells (HDPCs). NO produced by NOS can be cytotoxic at higher concentrations to HDPCs. However, the mechanism by which this cytotoxic pathway is activated in cells exposed to NO is not known. The purpose of this study was to elucidate the NO-induced cytotoxic mechanism in HDPCs. Sodium nitroprusside (SNP), a NO donor, reduced the viability of HDPCs in a dose- and time-dependent manner. We investigated the in vitro effects of nitric oxide on apoptosis of cultured HDPCs. Cells showed typical apoptotic morphology after exposure to SNP. Besides, the number of Annexin V positive cells was increased among the SNP-treated HDPCs. SNP enhanced the production of reactive oxygen species (ROS), and N-acetylcysteine (NAC) ameliorated the decrement of cell viability induced by SNP. However, a soluble guanylate cyclase inhibitor (ODQ) did not inhibited the decrement of cell viability induced by SNP. SNP increased cytochrome c release from the mitochondria to the cytosol and the ratio of Bax/Bcl-2 expression levels. Moreover, SNP-treated HDPCs elevated activities of caspase-3 and caspase-9. While pretreatment with inhibitors of caspase (z-VAD-fmk, z-DEVD-fmk) reversed the NO-induced apoptosis of HDPCs. From these results, it can be suggested that NO induces apoptosis of HDPCs through the mitochondria-dependent pathway mediated by ROS and Bcl-2 family, but not by the cyclic GMP pathway. PMID:24634593

Immunomodulatory properties of medicinal mushrooms: differential effects of water and ethanol extracts on NK cell-mediated cytotoxicity.

PubMed

Lu, Chia-Chen; Hsu, Ya-Jing; Chang, Chih-Jung; Lin, Chuan-Sheng; Martel, Jan; Ojcius, David M; Ko, Yun-Fei; Lai, Hsin-Chih; Young, John D

2016-10-01

Medicinal mushrooms have been used for centuries in Asian countries owing to their beneficial effects on health and longevity. Previous studies have reported that a single medicinal mushroom may produce both stimulatory and inhibitory effects on immune cells, depending on conditions, but the factors responsible for this apparent dichotomy remain obscure. We show here that water and ethanol extracts of cultured mycelium from various species (Agaricus blazei Murrill, Antrodia cinnamomea, Ganoderma lucidum and Hirsutella sinensis) produce opposite effects on NK cells. Water extracts enhance NK cell cytotoxic activity against cancer cells, whereas ethanol extracts inhibit cytotoxicity. Water extracts stimulate the expression and production of cytolytic proteins (perforin and granulysin) and NKG2D/NCR cell surface receptors, and activate intracellular signaling kinases (ERK, JNK and p38). In contrast, ethanol extracts inhibit expression of cytolytic and cell surface receptors. Our results suggest that the mode of extraction of medicinal mushrooms may determine the nature of the immunomodulatory effects produced on immune cells, presumably owing to the differential solubility of stimulatory and inhibitory mediators. These findings have important implications for the preparation of medicinal mushrooms to prevent and treat human diseases. © The Author(s) 2016.

Development of IgG Mediated Antibody Dependent Cell-mediated Cytotoxicity (ADCC) in the Serum and Genital Mucosa of HIV Seroconverters

PubMed Central

Aziz, Mariam; Mahmood, Fareeha; Mata, Mariana; Durkin, Helen G; Liu, Chenglong; Greenblatt, Ruth M; Nowicki, Marek; Golub, Elizabeth T; Anastos, Kathryn; French, Audrey L; Baum, Linda L

2015-01-01

Background We measured antibody-dependent cell mediated cytotoxicity (ADCC) activity in serum and genital fluids of heterosexually exposed women during HIV seroconversion. Methods Plasma and cervico-vaginal lavage (CVL) fluid from 11 seroconverters (SC) were analyzed biannually from one year pre- to 6 year post-seroconversion using a 51Cr-release assay to measure HIV-1 gp120 specific ADCC. Results No SC had significant HIV specific CVL ADCC activity before seroconversion or until 1.5 yr after seroconversion. One individual had a %Specific Release (SR) of 25.4 at 2 years, 26.7 at 3 years and 21.0 at 4 years after seroconversion in CVL. Another sample had 4.7% SR at 2 years, 5.3 at 3 years, 10.9 at 4 years, and 8.4 at 5 years after seroconversion in CVL. A third had no activity until 17% SR 5 years after seroconversion in CVL. A fourth showed activity of 36.5% SR at 6.5 years after seroconversion. Seven women had no ADCC activity in their CVL. Paired serum samples showed HIV specific ADCC activity prior to the appearance of CVL ADCC activity. Conclusions HIV specific ADCC activity in CVL rose 2 years after seroconversion; ADCC was present in the serum prior to this time. These data suggest that genital tract ADCC activity is not present until well after acute infection. PMID:26798561

Fas/FasL interaction mediates imbalanced cytokine/cytotoxicity responses of iNKT cells against Jurkat cells.

PubMed

Dou, Rui; Hong, Zhenya; Tan, Xiaosheng; Hu, Fenfen; Ding, Yajie; Wang, Wei; Liang, Zhihui; Zhong, Rongrong; Wu, Xiongwen; Weng, Xiufang

2018-07-01

The rapid antitumor cytokine production and direct cytotoxicity confer invariant NKT (iNKT) cells ideal candidates for cancer therapy. However, the therapeutic potential of iNKT cells in T-cell malignant diseases remains elusive, as antigen presentation by T cells (T-T presentation) has been suggested to induce hyporesponsiveness of iNKT cells. In this study, we found discrepancies in iNKT cell responses against two T cell-origin cell lines (Jurkat and Molt-4). Human iNKT cells exhibited more intensive cytotoxicity and less efficient cytokine production in response to Fas-bearing Jurkat cells than those to the Fas-negative tumor cells (Molt-4 and myeloid-derived K562). The imbalanced cytokine/cytotoxicity responses of iNKT cells against Jurkat cells were CD1d-dependent and relied mostly on Fas/FasL interaction. The impairment in cytokine production could be overcome by Fas/FasL blocking antibodies and exogenous IL-2. Elevated CD1d levels as well as CD1d and Fas co-localization were found in T-cell lymphomas. However, defects in frequency and function of circulating iNKT cells were observed in the patients, which could be partly rescued by exogenous IL-2. Collectively, the Fas/FasL-dependent aberrant iNKT cell responses and the reversibility of the defects suggest the distinct iNKT cell manipulation in CD1d- and Fas-bearing T cell malignancies. Copyright © 2018. Published by Elsevier Ltd.

Cystatin F as a regulator of immune cell cytotoxicity.

PubMed

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.

Regulation of NK92-MI cell cytotoxicity by substance P.

PubMed

Fu, W X; Qin, B; Zhou, A P; Yu, Q Y; Huang, Q J; Liang, Z F

2011-08-01

The neuropeptide substance P (SP) can regulate a number of immunological functions in vitro and in vivo and may regulate natural killer (NK) cell activity. Here, we investigated whether SP has a role in regulating NK92-MI cell function in vitro, and how it influences NK cell activity. We found that SP dose dependently increased the cytotoxicity of NK92-MI cells and had a maximal effect at a concentration of 10(-12) and 10(-10) m. Furthermore, the expression of cytotoxic-associated molecules (perforin, granzyme) and activating receptor NKp46 [a member of natural cytotoxicity receptors (NCRs)] was observed to be upregulated by SP at optimal concentration, at which SP enhanced the cytotoxicity of NK92-MI cells. Neurokinin-1 receptor (NK-1R), a functional receptor of SP, was found on NK92-MI cells, and the observed effects of SP on NK92-MI cells could be more partially blocked by an NK-1R antagonist. Our data suggest that SP induces NK92-MI cell cytotoxicity by directly increasing the expression of cytotoxic granules and upregulates NK92-MI cell receptor-mediated functions indirectly. Thus, SP may regulate NK cell function mainly through NK-1R. © 2011 The Authors. Scandinavian Journal of Immunology © 2011 Blackwell Publishing Ltd.

Microvesicle- and exosome-mediated drug delivery enhances the cytotoxicity of Paclitaxel in autologous prostate cancer cells.

PubMed

Saari, Heikki; Lázaro-Ibáñez, Elisa; Viitala, Tapani; Vuorimaa-Laukkanen, Elina; Siljander, Pia; Yliperttula, Marjo

2015-12-28

Extracellular vesicles (EVs) are naturally occurring membrane particles that mediate intercellular communication by delivering molecular information between cells. In this study, we investigated the effectiveness of two different populations of EVs (microvesicle- and exosome-enriched) as carriers of Paclitaxel to autologous prostate cancer cells. EVs were isolated from LNCaP- and PC-3 prostate cancer cell cultures using differential centrifugation and characterized by electron microscopy, nanoparticle tracking analysis, and Western blot. The uptake of microvesicles and exosomes by the autologous prostate cancer cells was assessed by flow cytometry and confocal microscopy. The EVs were loaded with Paclitaxel and the effectiveness of EV-mediated drug delivery was assessed with viability assays. The distribution of EVs and EV-delivered Paclitaxel in cells was inspected by confocal microscopy. Our main finding was that the loading of Paclitaxel to autologous prostate cancer cell-derived EVs increased its cytotoxic effect. This capacity was independent of the EV population and the cell line tested. Although the EVs without the drug increased cancer cell viability, the net effect of enhanced cytotoxicity remained. Both EV populations delivered Paclitaxel to the recipient cells through endocytosis, leading to the release of the drug from within the cells. The removal of EV surface proteins did not affect exosomes, while the drug delivery mediated by microvesicles was partially inhibited. Cancer cell-derived EVs can be used as effective carriers of Paclitaxel to their parental cells, bringing the drug into the cells through an endocytic pathway and increasing its cytotoxicity. However, due to the increased cell viability, the use of cancer cell-derived EVs must be further investigated before any clinical applications can be designed. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

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

PubMed

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.

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

PubMed Central

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

2015-01-01

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. PMID:26405606

Cytotoxicity of Manganese (III) Complex in Human Breast Adenocarcinoma Cell Line Is Mediated by the Generation of Reactive Oxygen Species Followed by Mitochondrial Damage.

PubMed

Al-Anbaky, Qudes; Al-Karakooly, Zeiyad; Kilaparty, Surya P; Agrawal, Megha; Albkuri, Yahya M; RanguMagar, Ambar B; Ghosh, Anindya; Ali, Nawab

2016-11-01

Manganese (Mn) complexes are widely studied because of their important catalytic properties in synthetic and biochemical reactions. A Mn (III) complex of an amidoamine ligand was synthesized using a tetradentate amidoamine ligand. In this study, the Mn (III) complex was evaluated for its biological activity by measuring its cytotoxicity in human breast adenocarcinoma cell line (MCF-7). Cytotoxic effects of the Mn (III) complex were determined using established biomarkers in an attempt to delineate the mechanism of action and the utility of the complex as a potential anticancer drug. The Mn (III) complex induces cell death in a dose- and time-dependent manner as shown by microculture tetrazolium assay, a measure of cytotoxic cell death. Our results demonstrated that cytotoxic effects were significantly increased at higher concentrations of Mn (III) complex and with longer time of treatment. The IC 50 (Inhibitor concentration that results in 50% cell death) value of Mn (III) complex in MCF-7 cells was determined to be 2.5 mmol/L for 24 hours of treatment. In additional experiments, we determined the Mn (III) complex-mediated cell death was due to both apoptotic and nonspecific necrotic cell death mechanisms. This was assessed by ethidium bromide/acridine orange staining and flow cytometry techniques. The Mn (III) complex produced reactive oxygen species (ROS) triggering the expression of manganese superoxide dismutase 1 and ultimately damaging the mitochondrial function as is evident by a decline in mitochondrial membrane potential. Treatment of the cells with free radical scavenger, N, N-dimethylthiourea decreased Mn (III) complex-mediated generation of ROS and attenuated apoptosis. Together, these results suggest that the Mn (III) complex-mediated MCF-7 cell death utilizes combined mechanism involving apoptosis and necrosis perhaps due to the generation of ROS. © The Author(s) 2016.

Mouse Cytotoxic T Cell-derived Granzyme B Activates the Mitochondrial Cell Death Pathway in a Bim-dependent Fashion*

PubMed Central

Catalán, Elena; Jaime-Sánchez, Paula; Aguiló, Nacho; Simon, Markus M.; Froelich, Christopher J.; Pardo, Julián

2015-01-01

Cytotoxic T cells (Tc) use perforin and granzyme B (gzmB) to kill virus-infected cells and cancer cells. Recent evidence suggests that human gzmB primarily induces apoptosis via the intrinsic mitochondrial pathway by either cleaving Bid or activating Bim leading to the activation of Bak/Bax and subsequent generation of active caspase-3. In contrast, mouse gzmB is thought to predominantly induce apoptosis by directly processing pro-caspase-3. However, in certain mouse cell types gzmB-mediated apoptosis mainly occurs via the mitochondrial pathway. To investigate whether Bim is involved under the latter conditions, we have now employed ex vivo virus-immune mouse Tc that selectively kill by using perforin and gzmB (gzmB+Tc) as effector cells and wild type as well as Bim- or Bak/Bax-deficient spontaneously (3T9) or virus-(SV40) transformed mouse embryonic fibroblast cells as targets. We show that gzmB+Tc-mediated apoptosis (phosphatidylserine translocation, mitochondrial depolarization, cytochrome c release, and caspase-3 activation) was severely reduced in 3T9 cells lacking either Bim or both Bak and Bax. This outcome was related to the ability of Tc cells to induce the degradation of Mcl-1 and Bcl-XL, the anti-apoptotic counterparts of Bim. In contrast, gzmB+Tc-mediated apoptosis was not affected in SV40-transformed mouse embryonic fibroblast cells lacking Bak/Bax. The data provide evidence that Bim participates in mouse gzmB+Tc-mediated apoptosis of certain targets by activating the mitochondrial pathway and suggest that the mode of cell death depends on the target cell. Our results suggest that the various molecular events leading to transformation and/or immortalization of cells have an impact on their relative resistance to the multiple gzmB+Tc-induced death pathways. PMID:25605735

Bioenergetic metabolites regulate base excision repair dependent cell death in response to DNA damage

PubMed Central

Tang, Jiang-bo; Goellner, Eva M.; Wang, Xiao-hong; Trivedi, Ram N.; Croix, Claudette M. St; Jelezcova, Elena; Svilar, David; Brown, Ashley R.; Sobol, Robert W.

2009-01-01

Base excision repair (BER) protein expression is important for resistance to DNA damage-induced cytotoxicity. Conversely, BER imbalance (Polß deficiency or repair inhibition) enhances cytotoxicity of radiation and chemotherapeutic DNA-damaging agents. Whereas inhibition of critical steps in the BER pathway result in the accumulation of cytotoxic DNA double-strand breaks, we report that DNA damage-induced cytotoxicity due to deficiency in the BER protein Polß triggers cell death dependent on PARP activation yet independent of poly(ADP-ribose) (PAR)-mediated AIF nuclear translocation or PARG, suggesting that cytotoxicity is not from PAR or PAR-catabolite signaling. Cell death is rescued by the NAD+ metabolite NMN and is synergistic with inhibition of NAD+ biosynthesis, demonstrating that DNA damage-induced cytotoxicity mediated via BER inhibition is primarily dependent on cellular metabolite bioavailability. We offer a mechanistic justification for the elevated alkylation-induced cytotoxicity of Polß deficient cells, suggesting a linkage between DNA repair, cell survival and cellular bioenergetics. PMID:20068071

Sirtuin 1-dependent resveratrol cytotoxicity and pro-differentiation activity on breast cancer cells.

PubMed

Deus, Cláudia M; Serafim, Teresa L; Magalhães-Novais, Silvia; Vilaça, Andreia; Moreira, Ana C; Sardão, Vilma A; Cardoso, Susana M; Oliveira, Paulo J

2017-03-01

Sirtuins regulate several processes associated with tumor development. Resveratrol was shown to stimulate sirtuin 1 and 3 (SIRT1/3) activities and to result in cytotoxicity for some tumor types. The relationship between modulation of sirtuin activities, cellular metabolic remodeling and resveratrol cytotoxicity mechanism on breast cancer cells is still an open question. Here, we evaluated whether sirtuin 1 and 3 are involved in resveratrol toxicity and whether resveratrol leads to a metabolic remodeling and cell differentiation. Results using the Extracellular Flux Analyzer indicated that resveratrol inhibits mitochondrial respiration in breast cancer cells. We also demonstrated here for the first time that resveratrol cytotoxic effects on breast cancer cells were modulated by SIRT1 and also involved mitochondrial complex I inhibition. Importantly, we also demonstrated that resveratrol reduced the pool of breast cancer cells with stemness markers through a SIRT1-dependent mechanism. Our data highlights the role of SIRT1 in regulating resveratrol induced differentiation and/or toxicity in breast cancer cells.

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

PubMed

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.

Optimization of Dendritic Cell-Mediated Cytotoxic T-Cell Activation by Tracking of Dendritic Cell Migration Using Reporter Gene Imaging.

PubMed

Lee, Hongje; Lee, Ho Won; La Lee, You; Jeon, Yong Hyun; Jeong, Shin Young; Lee, Sang-Woo; Lee, Jaetae; Ahn, Byeong-Cheol

2018-06-01

The aim of this study is to optimize the dendritic cell (DC)-mediated T-cell activation using reporter gene imaging and flow cytometric analysis in living mice. A murine dendritic cell line (DC2.4) co-expressing effluc and Thy1.1 genes were established by transfection with retroviral vectors. Thy1.1 positive cells were sorted by magnetic bead separation system (DC2.4/effluc). Cell proliferation assay and phenotype analysis to determine the effects of gene transduction on the function of dendritic cells between parental DC2.4 and DC2.4/effluc were performed. To optimize the DC-mediated immune response by cell number or frequency, different cell numbers (5 × 10 5 , 1 × 10 6 , and 2 × 10 6  DC2.4/effluc) or different frequencies of DC2.4/effluc (first, second, and third injections) were injected in the right footpad of mice. The migration of the DC2.4/effluc into the draining popliteal lymph node of mice was monitored by bioluminescence imaging (BLI). Flow cytometric analysis was performed with splenocytes to determine the cytotoxic T-cell population after injection of DC2.4/effluc. Parental DC2.4 and DC2.4/effluc exhibit no significant differences in their proliferation and phenotype. BLI signals were observed in the draining popliteal lymph node at day 1 after injection of DC2.4/effluc in 1 × 10 6 and 2 × 10 6 cells-injected groups. The highest BLI signal intensity was detected in 2 × 10 6 cells-injected mice. On day 11, the BLI signal was detected in only 2 × 10 6 cell-injected group but not in other groups. Optimized cell numbers (2 × 10 6 ) were injected in three animal groups with a different frequency (first, second, and third injection groups). The BLI signal was detected at day 1 and maintained until day 7 in the first injection group, but there is low signal intensity in the second and the third injection groups. Although the expression levels of Thy1.1 gene in the first injection group were very high, there reveals no expression of

Role of glycogen synthase kinase 3 beta (GSK3beta) in mediating the cytotoxic effects of the histone deacetylase inhibitor trichostatin A (TSA) in MCF-7 breast cancer cells.

PubMed

Alao, John P; Stavropoulou, Alexandra V; Lam, Eric W-F; Coombes, R Charles

2006-10-03

Histone deacetylase inhibitors (HDACIs) have been shown to induce apoptotic and autophagic cell death in vitro and in vivo. The molecular mechanisms that underlie these cytotoxic effects are not yet clearly understood. Recently, HDACIs were shown to induce Akt dephosphorylation by disrupting HDAC-protein phosphatase 1 (PP1) complexes. This disruption results in the increased association of PP1 with Akt, resulting in the dephosphorylation and consequent inactivation of the kinase. Akt enhances cellular survival through the phosphorylation-dependent inhibition of several pro-apoptotic proteins. Akt is an important negative regulator of GSK3beta, a kinase that has been shown to regulate apoptosis in response to various stimuli. In the present study, we investigated the role of GSK3beta in mediating the cytotoxic effects in MCF-7 breast cancer cells treated with trichostatin A (TSA), a prototype HDACI. We show that TSA induces Akt dephosphorylation in a PP1-dependent manner, resulting in activation of GSK3beta in MCF-7 cells. Similarly, knockdown of HDAC1 and-2 by small interfering RNA (siRNA) resulted in the dephosphorylation of Akt and GSK3beta. Selective inhibition of GSK3beta attenuated TSA induced cytotoxicity and resulted in enhanced proliferation following drug removal. Our findings identify GSK3beta as an important mediator of TSA-induced cytotoxicity in MCF-7 breast cancer cells.

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

EPA Pesticide Factsheets

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

Deficiency of activated STAT1 in head and neck cancer cells mediates TAP1-dependent escape from cytotoxic T lymphocytes

PubMed Central

Leibowitz, Michael S.; Filho, Pedro A. Andrade; Ferrone, Soldano; Ferris, Robert L.

2012-01-01

Squamous cell carcinoma of the head and neck (SCCHN) cells can escape recognition by tumor antigen (TA)-specific cytotoxic T lymphocytes (CTL) by downregulation of antigen processing machinery (APM) components, such as the transporter associated with antigen processing (TAP)-1/2 heterodimer. APM component upregulation by interferon gamma (IFN-γ) restores SCCHN cell recognition and susceptibility to lysis by CTL, but the mechanism underlying TAP1/2 downregulation in SCCHN cells is not known. Because IFN-γ activates signal transducer and activator of transcription (STAT)-1, we investigated phosphorylated (p)-STAT1 as a mediator of low basal TAP1/2 expression in SCCHN cells. SCCHN cells were found to express basal total STAT1 but low to undetectable levels of activated STAT1. The association of increased pSTAT1 levels and APM components likely reflects a cause–effect relationship, since STAT1 knockdown significantly reduced both IFN-γ-mediated APM component expression and TA-specific CTL recognition of IFN-γ-treated SCCHN cells. On the other hand, since oncogenic pSTAT3 is overexpressed in SCCHN cells and was found to heterodimerize with pSTAT1, we also tested whether pSTAT3 and pSTAT1:pSTAT3 heterodimers inhibited IFN-γ-induced STAT1 activation and APM component expression. First, STAT3 activation or depletion did not affect basal or IFN-γ-induced expression of pSTAT1 and APM components or recognition of SCCHN cells by TA-specific CTL. Second, pSTAT1:pSTAT3 heterodimers did not interfere with IFN-γ-induced STAT1 binding to the TAP1 promoter or APM protein expression. These findings demonstrate that APM component downregulation is regulated primarily by an IFN-γ-pSTAT1-mediated signaling pathway, independent of oncogenic STAT3 overexpression in SCCHN cells. PMID:21207025

Epistatic role of base excision repair and mismatch repair pathways in mediating cisplatin cytotoxicity

PubMed Central

Kothandapani, Anbarasi; Sawant, Akshada; Dangeti, Venkata Srinivas Mohan Nimai; Sobol, Robert W.; Patrick, Steve M.

2013-01-01

Base excision repair (BER) and mismatch repair (MMR) pathways play an important role in modulating cis-Diamminedichloroplatinum (II) (cisplatin) cytotoxicity. In this article, we identified a novel mechanistic role of both BER and MMR pathways in mediating cellular responses to cisplatin treatment. Cells defective in BER or MMR display a cisplatin-resistant phenotype. Targeting both BER and MMR pathways resulted in no additional resistance to cisplatin, suggesting that BER and MMR play epistatic roles in mediating cisplatin cytotoxicity. Using a DNA Polymerase β (Polβ) variant deficient in polymerase activity (D256A), we demonstrate that MMR acts downstream of BER and is dependent on the polymerase activity of Polβ in mediating cisplatin cytotoxicity. MSH2 preferentially binds a cisplatin interstrand cross-link (ICL) DNA substrate containing a mismatch compared with a cisplatin ICL substrate without a mismatch, suggesting a novel mutagenic role of Polβ in activating MMR in response to cisplatin. Collectively, these results provide the first mechanistic model for BER and MMR functioning within the same pathway to mediate cisplatin sensitivity via non-productive ICL processing. In this model, MMR participation in non-productive cisplatin ICL processing is downstream of BER processing and dependent on Polβ misincorporation at cisplatin ICL sites, which results in persistent cisplatin ICLs and sensitivity to cisplatin. PMID:23761438

Endothelial cell cytotoxicity of cotton bracts tannin and aqueous cotton bracts extract

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

Johnson, C.M.; Hanson, M.N.; Rohrbach, M.S.

1986-04-01

Using an in vitro cytotoxicity assay based on the release of /sup 51/Cr from cultured porcine thoracic aortic and pulmonary arterial endothelial cells, we have demonstrated that cotton bracts tannin is a potent endothelial cell cytotoxin. It produces dose-dependent lethal injury to both types of endothelial cells with the aortic cells, being somewhat more sensitive to tannin-mediated injury than the pulmonary arterial cells. Cytotoxic injury to the cells was biphasic. During the first 3 hr of exposure to tannin, no lethal injury was detected. However, during this period, profound changes in morphology were observed suggesting sublethal injury to the cellsmore » preceded the ultimate toxic damage. Comparison of the cytotoxicity dose curves for aqueous bracts extracts with those for tannin demonstrated that tannin was major cytotoxin present in bracts.« less

OSU-DY7, a novel D-tyrosinol derivative, mediates cytotoxicity in chronic lymphocytic leukaemia and Burkitt lymphoma through p38 mitogen-activated protein kinase pathway

PubMed Central

Bai, Li-Yuan; Ma, Yihui; Kulp, Samuel K.; Wang, Shu-Huei; Chiu, Chang-Fang; Frissora, Frank; Mani, Rajeswaran; Mo, Xiaokui; Jarjoura, David; Byrd, John C.; Chen, Ching-Shih; Muthusamy, Natarajan

2013-01-01

Summary Drug resistance and associated immune deregulation limit use of current therapies in chronic lymphocytic leukaemia (CLL), thus warranting alternative therapy development. Herein we demonstrate that OSU-DY7, a novel D-tyrosinol derivative targeting p38 mitogen-activated protein kinase (MAPK), mediates cytotoxicity in lymphocytic cell lines representing CLL (MEC-1), acute lymphoblastic leukaemia (697 cells), Burkitt lymphoma (Raji and Ramos) and primary B cells from CLL patients in a dose- and time-dependent manner. The OSU-DY7-induced cytotoxicity is dependent on caspase activation, as evidenced by induction of caspase-3 activation and poly (ADP-ribose) polymerase (PARP) cleavage and rescue of cytotoxicity by Z-VAD-FMK. Interestingly, OSU-DY7-induced cytotoxicity is mediated through activation of p38 MAPK, as evidenced by increased phosphorylation of p38 MAPK and downstream target protein MAPKAPK2. Pretreatment of B-CLL cells with SB202190, a specific p38 MAPK inhibitor, results in decreased MAPKAPK2 protein level with concomitant rescue of the cells from OSU-DY7-mediated cytotoxicity. Furthermore, OSU-DY7-induced cytotoxicity is associated with down regulation of p38 MAPK target BIRC5, that is rescued at protein and mRNA levels by SB202190. This study provides evidence for a role of OSU-DY7 in p38 MAPK activation and BIRC5 down regulation associated with apoptosis in B lymphocytic cells, thus warranting development of this alternative therapy for lymphoid malignancies. PMID:21470196

Neutrophils kill pulmonary endothelial cells by a hydrogen-peroxide-dependent pathway. An in vitro model of neutrophil-mediated lung injury.

PubMed

Martin, W J

1984-08-01

Neutrophil-mediated injury to lung parenchymal cells has been proposed as an important step in the pathogenesis of many acute and chronic lung disorders. As an in vitro model of neutrophil-mediated injury, this study used activated human neutrophils as effector cells in an 18-h cytotoxicity assay with 51Cr-labeled bovine pulmonary artery endothelial cells serving as target cells. Neutrophils effectively injured pulmonary endothelial cells, expressed as cytotoxic index (CI), of 63.8 +/- 5.4, and this injury could be significantly reduced by several agents, including 1% dimethyl sulfoxide (CI, 51.3 +/- 3.7), 50 micrograms/ml ascorbic acid (CI, 40.8 +/- 4.7), and especially 1,100 U/ml catalase (CI, 14.3 +/- 4.1). As cell-free models of neutrophil-mediated endothelial cell injury, H2O2 (30 microM), O2- (generated by 0.5 mU xanthine oxidase), and the myeloperoxidase-dependent (0.32 U) hypohalite ion were each capable of injuring the target cells with CI of 6.21 +/- 2.8, 53.6 +/- 5.3, and 21.2 +/- 1.5, respectively. Catalase was effective in reducing the injurious effect of each of these oxidant-generating systems (p less than 0.01, all comparisons), confirming the important role for H2O2 in the mediation of this injury. The data indicate that neutrophils are capable of killing pulmonary endothelial cells by a pathway largely dependent on the generation of H2O2, and suggest the possibility that removal of H2O2 from the alveolar structures in subjects with these disorder might be an effective future therapeutic approach.

Increased Soluble CD226 in Sera of Patients with Cutaneous T-Cell Lymphoma Mediates Cytotoxic Activity against Tumor Cells via CD155.

PubMed

Takahashi, Naomi; Sugaya, Makoto; Suga, Hiraku; Oka, Tomonori; Kawaguchi, Makiko; Miyagaki, Tomomitsu; Fujita, Hideki; Inozume, Takashi; Sato, Shinichi

2017-08-01

Immune checkpoint therapy, which targets regulatory pathways in T cells to enhance antitumor immune responses, has led to important clinical advances. CD155 is expressed in various types of cancer, and this surface molecule on tumor cells functions either as a co-stimulatory molecule or a co-inhibitory molecule, depending on its receptor. CD226, a CD155 ligand, is mainly expressed on natural killer cells and CD8 + T cells, playing important roles in natural killer cell-mediated cytotoxicity. In this study, we investigated the expression and function of CD155 and CD226 in cutaneous T-cell lymphoma (CTCL). CD155 was strongly expressed on tumor cells and CD155 mRNA expression levels were increased in CTCL lesional skin. CD226 expression on natural killer cells and CD8 + cells in peripheral blood of CTCL patients was decreased. On the other hand, serum CD226 levels were significantly elevated in CTCL patients, strongly reflecting disease activity, suggesting that soluble CD226 in sera was generated by shedding of its membrane form. Recombinant CD226 itself showed cytotoxic activity against CD155-expressing CTCL cells in vitro. These data suggest that soluble CD226 elevated in sera of CTCL patients would be important for tumor immunity by interacting with CD155 on tumor cells. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Lymphocyte-dependent antibody-mediated cytotoxicity in Hashimoto thyroiditis

PubMed Central

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

Particle-size-dependent cytokine responses and cell damage induced by silica particles and macrophages-derived mediators in endothelial cell.

PubMed

Rong, Yi; Zhou, Ting; Cheng, Wenjuan; Guo, Jiali; Cui, Xiuqing; Liu, Yuewei; Chen, Weihong

2013-11-01

Epidemiological evidence reports silica dust exposure has been associated with increased risk of cardiovascular diseases, but the mechanisms are largely unknown. In this study, endothelial cells were exposed to increasing concentrations of two sizes silica particles and the soluble mediators released by macrophages treated with the same particles for 24 h. Expression and release of cytokines (IL-1β, TNF-α and IL-6) were measured by using ELISA. Cytotoxicity was measured by MTT assay and LDH release. We show that both ways induced increases in cell toxicity and cytokines in a dose-dependent manner. For smaller particles, the soluble mediators are more capable of increasing cytokines compared with the effect of particles directly. For larger particles, evaluating results of these two ways are similar. Either way, smaller particles make the increasing action of cell toxicity and cytokines more remarkable. Our results indicate both silica particle and macrophage-derived mediators can induce endothelial cell injury and inflammation and demonstrate the potential importance of the particle sizes in this effect. Copyright © 2013. Published by Elsevier B.V.

Curcumin analog cytotoxicity against breast cancer cells: exploitation of a redox-dependent mechanism.

PubMed

Sun, Aiming; Lu, Yang J; Hu, Haipeng; Shoji, Mamoru; Liotta, Dennis C; Snyder, James P

2009-12-01

A series of novel curcumin analogs, symmetrical dienones, were previously shown to possess cytotoxic, anti-angiogenic and anti-tumor activities. Analogs 1 (EF24) and 2 (EF31) share the dienone scaffold and serve as Michael acceptors. We propose that the anti-cancer effects of 1 and 2 are mediated in part by redox-mediated induction of apoptosis. In order to support this concept, 1 and 2 were treated with L-glutathione (GSH) and cysteine-containing dipeptides under mild conditions to form colorless water-soluble adducts, which were identified by LC/MS. Comparison of the cytotoxic action of 1, 2 and the corresponding conjugates, 1-(GSH)(2) and 2-(GSH)(2), illustrated that the two classes of compounds exhibit essentially identical cell killing capabilities. Compared with the yellow, somewhat light sensitive and nearly water insoluble compounds 1 and 2, the glutathione conjugates represent a promising new series of stable and soluble anti-tumor pro-drugs.

β4-integrin-mediated cytotoxic activity of AexU in human prostate cancer PC3 cells

PubMed Central

KUMANO, MASAFUMI; MIYAKE, HIDEAKI; ABOLGHAIT, SAID K.; BEHNSAWY, HOSNY M.; FUJISAWA, MASATO

2013-01-01

The present study aimed to characterize the cytotoxic activity of AexU, an effector-mediating type three secretion system (TTSS) of gram-negative bacteria, in human prostate cancer cells, focusing on the association with β4-integrin expression. The cytotoxic effects of AexU either alone or in combination with chemotherapeutic agents were evaluated using several human prostate cancer cell lines. Human prostate cancer PC3 cells, in which an expression vector containing siRNA targeting β4-integrin had been introduced, were established (PC3/sh-In), and the cytotoxic effects of AexU on the PC3/sh-In cells were compared with the PC3 cells that were transfected with a control vector (PC3/C). The expression levels of β4-integrin in the PC3 cells were markedly higher compared with those in the LNCaP or DU145 cells, and the cytotoxic effects of AexU in the PC3 cells were more pronounced compared with those in the LNCaP or DU145 cells. The sensitivity of the PC3 cells to docetaxel and cisplatin was significantly enhanced following treatment with AexU, resulting in a decrease in the IC50 of the two agents by ~90%. The cytotoxic effect of AexU in the PC3/C cells was more marked compared with that in the PC3/sh-In cells, and the phosphorylation of Akt in the PC3/C cells appeared to be significantly more inhibited by the treatment with AexU compared with the PC3/sh-In cells. In conclusion, treatment with AexU may be a useful therapeutic option for prostate cancer when β4-integrin is overexpressed. The treatment appears to exert its effects through growth inhibition and by enhancing the sensitivity of the cancer cells to chemotherapeutic agents. PMID:24179545

Measuring T cell-mediated cytotoxicity using fluorogenic caspase substrates.

PubMed

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.

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

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

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 forebrainmore » 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.« less

Arf-like GTPase Arl8b regulates lytic granule polarization and natural killer cell-mediated cytotoxicity.

PubMed

Tuli, Amit; Thiery, Jerome; James, Ashley M; Michelet, Xavier; Sharma, Mahak; Garg, Salil; Sanborn, Keri B; Orange, Jordan S; Lieberman, Judy; Brenner, Michael B

2013-12-01

Natural killer (NK) lymphocytes contain lysosome-related organelles (LROs), known as lytic granules, which upon formation of immune synapse with the target cell, polarize toward the immune synapse to deliver their contents to the target cell membrane. Here, we identify a small GTP-binding protein, ADP-ribosylation factor-like 8b (Arl8b), as a critical factor required for NK cell-mediated cytotoxicity. Our findings indicate that Arl8b drives the polarization of lytic granules and microtubule-organizing centers (MTOCs) toward the immune synapse between effector NK lymphocytes and target cells. Using a glutathione S-transferase pull-down approach, we identify kinesin family member 5B (KIF5B; the heavy chain of kinesin-1) as an interaction partner of Arl8b from NK cell lysates. Previous studies showed that interaction between kinesin-1 and Arl8b is mediated by SifA and kinesin-interacting protein (SKIP) and the tripartite complex drives the anterograde movement of lysosomes. Silencing of both KIF5B and SKIP in NK cells, similar to Arl8b, led to failure of MTOC-lytic granule polarization to the immune synapse, suggesting that Arl8b and kinesin-1 together control this critical step in NK cell cytotoxicity.

Heme Mediates Cytotoxicity from Artemisinin and Serves as a General Anti-Proliferation Target

PubMed Central

Zhang, Shiming; Gerhard, Glenn S.

2009-01-01

Heme (Fe2+ protoporphyrin IX) is an essential molecule that has been implicated the potent antimalarial action of artemisinin and its derivatives, although the source and nature of the heme remain controversial. Artemisinins also exhibit selective cytotoxicity against cancer cells in vitro and in vivo. We demonstrate that intracellular heme is the physiologically relevant mediator of the cytotoxic effects of artemisinins. Increasing intracellular heme synthesis through the addition of aminolevulinic acid, protoporphyrin IX, or transferrin-bound iron increased the cytotoxicity of dihydroartemisinin, while decreasing heme synthesis through the addition of succinyl acetone decreased its cytotoxic activity. A simple and robust high throughput assay was developed to screen chemical compounds that were capable of interacting with heme. A natural products library was screened which identified the compound coralyne, in addition to artemisinin, as a heme interacting compound with heme synthesis dependent cytotoxic activity. These results indicate that cellular heme may serve a general target for the development of both anti-parasitic and anti-cancer therapeutics. PMID:19862332

Cell type-dependent uptake, localization, and cytotoxicity of 1.9 nm gold nanoparticles

PubMed Central

Coulter, Jonathan A; Jain, Suneil; Butterworth, Karl T; Taggart, Laura E; Dickson, Glenn R; McMahon, Stephen J; Hyland, Wendy B; Muir, Mark F; Trainor, Coleman; Hounsell, Alan R; O’Sullivan, Joe M; Schettino, Giuseppe; Currell, Fred J; Hirst, David G; Prise, Kevin M

2012-01-01

Background This follow-up study aims to determine the physical parameters which govern the differential radiosensitization capacity of two tumor cell lines and one immortalized normal cell line to 1.9 nm gold nanoparticles. In addition to comparing the uptake potential, localization, and cytotoxicity of 1.9 nm gold nanoparticles, the current study also draws on comparisons between nanoparticle size and total nanoparticle uptake based on previously published data. Methods We quantified gold nanoparticle uptake using atomic emission spectroscopy and imaged intracellular localization by transmission electron microscopy. Cell growth delay and clonogenic assays were used to determine cytotoxicity and radiosensitization potential, respectively. Mechanistic data were obtained by Western blot, flow cytometry, and assays for reactive oxygen species. Results Gold nanoparticle uptake was preferentially observed in tumor cells, resulting in an increased expression of cleaved caspase proteins and an accumulation of cells in sub G1 phase. Despite this, gold nanoparticle cytotoxicity remained low, with immortalized normal cells exhibiting an LD50 concentration approximately 14 times higher than tumor cells. The surviving fraction for gold nanoparticle-treated cells at 3 Gy compared with that of untreated control cells indicated a strong dependence on cell type in respect to radiosensitization potential. Conclusion Gold nanoparticles were most avidly endocytosed and localized within cytoplasmic vesicles during the first 6 hours of exposure. The lack of significant cytotoxicity in the absence of radiation, and the generation of gold nanoparticle-induced reactive oxygen species provide a potential mechanism for previously reported radiosensitization at megavoltage energies. PMID:22701316

Cytotoxic activity against human neuroblastoma and melanoma cells mediated by IgM antibodies derived from peripheral blood of healthy donors.

PubMed

Devarapu, Satish Kumar; Mamidi, Srinivas; Plöger, Frank; Dill, Othmar; Blixt, Ola; Kirschfink, Michael; Schwartz-Albiez, Reinhard

2016-06-15

A small percentage of healthy donors identified in the Western population carry antibodies in their peripheral blood which convey cytotoxic activity against certain human melanoma and neuroblastoma cell lines. We measured the cytotoxic activity of sera and plasmas from healthy donors on the human neuroblastoma cell line Kelly and various melanoma cell lines. Antibodies of IgM isotype, presumably belonging to the class of naturally occurring antibodies, exerted cytotoxic activity in a complement-dependent fashion. Apart from complement-dependent tumor cell lysis, we observed C3 opsonization in all tumor cell lines upon treatment with cytotoxic plasmas. Cell lines tested primarily expressed membrane complement regulatory proteins (mCRP) CD46, CD55 and CD59 to various extents. Blocking of mCRPs by monoclonal antibodies enhanced cell lysis and opsonization, though some melanoma cells remained resistant to complement attack. Epitopes recognized by cytotoxic antibodies were represented by gangliosides such as GD2 and GD3, as evidenced by cellular sialidase pretreatment and enhanced expression of distinct gangliosides. It remains to be clarified why only a small fraction of healthy persons carry these antitumor cytotoxic antibodies. © 2016 UICC.

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

PubMed

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.

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

PubMed

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.

A cell impedance measurement device for the cytotoxicity assay dependent on the velocity of supplied toxic fluid

NASA Astrophysics Data System (ADS)

Kang, Yoon-Tae; Kim, Min-Ji; Cho, Young-Ho

2018-04-01

We present a cell impedance measurement chip capable of characterizing the toxic response of cells depending on the velocity of the supplied toxic fluid. Previous impedance-based devices using a single open-top chamber have been limited to maintaining a constant supply velocity, and devices with a single closed-top chamber present difficulties in simultaneous cytotoxicity assay for varying levels of supply velocities. The present device, capable of generating constant and multiple levels of toxic fluid velocity simultaneously within a single stepwise microchannel, performs a cytotoxicity assay dependent on toxic fluid velocity, in order to find the effective velocity of toxic fluid to cells for maximizing the cytotoxic effect. We analyze the cellular toxic response of 5% ethanol media supplied to cancer cells within a toxic fluid velocity range of 0-8.3 mm s-1. We observe the velocity-dependent cell detachment rate, impedance, and death rate. We find that the cell detachment rate decreased suddenly to 2.4% at a velocity of 4.4 mm s-1, and that the change rates of cell resistance and cell capacitance showed steep decreases to 8% and 41%, respectively, at a velocity of 5.7 mm s-1. The cell death rate and impedance fell steeply to 32% at a velocity of 5.7 mm s-1. We conclude that: (1) the present device is useful in deciding on the toxic fluid velocity effective to cytotoxicity assay, since the cellular toxic response is dependent on the velocity of toxic fluid, and; (2) the cell impedance analysis facilitates a finer cellular response analysis, showing better correlation with the cell death rate, compared to conventional visual observation. The present device, capable of performing the combinational analysis of toxic fluid velocity and cell impedance, has potential for application to the fine cellular toxicity assay of drugs with proper toxic fluid velocity.

Influence of the Envelope gp120 Phe 43 Cavity on HIV-1 Sensitivity to Antibody-Dependent Cell-Mediated Cytotoxicity Responses

PubMed Central

Prévost, Jérémie; Zoubchenok, Daria; Richard, Jonathan; Veillette, Maxime; Pacheco, Beatriz; Coutu, Mathieu; Brassard, Nathalie; Parsons, Matthew S.; Ruxrungtham, Kiat; Bunupuradah, Torsak; Tovanabutra, Sodsai; Hwang, Kwan-Ki; Moody, M. Anthony; Haynes, Barton F.; Bonsignori, Mattia; Sodroski, Joseph; Kaufmann, Daniel E.; Shaw, George M.; Chenine, Agnès L.

2017-01-01

ABSTRACT HIV-1-infected cells presenting envelope glycoproteins (Env) in the CD4-bound conformation on their surface are preferentially targeted by antibody-dependent cellular-mediated cytotoxicity (ADCC). HIV-1 has evolved sophisticated mechanisms to avoid the exposure of Env ADCC epitopes by downregulating CD4 and by limiting the overall amount of Env on the cell surface. In HIV-1, substitution of large residues such as histidine or tryptophan for serine 375 (S375H/W) in the gp120 Phe 43 cavity, where Phe 43 of CD4 contacts gp120, results in the spontaneous sampling of an Env conformation closer to the CD4-bound state. While residue S375 is well conserved in the majority of group M HIV-1 isolates, CRF01_AE strains have a naturally occurring histidine at this position (H375). Interestingly, CRF01_AE is the predominant circulating strain in Thailand, where the RV144 trial took place. In this trial, which resulted in a modest degree of protection, ADCC responses were identified as being part of the correlate of protection. Here we investigate the influence of the Phe 43 cavity on ADCC responses. Filling this cavity with a histidine or tryptophan residue in Env with a natural serine residue at this position (S375H/W) increased the susceptibility of HIV-1-infected cells to ADCC. Conversely, the replacement of His 375 by a serine residue (H375S) within HIV-1 CRF01_AE decreased the efficiency of the ADCC response. Our results raise the intriguing possibility that the presence of His 375 in the circulating strain where the RV144 trial was held contributed to the observed vaccine efficacy. IMPORTANCE HIV-1-infected cells presenting Env in the CD4-bound conformation on their surface are preferentially targeted by ADCC mediated by HIV-positive (HIV+) sera. Here we show that the gp120 Phe 43 cavity modulates the propensity of Env to sample this conformation and therefore affects the susceptibility of infected cells to ADCC. CRF01_AE HIV-1 strains have an unusual Phe 43

Chimeric Anti-Human Podoplanin Antibody NZ-12 of Lambda Light Chain Exerts Higher Antibody-Dependent Cellular Cytotoxicity and Complement-Dependent Cytotoxicity Compared with NZ-8 of Kappa Light Chain.

PubMed

Kaneko, Mika K; Abe, Shinji; Ogasawara, Satoshi; Fujii, Yuki; Yamada, Shinji; Murata, Takeshi; Uchida, Hiroaki; Tahara, Hideaki; Nishioka, Yasuhiko; Kato, Yukinari

2017-02-01

Podoplanin (PDPN), a type I transmembrane 36-kDa glycoprotein, is expressed not only in normal cells, such as renal epithelial cells (podocytes), lymphatic endothelial cells, and pulmonary type I alveolar cells, but also in cancer cells, including brain tumors and lung squamous cell carcinomas. Podoplanin activates platelet aggregation by binding to C-type lectin-like receptor-2 (CLEC-2) on platelets, and the podoplanin/CLEC-2 interaction facilitates blood/lymphatic vessel separation. We previously produced neutralizing anti-human podoplanin monoclonal antibody (mAb), clone NZ-1 (rat IgG 2a , lambda), which neutralizes the podoplanin/CLEC-2 interaction and inhibits platelet aggregation and cancer metastasis. Human-rat chimeric antibody, NZ-8, was previously developed using variable regions of NZ-1 and human constant regions of heavy chain (IgG 1 ) and light chain (kappa chain). Although NZ-8 showed high antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against human podoplanin-expressing cancer cells, the binding affinity of NZ-8 was lower than that of NZ-1. Herein, we produced a novel human-rat chimeric antibody, NZ-12, the constant regions of which consist of IgG 1 heavy chain and lambda light chain. Using flow cytometry, we demonstrated that the binding affinity of NZ-12 was much higher than that of NZ-8. Furthermore, ADCC and CDC activities of NZ-12 were significantly increased against glioblastoma cell lines (LN319 and D397) and lung cancer cell line (PC-10). These results suggested that NZ-12 could become a promising therapeutic antibody against podoplanin-expressing brain tumors and lung cancers.

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

PubMed

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.

Withaferin A induces mitochondrial-dependent apoptosis in non-small cell lung cancer cells via generation of reactive oxygen species.

PubMed

Liu, Xi; Chen, Lei; Liang, Tao; Tian, Xiao-Dong; Liu, Yang; Zhang, Tao

2017-01-01

Withaferin A (WA) is a bioactive lactone, isolated from natural sources, mainly found in Withania somnifera, and was known to be highly effective against a variety of tumor cells both in vitro and in vivo. Accumulating experimental evidence suggests the involvement of reactive oxygen species (ROS) in WA-mediated cytotoxicity against cancer cells. Hence, the purpose of this study was to investigate the effect of WA in non-small cell lung cancer (NSCLC) cells and also the role of ROC in WA-mediated cytotoxicity. In the present study we investigated the cytotoxic potential of WA against NSCLC cell line A549 and also highlighted the mechanism of cytotoxicity of this compound. Non-carcinoma WI-38 and PBMC cell lines were used as controls. WA treatment resulted in a dose-dependent cytotoxicity in A549 cells, while the non-carcinoma cells WI-38 and PBMC were unaffected. Further experimental approaches revealed that ROS plays a major role in WAinduced apoptosis in NSCLC cells. WA induces oxidative damage to NSCLC cells with minimum toxicity to normal cells.

Sodium fluoride induces apoptosis in mouse embryonic stem cells through ROS-dependent and caspase- and JNK-mediated pathways

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

Nguyen Ngoc, Tam Dan; Son, Young-Ok; Lim, Shin-Saeng

2012-03-15

Sodium fluoride (NaF) is used as a source of fluoride ions in diverse applications. Fluoride salt is an effective prophylactic for dental caries and is an essential element required for bone health. However, fluoride is known to cause cytotoxicity in a concentration-dependent manner. Further, no information is available on the effects of NaF on mouse embryonic stem cells (mESCs). We investigated the mode of cell death induced by NaF and the mechanisms involved. NaF treatment greater than 1 mM reduced viability and DNA synthesis in mESCs and induced cell cycle arrest in the G{sub 2}/M phase. The addition of NaFmore » induced cell death mainly by apoptosis rather than necrosis. Catalase (CAT) treatment significantly inhibited the NaF-mediated cell death and also suppressed the NaF-mediated increase in phospho-c-Jun N-terminal kinase (p-JNK) levels. Pre-treatment with SP600125 or z-VAD-fmk significantly attenuated the NaF-mediated reduction in cell viability. In contrast, intracellular free calcium chelator, but not of sodium or calcium ion channel blockers, facilitated NaF-induced toxicity in the cells. A JNK specific inhibitor (SP600125) prevented the NaF-induced increase in growth arrest and the DNA damage-inducible protein 45α. Further, NaF-mediated loss of mitochondrial membrane potential was apparently inhibited by pifithrin-α or CAT inhibitor. These findings suggest that NaF affects viability of mESCs in a concentration-dependent manner, where more than 1 mM NaF causes apoptosis through hydroxyl radical-dependent and caspase- and JNK-mediated pathways. -- Highlights: ► The mode of NaF-induced cell death and the mechanisms involved were examined. ► NaF induced mainly apoptotic death of mouse embryonic stem cells (mESCs). ► NaF induced mitochondrial-mediated and caspase-dependent apoptosis. ► JNK- and p53-mediated pathways are involved in NaF-mediated apoptosis in the cells. ► ROS are the up-stream effector in NaF-mediated activation of JNK and p53

Adenovirus infection and cytotoxicity of primary mantle cell lymphoma cells.

PubMed

Medina, Daniel J; Sheay, Wendy; Osman, Mona; Goodell, Lauri; Martin, John; Rabson, Arnold B; Strair, Roger K

2005-11-01

Mantle cell lymphoma (MCL) is a distinct form of non-Hodgkin's lymphoma (NHL) derived from CD5+ B cells. MCL cells overexpress cyclin D1 as a consequence of translocation of the gene into the immunoglobulin heavy-chain gene locus. MCL is an aggressive form of NHL with frequent relapses after standard-dose chemotherapy. In this context, a variety of novel therapies for patients with MCL have been investigated. In this study, we use an expanded panel of attenuated adenoviruses to study adenovirus-mediated cytotoxicity of MCL cells. Our results demonstrate: 1) adenovirus infection of MCL cells despite the absence of receptor/coreceptor molecules known to be important for adenovirus infection of other cells types; 2) cytotoxicity of MCL cells after infection with specific adenovirus mutants; 3) a high degree of cytotoxicity after infection of some patient samples with viruses lacking the E1B 19k "antiapoptotic" gene; and 4) cytotoxicity after infection with viruses containing mutations in E1A pRb or p300 binding. The extent of cytotoxicity with the panel of viruses demonstrated interpatient variability, but 100% cytotoxicity, as determined by molecular analysis, was detected in some samples. These studies provide the foundation for: 1) the development of adenoviruses as cytotoxic agents for MCL and 2) analyses of key regulatory pathways operative in MCL cells.

Antitumour activity mediated by CD4+ cytotoxic T lymphocytes against MHC class II-negative mouse hepatocellular carcinoma induced by dendritic cell vaccine and interleukin-12.

PubMed

Homma, Sadamu; Komita, Hideo; Sagawa, Yukiko; Ohno, Tsuneya; Toda, Gotaro

2005-08-01

When BALA/c mice with BNL hepatocellular carcinoma (HCC) were treated with dendritic cells fused with BNL cells (DC/BNL) and recombinant murine interleukin (IL)-12, tumour development was significantly suppressed, whereas treatment with either DC/BNL or IL-12 alone did not show a tumour-suppressive effect. Antitumour activity induced by DC/BNL + IL-12 was abrogated by depletion of CD4+ T cells, but not by depletion of CD8+ T cells or natural killer cells. Splenic CD4+ T cells and CD8+ T cells from DC/BNL-treated mice showed cytotoxic activity against BNL cells after 3 days of incubation with DC/BNL, although BNL cells do not express major histocompatibility complex (MHC) class II molecules even after treatment with interferon (INF)-gamma. Furthermore, CD4+ T cells killed syngeneic-irrelevant CT26 cells and even allogeneic Hepa1-6 cells. This cytotoxicity was blocked by concanamycin A, but not by an anti-Fas ligand (FasL) monoclonal antibody, indicating that cytotoxic activity was mediated by perforin. Immunofluorescence microscopy demonstrated that abundant CD4+ T cells and MHC class II-positive macrophages, but not CD8(+) T cells, had infiltrated tumour tissue in mice treated with DC/BNL + IL-12. Flow cytometric analysis of tumour-infiltrating cells in mice treated with DC/BNL + IL-12 showed increases in CD4+ T cells and MHC class II+ CD11b+ cells but not in CD8+ T cells or MHC class I+ CD11b+ cells. Our results suggest that, in BNL-bearing mice treated with DC/BNL + IL-12, tumour macrophages activated by INF-gamma produced by IL-12-stimulated T cells might present BNL tumour antigens and activate DC/BNL-primed CD4+ cytotoxic T lymphocytes (CTLs) in a MHC class II-dependent manner, leading to perforin-mediated bystander killing of neighbouring MHC class II-negative tumour cells.

Antitumour activity mediated by CD4+ cytotoxic T lymphocytes against MHC class II-negative mouse hepatocellular carcinoma induced by dendritic cell vaccine and interleukin-12

PubMed Central

Homma, Sadamu; Komita, Hideo; Sagawa, Yukiko; Ohno, Tsuneya; Toda, Gotaro

2005-01-01

When BALA/c mice with BNL hepatocellular carcinoma (HCC) were treated with dendritic cells fused with BNL cells (DC/BNL) and recombinant murine interleukin (IL)-12, tumour development was significantly suppressed, whereas treatment with either DC/BNL or IL-12 alone did not show a tumour-suppressive effect. Antitumour activity induced by DC/BNL + IL-12 was abrogated by depletion of CD4+ T cells, but not by depletion of CD8+ T cells or natural killer cells. Splenic CD4+ T cells and CD8+ T cells from DC/BNL-treated mice showed cytotoxic activity against BNL cells after 3 days of incubation with DC/BNL, although BNL cells do not express major histocompatibility complex (MHC) class II molecules even after treatment with interferon (INF)-γ. Furthermore, CD4+ T cells killed syngeneic-irrelevant CT26 cells and even allogeneic Hepa1-6 cells. This cytotoxicity was blocked by concanamycin A, but not by an anti-Fas ligand (FasL) monoclonal antibody, indicating that cytotoxic activity was mediated by perforin. Immunofluorescence microscopy demonstrated that abundant CD4+ T cells and MHC class II-positive macrophages, but not CD8+ T cells, had infiltrated tumour tissue in mice treated with DC/BNL + IL-12. Flow cytometric analysis of tumour-infiltrating cells in mice treated with DC/BNL + IL-12 showed increases in CD4+ T cells and MHC class II+ CD11b+ cells but not in CD8+ T cells or MHC class I+ CD11b+ cells. Our results suggest that, in BNL-bearing mice treated with DC/BNL + IL-12, tumour macrophages activated by INF-γ produced by IL-12-stimulated T cells might present BNL tumour antigens and activate DC/BNL-primed CD4+ cytotoxic T lymphocytes (CTLs) in a MHC class II-dependent manner, leading to perforin-mediated bystander killing of neighbouring MHC class II-negative tumour cells. PMID:16011514

Protein disulfide isomerase mediates glutathione depletion-induced cytotoxicity.

PubMed

Okada, Kazushi; Fukui, Masayuki; Zhu, Bao-Ting

2016-08-26

Glutathione depletion is a distinct cause underlying many forms of pathogenesis associated with oxidative stress and cytotoxicity. Earlier studies showed that glutamate-induced glutathione depletion in immortalized murine HT22 hippocampal neuronal cells leads to accumulation of reactive oxygen species (ROS) and ultimately cell death, but the precise mechanism underlying these processes is not clear. Here we show that during the induction of glutathione depletion, nitric oxide (NO) accumulation precedes ROS accumulation. While neuronal NO synthase (nNOS) in untreated HT22 cells exists mostly as a monomer, glutathione depletion results in increased formation of the dimer nNOS, accompanied by increases in the catalytic activity. We identified that nNOS dimerization is catalyzed by protein disulfide isomerase (PDI). Inhibition of PDI's isomerase activity effectively abrogates glutathione depletion-induced conversion of monomer nNOS into dimer nNOS, accumulation of NO and ROS, and cytotoxicity. Furthermore, we found that PDI is present in untreated cells in an inactive S-nitrosylated form, which becomes activated following glutathione depletion via S-denitrosylation. These results reveal a novel role for PDI in mediating glutathione depletion-induced oxidative cytotoxicity, as well as its role as a valuable therapeutic target for protection against oxidative cytotoxicity. Copyright © 2016. Published by Elsevier Inc.

Receptor-mediated cytotoxicity of alpha-MSH fragments containing melphalan in a human melanoma cell line.

PubMed

Morandini, R; Süli-Vargha, H; Libert, A; Loir, B; Botyánszki, J; Medzihradszky, K; Ghanem, G

1994-01-02

Four alpha-MSH drug conjugates have been synthesized, 2 C-terminal (Pep 3 and 4) and 2 central fragments (Pep 1 and 2), the latter being the 4-10 sequence known to be the main alpha-MSH-receptor-recognition site. Melphalan was introduced into each sequence at different locations. Their ability to recognize alpha-MSH receptors as well as their cytotoxic effects were compared in 3 cell lines: melanoma, carcinoma and fibroblast cells. Pep 1 and 2 were able to specifically bind to MSH receptors on melanoma cells by displacing labelled alpha-MSH from its binding sites at concentrations similar to the 4-10 heptapeptide sequence known to contain the main receptor-recognition site. They subsequently penetrate the cell, most probably by a receptor internalization mechanism, since about half of their effect could be inhibited by competition at the receptor level. Significant and selective cytotoxic effects to melanoma cells could be observed after only 2 hr exposure to the drug conjugates. Interestingly, these 2 conjugates, differing only in melphalan position, showed completely different cytotoxicity in terms of IC50 values, Pep 1 being 24 times more toxic to all cells; but the 2 were equally specific to melanoma cells. However, they both were less toxic to all cells than melphalan itself. Furthermore, Pep 1 and 2 were able to block the receptor and, unlike Pep 3 and 4, their cytotoxic effect could be significantly inhibited by an alpha-MSH agonist. Pep 3 and 4 were 5 to 10 times less toxic than melphalan to melanoma and carcinoma cells and 50 times less to fibroblast cells, and did not show any cell-type selectivity. They were less toxic than Pep 1 to melanoma and carcinoma cells by a factor of 2, but equally toxic to fibroblasts. In contrast, they were more toxic than Pep 2 to fibroblasts, melanoma and carcinoma by a factor of 3, 10 and 24 respectively. Our data strongly suggest a receptor-mediated cytotoxicity mechanism occurring with alpha-MSH central fragments in human

Antagonism of cytotoxic chemotherapy in neuroblastoma cell lines by 13-cis-retinoic acid is mediated by the anti-apoptotic Bcl-2 family proteins

PubMed Central

Hadjidaniel, Michael Daniel; Reynolds, Charles Patrick

2010-01-01

13-cis-retinoic acid (13-cis-RA), is given at completion of cytotoxic therapy to control minimal residual disease in neuroblastoma. We investigated the effect of combining 13-cis-RA with cytotoxic agents employed in neuroblastoma therapy using a panel of 6 neuroblastoma cell lines. The effect of 13-cis-RA on the mitochondrial apoptotic pathway, was studied by flow cytometry, cytotoxicity by DIMSCAN, and protein expression by immuoblotting. Pre-treatment and direct combination of 13-cis-RA with etoposide, topotecan, cisplatin, melphalan, or doxorubicin markedly antagonized the cytotoxicity of those agents in 4 out of 6 tested neuroblastoma cell lines, increasing fractional cell survival by 1 to 3 logs. The inhibitory concentration of drugs (IC99) increased from clinically achievable levels to non-achievable levels: > 5-fold (cisplatin) to > 7-fold (etoposide). In SMS-KNCR neuroblastoma cells, 13-cis-RA upregulated expression of Bcl-2 and Bcl-xL RNA and protein, and this was associated with protection from etoposide-mediated apoptosis at the mitochondrial level. A small molecule inhibitor of the Bcl-2 family of proteins (ABT-737) restored mitochondrial membrane potential loss and apoptosis in response to cytotoxic agents in 13-cis-RA treated cells. Prior selection for resistance to RA did not diminish the response to cytotoxic treatment. Thus, combining 13-cis-RA with cytotoxic chemotherapy significantly reduced the cytotoxiciity for neuroblastoma in vitro, mediated at least in part via the anti-apoptotic Bcl-2 family of proteins. PMID:21159604

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.

PubMed

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.

Development of DS-5573a: A novel afucosylated mAb directed at B7-H3 with potent antitumor activity.

PubMed

Nagase-Zembutsu, Akiko; Hirotani, Kenji; Yamato, Michiko; Yamaguchi, Junko; Takata, Takehiko; Yoshida, Makoto; Fukuchi, Keisuke; Yazawa, Mitsuhiro; Takahashi, Shu; Agatsuma, Toshinori

2016-05-01

B7-H3 is highly overexpressed in a variety of human clinical tumors, and its expression is significantly associated with poor outcomes. In our study, we aimed to develop new antitumor mAbs by employing cancer cell immunization, and succeeded in generating a mouse anti-human B7-H3 antibody (M30) that shows antitumor activity. M30 was humanized (Hu-M30), and an afucosylated Hu-M30 (DS-5573a) was also generated. To assess the potency of DS-5573a as a therapeutic mAb, we characterized this mAb and evaluated its antitumor activity in vitro and in vivo. Flow cytometry analysis showed that B7-H3 proteins were expressed on various types of cancer cell lines broadly, and DS-5573a binds to IgC1 and IgC2 domains of human B7-H3. Antibody-dependent cellular cytotoxicity activity of DS-5573a was drastically enhanced against medium to high B7-H3-expressing cancer cell lines MDA-MB-231 and NCI-H322. DS-5573a also induced high antibody-dependent cellular cytotoxicity activity against low B7-H3-expressing cancer cell line COLO205, whereas Hu-M30 induced little activity against it. In addition, DS-5573a was found to be a novel anti-B7-H3 antibody which showed antibody-dependent cellular phagocytosis activity. Furthermore, DS-5573a showed dose-dependent and significant antitumor efficacy (0.03-3 mg/kg) in MDA-MB-231-bearing SCID mice (which have functional natural killer cells and macrophages), but little antitumor efficacy in NOG mice (which lack natural killer cells and have reduced macrophage function). These results suggest that antitumor activity of DS-5573a is mediated by effector cells, and this mAb could be a promising antitumor therapy for patients with a wide range of B7-H3-expressing tumors. © 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Cytotoxicity Induced by a Redox-silent Analog of Tocotrienol in Human Mesothelioma H2452 Cell Line via Suppression of Cap-dependent Protein Translation.

PubMed

Sato, Ayami; Ueno, Haruka; Takase, Akari; Ando, Akira; Sekine, Yuko; Yano, Tomohiro

2016-04-01

De novo synthesis of proteins is regulated by cap-dependent protein translation. Aberrant activation of the translation is a hallmark of many cancer types including malignant mesothelioma (MM). We previously reported that a redox-silent analog of α-tocotrienol, 6-O-carboxypropyl-α-tocotrienol (T3E) induces potent cytotoxicity against human MM cells. However, the detailed mechanism of cytotoxicity of T3E remains unclear. In this study, we investigated if T3E induced potent cytotoxicity aganist MM cells. T3E reduced the formation of the cap-dependent translation complex and induced inactivation of oncogene from rat sarcoma virus (RAS). These events were associated with T3E cytotoxicity in MM cells. Furthermore, atorvastatin, an inhibitor of RAS function, had similar effects on MM cells. Moreover, 4EGI-1, a specific inhibitor of the cap-dependent translation complex, induced severe cytotoxicity in MM cells. Overall, T3E had a cytotoxic effect on MM cells via disruption of the activated cap-dependent translation complex through inactivation of RAS. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Cystatin F Affects Natural Killer Cell Cytotoxicity

PubMed Central

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

2017-01-01

Cystatin F is a cysteine peptidase inhibitor which, unlike other cystatin family members, is targeted to endosomal/lysosomal compartments. It is synthesized as an inactive disulfide-linked dimer which is then converted to an active monomer by proteolytic cleavage of 15 N-terminal residues. Cystatin F has been suggested to regulate the cytotoxicity of natural killer (NK) cells by inhibiting the major granzyme convertases, cathepsins C and H. To test this hypothesis, we prepared variants of cystatin F and analyzed their uptake, subcellular trafficking, and peptidase inhibition, as well as their impact on the cytotoxicity of NK-92 cells and primary NK cells. The N-glycosylation pattern is responsible for the secretion, uptake, and subcellular sorting of cystatin F in HeLa and Hek293 cells, whereas the legumain binding site had no effect on these processes. Active, N-terminally truncated, monomeric cystatin F can also be internalized by recipient cells and targeted to endo/lysosomes, affecting also cells lacking the activating peptidase. Cystatin F mutants capable of cell internalization and trafficking through the endo/lysosomal pathway significantly decreased cathepsin C and H activities, both in situ, following transfection and in trans, using conditioned media. Further, incubation of IL-2 stimulated NK-92 and primary NK cells with full-length and N-terminally truncated cystatin F mutants led to suppression of their granule-mediated cytotoxicity. This effect was most significant with the N-terminally truncated mutants. These results suggest that cystatin F can be an important mediator within tumor microenvironment affecting the cytotoxicity of NK cells and consequently antitumor immune response. PMID:29180998

Cytotoxic Mechanisms Employed by Mouse T Cells to Destroy Pancreatic β-Cells

PubMed Central

Varanasi, Vineeth; Avanesyan, Lia; Schumann, Desiree M.; Chervonsky, Alexander V.

2012-01-01

Several cytotoxic mechanisms have been attributed to T cells participating in β-cell death in type 1 diabetes. However, sensitivity of β-cells to these mechanisms in vitro and in vivo is likely to be different. Moreover, CD4+ and CD8+ T cells may use distinct mechanisms to cause β-cell demise that possibly involve activation of third-party cytotoxic cells. We used the transfer of genetically modified diabetogenic T cells into normal, mutant, and bone marrow chimeric recipients to test the contribution of major cytotoxic mechanisms in β-cell death. We found that 1) the killing of β-cells by CD4+ T cells required activation of the recipient’s own cytotoxic cells via tumor necrosis factor-α (TNF-α); 2) CD8+ T-cell cytotoxic mechanisms destroying β-cells were limited to perforin and Fas ligand, as double knockouts of these molecules abrogated the ability of T cells to cause diabetes; and 3) individual CD8+ T-cell clones chose their cytotoxic weaponry by a yet unknown mechanism and destroyed their targets via either Fas-independent or Fas-dependent (∼40% of clones) pathways. Fas-dependent destruction was assisted by TNF-α. PMID:22773667

Extracellular thiol-assisted selenium uptake dependent on the xc− cystine transporter explains the cancer-specific cytotoxicity of selenite

PubMed Central

Olm, Eric; Fernandes, Aristi P.; Hebert, Christina; Rundlöf, Anna-Klara; Larsen, Erik H.; Danielsson, Olof; Björnstedt, Mikael

2009-01-01

The selenium salt selenite (SeO32−) is cytotoxic in low to moderate concentrations, with a remarkable specificity for cancer cells resistant to conventional chemotherapy. Our data show that selenium uptake and accumulation, rather than intracellular events, are crucial to the specific selenite cytotoxicity observed in resistant cancer cells. We show that selenium uptake depends on extracellular reduction, and that the extracellular environment is a key factor specific to selenite cytotoxicity. The extracellular reduction is mediated by cysteine, and the efficacy is determined by the uptake of cystine by the xc− antiporter and secretion of cysteine by multidrug resistance proteins, both of which are frequently overexpressed by resistant cancer cells. This mechanism provides molecular evidence for the existence of an inverse relationship between resistance to conventional chemotherapy and sensitivity to selenite cytotoxicity, and highlights the great therapeutic potential in treating multidrug-resistant cancer. PMID:19549867

Nanosilica induced dose-dependent cytotoxicity and cell type-dependent multinucleation in HepG2 and L-02 cells

NASA Astrophysics Data System (ADS)

Yu, Yongbo; Duan, Junchao; Li, Yang; Yu, Yang; Hu, Hejing; Wu, Jing; Zhang, Yannan; Li, Yanbo; CaixiaGuo; Zhou, Xianqing; Sun, Zhiwei

2016-11-01

The prevalent exposure to nanosilica gained concerns about health effects of these particles on human beings. Although nanosilica-induced multinucleation has been confirmed previously, the underlying mechanism was still not clear; this study was to investigate the origination of multinucleated cells caused by nanosilica (62 nm) in both HepG2 and L-02 cells. Cell viability and cellular uptake was determined by MTT assay and transmission electron microscope (TEM), respectively. Giemsa staining was applied to detect multinucleation. To clarify the origination of multinucleated cells, fluorescent probes, PKH26 and PKH67, time-lapse observation were further conducted by confocal microscopy. Results indicated that nanosilica particles were internalized into cells and induced cytotoxicity in a dose-dependent manner. Quantification analysis showed that nanosilica significantly increased the rates of binucleated and multinucleated cells, which suggested mitotic catastrophe induction. Moreover, dynamic visualization verified that multinucleation resulted from cell fusion in HepG2 cells not in L-02 cells after nanosilica exposure, suggesting cell type-dependent multinucleation formation. Both multinucleation and cell fusion were involved in genetic instability, which emphasized the significance to explore the multinucleation induced by nanosilica via environmental, occupational and consumer product exposure.

Non-cytotoxic differentiation treatment of renal cell cancer

PubMed Central

Negrotto, Soledad; Hu, Zhenbo; Alcazar, Oscar; Ng, Kwok Peng; Triozzi, Pierre; Lindner, Daniel; Rini, Brian; Saunthararajah, Yogen

2013-01-01

Current drug therapy for metastatic renal cell cancer (RCC) results in temporary disease control but not cure, necessitating continued investigation into alternative mechanistic approaches. Drugs that inhibit chromatin-modifying enzymes involved in transcription repression (chromatin-relaxing drugs) could have a role, by inducing apoptosis, and/or through differentiation pathways. At low doses, the cytosine analogue decitabine can be used to deplete DNA methyl-transferase 1 (DNMT1), modify chromatin and alter differentiation without causing apoptosis (cytotoxicity). Non-cytotoxic regimens of decitabine were evaluated for in vitro and in vivo efficacy against RCC cell lines, including a p53 mutated RCC cell line developed from a patient with treatment refractory metastatic RCC. The cell-division permissive mechanism of action, absence of early apoptosis or DNA damage, increase in expression of HNF4α (a key driver associated with the mesenchymal to epithelial transition), decrease in mesenchymal marker expression, increase in epithelial marker expression, and late increase in cyclin dependent kinase inhibitor CDKN1B (p27) protein, was consistent with differentiation-mediated cell cycle exit. In vivo blood counts and animal weights were consistent with minimal toxicity of therapy. The distinctive mechanism of action of a dose and schedule of decitabine designed for non-cytotoxic depletion of DNMT1 suggests a potential role in treating RCC. PMID:21303982

Granzyme B of cytotoxic T cells induces extramitochondrial reactive oxygen species production via caspase-dependent NADPH oxidase activation.

PubMed

Aguiló, Juan I; Anel, Alberto; Catalán, Elena; Sebastián, Alvaro; Acín-Pérez, Rebeca; Naval, Javier; Wallich, Reinhard; Simon, Markus M; Pardo, Julián

2010-07-01

Induction of reactive oxygen species (ROS) is a hallmark of granzyme B (gzmB)-mediated pro-apoptotic processes and target cell death. However, it is unclear to what extent the generated ROS derive from mitochondrial and/or extra-mitochondrial sources. To clarify this point, we have produced a mutant EL4 cell line, termed EL4-rho(0), which lacks mitochondrial DNA, associated with a decreased mitochondrial membrane potential and a defective ROS production through the electron transport chain of oxidative phosphorylation. When incubated with either recombinant gzmB plus streptolysin or ex vivo gzmB(+) cytotoxic T cells, EL4-rho(0) cells showed phosphatydylserine translocation, caspase 3 activation, Bak conformational change, cytochrome c release and apoptotic morphology comparable to EL4 cells. Moreover, EL4-rho(0) cells produced ROS at levels similar to EL4 under these conditions. GzmB-mediated ROS production was almost totally abolished in both cell lines by the pan-caspase inhibitor, Z-VAD-fmk. However, addition of apocynin, a specific inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, led to a significant reduction of ROS production and cell death only in EL4-rho(0) but not EL4 cells. These data suggest that gzmB-induced cell death is accompanied by a caspase-dependent pathway of extra-mitochondrial ROS production, most probably through activation of NADPH oxidase.

Real-time cell toxicity profiling of Tox21 10K compounds reveals cytotoxicity dependent toxicity pathway linkage

PubMed Central

Huang, Ruili; Lin, Ja-An; Sedykh, Alexander; Zhao, Jinghua; Tice, Raymond R.; Paules, Richard S.; Xia, Menghang; Auerbach, Scott S.

2017-01-01

Cytotoxicity is a commonly used in vitro endpoint for evaluating chemical toxicity. In support of the U.S. Tox21 screening program, the cytotoxicity of ~10K chemicals was interrogated at 0, 8, 16, 24, 32, & 40 hours of exposure in a concentration dependent fashion in two cell lines (HEK293, HepG2) using two multiplexed, real-time assay technologies. One technology measures the metabolic activity of cells (i.e., cell viability, glo) while the other evaluates cell membrane integrity (i.e., cell death, flor). Using glo technology, more actives and greater temporal variations were seen in HEK293 cells, while results for the flor technology were more similar across the two cell types. Chemicals were grouped into classes based on their cytotoxicity kinetics profiles and these classes were evaluated for their associations with activity in the Tox21 nuclear receptor and stress response pathway assays. Some pathways, such as the activation of H2AX, were associated with the fast-responding cytotoxicity classes, while others, such as activation of TP53, were associated with the slow-responding cytotoxicity classes. By clustering pathways based on their degree of association to the different cytotoxicity kinetics labels, we identified clusters of pathways where active chemicals presented similar kinetics of cytotoxicity. Such linkages could be due to shared underlying biological processes between pathways, for example, activation of H2AX and heat shock factor. Others involving nuclear receptor activity are likely due to shared chemical structures rather than pathway level interactions. Based on the linkage between androgen receptor antagonism and Nrf2 activity, we surmise that a subclass of androgen receptor antagonists cause cytotoxicity via oxidative stress that is associated with Nrf2 activation. In summary, the real-time cytotoxicity screen provides informative chemical cytotoxicity kinetics data related to their cytotoxicity mechanisms, and with our analysis, it is

The cytotoxic effects of brown Cuban propolis depend on the nemorosone content and may be mediated by mitochondrial uncoupling.

PubMed

Pardo Andreu, Gilberto L; Reis, Felippe H Z; Dalalio, Felipe M; Nuñez Figueredo, Yanier; Cuesta Rubio, Osmany; Uyemura, Sergio A; Curti, Carlos; Alberici, Luciane C

2015-02-25

Three main types of Cuban propolis directly related to their secondary metabolite composition have been identified: brown, red and yellow propolis; the former is majoritarian and is characterized by the presence of nemorosone. In this study, brown Cuban propolis extracts were found cytotoxic against HepG2 cells and primary rat hepatocytes, in close association with the nemorosone contents. In mitochondria isolated from rat liver the extracts displayed uncoupling activity, which was demonstrated by the increase in succinate-supported state 4 respiration rates, dissipation of mitochondrial membrane potential, Ca(2+) release from Ca(2+)-loaded mitochondria, and a marked ATP depletion. As in cells, the degree of such mitotoxic events was closely correlated to the nemorosone content. The propolis extracts that do not contain nemorosone were neither cytotoxic nor mitotoxic, except R-29, whose detrimental effect upon cells and mitochondria could be mediated by its isoflavonoids and chalcones components, well known mitochondrial uncouplers. Our results at least partly unravel the cytotoxic mechanism of Cuban propolis, particularly regarding brown propolis, and raise concerns about the toxicological implication of Cuban propolis consumption. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Natural killing and antibody-dependent cellular cytotoxicity are independent immune functions in the Minnesota miniature swine.

PubMed

Koren, H S; Amos, D B; Kim, Y B

1978-10-01

Peripheral blood lymphocytes from Minnesota miniature pigs were tested for natural killing (NK) and antibody-dependent cellular cytotoxicity (ADCC) in a 2- to 4-hr 51Cr release assay against human myeloid and lymphoid tumor target cells. Adult specific pathogen-free and germfree animals exhibited normal levels of activity in both assays. In addition, the NK and ADCC activities of peripheral blood lymphocytes from colostrum-deprived newborn piglets were examined. These animals were obtained by hysterectomy and previously shown to be immunologically "virgin." We found that these newborn piglets exhibited normal ADCC but lacked NK activity. The differences in the ontogeny of the two activities suggest that they are distinct. Preliminary effector cell characterization studies suggest that: (i) NK and ADCC in the pig are physically not separable; (ii) the majority of the cytotoxic activity on a cell-per-cell basis is mediated by the non-T lymphocyte fraction; and (iii) the rosetted T cells, which account for about 60% of the total pig peripheral blood lymphocytes, have low but demonstrable cytotoxic activity as well.

HA Antibody-Mediated FcγRIIIa Activity Is Both Dependent on FcR Engagement and Interactions between HA and Sialic Acids.

PubMed

Cox, Freek; Kwaks, Ted; Brandenburg, Boerries; Koldijk, Martin H; Klaren, Vincent; Smal, Bastiaan; Korse, Hans J W M; Geelen, Eric; Tettero, Lisanne; Zuijdgeest, David; Stoop, Esther J M; Saeland, Eirikur; Vogels, Ronald; Friesen, Robert H E; Koudstaal, Wouter; Goudsmit, Jaap

2016-01-01

Interactions with receptors for the Fc region of IgG (FcγRs) have been shown to contribute to the in vivo protection against influenza A viruses provided by broadly neutralizing antibodies (bnAbs) that bind to the viral hemagglutinin (HA) stem. In particular, Fc-mediated antibody-dependent cellular cytotoxicity (ADCC) has been shown to contribute to protection by stem-binding bnAbs. Fc-mediated effector functions appear not to contribute to protection provided by strain-specific HA head-binding antibodies. We used a panel of anti-stem and anti-head influenza A and B monoclonal antibodies with identical human IgG1 Fc domains and investigated their ability to mediate ADCC-associated FcγRIIIa activation. Antibodies which do not interfere with sialic acid binding of HA can mediate FcγRIIIa activation. However, the FcγRIIIa activation was inhibited when a mutant HA, unable to bind sialic acids, was used. Antibodies which block sialic acid receptor interactions of HA interfered with FcγRIIIa activation. The inhibition of FcγRIIIa activation by HA head-binding and sialic acid receptor-blocking antibodies was confirmed in plasma samples of H5N1 vaccinated human subjects. Together, these results suggest that in addition to Fc-FcγR binding, interactions between HA and sialic acids on immune cells are required for optimal Fc-mediated effector functions by anti-HA antibodies.

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

PubMed

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

NASA Astrophysics Data System (ADS)

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.

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

PubMed Central

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 cell-based fluorescent assay to detect the activity of AB toxins that inhibit protein synthesis

USDA-ARS?s Scientific Manuscript database

AB-type protein toxins, produced by numerous bacterial pathogens and some plants, elicit a cytotoxic effect involving the inhibition of protein synthesis. To develop an improved method to detect the inhibition of protein synthesis by AB-type toxins, the present study characterized a Vero cell line t...

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

PubMed

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.

Modulation of CD4(+) T cell-dependent specific cytotoxic CD8(+) T cells differentiation and proliferation by the timing of increase in the pathogen load.

PubMed

Tzelepis, Fanny; Persechini, Pedro M; Rodrigues, Mauricio M

2007-04-25

Following infection with viruses, bacteria or protozoan parasites, naïve antigen-specific CD8(+) T cells undergo a process of differentiation and proliferation to generate effector cells. Recent evidences suggest that the timing of generation of specific effector CD8(+) T cells varies widely according to different pathogens. We hypothesized that the timing of increase in the pathogen load could be a critical parameter governing this process. Using increasing doses of the protozoan parasite Trypanosoma cruzi to infect C57BL/6 mice, we observed a significant acceleration in the timing of parasitemia without an increase in mouse susceptibility. In contrast, in CD8 deficient mice, we observed an inverse relationship between the parasite inoculum and the timing of death. These results suggest that in normal mice CD8(+) T cells became protective earlier, following the accelerated development of parasitemia. The evaluation of specific cytotoxic responses in vivo to three distinct epitopes revealed that increasing the parasite inoculum hastened the expansion of specific CD8(+) cytotoxic T cells following infection. The differentiation and expansion of T. cruzi-specific CD8(+) cytotoxic T cells is in fact dependent on parasite multiplication, as radiation-attenuated parasites were unable to activate these cells. We also observed that, in contrast to most pathogens, the activation process of T. cruzi-specific CD8(+) cytotoxic T cells was dependent on MHC class II restricted CD4(+) T cells. Our results are compatible with our initial hypothesis that the timing of increase in the pathogen load can be a critical parameter governing the kinetics of CD4(+) T cell-dependent expansion of pathogen-specific CD8(+) cytotoxic T cells.

BST-2 Expression Modulates Small CD4-Mimetic Sensitization of HIV-1-Infected Cells to Antibody-Dependent Cellular Cytotoxicity

PubMed Central

Prévost, Jérémie; von Bredow, Benjamin; Ding, Shilei; Brassard, Nathalie; Medjahed, Halima; Coutu, Mathieu; Melillo, Bruno; Bibollet-Ruche, Frédéric; Hahn, Beatrice H.; Kaufmann, Daniel E.; Smith, Amos B.; Sodroski, Joseph; Sauter, Daniel; Kirchhoff, Frank; Gee, Katrina; Neil, Stuart J.; Evans, David T.

2017-01-01

ABSTRACT Antibodies recognizing conserved CD4-induced (CD4i) epitopes on human immunodeficiency virus type 1 (HIV-1) Env and able to mediate antibody-dependent cellular cytotoxicity (ADCC) have been shown to be present in sera from most HIV-1-infected individuals. These antibodies preferentially recognize Env in its CD4-bound conformation. CD4 downregulation by Nef and Vpu dramatically reduces exposure of CD4i HIV-1 Env epitopes and therefore reduce the susceptibility of HIV-1-infected cells to ADCC mediated by HIV-positive (HIV+) sera. Importantly, this mechanism of immune evasion can be circumvented with small-molecule CD4 mimetics (CD4mc) that are able to transition Env into the CD4-bound conformation and sensitize HIV-1-infected cells to ADCC mediated by HIV+ sera. However, HIV-1 developed additional mechanisms to avoid ADCC, including Vpu-mediated BST-2 antagonism, which decreases the overall amount of Env present at the cell surface. Accordingly, BST-2 upregulation in response to alpha interferon (IFN-α) was shown to increase the susceptibility of HIV-1-infected cells to ADCC despite the activity of Vpu. Here we show that BST-2 upregulation by IFN-β and interleukin-27 (IL-27) also increases the surface expression of Env and thus boosts the ability of CD4mc to sensitize HIV-1-infected cells to ADCC by sera from HIV-1-infected individuals. IMPORTANCE HIV-1 evolved sophisticated strategies to conceal Env epitopes from ADCC-mediating antibodies present in HIV+ sera. Vpu-mediated BST-2 downregulation was shown to decrease ADCC responses by limiting the amount of Env present at the cell surface. This effect of Vpu was shown to be attenuated by IFN-α treatment. Here we show that in addition to IFN-α, IFN-β and IL-27 also affect Vpu-mediated BST-2 downregulation and greatly enhance ADCC responses against HIV-1-infected cells in the presence of CD4mc. These findings may inform strategies aimed at HIV prevention and eradication. PMID:28331088

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

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

Wang, Yi-Fen; Shyu, Huey-Wen; Chang, Yi-Chuang

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 onlymore » 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.« less

Accessory proteins 8b and 8ab of severe acute respiratory syndrome coronavirus suppress the interferon signaling pathway by mediating ubiquitin-dependent rapid degradation of interferon regulatory factor 3.

PubMed

Wong, Hui Hui; Fung, To Sing; Fang, Shouguo; Huang, Mei; Le, My Tra; Liu, Ding Xiang

2018-02-01

Severe acute respiratory syndrome coronavirus (SARS-CoV) is an inefficient inducer of interferon (IFN) response. It expresses various proteins that effectively circumvent IFN production at different levels via distinct mechanisms. Through the construction of recombinant IBV expressing proteins 8a, 8b and 8ab encoded by SARS-CoV ORF8, we demonstrate that expression of 8b and 8ab enables the corresponding recombinant viruses to partially overcome the inhibitory actions of IFN activation to achieve higher replication efficiencies in cells. We also found that proteins 8b and 8ab could physically interact with IRF3. Overexpression of 8b and 8ab resulted in the reduction of poly (I:C)-induced IRF3 dimerization and inhibition of the IFN-β signaling pathway. This counteracting effect was partially mediated by protein 8b/8ab-induced degradation of IRF3 in a ubiquitin-proteasome-dependent manner. Taken together, we propose that SARS-CoV may exploit the unique functions of proteins 8b and 8ab as novel mechanisms to overcome the effect of IFN response during virus infection. Copyright © 2017 Elsevier Inc. All rights reserved.

Mechanistic Exploration of Cancer Stem Cell Marker Voltage-Dependent Calcium Channel α2δ1 Subunit-mediated Chemotherapy Resistance in Small-Cell Lung Cancer.

PubMed

Yu, Jiangyong; Wang, Shuhang; Zhao, Wei; Duan, Jianchun; Wang, Zhijie; Chen, Hanxiao; Tian, Yanhua; Wang, Di; Zhao, Jun; An, Tongtong; Bai, Hua; Wu, Meina; Wang, Jie

2018-05-01

Purpose: Chemoresistance in small-cell lung cancer (SCLC) is reportedly attributed to the existence of resistant cancer stem cells (CSC). Studies involving CSC-specific markers and related mechanisms in SCLC remain limited. This study explored the role of the voltage-dependent calcium channel α2δ1 subunit as a CSC marker in chemoresistance of SCLC, and explored the potential mechanisms of α2δ1-mediated chemoresistance and strategies of overcoming the resistance. Experimental Design: α2δ1-positive cells were identified and isolated from SCLC cell lines and patient-derived xenograft (PDX) models, and CSC-like properties were subsequently verified. Transcriptome sequencing and Western blotting were carried out to identify pathways involved in α2δ1-mediated chemoresistance in SCLC. In addition, possible interventions to overcome α2δ1-mediated chemoresistance were examined. Results: Different proportions of α2δ1 + cells were identified in SCLC cell lines and PDX models. α2δ1 + cells exhibited CSC-like properties (self-renewal, tumorigenic, differentiation potential, and high expression of genes related to CSCs and drug resistance). Chemotherapy induced the enrichment of α2δ1 + cells instead of CD133 + cells in PDXs, and an increased proportion of α2δ1 + cells corresponded to increased chemoresistance. Activation and overexpression of ERK in the α2δ1-positive H1048 cell line was identified at the protein level. mAb 1B50-1 was observed to improve the efficacy of chemotherapy and delay relapse as maintenance therapy in PDX models. Conclusions: SCLC cells expressing α2δ1 demonstrated CSC-like properties, and may contribute to chemoresistance. ERK may play a key role in α2δ1-mediated chemoresistance. mAb 1B50-1 may serve as a potential anti-SCLC drug. Clin Cancer Res; 24(9); 2148-58. ©2018 AACR . ©2018 American Association for Cancer Research.

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

PubMed

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.

Effects of murine leukemia virus env gene proteins on macrophage-mediated cytotoxicity in vitro

NASA Technical Reports Server (NTRS)

Chapes, S. K.; Takemoto, L. J.; Spooner, B. S. (Principal Investigator)

1991-01-01

F5b Tumor cells were incubated with concentrated culture supernatants taken from cells resistant (F5m) or sensitive (F5b) to contact-dependent macrophage cytotoxicity. Macrophage cell line B6MP102 and murine peritoneal macrophages killed targets incubated with supernatants taken from sensitive cells but poorly killed cells incubated in supernatants isolated from resistant cells. Membranes from cells resistant to macrophage killing, F5m, were fused into F5b cells. The fused F5b cells were killed significantly less than F5b cells fused with F5b cell membranes or untreated F5b cells. The decreased killing of F5b cells corresponded to increased concentrations of gp70(a) molecules on F5b cells. Affinity purified gp70(a) was added to cytotoxicity assays but failed to inhibit macrophage cytotoxicity. P15E molecules were detectable on both F5b and F5m cells. In addition, a synthetic peptide found to exhibit the inhibitory properties of p15E was added to cytotoxicity assays. P15E synthetic peptide also did not inhibit macrophage cytotoxicity. Therefore, env gene proteins of murine leukemia virus do not appear responsible for inducing tumor cell resistance to activated macrophage contact-dependent cytotoxicity.

Impaired respiration elicits SrrAB-dependent programmed cell lysis and biofilm formation in Staphylococcus aureus

PubMed Central

Mashruwala, Ameya A; van de Guchte, Adriana; Boyd, Jeffrey M

2017-01-01

Biofilms are communities of microorganisms attached to a surface or each other. Biofilm-associated cells are the etiologic agents of recurrent Staphylococcus aureus infections. Infected human tissues are hypoxic or anoxic. S. aureus increases biofilm formation in response to hypoxia, but how this occurs is unknown. In the current study we report that oxygen influences biofilm formation in its capacity as a terminal electron acceptor for cellular respiration. Genetic, physiological, or chemical inhibition of respiratory processes elicited increased biofilm formation. Impaired respiration led to increased cell lysis via divergent regulation of two processes: increased expression of the AtlA murein hydrolase and decreased expression of wall-teichoic acids. The AltA-dependent release of cytosolic DNA contributed to increased biofilm formation. Further, cell lysis and biofilm formation were governed by the SrrAB two-component regulatory system. Data presented support a model wherein SrrAB-dependent biofilm formation occurs in response to the accumulation of reduced menaquinone. DOI: http://dx.doi.org/10.7554/eLife.23845.001 PMID:28221135

An estimation of the frequency of precursor cells which generate cytotoxic lymphocytes

PubMed Central

1976-01-01

The cell-mediated immune response has been generated in vitro with a polyacrylamide culture system which allows the segregation of foci (clones?) of cytotoxic lymphocytes. Using the method of limiting dilutions, the frequency of precursor cells in CBA spleen cells able to generate a cytotoxic response against DBA mastocytoma is estimated at 1 per 1,700 cells. PMID:1083894

CD8(+) T-cell Cytotoxic Capacity Associated with Human Immunodeficiency Virus-1 Control Can Be Mediated through Various Epitopes and Human Leukocyte Antigen Types.

PubMed

Migueles, Stephen A; Mendoza, Daniel; Zimmerman, Matthew G; Martins, Kelly M; Toulmin, Sushila A; Kelly, Elizabeth P; Peterson, Bennett A; Johnson, Sarah A; Galson, Eric; Poropatich, Kate O; Patamawenu, Andy; Imamichi, Hiromi; Ober, Alexander; Rehm, Catherine A; Jones, Sara; Hallahan, Claire W; Follmann, Dean A; Connors, Mark

2015-01-01

Understanding natural immunologic control over Human Immunodeficiency Virus (HIV)-1 replication, as occurs in rare long-term nonprogressors/elite controllers (LTNP/EC), should inform the design of efficacious HIV vaccines and immunotherapies. Durable control in LTNP/EC is likely mediated by highly functional virus-specific CD8(+) T-cells. Protective Human Leukocyte Antigen (HLA) class I alleles, like B*27 and B*57, are present in most, but not all LTNP/EC, providing an opportunity to investigate features shared by their HIV-specific immune responses. To better understand the contribution of epitope targeting and conservation to immune control, we compared the CD8(+) T-cell specificity and function of B*27/57(neg) LTNP/EC (n = 23), B*27/57(pos) LTNP/EC (n = 23) and B*27/57(neg) progressors (n = 13). Fine mapping revealed 11 previously unreported immunodominant responses. Although B*27/57(neg) LTNP/EC did not target more highly conserved epitopes, their CD8(+) T-cell cytotoxic capacity was significantly higher than progressors. Similar to B*27/57(pos) LTNP/EC, this superior cytotoxicity was mediated by preferential expansion of immunodominant responses and lysis through the predicted HLA. These findings suggest that increased CD8(+) T-cell cytotoxic capacity is a common mechanism of control in most LTNP/EC regardless of HLA type. They also suggest that potent cytotoxicity can be mediated through various epitopes and HLA molecules and could, in theory, be induced in most people.

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

PubMed

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

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

Ko, Jen-Chung; Department of Nursing, Yuanpei University, HsinChu, Taiwan; Graduate Institute of Technology Law, National Chiao Tung University, Taiwan

2011-09-15

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)more » 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

CXCR5+CD8+ T cells present elevated capacity in mediating cytotoxicity toward autologous tumor cells through interleukin 10 in diffuse large B-cell lymphoma.

PubMed

Tang, Jiahong; Zha, Jie; Guo, Xutao; Shi, Pengcheng; Xu, Bing

2017-09-01

Diffuse large B-cell lymphoma (DLBCL) is a common and aggressive subtype of non-Hodgkin's lymphomas, with limited treatment options in refractory and relapsed patients. Growing evidence supports the notion that CD8 + T cell immunity could be utilized to eliminate B cell lymphomas. CXCR5 + CD8 + T cell is a novel cell subtype and share CXCR5 expression with CD19 + tumor cells. In this study, we investigated the frequency and function of existing CXCR5 + CD8 + T cells in DLBCL patients. We found that DLBCL patients as a group demonstrated significantly higher level of CXCR5 + CD8 + T cells than healthy individuals, with huge variability in each patient. Using anti-CD3/CD28-stimulated CD8 + T cells as effector (E) cells and autologous CD19 + tumor cells as target (T) cells, at high E:T ratio, no difference between the intensities of CXCR5 + CD8 + T cell- and CXCR5 - CD8 + T cell-mediated cytotoxicity were observed. However, at intermediate and low E:T ratios, the CXCR5 + CD8 + T cells presented stronger cytotoxicity than CXCR5 - CD8 + T cells. The expressions of granzyme A, granzyme B, and perforin were significantly higher in CXCR5 + CD8 + T cells than in CXCR5 - CD8 + T cells, with no significant difference in the level of degranulation. Tumor cells in DLBCL were known to secrete high level of interleukin 10 (IL-10). We therefore blocked the IL-10/IL-10R pathway, and found that the expressions of granzyme A, granzyme B, and perforin by CXCR5 + CD8 + T cells were significantly elevated. Together, these results suggest that CXCR5 + CD8 + T cells are potential candidates of CD8 + T cell-based immunotherapies, could mediate elimination of autologous tumor cells in DLBCL patients, but are also susceptible to IL-10-mediated suppression. Copyright © 2017. Published by Elsevier B.V.

Combined glyco- and protein-Fc engineering simultaneously enhance cytotoxicity and half-life of a therapeutic antibody.

PubMed

Monnet, Céline; Jorieux, Sylvie; Souyris, Nathalie; Zaki, Ouafa; Jacquet, Alexandra; Fournier, Nathalie; Crozet, Fabien; de Romeuf, Christophe; Bouayadi, Khalil; Urbain, Rémi; Behrens, Christian K; Mondon, Philippe; Fontayne, Alexandre

2014-01-01

While glyco-engineered monoclonal antibodies (mAbs) with improved antibody-dependent cell-mediated cytotoxicity (ADCC) are reaching the market, extensive efforts have also been made to improve their pharmacokinetic properties to generate biologically superior molecules. Most therapeutic mAbs are human or humanized IgG molecules whose half-life is dependent on the neonatal Fc receptor FcRn. FcRn reduces IgG catabolism by binding to the Fc domain of endocytosed IgG in acidic lysosomal compartments, allowing them to be recycled into the blood. Fc-engineered mAbs with increased FcRn affinity resulted in longer in vivo half-life in animal models, but also in healthy humans. These Fc-engineered mAbs were obtained by alanine scanning, directed mutagenesis or in silico approach of the FcRn binding site. In our approach, we applied a random mutagenesis technology (MutaGen™) to generate mutations evenly distributed over the whole Fc sequence of human IgG1. IgG variants with improved FcRn-binding were then isolated from these Fc-libraries using a pH-dependent phage display selection process. Two successive rounds of mutagenesis and selection were performed to identify several mutations that dramatically improve FcRn binding. Notably, many of these mutations were unpredictable by rational design as they were located distantly from the FcRn binding site, validating our random molecular approach. When produced on the EMABling(®) platform allowing effector function increase, our IgG variants retained both higher ADCC and higher FcRn binding. Moreover, these IgG variants exhibited longer half-life in human FcRn transgenic mice. These results clearly demonstrate that glyco-engineering to improve cytotoxicity and protein-engineering to increase half-life can be combined to further optimize therapeutic mAbs.

B cells are critical to T-cell-mediated antitumor immunity induced by a combined immune-stimulatory/conditionally cytotoxic therapy for glioblastoma.

PubMed

Candolfi, Marianela; Curtin, James F; Yagiz, Kader; Assi, Hikmat; Wibowo, Mia K; Alzadeh, Gabrielle E; Foulad, David; Muhammad, A K M G; Salehi, Sofia; Keech, Naomi; Puntel, Mariana; Liu, Chunyan; Sanderson, Nicholas R; Kroeger, Kurt M; Dunn, Robert; Martins, Gislaine; Lowenstein, Pedro R; Castro, Maria G

2011-10-01

We have demonstrated that modifying the tumor microenvironment through intratumoral administration of adenoviral vectors (Ad) encoding the conditional cytotoxic molecule, i.e., HSV1-TK and the immune-stimulatory cytokine, i.e., fms-like tyrosine kinase 3 ligand (Flt3L) leads to T-cell-dependent tumor regression in rodent models of glioblastoma. We investigated the role of B cells during immune-mediated glioblastoma multiforme regression. Although treatment with Ad-TK+Ad-Flt3L induced tumor regression in 60% of wild-type (WT) mice, it completely failed in B-cell-deficient Igh6(-/-) mice. Tumor-specific T-cell precursors were detected in Ad-TK+Ad-Flt3L-treated WT mice but not in Igh6(-/-) mice. The treatment also failed in WT mice depleted of total B cells or marginal zone B cells. Because we could not detect circulating antibodies against tumor cells and the treatment was equally efficient in WT mice and in mice with B-cell-specific deletion of Prdm 1 (encoding Blimp-1), in which B cells are present but unable to fully differentiate into antibody-secreting plasma cells, tumor regression in this model is not dependent on B cells' production of tumor antigen-specific immunoglobulins. Instead, B cells seem to play a role as antigen-presenting cells (APCs). Treatment with Ad-TK+Ad-Flt3L led to an increase in the number of B cells in the cervical lymph nodes, which stimulated the proliferation of syngeneic T cells and induced clonal expansion of antitumor T cells. Our data show that B cells act as APCs, playing a critical role in clonal expansion of tumor antigen-specific T cells and brain tumor regression.

Surface-charge-dependent cell localization and cytotoxicity of cerium oxide nanoparticles.

PubMed

Asati, Atul; Santra, Santimukul; Kaittanis, Charalambos; Perez, J Manuel

2010-09-28

Cerium oxide nanoparticles (nanoceria) have shown great potential as antioxidant and radioprotective agents for applications in cancer therapy. Recently, various polymer-coated nanoceria preparations have been developed to improve their aqueous solubility and allow for surface functionalization of these nanoparticles. However, the interaction of polymer-coated nanoceria with cells, their uptake mechanism, and subcellular localization are poorly understood. Herein, we engineered polymer-coated cerium oxide nanoparticles with different surface charges (positive, negative, and neutral) and studied their internalization and toxicity in normal and cancer cell lines. The results showed that nanoceria with a positive or neutral charge enters most of the cell lines studied, while nanoceria with a negative charge internalizes mostly in the cancer cell lines. Moreover, upon entry into the cells, nanoceria is localized to different cell compartments (e.g., cytoplasm and lysosomes) depending on the nanoparticle's surface charge. The internalization and subcellular localization of nanoceria plays a key role in the nanoparticles' cytotoxicity profile, exhibiting significant toxicity when they localize in the lysosomes of the cancer cells. In contrast, minimal toxicity is observed when they localize into the cytoplasm or do not enter the cells. Taken together, these results indicate that the differential surface-charge-dependent localization of nanoceria in normal and cancer cells plays a critical role in the nanoparticles' toxicity profile.

Cisplatin Induces a Mitochondrial-ROS Response That Contributes to Cytotoxicity Depending on Mitochondrial Redox Status and Bioenergetic Functions

PubMed Central

Marullo, Rossella; Werner, Erica; Degtyareva, Natalya; Moore, Bryn; Altavilla, Giuseppe; Ramalingam, Suresh S.; Doetsch, Paul W.

2013-01-01

Cisplatin is one of the most effective and widely used anticancer agents for the treatment of several types of tumors. The cytotoxic effect of cisplatin is thought to be mediated primarily by the generation of nuclear DNA adducts, which, if not repaired, cause cell death as a consequence of DNA replication and transcription blockage. However, the ability of cisplatin to induce nuclear DNA (nDNA) damage per se is not sufficient to explain its high degree of effectiveness nor the toxic effects exerted on normal, post-mitotic tissues. Oxidative damage has been observed in vivo following exposure to cisplatin in several tissues, suggesting a role for oxidative stress in the pathogenesis of cisplatin-induced dose-limiting toxicities. However, the mechanism of cisplatin-induced generation of ROS and their contribution to cisplatin cytotoxicity in normal and cancer cells is still poorly understood. By employing a panel of normal and cancer cell lines and the budding yeast Saccharomyces cerevisiae as model system, we show that exposure to cisplatin induces a mitochondrial-dependent ROS response that significantly enhances the cytotoxic effect caused by nDNA damage. ROS generation is independent of the amount of cisplatin-induced nDNA damage and occurs in mitochondria as a consequence of protein synthesis impairment. The contribution of cisplatin-induced mitochondrial dysfunction in determining its cytotoxic effect varies among cells and depends on mitochondrial redox status, mitochondrial DNA integrity and bioenergetic function. Thus, by manipulating these cellular parameters, we were able to enhance cisplatin cytotoxicity in cancer cells. This study provides a new mechanistic insight into cisplatin-induced cell killing and may lead to the design of novel therapeutic strategies to improve anticancer drug efficacy. PMID:24260552

SK3/TRPC1/Orai1 complex regulates SOCE-dependent colon cancer cell migration: a novel opportunity to modulate anti-EGFR mAb action by the alkyl-lipid Ohmline

PubMed Central

Guéguinou, Maxime; Harnois, Thomas; Crottes, David; Uguen, Arnaud; Deliot, Nadine; Gambade, Audrey; Chantôme, Aurélie; Haelters, Jean Pierre; Jaffrès, Paul Alain; Jourdan, Marie Lise; Weber, Günther; Soriani, Olivier; Bougnoux, Philippe; Mignen, Olivier; Bourmeyster, Nicolas; Constantin, Bruno; Lecomte, Thierry

2016-01-01

Background Barely 10-20% of patients with metastatic colorectal cancer (mCRC) receive a clinical benefit from the use of anti-EGFR monoclonal antibodies (mAbs). We hypothesized that this could depends on their efficiency to reduce Store Operated Calcium Entry (SOCE) that are known to enhance cancer cells. Results In the present study, we demonstrate that SOCE promotes migration of colon cancer cell following the formation of a lipid raft ion channel complex composed of TRPC1/Orai1 and SK3 channels. Formation of this complex is stimulated by the phosphorylation of the reticular protein STIM1 by EGF and activation of the Akt pathway. Our data show that, in a positive feedback loop SOCE activates both Akt pathway and SK3 channel activity which lead to SOCE amplification. This amplification occurs through the activation of Rac1/Calpain mediated by Akt. We also show that Anti-EGFR mAbs can modulate SOCE and cancer cell migration through the Akt pathway. Interestingly, the alkyl-lipid Ohmline, which we previously showed to be an inhibitor of SK3 channel, can dissociated the lipid raft ion channel complex through decreased phosphorylation of Akt and modulation of mAbs action. Conclusions This study demonstrates that the inhibition of the SOCE-dependent colon cancer cell migration trough SK3/TRPC1/Orai1 channel complex by the alkyl-lipid Ohmline may be a novel strategy to modulate Anti-EGFR mAb action in mCRC. PMID:27102434

Myelin-reactive antibodies initiate T cell-mediated CNS autoimmune disease by opsonization of endogenous antigen.

PubMed

Kinzel, Silke; Lehmann-Horn, Klaus; Torke, Sebastian; Häusler, Darius; Winkler, Anne; Stadelmann, Christine; Payne, Natalie; Feldmann, Linda; Saiz, Albert; Reindl, Markus; Lalive, Patrice H; Bernard, Claude C; Brück, Wolfgang; Weber, Martin S

2016-07-01

In the pathogenesis of central nervous system (CNS) demyelinating disorders, antigen-specific B cells are implicated to act as potent antigen-presenting cells (APC), eliciting waves of inflammatory CNS infiltration. Here, we provide the first evidence that CNS-reactive antibodies (Ab) are similarly capable of initiating an encephalitogenic immune response by targeting endogenous CNS antigen to otherwise inert myeloid APC. In a transgenic mouse model, constitutive production of Ab against myelin oligodendrocyte glycoprotein (MOG) was sufficient to promote spontaneous experimental autoimmune encephalomyelitis (EAE) in the absence of B cells, when mice endogenously contained MOG-recognizing T cells. Adoptive transfer studies corroborated that anti-MOG Ab triggered activation and expansion of peripheral MOG-specific T cells in an Fc-dependent manner, subsequently causing EAE. To evaluate the underlying mechanism, anti-MOG Ab were added to a co-culture of myeloid APC and MOG-specific T cells. At otherwise undetected concentrations, anti-MOG Ab enabled Fc-mediated APC recognition of intact MOG; internalized, processed and presented MOG activated naïve T cells to differentiate in an encephalitogenic manner. In a series of translational experiments, anti-MOG Ab from two patients with an acute flare of CNS inflammation likewise facilitated detection of human MOG. Jointly, these observations highlight Ab-mediated opsonization of endogenous CNS auto-antigen as a novel disease- and/or relapse-triggering mechanism in CNS demyelinating disorders.

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

EPA Science Inventory

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...

Clinical-Grade Human Multipotent Adult Progenitor Cells Block CD8+ Cytotoxic T Lymphocytes

PubMed Central

Dekimpe, Emily; Van Woensel, Matthias; Roobrouck, Valerie D.; Bullens, Dominique M.; Pinxteren, Jef; Verfaillie, Catherine M.; Van Gool, Stefaan W.

2016-01-01

MultiStem cells are clinical-grade multipotent adult bone marrow-derived progenitor cells (MAPCs), with extensive replication potential and broader differentiation capacity compared with mesenchymal stem cells. Human MAPCs suppress T-cell proliferation induced by alloantigens and mutually interact with allogeneic natural killer cells. In this study, the interaction between MultiStem and CD8+ cytotoxic T lymphocytes (CTLs) was addressed for the first time. In an in vitro setting, the immunogenicity of MultiStem, the susceptibility of MultiStem toward CTL-mediated lysis, and its effects on CTL function were investigated. MultiStem was nonimmunogenic for alloreactive CTL induction and was—even after major histocompatibility complex class I upregulation—insensitive to alloantigen-specific CTL-mediated lysis. Furthermore, MultiStem reduced CTL proliferation and significantly decreased perforin expression during the T-cell activation phase. As a consequence, MultiStem dose-dependently impaired the induction of CTL function. These effects of MultiStem were mediated predominantly through contact-dependent mechanisms. Moreover, MultiStem cells considerably influenced the expression of T-cell activation markers CD25, CD69, and human leukocyte antigen-DR. The MultiStem-induced CD8−CD69+ T-cell population displayed a suppressive effect on the induction of CTL function during a subsequent mixed-lymphocyte culture. Finally, the killer activity of activated antigen-specific CTLs during their cytolytic effector phase was also diminished in the presence of MultiStem. This study confirms that these clinical-grade MAPCs are an immune-modulating population that inhibits CTL activation and effector responses and are, consequently, a highly valuable cell population for adoptive immunosuppressive therapy in diseases where damage is induced by CTLs. Significance Because multipotent adult progenitor cells (MAPCs) are among the noteworthy adult mesenchymal stem cell populations for

Clinical-Grade Human Multipotent Adult Progenitor Cells Block CD8+ Cytotoxic T Lymphocytes.

PubMed

Plessers, Jeroen; Dekimpe, Emily; Van Woensel, Matthias; Roobrouck, Valerie D; Bullens, Dominique M; Pinxteren, Jef; Verfaillie, Catherine M; Van Gool, Stefaan W

2016-12-01

: MultiStem cells are clinical-grade multipotent adult bone marrow-derived progenitor cells (MAPCs), with extensive replication potential and broader differentiation capacity compared with mesenchymal stem cells. Human MAPCs suppress T-cell proliferation induced by alloantigens and mutually interact with allogeneic natural killer cells. In this study, the interaction between MultiStem and CD8 + cytotoxic T lymphocytes (CTLs) was addressed for the first time. In an in vitro setting, the immunogenicity of MultiStem, the susceptibility of MultiStem toward CTL-mediated lysis, and its effects on CTL function were investigated. MultiStem was nonimmunogenic for alloreactive CTL induction and was-even after major histocompatibility complex class I upregulation-insensitive to alloantigen-specific CTL-mediated lysis. Furthermore, MultiStem reduced CTL proliferation and significantly decreased perforin expression during the T-cell activation phase. As a consequence, MultiStem dose-dependently impaired the induction of CTL function. These effects of MultiStem were mediated predominantly through contact-dependent mechanisms. Moreover, MultiStem cells considerably influenced the expression of T-cell activation markers CD25, CD69, and human leukocyte antigen-DR. The MultiStem-induced CD8 - CD69 + T-cell population displayed a suppressive effect on the induction of CTL function during a subsequent mixed-lymphocyte culture. Finally, the killer activity of activated antigen-specific CTLs during their cytolytic effector phase was also diminished in the presence of MultiStem. This study confirms that these clinical-grade MAPCs are an immune-modulating population that inhibits CTL activation and effector responses and are, consequently, a highly valuable cell population for adoptive immunosuppressive therapy in diseases where damage is induced by CTLs. Because multipotent adult progenitor cells (MAPCs) are among the noteworthy adult mesenchymal stem cell populations for immune therapy

Correlation between luminescence intensity and cytotoxicity in cell-based cytotoxicity assay using luciferase.

PubMed

Wakuri, S; Yamakage, K; Kazuki, Y; Kazuki, K; Oshimura, M; Aburatani, S; Yasunaga, M; Nakajima, Y

2017-04-01

The luciferase reporter assay has become one of the conventional methods for cytotoxicity evaluation. Typically, the decrease of luminescence expressed by a constitutive promoter is used as an index of cytotoxicity. However, to our knowledge, there have been no reports of the correlation between cytotoxicity and luminescence intensity. In this study, to accurately verify the correlation between them, beetle luciferase was stably expressed in human hepatoma HepG2 cells harboring the multi-integrase mouse artificial chromosome vector. We showed that the cytotoxicity assay using luciferase does not depend on the stability of luciferase protein and the kind of constitutive promoter. Next, HepG2 cells in which green-emitting beetle luciferase was expressed under the control of CAG promoter were exposed to 58 compounds. The luminescence intensity and cytotoxicity curves of cells exposed to 48 compounds showed similar tendencies, whereas those of cells exposed to 10 compounds did not do so, although the curves gradually approached each other with increasing exposure time. Finally, we demonstrated that luciferase expressed under the control of a constitutive promoter can be utilized both as an internal control reporter for normalizing a test reporter and for monitoring cytotoxicity when two kinds of luciferases are simultaneously used in the cytotoxicity assay. Copyright © 2017 Elsevier Inc. All rights reserved.

CD8+ T-cell Cytotoxic Capacity Associated with Human Immunodeficiency Virus-1 Control Can Be Mediated through Various Epitopes and Human Leukocyte Antigen Types

PubMed Central

Migueles, Stephen A.; Mendoza, Daniel; Zimmerman, Matthew G.; Martins, Kelly M.; Toulmin, Sushila A.; Kelly, Elizabeth P.; Peterson, Bennett A.; Johnson, Sarah A.; Galson, Eric; Poropatich, Kate O.; Patamawenu, Andy; Imamichi, Hiromi; Ober, Alexander; Rehm, Catherine A.; Jones, Sara; Hallahan, Claire W.; Follmann, Dean A.; Connors, Mark

2014-01-01

Understanding natural immunologic control over Human Immunodeficiency Virus (HIV)-1 replication, as occurs in rare long-term nonprogressors/elite controllers (LTNP/EC), should inform the design of efficacious HIV vaccines and immunotherapies. Durable control in LTNP/EC is likely mediated by highly functional virus-specific CD8+ T-cells. Protective Human Leukocyte Antigen (HLA) class I alleles, like B*27 and B*57, are present in most, but not all LTNP/EC, providing an opportunity to investigate features shared by their HIV-specific immune responses. To better understand the contribution of epitope targeting and conservation to immune control, we compared the CD8+ T-cell specificity and function of B*27/57neg LTNP/EC (n = 23), B*27/57pos LTNP/EC (n = 23) and B*27/57neg progressors (n = 13). Fine mapping revealed 11 previously unreported immunodominant responses. Although B*27/57neg LTNP/EC did not target more highly conserved epitopes, their CD8+ T-cell cytotoxic capacity was significantly higher than progressors. Similar to B*27/57pos LTNP/EC, this superior cytotoxicity was mediated by preferential expansion of immunodominant responses and lysis through the predicted HLA. These findings suggest that increased CD8+ T-cell cytotoxic capacity is a common mechanism of control in most LTNP/EC regardless of HLA type. They also suggest that potent cytotoxicity can be mediated through various epitopes and HLA molecules and could, in theory, be induced in most people. PMID:26137533

Leptin Protects Host Cells from Entamoeba histolytica Cytotoxicity by a STAT3-Dependent Mechanism

PubMed Central

Verkerke, Hans P.; Paul, Shom N.; Mackey, Aaron J.; Petri, William A.

2012-01-01

The adipocytokine leptin links nutritional status to immune function. Leptin signaling protects from amebiasis, but the molecular mechanism is not understood. We developed an in vitro model of ameba-host cell interaction to test the hypothesis that leptin prevents ameba-induced apoptosis in host epithelial cells. We demonstrated that activation of mammalian leptin signaling increased cellular resistance to amebic cytotoxicity, including caspase-3 activation. Exogenous expression of the leptin receptor conferred resistance in susceptible cells, and leptin stimulation enhanced protection. A series of leptin receptor signaling mutants showed that resistance to amebic cytotoxicity was dependent on activation of STAT3 but not the Src homology-2 domain-containing tyrosine phosphatase (SHP-2) or STAT5. A common polymorphism in the leptin receptor (Q223R) that increases susceptibility to amebiasis in humans and mice was found to increase susceptibility to amebic cytotoxicity in single cells. The Q223R polymorphism also decreased leptin-dependent STAT3 activation by 21% relative to that of the wild-type (WT) receptor (P = 0.035), consistent with a central role of STAT3 signaling in protection. A subset of genes uniquely regulated by STAT3 in response to leptin was identified. Most notable were the TRIB1 and suppressor of cytokine signaling 3 (SOCS3) genes, which have opposing roles in the regulation of apoptosis. Overall apoptotic genes were highly enriched in this gene set (P < 1E−05), supporting the hypothesis that leptin regulation of host apoptotic genes via STAT3 is responsible for protection. This is the first demonstration of a mammalian signaling pathway that restricts amebic pathogenesis and represents an important advance in our mechanistic understanding of how leptin links nutrition and susceptibility to infection. PMID:22331430

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

PubMed

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 Fibroblast-Like Cells of Mesenchymal Origin of Cytotoxic Activity of Lymphocytes against NK-Sensitive Target Cells.

PubMed

Lupatov, A Yu; Kim, Ya S; Bystrykh, O A; Vakhrushev, I V; Pavlovich, S V; Yarygin, K N; Sukhikh, G T

2017-02-01

We studied immunosuppressive properties of skin fibroblasts and mesenchymal stromal cells against NK cells. In vitro experiments showed that mesenchymal stromal cells isolated from human umbilical cord and human skin fibroblasts can considerably attenuate cytotoxic activity of NK cells against Jurkat cells sensitive to NK-mediated lysis. NK cells cultured in lymphocyte population exhibited higher cytotoxic activity than isolated NK cells. Mesenchymal stromal cells or fibroblasts added 1:1 to lymphocyte culture almost completely suppressed NK cell cytotoxicity. This suggests that fibroblast-like cells can suppress not only isolated NK cells, but also NK cells in natural cell microenvironment.

CD16A Activation of NK Cells Promotes NK Cell Proliferation and Memory-Like Cytotoxicity against Cancer Cells.

PubMed

Pahl, Jens H W; Koch, Joachim; Götz, Jana-Julia; Arnold, Annette; Reusch, Uwe; Gantke, Thorsten; Rajkovic, Erich; Treder, Martin; Cerwenka, Adelheid

2018-05-01

CD16A is a potent cytotoxicity receptor on human natural killer (NK) cells, which can be exploited by therapeutic bispecific antibodies. So far, the effects of CD16A-mediated activation on NK cell effector functions beyond classical antibody-dependent cytotoxicity have remained poorly elucidated. Here, we investigated NK cell responses after exposure to therapeutic antibodies such as the tetravalent bispecific antibody AFM13 (CD30/CD16A), designed for the treatment of Hodgkin lymphoma and other CD30 + lymphomas. Our results reveal that CD16A engagement enhanced subsequent IL2- and IL15-driven NK cell proliferation and expansion. This effect involved the upregulation of CD25 (IL2Rα) and CD132 (γ c ) on NK cells, resulting in increased sensitivity to low-dose IL2 or to IL15. CD16A engagement initially induced NK cell cytotoxicity. The lower NK cell reactivity observed 1 day after CD16A engagement could be recovered by reculture in IL2 or IL15. After reculture in IL2 or IL15, these CD16A-experienced NK cells exerted more vigorous IFNγ production upon restimulation with tumor cells or cytokines. Importantly, after reculture, CD16A-experienced NK cells also exerted increased cytotoxicity toward different tumor targets, mainly through the activating NK cell receptor NKG2D. Our findings uncover a role for CD16A engagement in priming NK cell responses to restimulation by cytokines and tumor cells, indicative of a memory-like functionality. Our study suggests that combination of AFM13 with IL2 or IL15 may boost NK cell antitumor activity in patients by expanding tumor-reactive NK cells and enhancing NK cell reactivity, even upon repeated tumor encounters. Cancer Immunol Res; 6(5); 517-27. ©2018 AACR . ©2018 American Association for Cancer Research.

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

PubMed

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.

Endonuclease G mediates α-synuclein cytotoxicity during Parkinson's disease.

PubMed

Büttner, Sabrina; Habernig, Lukas; Broeskamp, Filomena; Ruli, Doris; Vögtle, F Nora; Vlachos, Manolis; Macchi, Francesca; Küttner, Victoria; Carmona-Gutierrez, Didac; Eisenberg, Tobias; Ring, Julia; Markaki, Maria; Taskin, Asli Aras; Benke, Stefan; Ruckenstuhl, Christoph; Braun, Ralf; Van den Haute, Chris; Bammens, Tine; van der Perren, Anke; Fröhlich, Kai-Uwe; Winderickx, Joris; Kroemer, Guido; Baekelandt, Veerle; Tavernarakis, Nektarios; Kovacs, Gabor G; Dengjel, Jörn; Meisinger, Chris; Sigrist, Stephan J; Madeo, Frank

2013-11-27

Malfunctioning of the protein α-synuclein is critically involved in the demise of dopaminergic neurons relevant to Parkinson's disease. Nonetheless, the precise mechanisms explaining this pathogenic neuronal cell death remain elusive. Endonuclease G (EndoG) is a mitochondrially localized nuclease that triggers DNA degradation and cell death upon translocation from mitochondria to the nucleus. Here, we show that EndoG displays cytotoxic nuclear localization in dopaminergic neurons of human Parkinson-diseased patients, while EndoG depletion largely reduces α-synuclein-induced cell death in human neuroblastoma cells. Xenogenic expression of human α-synuclein in yeast cells triggers mitochondria-nuclear translocation of EndoG and EndoG-mediated DNA degradation through a mechanism that requires a functional kynurenine pathway and the permeability transition pore. In nematodes and flies, EndoG is essential for the α-synuclein-driven degeneration of dopaminergic neurons. Moreover, the locomotion and survival of α-synuclein-expressing flies is compromised, but reinstalled by parallel depletion of EndoG. In sum, we unravel a phylogenetically conserved pathway that involves EndoG as a critical downstream executor of α-synuclein cytotoxicity.

An endogenous 55 kDa TNF receptor mediates cell death in a neural cell line.

PubMed

Sipe, K J; Srisawasdi, D; Dantzer, R; Kelley, K W; Weyhenmeyer, J A

1996-06-01

Tumor necrosis factor-alpha (TNF) is associated with developmental and injury-related events in the central nervous system (CNS). In the present study, we have examined the role of TNF on neurons using the clonal murine neuroblastoma line, N1E-115 (N1E). N1E cells represent a well-defined model for studying neuronal development since they can be maintained as either undifferentiated, mitotically active neuroblasts or as differentiated, mature neurons. Northern and reverse transcription-polymerase chain reaction (RT-PCR) analyses revealed that both undifferentiated and differentiated N1Es express transcripts for the 55 kDa TNF receptor (TNFR), but not the 75 kDa TNFR. The biological activity of the expressed TNF receptor was demonstrated by a dose dependent cytotoxicity to either recombinant murine or human TNF when the cells were incubated with the transcriptional inhibitor actinomycin D. The lack of the 75 kDa receptor mRNA expression and the dose dependent response to rHuTNF, an agonist specific for the murine 55 kDa receptor, suggest that the TNF induced cytotoxicity is mediated through the 55 kDa receptor in both the undifferentiated and differentiated N1Es. Light microscopic observations, flow cytometric analysis of hypodiploid DNA, and electrophoretic analysis of nucleosomal DNA fragmentation of N1Es treated with actinomycin D and TNF revealed features characteristic of both necrotic and apoptotic cell death. These findings demonstrate that blast and mature N1E cells express the 55 kDa TNF receptor which is responsible for inducing both necrotic and apoptotic death in these cells. The observation that actinomycin D renders N1E cells susceptible to the cytotoxic effects of TNF indicates that a sensitization step, such as removal of an endogenous protective factor or viral-mediated inhibition of transcription, may be necessary for TNF cytotoxicity in neurons.

Cytotoxicity of flavones and flavonols to a human esophageal squamous cell carcinoma cell line (KYSE-510) by induction of G2/M arrest and apoptosis.

PubMed

Zhang, Qiang; Zhao, Xin-Huai; Wang, Zhu-Jun

2009-08-01

In this study, cytotoxic effects of structurally related flavones and flavonols on a human esophageal squamous cell carcinoma cell line (KYSE-510) were determined, and the molecular mechanisms responsible for their cytotoxic effects were studied. The results of MTT assay showed that flavones (luteolin, apigenin, chrysin) and flavonols (quercetin, kaempferol, myricetin) were able to induce cytotoxicity in KYSE-510 cells in a dose- and time-dependent manner, and the cytotoxic potency of these compounds was in the order of: luteolin>quercetin>chrysin>kaempferol>apigenin>myricetin. Flow cytometry and DNA fragmentation analysis indicated that the cytotoxicity induced by flavones and flavonols was mediated by G(2)/M cell cycle arrest and apoptosis. Furthermore, the expression of genes related to cell cycle arrest and apoptosis was assessed by oligonucleotide microarray, real-time RT-PCR and Western blot. It was shown that the treatment of KYSE-510 cells with these compounds caused G(2)/M arrest through up-regulation of p21(waf1) and down-regulation of cyclin B1 at the mRNA and protein levels, and induced p53-independent mitochondrial-mediated apoptosis through up-regulation of PIG3 and cleavage of caspase-9 and caspase-3. The results of western blot analysis further showed that increases of p63 and p73 protein translation or stability might be contributed to the regulation of p21(waf1), cyclin B1 and PIG3.

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

PubMed

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.

Proteomic comparison of 3D and 2D glioma models reveals increased HLA-E expression in 3D models is associated with resistance to NK cell-mediated cytotoxicity.

PubMed

He, Weiqi; Kuang, Yongqin; Xing, Xuemin; Simpson, Richard J; Huang, Haidong; Yang, Tao; Chen, Jingmin; Yang, Libin; Liu, Enyu; He, Weifeng; Gu, Jianwen

2014-05-02

Three-dimensional cell culture techniques can better reflect the in vivo characteristics of tumor cells compared with traditional monolayer cultures. Compared with their 2D counterparts, 3D-cultured tumor cells showed enhanced resistance to the cytotoxic T cell-mediated immune response. However, it remains unclear whether 3D-cultured tumor cells have an enhanced resistance to NK cell cytotoxicity. In this study, a total of 363 differentially expressed proteins were identified between the 2D- and 3D-cultured U251 cells by comparative proteomics, and an immune-associated protein-protein interaction (PPI) network based on these differential proteins was constructed by bioinformatics. Within the network, HLA-E, as a molecule for inhibiting NK cell activation, was significantly up-regulated in the 3D-cultured tumor cells. Then, we found that the 3D-cultured U251 cells exhibited potent resistance to NK cell cytotoxicity in vitro and were prone to tumor formation in vivo. The resistance of the 3D-cultured tumor cells to NK cell lysis was mediated by the HLA-E/NKG2A interaction because the administration of antibodies that block either HLA-E or NKG2A completely eliminated this resistance and significantly decreased tumor formation. Taken together, our findings indicate that HLA-E up-regulation in 3D-cultured cells may result in enhanced tumor resistance to NK cell-mediated immune response.

TLR8 stimulation enhances cetuximab-mediated natural killer cell lysis of head and neck cancer cells and dendritic cell cross priming of EGFR-specific CD8+ T cells

PubMed Central

Stephenson, Ryan M.; Lim, Chwee Ming; Matthews, Maura; Dietsch, Gregory; Hershberg, Robert; Ferris, Robert L.

2013-01-01

Background Cetuximab is an anti-epidermal growth factor receptor (EGFR) monoclonal antibody (mAb) that prolongs survival in the treatment of head and neck cancer (HNC), but only in 10–20% of patients. An immunological mechanism of action such as natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC) has been suggested. We investigated the effects of activating toll-like receptor (TLR)-8 to enhance activity of cetuximab-stimulated, FcγR bearing cells. Objective To determine the capability of TLR8-stimulation to enhance the activation and function of NK cells and dendritic cells (DC) in the presence of cetuximab-coated HNC cells. Methods Peripheral blood mononuclear cells (PBMC), NK, DC and CD8+ T cells were isolated and analyzed using 51Cr release ADCC, flow cytometry analysis, cytokine ELISA, and EGFR853–861 tetramer staining. Results TLR8 stimulation of unfractionated PBMC led to enhanced cetuximab-mediated ADCC in healthy donors (p<0.01) and HNC patients (p<0.001), which was dependent on NK cells. Secretion of Th1 cytokines TNFα(p<0.0001), IFNγ(p<0.0001), and IL-12p40(p<0.005) was increased. TLR8 stimulation of PBMC augmented cetuximab-enhanced NK cell degranulation (p<0.001). TLR8 stimulated NK cells enhanced DC maturation markers CD80, CD83, and CD86 in co-culture with cetuximab-treated HNC cells. TLR8 stimulation of NK-DC co-cultures significantly increased DC priming of EGFR-specific CD8+ T cells in the presence of cetuximab. Discussion VTX-2337 and cetuximab combination therapy can activate innate and adaptive anti-cancer immune responses. Further investigation in human trials will be important for determining the clinical benefit of this combination, and for determining biomarkers of response. PMID:23685782

Human embryonic stem cell-derived NK cells acquire functional receptors and cytolytic activity.

PubMed

Woll, Petter S; Martin, Colin H; Miller, Jeffrey S; Kaufman, Dan S

2005-10-15

Human embryonic stem cells (hESCs) provide a unique resource to analyze early stages of human hematopoiesis. However, little is known about the ability to use hESCs to evaluate lymphocyte development. In the present study, we use a two-step culture method to demonstrate efficient generation of functional NK cells from hESCs. The CD56(+)CD45(+) hESC-derived lymphocytes express inhibitory and activating receptors typical of mature NK cells, including killer cell Ig-like receptors, natural cytotoxicity receptors, and CD16. Limiting dilution analysis suggests that these cells can be produced from hESC-derived hemopoietic progenitors at a clonal frequency similar to CD34(+) cells isolated from cord blood. The hESC-derived NK cells acquire the ability to lyse human tumor cells by both direct cell-mediated cytotoxicity and Ab-dependent cellular cytotoxicity. Additionally, activated hESC-derived NK cells up-regulate cytokine production. hESC-derived lymphoid progenitors provide a novel means to characterize specific cellular and molecular mechanisms that lead to development of specific human lymphocyte populations. These cells may also provide a source for innovative cellular immune therapies.

In vitro cytotoxicity induced by Clostridium perfringens isolate carrying a chromosomal cpe gene is exclusively dependent on sporulation and enterotoxin production.

PubMed

Yasugi, Mayo; Sugahara, Yuki; Hoshi, Hidenobu; Kondo, Kaori; Talukdar, Prabhat K; Sarker, Mahfuzur R; Yamamoto, Shigeki; Kamata, Yoichi; Miyake, Masami

2015-08-01

Clostridium perfringens type A is a common source of food poisoning (FP) and non-food-borne (NFB) gastrointestinal diseases in humans. In the intestinal tract, the vegetative cells sporulate and produce a major pathogenic factor, C. perfringens enterotoxin (CPE). Most type A FP isolates carry a chromosomal cpe gene, whereas NFB type A isolates typically carry a plasmid-encoded cpe. In vitro, the purified CPE protein binds to a receptor and forms pores, exerting a cytotoxic activity in epithelial cells. However, it remains unclear if CPE is indispensable for C. perfringens cytotoxicity. In this study, we examined the cytotoxicity of cpe-harboring C. perfringens isolates co-cultured with human intestinal epithelial Caco-2 cells. The FP strains showed severe cytotoxicity during sporulation and CPE production, but not during vegetative cell growth. While Caco-2 cells were intact during co-culturing with cpe-null mutant derivative of strain SM101 (a FP strain carrying a chromosomal cpe gene), the wild-type level cytotoxicity was observed with cpe-complemented strain. In contrast, both wild-type and cpe-null mutant derivative of the NFB strain F4969 induced Caco-2 cell death during both vegetative and sporulation growth. Collectively, the Caco-2 cell cytotoxicity caused by C. perfringens strain SM101 is considered to be exclusively dependent on CPE production, whereas some additional toxins should be involved in F4969-mediated in vitro cytotoxicity. Copyright © 2015 Elsevier Ltd. All rights reserved.

Cytotoxic effect of disulfiram/copper on human glioblastoma cell lines and ALDH-positive cancer-stem-like cells

PubMed Central

Liu, P; Brown, S; Goktug, T; Channathodiyil, P; Kannappan, V; Hugnot, J-P; Guichet, P-O; Bian, X; Armesilla, A L; Darling, J L; Wang, W

2012-01-01

Background: Glioblastoma multiforme (GBM) cells are resistant to anticancer drugs. Cancer stem cells (CSCs) are a key mediator of chemoresistance. We have reported that disulfiram (DS), an aldehyde dehydrogenase (ALDH) inhibitor, targets breast CSC-like cells. In this study, the effect of DS and combination of DS and gemcitabine (dFdC) on GBM cells and GBM stem-like cells was investigated. Methods: 1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT), combination index (CI)-isobologram, western blot, luciferase reporter gene assay, electrophoretic mobility-shift assay and ALDH analysis were used in this study. Results: Disulfiram is cytotoxic in GBM cell lines in a copper (Cu)-dependent manner. Disulfiram/copper enhances the cytotoxicity of dFdC. Combination index-isobologram analysis indicates a synergistic effect between DS/Cu and dFdC. Disulfiram/copper induces reactive oxygen species (ROS), activates JNK and p38 pathways and inhibits nuclear factor-kappa B activity in GBM cell lines. Disulfiram/copper may trigger intrinsic apoptotic pathway via modulation of the Bcl2 family. Disulfiram/copper abolishes stem-like cell population in GBM cell lines. Conclusion: Our findings indicate that the cytotoxicity of DS/Cu and the enhancing effect of DS/Cu on the cytotoxicity of dFdC in GBM stem-like cells may be caused by induction of ROS and inhibition of both ALDH and the NFkB pathway. Both DS and dFdC can traverse the blood–brain barrier. Further study may lead them into GBM chemotherapy. PMID:23033007

Novel role of TRPV2 in promoting the cytotoxicity of H2O2-mediated oxidative stress in human hepatoma cells.

PubMed

Ma, Wenbo; Li, Caiyue; Yin, Shikui; Liu, Jingxin; Gao, Chao; Lin, Zuoxian; Huang, Rongqi; Huang, Jufang; Li, Zhiyuan

2015-12-01

Oxidative stress is important for the initiation and progression of cancers, which confers the cells with a survival advantage by inducing oxidative adaption and drug resistance. Therefore, developing strategies to promote oxidative stress-induced cytotoxicity could be important for cancer therapy. Herein, we found that H2O2-mediated oxidative stress increases TRPV2 expression in human hepatoma (HepG2 and Huh-7) cells. This occurred at the mRNA and protein levels in a dose-dependent manner. The significance of TRPV2 in promoting H2O2-induced cell death was demonstrated in gain and loss of function studies with overexpression and knockdown of TRPV2, respectively. Mechanistically, H2O2-induced cell death involves inhibition of pro-survival signaling proteins (Akt, Nrf2) and activation of pro-death signaling proteins (p38, JNK1). Overexpression of TRPV2 in H2O2-treated hepatoma cells aggravates the inhibition of Akt and Nrf2, while it enhances the activation of p38 and JNK1 at the early stage of cell death. Interestingly, increased expression of TRPV2 in HepG2 cells improved the efficacy of stress-associated chemicals to induce cell death. Our findings suggest that TRPV2 acts as an important enhancer for H2O2-induced cytotoxicity. This process occurred by the inhibition of Akt and Nrf2 as well as the early activation of p38 and JNK1. These findings have important implications for inhibition of oxidative adaption and drug resistance. Copyright © 2015 Elsevier Inc. All rights reserved.

Cell Specific Cytotoxicity and Uptake of Graphene Nanoribbons

PubMed Central

Chowdhury, Sayan Mullick; Lalwani, Gaurav; Zhang, Kevin; Yang, Jeong Yun; Neville, Kayla; Sitharaman, Balaji

2012-01-01

The synthesis of oxidized graphene nanoribbons (O-GNR) via longitudinal unzipping of carbon nanotubes opens avenues for their further development for a variety of biomedical applications. Evaluation of the cyto- and bio-compatibility is necessary to develop any new material for in vivo biomedical applications. In this study, we report the cytotoxicity screening of O-GNRs water-solubilized with PEG-DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)]), using six different assays, in four representative cell lines; Henrietta Lacks cells (HeLa) derived from cervical cancer tissue, National Institute of Health 3T3 mouse fibroblast cells (NIH-3T3), Sloan Kettering breast cancer cells (SKBR3) and Michigan cancer foundation-7 breast cancer cells (MCF7). These cell lines significantly differed in their response to O-GNR-PEG-DSPE formulations; assessed and evaluated using various endpoints (lactate dehydrogenase (LDH) release, cellular metabolism, lysosomal integrity and cell proliferation) for cytotoxicity. In general, all the cells showed a dose-dependent (10–400 μg/ml) and time-dependent (12–48 h) decrease in cell viability. However, the degree of cytotoxicity was significantly lower in MCF7 or SKBR3 cells compared to HeLa cells. These cells were 100% viable upto 48 hours, when incubated at 10μg/ml O-GNR-PEG-DSPE concentration, and showed decrease in cell viability above this concentration with ~78% of cells viable at the highest concentration (400 μg/ml). In contrast, significant cell death (5–25% cell death depending on the time point, and the assay) was observed for HeLa cells even at a low concentration of 10μg/ml. The decrease in cell viability was steep with increase in concentration with the CD50 values ≥ 100μg/ml depending on the assay, and time point. Transmission electron microscopy of the various cells treated with the O-GNR solutions show higher uptake of the O-GNR-PEG-DSPEs into HeLa cells compared to other cell

Augmented IFN-γ and TNF-α Induced by Probiotic Bacteria in NK Cells Mediate Differentiation of Stem-Like Tumors Leading to Inhibition of Tumor Growth and Reduction in Inflammatory Cytokine Release; Regulation by IL-10

PubMed Central

Bui, Vickie T.; Tseng, Han-Ching; Kozlowska, Anna; Maung, Phyu Ou; Kaur, Kawaljit; Topchyan, Paytsar; Jewett, Anahid

2015-01-01

Our previous reports demonstrated that the magnitude of natural killer (NK) cell-mediated cytotoxicity correlate directly with the stage and level of differentiation of tumor cells. In addition, we have shown previously that activated NK cells inhibit growth of cancer cells through induction of differentiation, resulting in the resistance of tumor cells to NK cell-mediated cytotoxicity through secreted cytokines, as well as direct NK-tumor cell contact. In this report, we show that in comparison to IL-2 + anti-CD16mAb-treated NK cells, activation of NK cells by probiotic bacteria (sAJ2) in combination with IL-2 and anti-CD16mAb substantially decreases tumor growth and induces maturation, differentiation, and resistance of oral squamous cancer stem cells, MIA PaCa-2 stem-like/poorly differentiated pancreatic tumors, and healthy stem cells of apical papillae through increased secretion of IFN-γ and TNF-α, as well as direct NK-tumor cell contact. Tumor resistance to NK cell-mediated killing induced by IL-2 + anti-CD16mAb + sAJ2-treated NK cells is induced by combination of IFN-γ and TNF-α since antibodies to both, and not each cytokine alone, were able to restore tumor sensitivity to NK cells. Increased surface expression of CD54, B7H1, and MHC-I on NK-differentiated tumors was mediated by IFN-γ since the addition of anti-IFN-γ abolished their increase and restored the ability of NK cells to trigger cytokine and chemokine release; whereas differentiated tumors inhibited cytokine release by the NK cells. Monocytes synergize with NK cells in the presence of probiotic bacteria to induce regulated differentiation of stem cells through secretion of IL-10 resulting in resistance to NK cell-mediated cytotoxicity and inhibition of cytokine release. Therefore, probiotic bacteria condition activated NK cells to provide augmented differentiation of cancer stem cells resulting in inhibition of tumor growth, and decreased inflammatory cytokine release. PMID

Effects of in vivo hydrocortisone on lymphocyte-mediated cytotoxicity

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

Katz, P.; Zaytoun, A.M.; Lee, J.H. Jr.

To examine the effects of in vivo hydrocortisone sodium succinate (HC) on natural killer (NK) cell and antibody-dependent cellular cytotoxicity (ADCC), 11 normal adults received a single intravenous bolus of 400 mg hydrocortisone. Lymphocytes were tested for NK activity and ADCC using 51chromium (51Cr)-release and single cell cytotoxicity assays against Molt-4 and sensitized RL O leads to target cells, respectively. Four hours after injection, both NK and ADCC activity were transiently increased in the 51Cr-release system. At 4 hours, there was a twofold increase in the relative frequency of potentially cytotoxic target binding cells but the absolute number of thesemore » cells did not change. However, the percentage lysis of bound targets at 4 hours was not altered. These data suggest that: 1) lymphocytes participating in NK and ADCC reactions are refractory to the kinetic and functional effects of HC; 2) the increased lytic activity observed at 4 hours is due to a selective depletion of noncytotoxic cells from the circulation; and 3) NK and ADCC activity did not differ in their responses to HC.« less

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

PubMed

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

2001-01-01

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

Inhibitory effects of low molecular weight heparin on mediator release by mast cells: preferential inhibition of cytokine production and mast cell-dependent cutaneous inflammation

PubMed Central

BARAM, D; RASHKOVSKY, M; HERSHKOVIZ, R; DRUCKER, I; RESHEF, T; BEN-SHITRIT, S; MEKORI, Y A

1997-01-01

There has been substantial evidence that suggests that heparin may modulate various aspects of immune function and inflammation in addition to its well known anticoagulant activity. In this regard heparin was found to suppress cell-mediated immune responses or asthmatic reactions to allergen challenge. In the present study we analyse the effects of low molecular weight heparin (LMWH) on mast cell degranulation and cytokine production in vitro and on the elicitation of IgE-mediated mast cell-dependent late cutaneous allergic inflammation in vivo. We have established that LMWH preferentially inhibited tumour necrosis factor-alpha (TNF-α) and IL-4 production without having any significant effect on mast cell degranulation. These effects have been observed in mast cells derived from three different origins that were activated by either immunological or non-immunological stimuli. We have shown that there is inhibition of TNF-α production (and not neutralization of activity), as elimination of the drug after a short preincubation and addition of LMWH to rTNF-α had no effect on TNF-α-mediated cytotoxic activity. These results were also confirmed by ELISA. In vivo, s.c. injection of the LMWH inhibited the leucocyte infiltration associated with the late cutaneous response which followed passive cutaneous anaphylaxis (PCA) reaction, without affecting mast cell numbers or degranulation. These data suggest that LMWH may have an inhibitory role in mast cell-mediated allergic inflammation, and thus might be considered as a possible therapeutic modality. PMID:9409655

Dynamin-related protein 1 mediates mitochondria-dependent apoptosis in chlorpyrifos-treated SH-SY5Y cells.

PubMed

Park, Jae Hyeon; Ko, Juyeon; Hwang, Jungwook; Koh, Hyun Chul

2015-12-01

Recent studies have demonstrated that dynamin-related protein 1 (Drp1), a mitochondrial fission protein, mediates mitochondria-dependent apoptosis through mitochondrial division. However, little is known about the mechanism by which Drp1 modulates apoptosis in response to chlorpyrifos (CPF)-induced toxicity. In this study, we determined that CPF-induced mitochondrial apoptosis is mediated by Drp1 translocation in SH-SY5Y human neuroblastoma cells. Our results showed that CPF treatment induced intrinsic apoptosis by activating caspase-9, caspase-3, and cytochrome c release in SH-SY5Y cells. Cytosolic Drp1 translocated to the mitochondria in CPF-treated cells and was phosphorylated at Ser616. Treating cells with CPF induced the generation of reactive oxygen species (ROS) and activation of mitogen-activated protein kinases (MAPKs). Inhibiting this ROS generation and MAPK activation abolished CPF-induced expression of phospho-Drp1. Furthermore, Drp1 was required for p53 to translocate to the mitochondria under CPF-induced oxidative stress. Treating cells with mitochondrial-division inhibitor-1 (mdivi-1), which blocks Drp1 translocation, increased the viability of CPF-treated cells by abrogating Drp1 translocation and caspase-3 activation. Specifically, pretreating cells with mdivi-1 inhibited Bax translocation to the mitochondria by blocking p53 signaling. Taken together, these data reveal a novel mechanism by which Drp1 activates mitochondrial-dependent apoptosis and indicate that inhibiting Dpr1 function can protect against CPF-induced cytotoxicity. We propose that inhibiting Drp1 is a possible therapeutic approach for pesticide-induced toxicity when hyperactivated Drp1 contributes to pathology. Crown Copyright © 2015. Published by Elsevier B.V. All rights reserved.

Myricetin-induced apoptosis of triple-negative breast cancer cells is mediated by the iron-dependent generation of reactive oxygen species from hydrogen peroxide.

PubMed

Knickle, Allison; Fernando, Wasundara; Greenshields, Anna L; Rupasinghe, H P Vasantha; Hoskin, David W

2018-05-06

Myricetin is a dietary phytochemical with anticancer activity; however, the effect of myricetin on breast cancer cells remains unclear. Here, we show that myricetin inhibited the growth of triple-negative breast cancer (TNBC) cells but was less inhibitory for normal cells. The effect of myricetin was comparable to epigallocatechin gallate and doxorubicin, and greater than resveratrol and cisplatin. Myricetin-treated TNBC cells showed evidence of early and late apoptosis/necrosis, which was associated with intracellular reactive oxygen species (ROS) accumulation, extracellular regulated kinase 1/2 and p38 mitogen-activated protein kinase activation, mitochondrial membrane destabilization and cytochrome c release, and double-strand DNA breaks. The antioxidant N-acetyl-cysteine protected myricetin-treated TNBC cells from cytotoxicity due to DNA damage. Myricetin also induced hydrogen peroxide (H 2 O 2 ) production in cell-free culture medium, as well as in the presence of TNBC cells and normal cells. In addition, deferriprone-mediated inhibition of intracellular ROS generation via the iron-dependent Fenton reaction and inhibition of extracellular ROS accumulation with superoxide dismutase plus catalase prevented myricetin-induced cytotoxicity in TNBC cell cultures. We conclude that the cytotoxic effect of myricetin on TNBC cells was due to oxidative stress initiated by extracellular H 2 O 2 formed by autoxidation of myricetin, leading to intracellular ROS production via the Fenton reaction. Copyright © 2018. Published by Elsevier Ltd.

Cytotoxic-T-lymphocyte antigen 4 receptor signaling for lymphocyte adhesion is mediated by C3G and Rap1.

PubMed

Kloog, Yoel; Mor, Adam

2014-03-01

T-lymphocyte adhesion plays a critical role in both inflammatory and autoimmune responses. The small GTPase Rap1 is the key coordinator mediating T-cell adhesion to endothelial cells, antigen-presenting cells, and virus-infected cells. We describe a signaling pathway, downstream of the cytotoxic T-lymphocyte antigen 4 (CTLA-4) receptor, leading to Rap1-mediated adhesion. We identified a role for the Rap1 guanine nucleotide exchange factor C3G in the regulation of T-cell adhesion and showed that this factor is required for both T-cell receptor (TCR)-mediated and CTLA-4-mediated T-cell adhesion. Our data indicated that C3G translocates to the plasma membrane downstream of TCR signaling, where it regulates activation of Rap1. We also showed that CTLA-4 receptor signaling mediates tyrosine phosphorylation in the C3G protein, and that this is required for augmented activation of Rap1 and increased adhesion mediated by leukocyte function-associated antigen type 1 (LFA-1). Zap70 is required for C3G translocation to the plasma membrane, whereas the Src family member Hck facilitates C3G phosphorylation. These findings point to C3G and Hck as promising potential therapeutic targets for the treatment of T-cell-dependent autoimmune disorders.

Cytotoxic-T-Lymphocyte Antigen 4 Receptor Signaling for Lymphocyte Adhesion Is Mediated by C3G and Rap1

PubMed Central

Kloog, Yoel

2014-01-01

T-lymphocyte adhesion plays a critical role in both inflammatory and autoimmune responses. The small GTPase Rap1 is the key coordinator mediating T-cell adhesion to endothelial cells, antigen-presenting cells, and virus-infected cells. We describe a signaling pathway, downstream of the cytotoxic T-lymphocyte antigen 4 (CTLA-4) receptor, leading to Rap1-mediated adhesion. We identified a role for the Rap1 guanine nucleotide exchange factor C3G in the regulation of T-cell adhesion and showed that this factor is required for both T-cell receptor (TCR)-mediated and CTLA-4-mediated T-cell adhesion. Our data indicated that C3G translocates to the plasma membrane downstream of TCR signaling, where it regulates activation of Rap1. We also showed that CTLA-4 receptor signaling mediates tyrosine phosphorylation in the C3G protein, and that this is required for augmented activation of Rap1 and increased adhesion mediated by leukocyte function-associated antigen type 1 (LFA-1). Zap70 is required for C3G translocation to the plasma membrane, whereas the Src family member Hck facilitates C3G phosphorylation. These findings point to C3G and Hck as promising potential therapeutic targets for the treatment of T-cell-dependent autoimmune disorders. PMID:24396067

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

PubMed

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.

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

NASA Technical Reports Server (NTRS)

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

1994-01-01

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

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

PubMed

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.

CRTAM determines the CD4+ cytotoxic T lymphocyte lineage

PubMed Central

Takeuchi, Arata; Badr, Mohamed El Sherif Gadelhaq; Miyauchi, Kosuke; Ishihara, Chitose; Onishi, Reiko; Guo, Zijin; Sasaki, Yoshiteru; Ike, Hiroshi; Takumi, Akiko; Tsuji, Noriko M.; Murakami, Yoshinori; Katakai, Tomoya; Kubo, Masato

2016-01-01

Naive T cells differentiate into various effector T cells, including CD4+ helper T cell subsets and CD8+ cytotoxic T cells (CTL). Although cytotoxic CD4+ T cells (CD4+CTL) also develop from naive T cells, the mechanism of development is elusive. We found that a small fraction of CD4+ T cells that express class I–restricted T cell–associated molecule (CRTAM) upon activation possesses the characteristics of both CD4+ and CD8+ T cells. CRTAM+ CD4+ T cells secrete IFN-γ, express CTL-related genes, such as eomesodermin (Eomes), Granzyme B, and perforin, after cultivation, and exhibit cytotoxic function, suggesting that CRTAM+ T cells are the precursor of CD4+CTL. Indeed, ectopic expression of CRTAM in T cells induced the production of IFN-γ, expression of CTL-related genes, and cytotoxic activity. The induction of CD4+CTL and IFN-γ production requires CRTAM-mediated intracellular signaling. CRTAM+ T cells traffic to mucosal tissues and inflammatory sites and developed into CD4+CTL, which are involved in mediating protection against infection as well as inducing inflammatory response, depending on the circumstances, through IFN-γ secretion and cytotoxic activity. These results reveal that CRTAM is critical to instruct the differentiation of CD4+CTL through the induction of Eomes and CTL-related gene. PMID:26694968

Cell specific cytotoxicity and uptake of graphene nanoribbons.

PubMed

Mullick Chowdhury, Sayan; Lalwani, Gaurav; Zhang, Kevin; Yang, Jeong Y; Neville, Kayla; Sitharaman, Balaji

2013-01-01

The synthesis of oxidized graphene nanoribbons (O-GNR) via longitudinal unzipping of carbon nanotubes opens avenues for their further development for a variety of biomedical applications. Evaluation of the cyto- and bio-compatibility is necessary to develop any new material for in vivo biomedical applications. In this study, we report the cytotoxicity screening of O-GNRs water-solubilized with PEG-DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)]), using six different assays, in four representative cell lines; Henrietta Lacks cells (HeLa) derived from cervical cancer tissue, National Institute of Health 3T3 mouse fibroblast cells (NIH-3T3), Sloan Kettering breast cancer cells (SKBR3) and Michigan cancer foundation-7 breast cancer cells (MCF7). These cell lines significantly differed in their response to O-GNR-PEG-DSPE formulations; assessed and evaluated using various endpoints (lactate dehydrogenase (LDH) release, cellular metabolism, lysosomal integrity and cell proliferation) for cytotoxicity. In general, all the cells showed a dose-dependent (10-400 μg/ml) and time-dependent (12-48 h) decrease in cell viability. However, the degree of cytotoxicity was significantly lower in MCF7 or SKBR3 cells compared to HeLa cells. These cells were 100% viable upto 48 h, when incubated at 10 μg/ml O-GNR-PEG-DSPE concentration, and showed decrease in cell viability above this concentration with ~78% of cells viable at the highest concentration (400 μg/ml). In contrast, significant cell death (5-25% cell death depending on the time point, and the assay) was observed for HeLa cells even at a low concentration of 10 μg/ml. The decrease in cell viability was steep with increase in concentration with the CD(50) values ≥ 100 μg/ml depending on the assay, and time point. Transmission electron microscopy of the various cells treated with the O-GNR solutions show higher uptake of the O-GNR-PEG-DSPEs into HeLa cells compared to other cell types

A mathematical model of antibody-dependent cellular cytotoxicity (ADCC).

PubMed

Hoffman, F; Gavaghan, D; Osborne, J; Barrett, I P; You, T; Ghadially, H; Sainson, R; Wilkinson, R W; Byrne, H M

2018-01-07

Immunotherapies exploit the immune system to target and kill cancer cells, while sparing healthy tissue. Antibody therapies, an important class of immunotherapies, involve the binding to specific antigens on the surface of the tumour cells of antibodies that activate natural killer (NK) cells to kill the tumour cells. Preclinical assessment of molecules that may cause antibody-dependent cellular cytotoxicity (ADCC) involves co-culturing cancer cells, NK cells and antibody in vitro for several hours and measuring subsequent levels of tumour cell lysis. Here we develop a mathematical model of such an in vitro ADCC assay, formulated as a system of time-dependent ordinary differential equations and in which NK cells kill cancer cells at a rate which depends on the amount of antibody bound to each cancer cell. Numerical simulations generated using experimentally-based parameter estimates reveal that the system evolves on two timescales: a fast timescale on which antibodies bind to receptors on the surface of the tumour cells, and NK cells form complexes with the cancer cells, and a longer time-scale on which the NK cells kill the cancer cells. We construct approximate model solutions on each timescale, and show that they are in good agreement with numerical simulations of the full system. Our results show how the processes involved in ADCC change as the initial concentration of antibody and NK-cancer cell ratio are varied. We use these results to explain what information about the tumour cell kill rate can be extracted from the cytotoxicity assays. Copyright © 2017 Elsevier Ltd. All rights reserved.

Leukocyte function-associated antigen-1-dependent lysis of Fas+ (CD95+/Apo-1+) innocent bystanders by antigen-specific CD8+ CTL.

PubMed

Kojima, H; Eshima, K; Takayama, H; Sitkovsky, M V

1997-09-15

Exquisite specificity toward Ag-bearing cells (cognate targets) is one of the most important properties of CD8+ CTL-mediated cytotoxicity. Using highly Ag-specific CD8+ CTL lines and clones, which spare noncognate, Ag-free targets, we found that in the presence of Ag-bearing targets the CTL acquire the ability to lyse noncognate target cells (bystanders). It is shown that the unexpectedly rapid and efficient lysis of bystanders by Ag-activated CTL is mediated by a Fas ligand (FasL)/Fas-based mechanism and does not depend on perforin. The CTL lysed Fas-expressing bystanders, but spared the Fas-negative or anti-Fas mAb-resistant bystander cells. Accordingly, the FasL-deficient gld/gld CTL did not kill bystanders, while perforin-deficient CTL did. Unlike anti-Fas mAb-induced cell death, the lysis of bystanders was not only FasL/Fas dependent but also required adhesion molecule LFA-1 on the surface of the activated CTL. Lysis of bystanders is viewed as acceptable "collateral" damage, but the persistent presence of activated CTL could result in immunopathologies involving functional Fas-expressing tissues.

Hexamethoxylated Monocarbonyl Analogues of Curcumin Cause G2/M Cell Cycle Arrest in NCI-H460 Cells via Michael Acceptor-Dependent Redox Intervention.

PubMed

Li, Yan; Zhang, Li-Ping; Dai, Fang; Yan, Wen-Jing; Wang, Hai-Bo; Tu, Zhi-Shan; Zhou, Bo

2015-09-09

Curcumin, derived from the dietary spice turmeric, holds promise for cancer prevention. This prompts much interest in investigating the action mechanisms of curcumin and its analogues. Two symmetrical hexamethoxy-diarylpentadienones (1 and 2) as cucumin analogues were reported to possess significantly enhanced cytotoxicity compared with the parent molecule. However, the detailed mechanisms remain unclear. In this study, compounds 1 and 2 were identified as the G2/M cell cycle arrest agents to mediate the cytotoxicity toward NCI-H460 cells via Michael acceptor-dependent redox intervention. Compared with curcumin, they could more easily induce a burst of reactive oxygen species (ROS) and collapse of the redox buffering system. One possible reason is that they could more effectively target intracellular TrxR to convert this antioxidant enzyme into a ROS promoter. Additionally, they caused up-regulation of p53 and p21 and down-regulation of redox-sensitive Cdc25C along with cyclin B1/Cdk1 in a Michael acceptor- and ROS-dependent fashion. Interestingly, in comparison with compound 2, compound 1 displayed a relatively weak ability to generate ROS but increased cell cycle arrest activity and cytotoxicity probably due to its Michael acceptor-dependent microtubule-destabilizing effect and greater GST-inhibitory activity, as well as its enhanced cellular uptake. This work provides useful information for understanding Michael acceptor-dependent and redox-mediated cytotoxic mechanisms of curcumin and its active analogues.

Downregulation of membrane complement inhibitors CD55 and CD59 by siRNA sensitises uterine serous carcinoma overexpressing Her2/neu to complement and antibody-dependent cell cytotoxicity in vitro: implications for trastuzumab-based immunotherapy.

PubMed

Bellone, S; Roque, D; Cocco, E; Gasparrini, S; Bortolomai, I; Buza, N; Abu-Khalaf, M; Silasi, D-A; Ratner, E; Azodi, M; Schwartz, P E; Rutherford, T J; Pecorelli, S; Santin, A D

2012-04-24

We evaluated the expression of CD46, CD55 and CD59 membrane-bound complement-regulatory proteins (mCRPs) in primary uterine serous carcinoma (USC) and the ability of small interfering RNA (siRNA) against these mCRPs to sensitise USC to complement-dependent cytotoxicity (CDC) and antibody (trastuzumab)-dependent cellular cytotoxicity (ADCC) in vitro. Membrane-bound complement-regulatory proteins expression was evaluated using real-time PCR (RT-PCR) and flow cytometry, whereas Her2/neu expression and c-erbB2 gene amplification were assessed using immunohistochemistry, flow cytometry and fluorescent in-situ hybridisation. The biological effect of siRNA-mediated knockdown of mCRPs on HER2/neu-overexpressing USC cell lines was evaluated in CDC and ADCC 4-h chromium-release assays. High expression of mCRPs was found in USC cell lines when compared with normal endometrial cells (P<0.05). RT-PCR and FACS analyses demonstrated that anti-mCRP siRNAs were effective in reducing CD46, CD55 and CD59 expression on USC (P<0.05). Baseline complement-dependent cytotoxicity (CDC) against USC cell lines was low (mean ± s.e.m.=6.8 ± 0.9%) but significantly increased upon CD55 and CD59 knockdown (11.6 ± 0.8% and 10.7 ± 0.9%, respectively, P<0.05). Importantly, in the absence of complement, both CD55 and CD59, but not CD46, knockdowns significantly augmented ADCC against USC overexpressing Her2/neu. Uterine serous carcinoma express high levels of the mCRPs CD46, CD55 and CD59. Small interfering RNA inhibition of CD55 and CD59, but not CD46, sensitises USC to both CDC and ADCC in vitro, and if specifically targeted to tumour cells, may significantly increase trastuzumab-mediated therapeutic effect in vivo.

Repression of GSK3 restores NK cell cytotoxicity in AML patients

PubMed Central

Parameswaran, Reshmi; Ramakrishnan, Parameswaran; Moreton, Stephen A.; Xia, Zhiqiang; Hou, Yongchun; Lee, Dean A.; Gupta, Kalpana; deLima, Marcos; Beck, Rose C.; Wald, David N.

2016-01-01

Natural killer cells from acute myeloid leukaemia patients (AML-NK) show a dramatic impairment in cytotoxic activity. The exact reasons for this dysfunction are not fully understood. Here we show that the glycogen synthase kinase beta (GSK3β) expression is elevated in AML-NK cells. Interestingly, GSK3 overexpression in normal NK cells impairs their ability to kill AML cells, while genetic or pharmacological GSK3 inactivation enhances their cytotoxic activity. Mechanistic studies reveal that the increased cytotoxic activity correlates with an increase in AML-NK cell conjugates. GSK3 inhibition promotes the conjugate formation by upregulating LFA expression on NK cells and by inducing ICAM-1 expression on AML cells. The latter is mediated by increased NF-κB activation in response to TNF-α production by NK cells. Finally, GSK3-inhibited NK cells show significant efficacy in human AML mouse models. Overall, our work provides mechanistic insights into the AML-NK dysfunction and a potential NK cell therapy strategy. PMID:27040177

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

PubMed

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.

Experimental and Computational Analysis of Polyglutamine-Mediated Cytotoxicity

PubMed Central

Tang, Matthew Y.; Proctor, Carole J.; Woulfe, John; Gray, Douglas A.

2010-01-01

Expanded polyglutamine (polyQ) proteins are known to be the causative agents of a number of human neurodegenerative diseases but the molecular basis of their cytoxicity is still poorly understood. PolyQ tracts may impede the activity of the proteasome, and evidence from single cell imaging suggests that the sequestration of polyQ into inclusion bodies can reduce the proteasomal burden and promote cell survival, at least in the short term. The presence of misfolded protein also leads to activation of stress kinases such as p38MAPK, which can be cytotoxic. The relationships of these systems are not well understood. We have used fluorescent reporter systems imaged in living cells, and stochastic computer modeling to explore the relationships of polyQ, p38MAPK activation, generation of reactive oxygen species (ROS), proteasome inhibition, and inclusion body formation. In cells expressing a polyQ protein inclusion, body formation was preceded by proteasome inhibition but cytotoxicity was greatly reduced by administration of a p38MAPK inhibitor. Computer simulations suggested that without the generation of ROS, the proteasome inhibition and activation of p38MAPK would have significantly reduced toxicity. Our data suggest a vicious cycle of stress kinase activation and proteasome inhibition that is ultimately lethal to cells. There was close agreement between experimental data and the predictions of a stochastic computer model, supporting a central role for proteasome inhibition and p38MAPK activation in inclusion body formation and ROS-mediated cell death. PMID:20885783

Ras-related C3 Botulinum Toxin Substrate (Rac) and Src Family Kinases (SFK) Are Proximal and Essential for Phosphatidylinositol 3-Kinase (PI3K) Activation in Natural Killer (NK) Cell-mediated Direct Cytotoxicity against Cryptococcus neoformans*

PubMed Central

Xiang, Richard F.; Stack, Danuta; Huston, Shaunna M.; Li, Shu Shun; Ogbomo, Henry; Kyei, Stephen K.; Mody, Christopher H.

2016-01-01

The activity of Rac in leukocytes is essential for immunity. However, its role in NK cell-mediated anti-microbial signaling remains unclear. In this study, we investigated the role of Rac in NK cell mediated anti-cryptococcal killing. We found that Cryptococcus neoformans independently activates both Rac and SFK pathways in NK cells, and unlike in tumor killing, Cryptococcus initiated a novel Rac → PI3K → Erk cytotoxicity cascade. Remarkably, Rac was not required for conjugate formation, despite its essential role in NK cytotoxicity against C. neoformans. Taken together, our data show that, unlike observations with tumor cells, NK cells use a novel Rac cytotoxicity pathway in conjunction with SFK, to kill C. neoformans. PMID:26867574

Epidermal Cadm1 expression promotes autoimmune alopecia via enhanced T cell adhesion and cytotoxicity.

PubMed

Giangreco, Adam; Hoste, Esther; Takai, Yoshimi; Rosewell, Ian; Watt, Fiona M

2012-02-01

Autoimmune alopecia is characterized by an extensive epidermal T cell infiltrate that mediates hair follicle destruction. We have investigated the role of cell adhesion molecule 1 (Cadm1; Necl2) in this disease. Cadm1 is expressed by epidermal cells and mediates heterotypic adhesion to lymphocytes expressing class 1-restricted T cell-associated molecule (CRTAM). Using a murine autoimmune alopecia model, we observed an increase in early-activated cytotoxic (CD8-restricted, CRTAM-expressing) T cells, which preferentially associated with hair follicle keratinocytes expressing Cadm1. Coculture with Cadm1-transduced MHC-matched APCs stimulated alopecic lymph node cells to release IL-2 and IFN-γ. Overexpression of Cadm1 in cultured human keratinocytes did not promote cytokine secretion, but led to increased adhesion of alopecic cytotoxic T cells and enhanced T cell cytotoxicity in an MHC-independent manner. Epidermal overexpression of Cadm1 in transgenic mice led to increased autoimmune alopecia susceptibility relative to nontransgenic littermate controls. Our findings reveal that Cadm1 expression in the hair follicle plays a role in autoimmune alopecia.

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

PubMed

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.

Characterization of Thermally Activated Metalloenediyne Cytotoxicity in Human Melanoma Cells.

PubMed

Keller, Eric J; Porter, Meghan; Garrett, Joy E; Varie, Meredith; Wang, Haiyan; Pollok, Karen E; Turchi, John J; Zaleski, Jeffrey M; Dynlacht, Joseph R

2018-05-15

Enediynes are a highly cytotoxic class of compounds. However, metallation of these compounds may modulate their activation, and thus their cytotoxicity. We previously demonstrated that cytotoxicity of two different metalloenediynes, including (Z)-N,N'-bis[1-pyridyl-2-yl-meth-(E)-ylidene]octa-4-ene-2,6-diyne-1,8-diamine] (PyED), is potentiated when the compounds are administered to HeLa cells during hyperthermia treatment at concentrations that are minimally or not cytotoxic at 37°C. In this study, we further characterized the concentration, time and temperature dependence of cytotoxicity of PyED on human U-1 melanoma cells. We also investigated the potential mechanisms by which PyED cytotoxicity is enhanced during hyperthermia treatment. Cell killing with PyED was dependent on concentration, temperature during treatment and time of exposure. Potentiation of cytotoxicity was observed when cells were treated with PyED at temperatures ≥39.5°C, and enhancement of cell killing increased with temperature and with increasing time at a given temperature. All cells treated with PyED were shown to have DNA damage, but substantially more damage was observed in cells treated with PyED during heating. DNA repair was also inhibited in cells treated with the drug during hyperthermia. Thus, potentiation of PyED cytotoxicity by hyperthermia may be due to enhancement of drug-induced DNA lesions, and/or the inhibition of repair of sublethal DNA damage. While the selective thermal activation of PyED supports the potential clinical utility of metalloenediynes as cancer thermochemotherapeutic agents, therapeutic gain could be optimized by identifying compounds that produce minimal toxicity at 37°C but which become activated and show enhancement of cytotoxicity within a tumor subjected to localized hyperthermic or thermal ablative treatment, or which might act as bifunctional agents. We thus also describe the development and initial characterization of a novel cofactor complex of Py

A Cytotoxic Type III Secretion Effector of Vibrio parahaemolyticus Targets Vacuolar H+-ATPase Subunit c and Ruptures Host Cell Lysosomes

PubMed Central

Matsuda, Shigeaki; Okada, Natsumi; Kodama, Toshio; Honda, Takeshi; Iida, Tetsuya

2012-01-01

Vibrio parahaemolyticus is one of the human pathogenic vibrios. During the infection of mammalian cells, this pathogen exhibits cytotoxicity that is dependent on its type III secretion system (T3SS1). VepA, an effector protein secreted via the T3SS1, plays a major role in the T3SS1-dependent cytotoxicity of V. parahaemolyticus. However, the mechanism by which VepA is involved in T3SS1-dependent cytotoxicity is unknown. Here, we found that protein transfection of VepA into HeLa cells resulted in cell death, indicating that VepA alone is cytotoxic. The ectopic expression of VepA in yeast Saccharomyces cerevisiae interferes with yeast growth, indicating that VepA is also toxic in yeast. A yeast genome-wide screen identified the yeast gene VMA3 as essential for the growth inhibition of yeast by VepA. Although VMA3 encodes subunit c of the vacuolar H+-ATPase (V-ATPase), the toxicity of VepA was independent of the function of V-ATPases. In HeLa cells, knockdown of V-ATPase subunit c decreased VepA-mediated cytotoxicity. We also demonstrated that VepA interacted with V-ATPase subunit c, whereas a carboxyl-terminally truncated mutant of VepA (VepAΔC), which does not show toxicity, did not. During infection, lysosomal contents leaked into the cytosol, revealing that lysosomal membrane permeabilization occurred prior to cell lysis. In a cell-free system, VepA was sufficient to induce the release of cathepsin D from isolated lysosomes. Therefore, our data suggest that the bacterial effector VepA targets subunit c of V-ATPase and induces the rupture of host cell lysosomes and subsequent cell death. PMID:22829766

Bombesin receptor-mediated imaging and cytotoxicity: review and current status

PubMed Central

Sancho, Veronica; Di Florio, Alessia; Moody, Terry W.; Jensen, Robert T.

2010-01-01

The three mammalian bombesin (Bn) receptors (gastrin-releasing peptide [GRP] receptor, neuromedin B [NMB] receptor, BRS-3) are one of the classes of G protein-coupled receptors that are most frequently over-express/ectopically expressed by common, important malignancies. Because of the clinical success of somatostatin receptor-mediated imaging and cytotoxicity with neuroendocrine tumors, there is now increasing interest in pursuing a similar approach with Bn receptors. In the last few years then have been more than 200 studies in this area. In the present paper, the in vitro and in vivo results, as well as results of human studies from many of these studies are reviewed and the current state of Bn receptor-mediated imaging or cytotoxicity is discussed. Both Bn receptor-mediated imaging studies as well as Bn receptor-mediated tumoral cytotoxic studies using radioactive and non-radioactive Bn-based ligands are covered. PMID:21034419

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

PubMed

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.

Antioxidative and antigenotoxic effect of vitamin E against patulin cytotoxicity and genotoxicity in HepG2 cells.

PubMed

Ayed-Boussema, Imen; Abassi, Haila; Bouaziz, Chayma; Hlima, Wiem Ben; Ayed, Yosra; Bacha, Hassen

2013-06-01

Patulin (PAT) is a mycotoxin produced in fruits, mainly in apples, by certain species of Penicillium, Aspergillus, and Byssochlamys. It has been shown that PAT is cytotoxic, genotoxic, and mutagenic in different cell types. Several studies incriminate the oxidative stress as a mechanism of PAT-mediated toxicity. In this context, our aim was to investigate the protective role of Vitamin E (Vit E), an antioxidant agent, against PAT induced cytotoxicity and genotoxicity in cultured HepG2 cells. The obtained results showed that addition of Vit E in cells treated with PAT significantly reduce cell mortality induced by this toxin. In the same conditions, Vit E decreased the intracellular level of ROS, reduced PAT induced p53 expression, and reversed PAT induced DNA damage. In addition, Vit E prevented significantly the percentage of chromosome aberrations induced by PAT in HepG2 cells in a concentration dependant manner. These results suggest that Vit E, an exogenous antioxidant agent, plays an important role in defense against PAT-induced cytotoxicity and genotoxicity, which confirms the involvement of oxidative stress in the induction of DNA damage by PAT in HepG2 cells. Copyright © 2011 Wiley Periodicals, Inc.

The Genotoxic and Cytotoxic Effects of Bisphenol-A (BPA) in MCF-7 Cell Line and Amniocytes.

PubMed

Aghajanpour-Mir, Seyed Mohsen; Zabihi, Ebrahim; Akhavan-Niaki, Haleh; Keyhani, Elahe; Bagherizadeh, Iman; Biglari, Sajjad; Behjati, Farkhondeh

2016-01-01

Bisphenol-A (BPA) is an industrial xenoestrogen used widely in our living environment. Recently, several studies suggested that BPA has destructive effects on DNA and chromosomes in normal body cells via estrogen receptors (ER). Therefore, BPA could be considered as an important mediator in many diseases such as cancer. However, there are still many controversial issues which need clarification. In this study, we investigated the BPA-induced chromosomal damages in MCF-7 cell line, ER-positive and negative amniocyte cells. Cytotoxicity and genotoxicity effects of BPA were also compared between these three cell groups. Expression of estrogen receptors was determined using immunocytochemistry technique. The cell cytotoxicity of BPA was measured by MTT assay. Classic cytogenetic technique was carried out for the investigation of chromosome damage. BPA, in addition to cytotoxicity, had remarkable genotoxicity at concentrations close to the traceable levels in tissues or biological fluids. Although some differences were observed in the amount of damages between ER-positive and negative fetal cells, interestingly, these differences were not significant. The present study showed that BPA could lead to chromosomal aberrations in both ER-dependent and independent pathways at some concentrations or in cell types yet not reported. Also, BPA could probably be considered as a facilitator for some predisposed cells to be cancerous by raising the chromosome instability levels. Finally, estrogen receptor seems to have a different role in cytotoxicity and genotoxicity effects.

Oxygen and indocyanine green loaded phase-transition nanoparticle-mediated photo-sonodynamic cytotoxic effects on rheumatoid arthritis fibroblast-like synoviocytes.

PubMed

Tang, Qin; Cui, Jianyu; Tian, Zhonghua; Sun, Jiangchuan; Wang, Zhigang; Chang, Shufang; Zhu, Shenyin

2017-01-01

Photodynamic therapy and sonodynamic therapy are developing, minimally invasive, and site-specific modalities for cancer therapy. A combined strategy PSDT (photodynamic therapy followed by sonodynamic therapy) has been proposed in this study. Here, we aimed to develop novel biodegradable poly(DL-lactide- co -glycolic acid) phase-transition nanoparticles simultaneously loaded with oxygen and indocyanine green (OI-NPs) and to investigate the cytotoxic effects and the potential mechanisms of OI-NP-mediated PSDT on MH7A synoviocytes. The OI-NPs were prepared using a modified double emulsion method and the physicochemical properties were determined. The cellular uptake of OI-NPs was detected by confocal microscopy and flow cytometry. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide assay, flow cytometry, and Hoechst 33342/propidium iodide double staining were used to determine the cytotoxic effect of OI-NP-mediated PSDT on MH7A cells. Fluorescence microscope and fluorescence microplate reader were used to detect reactive oxygen species (ROS) generation. The OI-NPs were a stable and efficient carrier to deliver oxygen and indocyanine green, and enhanced cellular uptake was observed in MH7A cells with the nanoparticles. OI-NP-mediated PSDT caused more serious cell damage and more evident cell apoptosis, compared with other groups. Furthermore, increased generation of intracellular ROS was detected in MH7A cells treated with PSDT. Interestingly, the OI-NP-mediated PSDT-induced cell viability loss was effectively rescued by pretreatment with the ROS scavenger N -acetylcysteine. Multifunctional OI-NPs were successfully developed and characterized for the combined delivery of oxygen and indocyanine green, and OI-NP-mediated PSDT would be a potential cytotoxic treatment for MH7A cells. This study may provide a novel strategy for the treatment of RA and develop a model of theranostic application through phase-transition nanoparticle-mediated PSDT in the future.

Oxygen and indocyanine green loaded phase-transition nanoparticle-mediated photo-sonodynamic cytotoxic effects on rheumatoid arthritis fibroblast-like synoviocytes

PubMed Central

Tang, Qin; Cui, Jianyu; Tian, Zhonghua; Sun, Jiangchuan; Wang, Zhigang; Chang, Shufang; Zhu, Shenyin

2017-01-01

Background Photodynamic therapy and sonodynamic therapy are developing, minimally invasive, and site-specific modalities for cancer therapy. A combined strategy PSDT (photodynamic therapy followed by sonodynamic therapy) has been proposed in this study. Here, we aimed to develop novel biodegradable poly(DL-lactide-co-glycolic acid) phase-transition nanoparticles simultaneously loaded with oxygen and indocyanine green (OI-NPs) and to investigate the cytotoxic effects and the potential mechanisms of OI-NP–mediated PSDT on MH7A synoviocytes. Methods The OI-NPs were prepared using a modified double emulsion method and the physicochemical properties were determined. The cellular uptake of OI-NPs was detected by confocal microscopy and flow cytometry. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide assay, flow cytometry, and Hoechst 33342/propidium iodide double staining were used to determine the cytotoxic effect of OI-NP–mediated PSDT on MH7A cells. Fluorescence microscope and fluorescence microplate reader were used to detect reactive oxygen species (ROS) generation. Results The OI-NPs were a stable and efficient carrier to deliver oxygen and indocyanine green, and enhanced cellular uptake was observed in MH7A cells with the nanoparticles. OI-NP–mediated PSDT caused more serious cell damage and more evident cell apoptosis, compared with other groups. Furthermore, increased generation of intracellular ROS was detected in MH7A cells treated with PSDT. Interestingly, the OI-NP–mediated PSDT-induced cell viability loss was effectively rescued by pretreatment with the ROS scavenger N-acetylcysteine. Conclusion Multifunctional OI-NPs were successfully developed and characterized for the combined delivery of oxygen and indocyanine green, and OI-NP–mediated PSDT would be a potential cytotoxic treatment for MH7A cells. This study may provide a novel strategy for the treatment of RA and develop a model of theranostic application through phase

Glycovariant anti-CD37 monospecific protein therapeutic exhibits enhanced effector cell-mediated cytotoxicity against chronic and acute B cell malignancies

PubMed Central

Rafiq, Sarwish; Siadak, Anthony; Butchar, Jonathan P.; Cheney, Carolyn; Lozanski, Gerard; Jacob, Naduparambil K.; Lapalombella, Rosa; McGourty, Jackie; Moledor, Meghan; Lowe, Richard; Setter, Ben; Jones, Jeffrey; Flynn, Joseph M.; Andritsos, Leslie; Devine, Steven; Mo, Xiaokui; Jarjoura, David; Tridandapani, Susheela; Algate, Paul; Byrd, John C.; Muthusamy, Natarajan

2013-01-01

TRU-016 is a SMIPTM (monospecific protein therapeutic) molecule against the tetraspanin transmembrane family protein CD37 that is currently in Phase 2 trials in Chronic Lymphocytic Leukemia (CLL) and Non-Hodgkin Lymphoma (NHL). In an attempt to enhance the ADCC function of SMIP-016, the chimeric version of TRU-016, SMIP-016GV was engineered with a modification in a glycosylation site in the Fc domain. The wild-type and glycovariant SMIP proteins mediate comparable Type I antibody-like direct cytotoxicity in the presence of anti-human Fc crosslinker and show a similar tyrosine phosphorylation pattern post-treatment. However, NK cells stimulated with the SMIP-016GV exhibit enhanced activation and release 3-fold more interferon-γ compared with SMIP-016. SMIP-016GV shows enhanced ADCC function against cells expressing CD37 with NK cell effectors derived from both normal and CLL-affected individuals. Enhanced ADCC is observed against CLL cells and is sustained at concentrations of SMIP-016GV as low at 5E−6 µg/mL on cells expressing minimal CD37 antigen. In support of the biological relevance of this, SMIP-016GV mediates effective ADCC against primary acute lymphoblastic leukemia (ALL) cells with low surface expression of CD37. Collectively, these data suggest potential use of the novel therapeutic agent SMIP-016GV with enhanced effector function for B cell malignancies, including CLL and ALL therapy. PMID:23883821

Phosphatase inhibition augments anti-CD22-mediated signaling and cytotoxicity in non-hodgkin's lymphoma cells.

PubMed

O'Donnell, Robert T; Pearson, David; McKnight, Hayes C; Ma, Ya Peng; Tuscano, Joseph M

2009-07-01

CD22 is a cell-surface molecule found on most B-cell lymphomas (NHL). HB22.7 is an anti-CD22 antibody that blocks CD22 ligand binding, initiates signaling, and kills NHL cells. The SHP-1 tyrosine phosphatase is disproportionately associated with the cytoplasmic domain of CD22. Sodium orthovanadate (NaV) and dephostatin (DP) are phosphatase inhibitors. The interaction of SHP-1 with CD22 presents an opportunity to manipulate CD22-mediated signaling effects. NaV caused dose dependent killing of NHL cells in vitro; when HB22.7 was given with NaV, antibody-mediated cell death increased. NaV caused a substantial increase in CD22-mediated SAPK and ERK-1/2 activation when CD22 was crosslinked by HB22.7; NaV did not significantly affect IgM-mediated signals. Studies using Raji NHL cells stably transfected with a SHP-1 dominant negative (DN) confirmed that these observations were due to SHP-1 inhibition. The relatively specific association of SHP-1 with CD22 suggests that CD22-specific signaling may be altered by phosphatase inhibition in ways that could prove useful for anti-CD22-based immunotherapy.

A comparative assessment of cytotoxicity of commonly used agricultural insecticides to human and insect cells.

PubMed

Yun, Xinming; Huang, Qingchun; Rao, Wenbing; Xiao, Ciying; Zhang, Tao; Mao, Zhifan; Wan, Ziyi

2017-03-01

The cytotoxic potential of 13 commonly used agricultural insecticides was examined using cell-based systems with three human HepG2, Hek293, HeLa cells and three insect Tn5B1-4, Sf-21, and Drosophila S2 cells. Data showed that (1) an enhancement of some insecticides (e.g. pyrethroids) on cells proliferation; (2) an inhibition of some insecticides on cells viability; (3) various levels of susceptibility of different cells to the same insecticide; and (4) the cell type dependent sensitivity to different insecticides. The degree of cytotoxicity of insecticides on human cells was significantly lower than that on insect cells (P<0.05). Methomyl, even 20μg/ml, showed little cytotoxicity at 24h exposure whereas emamectin benzoate possessed the strongest cytotoxic potential in a dose-dependent fashion. The results revealed comparable cytotoxic property of agricultural insecticides against intact cells. Copyright © 2016 Elsevier Inc. All rights reserved.

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

PubMed

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 protein corona-graphene oxide nanoribbons on human epithelial cells

NASA Astrophysics Data System (ADS)

Mbeh, Doris A.; Akhavan, Omid; Javanbakht, Taraneh; Mahmoudi, Morteza; Yahia, L.'Hocine

2014-11-01

Graphene oxide nanoribbons (GONRs) were synthesized using an oxidative unzipping of multi-walled carbon nanotubes. The interactions of the GONRs with various concentrations of fetal bovine serum or human plasma serum indicated that the GONRs were functionalized substantially by the albumin originated from the two different protein sources. Then, concentration-dependent cytotoxicity of the protein-functionalized GONRs on human epithelial cells was studied. Although the GONRs with concentrations ≤50 μg/mL did not exhibit significant cytotoxicity on the cells (with the cell viability >85%), the concentration of 100 μg/mL exhibited significant cytotoxicity including prevention of cell proliferation and induction of cell apoptosis. These results can provide more in-depth understanding about cytotoxic effects of graphene nanostructures which can be functionalized by the proteins of media.

Interleukin-17A Promotes CD8+ T Cell Cytotoxicity To Facilitate West Nile Virus Clearance.

PubMed

Acharya, Dhiraj; Wang, Penghua; Paul, Amber M; Dai, Jianfeng; Gate, David; Lowery, Jordan E; Stokic, Dobrivoje S; Leis, A Arturo; Flavell, Richard A; Town, Terrence; Fikrig, Erol; Bai, Fengwei

2017-01-01

CD8 + T cells are crucial components of immunity and play a vital role in recovery from West Nile virus (WNV) infection. Here, we identify a previously unrecognized function of interleukin-17A (IL-17A) in inducing cytotoxic-mediator gene expression and promoting CD8 + T cell cytotoxicity against WNV infection in mice. We find that IL-17A-deficient (Il17a -/- ) mice are more susceptible to WNV infection and develop a higher viral burden than wild-type (WT) mice. Interestingly, the CD8 + T cells isolated from Il17a -/- mice are less cytotoxic and express lower levels of cytotoxic-mediator genes, which can be restored by supplying recombinant IL-17A in vitro and in vivo Importantly, treatment of WNV-infected mice with recombinant IL-17A, as late as day 6 postinfection, significantly reduces the viral burden and increases survival, suggesting a therapeutic potential for IL-17A. In conclusion, we report a novel function of IL-17A in promoting CD8 + T cell cytotoxicity, which may have broad implications in other microbial infections and cancers. Interleukin-17A (IL-17A) and CD8 + T cells regulate diverse immune functions in microbial infections, malignancies, and autoimmune diseases. IL-17A is a proinflammatory cytokine produced by diverse cell types, while CD8 + T cells (known as cytotoxic T cells) are major cells that provide immunity against intracellular pathogens. Previous studies have demonstrated a crucial role of CD8 + T cells in recovery from West Nile virus (WNV) infection. However, the role of IL-17A during WNV infection remains unclear. Here, we demonstrate that IL-17A protects mice from lethal WNV infection by promoting CD8 + T cell-mediated clearance of WNV. In addition, treatment of WNV-infected mice with recombinant IL-17A reduces the viral burden and increases survival of mice, suggesting a potential therapeutic. This novel IL-17A-CD8 + T cell axis may also have broad implications for immunity to other microbial infections and cancers, where CD8 + T cell

Piperine impairs cell cycle progression and causes reactive oxygen species-dependent apoptosis in rectal cancer cells.

PubMed

Yaffe, Paul B; Doucette, Carolyn D; Walsh, Mark; Hoskin, David W

2013-02-01

Piperine, an alkaloid phytochemical found in the fruit of black and long pepper plants, is reported to inhibit the growth of cancer cells; however, the mechanism of action in human cancer cells is not clear. In this study we investigated the effect of piperine on the growth of HRT-18 human rectal adenocarcinoma cells. MTT assays showed that piperine inhibited the metabolic activity of HRT-18 cells in a dose- and time-dependent fashion, suggesting a cytostatic and/or cytotoxic effect. Flow cytometric analysis of Oregon Green 488-stained and propidium iodide-stained HRT-18 cells showed that piperine inhibited cell cycle progression. Piperine also caused HRT-18 cells to die by apoptosis, as determined by Annexin-V-FLUOS staining and characteristic changes in cell morphology. Flow cytometric analysis of dihydroethidium- and 2',7'-dichlorofluorescein diacetate-stained HRT-18 cells showed increased production of reactive oxygen species in piperine-treated cells. Furthermore, the antioxidant N-acetylcysteine reduced apoptosis in cultures of piperine-treated HRT-18 cells, indicating that piperine-induced cytotoxicity was mediated at least in part by reactive oxygen species. The cytostatic and cytotoxic effects of piperine on rectal cancer cells suggest that this dietary phytochemical may be useful in cancer treatment. Copyright © 2012 Elsevier Inc. All rights reserved.

ChLpMab-23: Cancer-Specific Human-Mouse Chimeric Anti-Podoplanin Antibody Exhibits Antitumor Activity via Antibody-Dependent Cellular Cytotoxicity.

PubMed

Kaneko, Mika K; Nakamura, Takuro; Kunita, Akiko; Fukayama, Masashi; Abe, Shinji; Nishioka, Yasuhiko; Yamada, Shinji; Yanaka, Miyuki; Saidoh, Noriko; Yoshida, Kanae; Fujii, Yuki; Ogasawara, Satoshi; Kato, Yukinari

2017-06-01

Podoplanin is expressed in many cancers, including oral cancers and brain tumors. The interaction between podoplanin and its receptor C-type lectin-like receptor 2 (CLEC-2) has been reported to be involved in cancer metastasis and tumor malignancy. We previously established many monoclonal antibodies (mAbs) against human podoplanin using the cancer-specific mAb (CasMab) technology. LpMab-23 (IgG 1 , kappa), one of the mouse anti-podoplanin mAbs, was shown to be a CasMab. However, we have not shown the usefulness of LpMab-23 for antibody therapy against podoplanin-expressing cancers. In this study, we first determined the minimum epitope of LpMab-23 and revealed that Gly54-Leu64 peptide, especially Gly54, Thr55, Ser56, Glu57, Asp58, Arg59, Tyr60, and Leu64 of podoplanin, is a critical epitope of LpMab-23. We further produced human-mouse chimeric LpMab-23 (chLpMab-23) and investigated whether chLpMab-23 exerts antibody-dependent cellular cytotoxicity (ADCC) and antitumor activity. In flow cytometry, chLpMab-23 showed high sensitivity against a podoplanin-expressing glioblastoma cell line, LN319, and an oral cancer cell line, HSC-2. chLpMab-23 also showed ADCC activity against podoplanin-expressing CHO cells (CHO/podoplanin). In xenograft models with HSC-2 and CHO/podoplanin, chLpMab-23 exerts antitumor activity using human natural killer cells, indicating that chLpMab-23 could be useful for antibody therapy against podoplanin-expressing cancers.

Propofol inhibits gap junctions by attenuating sevoflurane-induced cytotoxicity against rat liver cells in vitro.

PubMed

Huang, Fei; Li, Shangrong; Gan, Xiaoliang; Wang, Ren; Chen, Zhonggang

2014-04-01

Liver abnormalities are seen in a small proportion of patients following anaesthesia with sevoflurane. To investigate whether the cytotoxicity of sevoflurane against rat liver cells was mediated by gap junction intercellular communications, and the effect of propofol on sevoflurane-induced cytotoxicity. Experimental study. The study was carried out in the central laboratory of The Third Affiliated Hospital, Sun Yat-sen University. BRL-3A rat liver cells. Immortal rat liver cells BRL-3A were grown at low and high density. Colony-forming assays were performed to determine clonogenic growth of these cells. To investigate the effect of oleamide and propofol on gap junction function, we measured fluorescence transmission between cells using parachute dye-coupling assays. Immunoblotting assays were performed to determine connexin32 and connexin43 expression. Our colony formation assays revealed that, in low-density culture, sevoflurane caused no apparent inhibition of clonogenic growth of BRL-3A cells. In high-density culture, 2.2 to 4.4% sevoflurane markedly inhibited clonogenic growth of BRL-3A cells with 67.6 (0.34)% and 61.2 (0.17)% of the cells being viable, respectively (P = 0.003 vs. low-density culture), suggesting cell density dependency of sevoflurane-induced cytotoxicity. Our colony formation assays revealed that propofol markedly attenuated the suppression by sevoflurane of the clonogenic growth of BRL-3A cells (viability: propofol and sevoflurane, 91.5 (0.014)% vs. sevoflurane, 56.6 (0.019)%; P <0.01). Blocking gap junctions with 10 μmol l oleamide significantly attenuated 4.4% sevoflurane-induced suppression with a viability of 83.6 ± 0.138% (oleamide and sevoflurane vs. sevoflurane, P < 0.01). Immunoblotting assays further showed that propofol (3.2 μg ml) markedly reduced CX32 levels and significantly inhibited gap junctional intercellular communications as revealed by parachute dye-coupling assays. Values are mean (SD). This study

Shape-dependent antibacterial effects of non-cytotoxic gold nanoparticles

PubMed Central

Penders, Jelle; Stolzoff, Michelle; Hickey, Daniel J; Andersson, Martin; Webster, Thomas J

2017-01-01

Gold nanoparticles (AuNPs) of various shapes (including spheres, stars and flowers), with similar dimensions, were synthesized and evaluated for their antibacterial effects toward Staphylococcus aureus, a bacterium responsible for numerous life-threatening infections worldwide. Optical growth curve measurements and Gompertz modeling showed significant AuNP shape- and concentration-dependent decreases in bacterial growth with increases in bacterial growth lag time. To evaluate prospective use in in vivo systems, the cytotoxicity of the same AuNPs was evaluated toward human dermal fibroblasts in vitro by 3-(4,5 dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) viability assays and confocal microscopy. No indication of any mammalian cell toxicity or morphological effects was found. Additionally, it was observed that the AuNPs were readily internalized in fibroblasts after 4 days of incubation. Most importantly, the results of the present study showed that gold nanoflowers in particular possessed the most promising non-cytotoxic mammalian cell behavior with the greatest shape-dependent antibacterial activity-promising properties for their future investigation in a wide range of anti-infection applications. PMID:28408817

Targeting MET kinase with the small-molecule inhibitor amuvatinib induces cytotoxicity in primary myeloma cells and cell lines

PubMed Central

2013-01-01

Background MET is a receptor tyrosine kinase that is activated by the ligand HGF and this pathway promotes cell survival, migration, and motility. In accordance with its oncogenic role, MET is constitutively active, mutated, or over-expressed in many cancers. Corollary to its impact, inhibition of MET kinase activity causes reduction of the downstream signaling and demise of cells. In myeloma, a B-cell plasma malignancy, MET is neither mutated nor over-expressed, however, HGF is increased in plasma or serum obtained from myeloma patients and this was associated with poor prognosis. The small-molecule, amuvatinib, inhibits MET receptor tyrosine kinase. Based on this background, we hypothesized that targeting the HGF/MET signaling pathway is a rational approach to myeloma therapy and that myeloma cells would be sensitive to amuvatinib. Methods Expression of MET and HGF mRNAs in normal versus malignant plasma cells was compared during disease progression. Cell death and growth as well as MET signaling pathway were assessed in amuvatinib treated primary myeloma cells and cell lines. Results There was a progressive increase in the transcript levels of HGF (but not MET) from normal plasma cells to refractory malignant plasma cells. Amuvatinib readily inhibited MET phosphorylation in primary CD138+ cells from myeloma patients and in concordance, increased cell death. A 48-hr amuvatinib treatment in high HGF-expressing myeloma cell line, U266, resulted in growth inhibition. Levels of cytotoxicity were time-dependent; at 24, 48, and 72 h, amuvatinib (25 μM) resulted in 28%, 40%, and 55% cell death. Consistent with these data, there was an amuvatinib-mediated decrease in MET phosphorylation in the cell line. Amuvatinib at concentrations of 5, 10, or 25 μM readily inhibited HGF-dependent MET, AKT, ERK and GSK-3-beta phosphorylation. MET-mediated effects were not observed in myeloma cell line that has low MET and/or HGF expression. Conclusions These data suggest that at

EGFR-targeted granzyme B expressed in NK cells enhances natural cytotoxicity and mediates specific killing of tumor cells.

PubMed

Oberoi, Pranav; Jabulowsky, Robert A; Bähr-Mahmud, Hayat; Wels, Winfried S

2013-01-01

Natural killer (NK) cells are highly specialized effectors of the innate immune system that hold promise for adoptive cancer immunotherapy. Their cell killing activity is primarily mediated by the pro-apoptotic serine protease granzyme B (GrB), which enters targets cells with the help of the pore-forming protein perforin. We investigated expression of a chimeric GrB fusion protein in NK cells as a means to augment their antitumoral activity. For selective targeting to tumor cells, we fused the epidermal growth factor receptor (EGFR) peptide ligand transforming growth factor α (TGFα) to human pre-pro-GrB. Established human NKL natural killer cells transduced with a lentiviral vector expressed this GrB-TGFα (GrB-T) molecule in amounts comparable to endogenous wildtype GrB. Activation of the genetically modified NK cells by cognate target cells resulted in the release of GrB-T together with endogenous granzymes and perforin, which augmented the effector cells' natural cytotoxicity against NK-sensitive tumor cells. Likewise, GrB-T was released into the extracellular space upon induction of degranulation with PMA and ionomycin. Secreted GrB-T fusion protein displayed specific binding to EGFR-overexpressing tumor cells, enzymatic activity, and selective target cell killing in the presence of an endosomolytic activity. Our data demonstrate that ectopic expression of a targeted GrB fusion protein in NK cells is feasible and can enhance antitumoral activity of the effector cells.

Spontaneous and natural cytotoxicity receptor-mediated cytotoxicity are effector functions of distinct natural killer subsets in hepatitis C virus-infected chimpanzees.

PubMed

Verstrepen, B E; Nieuwenhuis, I G; Mooij, P; Bogers, W M; Boonstra, A; Koopman, G

2016-07-01

In humans, CD16 and CD56 are used to identify functionally distinct natural killer (NK) subsets. Due to ubiquitous CD56 expression, this marker cannot be used to distinguish between NK cell subsets in chimpanzees. Therefore, functional analysis of distinct NK subsets during hepatitis C virus (HCV) infection has never been performed in these animals. In the present study an alternative strategy was used to identify four distinct NK subsets on the basis of the expression of CD16 and CD94. The expression of activating and inhibiting surface receptors showed that these subsets resemble human NK subsets. CD107 expression was used to determine degranulation of the different subsets in naive and HCV-infected chimpanzees. In HCV-infected chimpanzees increased spontaneous cytotoxicity was observed in CD94(high/dim) CD16(pos) and CD94(low) CD16(pos) subsets. By contrast, increased natural cytotoxicity receptor (NCR)- mediated degranulation after NKp30 and NKp44 triggering was demonstrated in the CD94(dim) CD16(neg) subset. Our findings suggest that spontaneous and NCR-mediated cytotoxicity are effector functions of distinct NK subsets in HCV-infected chimpanzees. © 2016 British Society for Immunology.

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

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

Kurayoshi, Kenta; Ozono, Eiko; Iwanaga, Ritsuko

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 (suicidemore » 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

Cytotoxicity of compounds from Xylopia aethiopica towards multi-factorial drug-resistant cancer cells.

PubMed

Kuete, Victor; Sandjo, Louis P; Mbaveng, Armelle T; Zeino, Maen; Efferth, Thomas

2015-12-15

Multidrug resistance (MDR) in cancer represent a major hurdle in chemotherapy. Previously, the methanol extract of the medicinal spice Xylopia aethiopica displayed considerable cytotoxicity against multidrug resistant (MDR) cancer cell lines. The present study was designed to assess the cytotoxicity of compounds, 16α-hydroxy-ent-kauran-19-oic acid (2), 3,4',5-trihydroxy-6″,6″-dimethylpyrano[2,3-g]flavone (3), isotetrandrine (5) and trans-tiliroside (6) derived from the methanol crude extract of Xylopia aethiopica against 9 drug-sensitive and -resistant cancer cell lines. The resazurin reduction assay was used to evaluate the cytotoxicity of these compounds, whilst caspase-Glo assay was used to detect caspase activation. Cell cycle, mitochondrial membrane potential (MMP) and levels of reactive oxygen species (ROS) were all analyzed via flow cytometry. Flavonoid 3 and alkaloid 5 also displayed IC50 values ranging from 2.61 µM (towards leukemia CCRF-CEM cells) to 18.60 µM (towards gliobastoma multiforme U87MG.ΔEGFR cells) and from 1.45 µM (towards HepG2 cells) to 7.28 µM (towards MDA-MB-231-pcDNA cells), respectively. IC50 values ranged from 0.20 µM (against CCRF-CEM cells) to 195.12 µM (against CEM/ADR5000 cells) for doxorubicin. Compound 3 induced apoptosis in leukemia CCRF-CEM cells mediated by the disruption of the MMP, whilst 5 induced apoptosis mediated by ROS production. Compounds 2 and 5 represent potential cytotoxic phytochemicals that deserve more investigations to develop novel antineoplastic drugs against multifactorial drug-resistant cancers. Copyright © 2015 Elsevier GmbH. All rights reserved.

The mechanism of T-cell mediated cytotoxicity. VI. T-cell projections and their role in target cell killing.

PubMed Central

Sanderson, C J; Glauert, A M

1979-01-01

Electron micrographs of material fixed during the first 10 min of a T-cell cytotoxic system showed T-cell projections and T-cell burrowing into target cells. These observations were made possible by using a system with a very high rate of killing. The projections vary in shape and size, and can push deeply into the target cell, distorting organelles in their path, including the nucleus. The projections contain fine fibrillar material, to the exclusion of organelles. They push the target cell membrane in front of them to form pockets approximating to the shape of the projection. Areas of close contact occur between the projections and the target cell membrane, particularly at the leading edges. The likelihood that these projections develop as a result of contact with specific antigen, and are involved in the cytotoxic mechanism is discussed. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 Figure 16 PMID:311336

Metabolite profiles of Stachybotrys isolates from water-damaged buildings and their induction of inflammatory mediators and cytotoxicity in macrophages.

PubMed

Nielsen, Kristian Fog; Huttunen, Kati; Hyvärinen, Anne; Andersen, Birgitte; Jarvis, Bruce B; Hirvonen, Maija-Riitta

2002-01-01

The metabolite profiles of 20 Stachybotrys spp. isolates from Finnish water-damaged buildings were compared with their biological activities. Effects of purified compounds on cytotoxicity and production of inflammatory mediators such as nitric oxide, IL-6 and TNFalpha in murine RAW264.7 macrophage cells were studied. The 11 isolates belonging to the satratoxin-producing chemotype were highly cytotoxic to the macrophages. The isolates inducing inflammatory mediators all belonged to the atranone-producing chemotype, but pure atranones B, and D did not elicit a response in the bioassay. Altogether, cytotoxicity of Stachybotrys sp. isolates appear to be related to satratoxin production whereas the specific component inducing inflammatory responses in atranone-producing isolates remains obscure.

Uremic toxins enhance statin-induced cytotoxicity in differentiated human rhabdomyosarcoma cells.

PubMed

Uchiyama, Hitoshi; Tsujimoto, Masayuki; Shinmoto, Tadakazu; Ogino, Hitomi; Oda, Tomoko; Yoshida, Takuya; Furukubo, Taku; Izumi, Satoshi; Yamakawa, Tomoyuki; Tachiki, Hidehisa; Minegaki, Tetsuya; Nishiguchi, Kohshi

2014-09-03

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.

PubMed

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.

Natural Mineral Particles Are Cytotoxic to Rainbow Trout Gill Epithelial Cells In Vitro

PubMed Central

de Capitani, Christian; Burkhardt-Holm, Patricia; Pietsch, Constanze

2014-01-01

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 species

Size-dependent cytotoxicity of yttrium oxide nanoparticles on primary osteoblasts in vitro

NASA Astrophysics Data System (ADS)

Zhou, Guoqiang; Li, Yunfei; Ma, Yanyan; Liu, Zhu; Cao, Lili; Wang, Da; Liu, Sudan; Xu, Wenshi; Wang, Wenying

2016-05-01

Yttrium oxide nanoparticles are an excellent host material for the rare earth metals and have high luminescence efficiency providing a potential application in photodynamic therapy and biological imaging. In this study, the effects of yttrium oxide nanoparticles with four different sizes were investigated using primary osteoblasts in vitro. The results demonstrated that the cytotoxicity generated by yttrium oxide nanoparticles depended on the particle size, and smaller particles possessed higher toxicological effects. For the purpose to elucidate the relationship between reactive oxygen species generation and cell damage, cytomembrane integrity, intracellular reactive oxygen species level, mitochondrial membrane potential, cell apoptosis rate, and activity of caspase-3 in cells were then measured. Increased reactive oxygen species level was also observed in a size-dependent way. Thus, our data demonstrated that exposure to yttrium oxide nanoparticles resulted in a size-dependent cytotoxicity in cultured primary osteoblasts, and reactive oxygen species generation should be one possible damage pathway for the toxicological effects produced by yttrium oxide particles. The results may provide useful information for more rational applications of yttrium oxide nanoparticles in the future.

Cytotoxicity of β-D-glucose/sucrose-coated silver nanoparticles depends on cell type, nanoparticles concentration and time of incubation

NASA Astrophysics Data System (ADS)

Vergallo, Cristian; Panzarini, Elisa; Carata, Elisabetta; Ahmadi, Meysam; Mariano, Stefania; Tenuzzo, Bernardetta Anna; Dini, Luciana

2016-06-01

The use of silver NanoParticles (AgNPs) in several consumer commercialized products, like food contact materials, medical devices and cosmetics has increased significantly, owing to their antibacterial and antifungal properties. Even though the NPs are widely diffused, due to the great variety in size, coating or shape, controversial data on their possible detrimental health effects still exist. Herein, by performing an easy and fast green method synthesis, we used β-D-glucose/sucrose to stabilize AgNPs and avoid the release of cytotoxic soluble silver ions Ag+ in the culture medium. The cytotoxic effects of these β-D-Glucose/Sucrose-Coated AgNPs (AgNPs-GS) was assessed on two cell culture models, which are human liver HepG2 and human Peripheral Blood Lymphocytes (PBLs) cells. AgNPs-GS, as determined by Transmission Electron Microscopy (TEM) analyses, had an average diameter of 30±5 nm, a spherical shape and were well-dispersed in the freshly-prepared solution. In addition, they were found spectrophotometrically stable throughout the experiment. Cytotoxicity, determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay, was evaluated by using two AgNPs-GS amounts, indicated as highest (10×103 of NPs/cell) and lowest (2×103 NPs/cell) concentration for 6, 12 and 24 h. The highest concentration of AgNPs-GS was significantly cytotoxic for both HepG2 and PBLs cells at all times, when compared with the negative control; conversely, the lowest amount of AgNPs-GS was toxic only for HepG2 cells. A significant increase of Reactive Oxygen Species (ROS) levels, determined by Nitro Blue Tetrazolium (NBT) reduction assay, was observed only in PBLs after treatment with NPs, by reaching maximum levels after the incubation with the lowest amount of NPs for 24 h. Significant morphological changes, depending on NPs/cell amount, characteristic of cell toxicity, like shape, cytoplasm, and nucleus alterations, were observed in lymphocytes and Hep

Asbestos-induced endothelial cell activation and injury. Demonstration of fiber phagocytosis and oxidant-dependent toxicity.

PubMed

Garcia, J G; Gray, L D; Dodson, R F; Callahan, K S

1988-10-01

Vascular endothelial cell injury is important in the development of a variety of chronic interstitial lung disorders. However, the involvement of such injury in the inflammatory response associated with the inhalation of asbestos fibers is unclear and the mechanism of asbestos fiber cytotoxicity remains unknown. In the present study, human umbilical vein endothelial cells were challenged with amosite asbestos and several parameters of cellular function were examined. Electron microscopic examination revealed that endothelial cell exposure to asbestos resulted in active phagocytosis of these particulates. Biochemical evidence of dose-dependent asbestos-mediated endothelial cell activation was indicated by increased metabolism of arachidonic acid. For example, amosite asbestos (500 micrograms/ml) produced a ninefold increase in prostacyclin (PGI2) levels over those levels in non-exposed cells. Incubation of human endothelial cells with asbestos fibers induced specific 51Cr release in both a dose- and time-dependent fashion indicative of cellular injury. Injury induced by amosite asbestos was not significantly attenuated by treatment of the endothelial cell monolayer with either the iron chelator deferoxamine, which prevents hydroxyl radical (.OH) formation, or by the superoxide anion (O2-) scavenger, superoxide dismutase. However, significant dose-dependent protection was observed with the hydrogen peroxide (H2O2) scavenger, catalase. Chelation of elemental iron present within amosite asbestos fibers by deferoxamine produced a 33% reduction in asbestos cytotoxicity, suggesting a potential role for hydroxyl radical-mediated injury via the iron-catalyzed Haber-Weiss reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Activin A, B and AB decrease progesterone production by down-regulating StAR in human granulosa cells.

PubMed

Chang, Hsun-Ming; Cheng, Jung-Chien; Huang, He-Feng; Shi, Feng-Tao; Leung, Peter C K

2015-09-05

Activins are homo- or heterodimers of inhibin β subunits that play important roles in the reproductive system. Our previous work has shown that activins A (βAβA), B (βBβB) and AB (βAβB) induce aromatase/estradiol, but suppress StAR/progesterone production in human granulosa-lutein cells. However, the underlying molecular determinants of these effects have not been examined. In this continuing study, we used immortalized human granulosa cells (SVOG) to investigate the effects of activins in regulating StAR/progesterone and the potential mechanisms of action. In SVOG cells, activins A, B and AB produced comparable down-regulation of StAR expression and progesterone production. In addition, all three activin isoforms induced equivalent phosphorylation of both SMAD2 and SMAD3. Importantly, the activin-induced down-regulation of StAR, increase in SMAD2/3 phosphorylation, and decrease in progesterone were abolished by the TGF-β type I receptor inhibitor SB431542. Interestingly, the small interfering RNA-mediated knockdown of ALK4 but not ALK5 reversed the activin-induced suppression of StAR. Furthermore, the knockdown of SMAD4 or SMAD2 but not SMAD3 abolished the inhibitory effects of all three activin isoforms on StAR expression. These results provide evidence that activins A, B and AB down-regulate StAR expression and decrease progesterone production in human granulosa cells, likely via an ALK4-mediated SMAD2/SMAD4-dependent pathway. Our findings provide important insights into the molecular mechanisms underlying the regulatory effects of activins on human granulosa cell steroidogenesis. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

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

PubMed

Winkler, Mark T; Bushey, Ryan T; Gottlin, Elizabeth B; Campa, Michael J; Guadalupe, Eross S; Volkheimer, Alicia D; Weinberg, J Brice; Patz, Edward F

2017-01-01

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.

The hypertension drug, verapamil, activates Nrf2 by promoting p62-dependent autophagic Keap1 degradation and prevents acetaminophen-induced cytotoxicity.

PubMed

Lee, Da Hyun; Park, Jeong Su; Lee, Yu Seol; Sung, Su Haeng; Lee, Yong-Ho; Bae, Soo Han

2017-02-01

Nuclear factor erythroid 2-related factor 2 (Nrf2) provides a cellular defense against oxidative stress by inducing the expression of antioxidant and detoxification enzymes. The calcium antagonist, verapamil, is an FDA-approved drug prescribed for the treatment of hypertension. Here, we show that verapamil acts as a potent Nrf2 activator without causing cytotoxicity, through degradation of Kelch-like ECH-associated protein 1 (Keap1), a Nrf2 repressor. Furthermore, verapamilinduced Keap1 degradation is prominently mediated by a p62-dependent autophagic pathway. Correspondingly, verapamil protects cells from acetaminophen-induced oxidative damage through Nrf2 activation. These results demonstrated the underlying mechanisms for the protective role of verapamil against acetaminophen-induced cytotoxicity. [BMB Reports 2017; 50(2): 91-96].

Zoledronic acid renders human M1 and M2 macrophages susceptible to Vδ2+ γδ T cell cytotoxicity in a perforin-dependent manner.

PubMed

Fowler, Daniel W; Copier, John; Dalgleish, Angus G; Bodman-Smith, Mark D

2017-09-01

Vδ2 + T cells are a subpopulation of γδ T cells in humans that are cytotoxic towards cells which accumulate isopentenyl pyrophosphate. The nitrogen-containing bisphosphonate, zoledronic acid (ZA), can induce tumour cell lines to accumulate isopentenyl pyrophosphate, thus rendering them more susceptible to Vδ2 + T cell cytotoxicity. However, little is known about whether ZA renders other, non-malignant cell types susceptible. In this study we focussed on macrophages (Mϕs), as these cells have been shown to take up ZA. We differentiated peripheral blood monocytes from healthy donors into Mϕs and then treated them with IFN-γ or IL-4 to generate M1 and M2 Mϕs, respectively. We characterised these Mϕs based on their phenotype and cytokine production and then tested whether ZA rendered them susceptible to Vδ2 + T cell cytotoxicity. Consistent with the literature, IFN-γ-treated Mϕs expressed higher levels of the M1 markers CD64 and IL-12p70, whereas IL-4-treated Mϕs expressed higher levels of the M2 markers CD206 and chemokine (C-C motif) ligand 18. When treated with ZA, both M1 and M2 Mϕs became susceptible to Vδ2 + T cell cytotoxicity. Vδ2 + T cells expressed perforin and degranulated in response to ZA-treated Mϕs as shown by mobilisation of CD107a and CD107b to the cell surface. Furthermore, cytotoxicity towards ZA-treated Mϕs was sensitive-at least in part-to the perforin inhibitor concanamycin A. These findings suggest that ZA can render M1 and M2 Mϕs susceptible to Vδ2 + T cell cytotoxicity in a perforin-dependent manner, which has important implications regarding the use of ZA in cancer immunotherapy.

An ab initio mechanism for efficient population of triplet states in cytotoxic sulfur substituted DNA bases: the case of 6-thioguanine.

PubMed

Martínez-Fernández, Lara; González, Leticia; Corral, Inés

2012-02-18

The deactivation mechanism of the cytotoxic 6-thioguanine, the 6-sulfur-substituted analogue of the canonical DNA base, is unveiled by ab initio calculations. Oxygen-by-sulfur substitution leads to efficient population of triplet states-the first step for generating singlet oxygen-which is responsible for its cytotoxicity. This journal is © The Royal Society of Chemistry 2012

Sorafenib induces cathepsin B-mediated apoptosis of bladder cancer cells by regulating the Akt/PTEN pathway. The Akt inhibitor, perifosine, enhances the sorafenib-induced cytotoxicity against bladder cancer cells

PubMed Central

Amantini, Consuelo; Morelli, Maria Beatrice; Santoni, Matteo; Soriani, Alessandra; Cardinali, Claudio; Farfariello, Valerio; Eleuteri, Anna Maria; Bonfili, Laura; Mozzicafreddo, Matteo; Nabissi, Massimo; Cascinu, Stefano; Santoni, Giorgio

2015-01-01

Sorafenib, a tyrosine kinase inhibitor, has been demonstrated to exert anti-tumor effects. However, the molecular mechanisms underlying its effects on bladder cancer remain unknown. Here, we evaluated the mechanisms responsible for the sorafenib-induced anti-tumor effects on 5637 and T24 bladder cancer cells. We demonstrated that sorafenib reduces cell viability, stimulates lysosome permeabilization and induces apoptosis of bladder cancer cells. These effects are dependent by the activation of cathepsin B released from lysosomes. The sorafenib-increased cathepsin B activity induced the proteolysis of Bid into tBid that stimulates the intrinsic pathway of apoptosis characterized by mitochondrial membrane depolarization, oxygen radical generation and cytochrome c release. Moreover, we found that cathepsin B enzymatic activity, induced by sorafenib, is dependent on its dephosphorylation via PTEN activation and Akt inactivation. Pretreatment with orthovanadate rescued bladder cancer cells from apoptosis. In addition, the Akt inhibitor perifosine increased the sensitivity of bladder cancer cells to sorafenib-induced cytotoxicity. Overall, our results show that apoptotic cell death induced by sorafenib in bladder cancer cells is dependent on cathepsin B activity and involved PTEN and Akt signaling pathways. The Akt inhibitor perifosine increased the cytotoxic effects of sorafenib in bladder cancer cells. PMID:26097873

A semi high-throughput method for screening small bispecific antibodies with high cytotoxicity.

PubMed

Sugiyama, Aruto; Umetsu, Mitsuo; Nakazawa, Hikaru; Niide, Teppei; Onodera, Tomoko; Hosokawa, Katsuhiro; Hattori, Shuhei; Asano, Ryutaro; Kumagai, Izumi

2017-06-06

Small bispecific antibodies that induce T-cell-mediated cytotoxicity have the potential to damage late-stage tumor masses to a clinically relevant degree, but their cytotoxicity is critically dependent on their structural and functional properties. Here, we constructed an optimized procedure for identifying highly cytotoxic antibodies from a variety of the T-cell-recruiting antibodies engineered from a series of antibodies against cancer antigens of epidermal growth factor receptor family and T-cell receptors. By developing and applying a set of rapid operations for expression vector construction and protein preparation, we screened the cytotoxicity of 104 small antibodies with diabody format and identified some with 10 3 -times higher cytotoxicity than that of previously reported active diabody. The results demonstrate that cytotoxicity is enhanced by synergistic effects between the target, epitope, binding affinity, and the order of heavy-chain and light-chain variable domains. We demonstrate the importance of screening to determine the critical rules for highly cytotoxic antibodies.

Microwave-induced Apoptosis and Cytotoxicity of NK Cells through ERK1/2 Signaling.

PubMed

Zhao, Li; Li, Jing; Hao, Yan Hui; Gao, Ya Bing; Wang, Shui Ming; Zhang, Jing; Dong, Ji; Zhou, Hong Mei; Liu, Shu Chen; Peng, Rui Yun

2017-05-01

To investigate microwave-induced morphological and functional injury of natural killer (NK) cells and uncover their mechanisms. NK-92 cells were exposed to 10, 30, and 50 mW/cm2 microwaves for 5 min. Ultrastructural changes, cellular apoptosis and cell cycle regulation were detected at 1 h and 24 h after exposure. Cytotoxic activity was assayed at 1 h after exposure, while perforin and NKG2D expression were detected at 1 h, 6 h, and 12 h after exposure. To clarify the mechanisms, phosphorylated ERK (p-ERK) was detected at 1 h after exposure. Moreover, microwave-induced cellular apoptosis and cell cycle regulation were analyzed after blockade of ERK signaling by using U0126. Microwave-induced morphological and ultrastructural injury, dose-dependent apoptosis (P < 0.001) and cell cycle arrest (P < 0.001) were detected at 1 h after microwave exposure. Moreover, significant apoptosis was still detected at 24 h after 50 mW/cm2 microwave exposure (P < 0.01). In the 30 mW/cm2 microwave exposure model, microwaves impaired the cytotoxic activity of NK-92 cells at 1 h and down regulated perforin protein both at 1 h and 6 h after exposure (P < 0.05). Furthermore, p-ERK was down regulated at 1 h after exposure (P < 0.05), while ERK blockade significantly promoted microwave-induced apoptosis (P < 0.05) and downregulation of perforin (P < 0.01). Microwave dose-dependently induced morphological and functional injury in NK-92 cells, possibly through ERK-mediated regulation of apoptosis and perforin expression. Copyright © 2017 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

Rapid desensitization of mice with anti-FcγRIIb/FcγRIII mAb safely prevents IgG-mediated anaphylaxis.

PubMed

Khodoun, Marat V; Kucuk, Zeynep Yesim; Strait, Richard T; Krishnamurthy, Durga; Janek, Kevin; Clay, Corey D; Morris, Suzanne C; Finkelman, Fred D

2013-12-01

Stimulatory IgG receptors (FcγRs) on bone marrow-derived cells contribute to the pathogenesis of several autoimmune and inflammatory disorders. Monoclonal antibodies that block FcγRs might suppress these diseases, but they can induce anaphylaxis. We wanted to determine whether a rapid desensitization approach can safely suppress IgG/FcγR-mediated anaphylaxis. Mice were injected with serially increasing doses of 2.4G2, a rat mAb that blocks the inhibitory FcγR, FcγRIIb, and the stimulatory receptor, FcγRIII. Rectal temperature was used to detect the development of anaphylaxis. Passive and active IgG-mediated anaphylaxis were evaluated in mice that had been rapidly desensitized with 2.4G2 or mock-desensitized in mice in which monocyte/macrophages, basophils, or neutrophils had been depleted or desensitized and in mice in which FcγRI, FcγRIII, and/or FcγRIV had been deleted or blocked. Rapid desensitization with 2.4G2 prevented 2.4G2-induced shock and completely suppressed IgG-mediated anaphylaxis. Rapid desensitization of ovalbumin-sensitized mice with 2.4G2 was safer and more effective than rapid desensitization with ovalbumin. 2.4G2 treatment completely blocked FcγRIII and removed most FcγRI and FcγRIV from nucleated peripheral blood cells. Because IgG(2a)-mediated anaphylaxis was partially FcγRI and FcγRIV dependent, the effects of 2.4G2 on FcγRI and FcγRIV were probably crucial for its complete inhibition of IgG(2a)-mediated anaphylaxis. IgG(2a)-mediated anaphylaxis was partially inhibited by depletion or desensitization of monocyte/macrophages, basophils, or neutrophils. IgG-mediated anaphylaxis can be induced by ligation of FcγRI, FcγRIII, or FcγRIV on monocycte/macrophages, basophils, or neutrophils and can be safely suppressed by rapid desensitization with anti-FcγRII/RIII mAb. A similar approach may safely suppress other FcγR-dependent immunopathology. Published by Mosby, Inc.

Mechanisms of Dihydroartemisinin and Dihydroartemisinin/Holotransferrin Cytotoxicity in T-Cell Lymphoma Cells

PubMed Central

Zhao, Xindong; Zhao, Chunting; Zhao, Hongguo; Huo, Lanfen

2015-01-01

The validated therapeutic effects of dihydroartemisinin (DHA) in solid tumors have encouraged us to explore its potential in treating T-cell lymphoma. We found that Jurkat cells (a T-cell lyphoma cell line) were sensitive to DHA treatment with a IC50 of dihydroartemisinin. The cytotoxic effect of DHA in Jurkat cells showed a dose- and time- dependent manner. Interestingly, the cytotoxic effect of DHA was further enhanced by holotransferrin (HTF) due to the high expression of transferrin receptors in T-cell lymphoma. Mechanistically, DHA significantly increased the production of intracellular reactive oxygen species, which led to cell cycle arrest and apoptosis. The DHA treatment also inhibited the expression of protumorgenic factors including VEGF and telomerase catalytic subunit. Our results have proved the therapeutic effect of DHA in T-cell lymphoma. Especially in combination with HTF, DHA may provide a novel efficient approach in combating the deadly disease. PMID:26502166

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

PubMed Central

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

Nuclear DAMP complex-mediated RAGE-dependent macrophage cell death

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

Chen, Ruochan; Department of Infectious Diseases and State Key Lab of Viral Hepatitis, Xiangya Hospital, Central South University, Changsha, Hunan 410008; Fu, Sha

High mobility group box 1 (HMGB1), histone, and DNA are essential nuclear components involved in the regulation of chromosome structure and function. In addition to their nuclear function, these molecules act as damage-associated molecular patterns (DAMPs) alone or together when released extracellularly. The synergistic effect of these nuclear DNA-HMGB1-histone complexes as DAMP complexes (nDCs) on immune cells remains largely unexplored. Here, we demonstrate that nDCs limit survival of macrophages (e.g., RAW264.7 and peritoneal macrophages) but not cancer cells (e.g., HCT116, HepG2 and Hepa1-6). nDCs promote production of inflammatory tumor necrosis factor α (TNFα) release, triggering reactive oxygen species-dependent apoptosis andmore » necrosis. Moreover, the receptor for advanced glycation end products (RAGE), but not toll-like receptor (TLR)-4 and TLR-2, was required for Akt-dependent TNFα release and subsequent cell death following treatment with nDCs. Genetic depletion of RAGE by RNAi, antioxidant N-Acetyl-L-cysteine, and TNFα neutralizing antibody significantly attenuated nDC-induced cell death. These findings provide evidence supporting novel signaling mechanisms linking nDCs and inflammation in macrophage cell death. - Highlights: • Nuclear DAMP complexes (nDCs) selectively induce cell death in macrophages, but not cancer cells. • TNFα-mediated oxidative stress is required for nDC-induced death. • RAGE-mediated Akt activation is required for nDC-induced TNFα release. • Blocking RAGE and TNFα inhibits nDC-induced macrophage cell death.« less

Enhancement of dendritic cell-based vaccine potency by anti-apoptotic siRNAs targeting key pro-apoptotic proteins in cytotoxic CD8(+) T cell-mediated cell death.

PubMed

Kim, Jin Hee; Kang, Tae Heung; Noh, Kyung Hee; Bae, Hyun Cheol; Kim, Seok-Ho; Yoo, Young Do; Seong, Seung-Yong; Kim, Tae Woo

2009-01-29

Dendritic cells (DCs) have become an important measure for the treatment of malignancies. Current DC preparations, however, generate short-lived DCs because they are subject to cell death from various apoptotic pressures. Antigen-specific CD8(+) cytotoxic T lymphocytes (CTLs) is one of the main obstacles to limit the DC-mediated immune priming since CTLs can recognize the target antigen expressing DCs as target cells and kill the DCs. CTLs secret perforin and serine protease granzymes during CTL killing. Perforin and serine protease granzymes induce the release of a number of mitochondrial pro-apoptotic factors, which are controlled by members of the BCL-2 family, such as BAK, BAX and BIM. FasL linking to Fas on DCs triggers the activation of caspase-8, which eventually leads to mitochondria-mediated apoptosis via truncation of BID. In this study, we tried to enhance the DC priming capacity by prolonging DC survival using anti-apoptotic siRNA targeting these key pro-apoptotic molecules in CTL killing. Human papillomavirus (HPV)-16 E7 antigen presenting DCs that were transfected with these anti-apoptotic siRNAs showed increased resistance to T cell-mediated death, leading to enhanced E7-specific CD8(+) T cell activation in vitro and in vivo. Among them, siRNA targeting BIM (siBIM) generated strongest E7-specific E7-specific CD8(+) T cell immunity. More importantly, vaccination with E7 presenting DCs transfected with siBIM was capable of generating a marked therapeutic effect in vaccinated mice. Our data indicate that ex vivo manipulation of DCs with siBIM may represent a plausible strategy for enhancing dendritic cell-based vaccine potency.

Sticholysin II-mediated cytotoxicity involves the activation of regulated intracellular responses that anticipates cell death.

PubMed

Soto, Carmen; Bergado, Gretchen; Blanco, Rancés; Griñán, Tania; Rodríguez, Hermis; Ros, Uris; Pazos, Fabiola; Lanio, María Eliana; Hernández, Ana María; Álvarez, Carlos

2018-05-01

Sticholysin II (StII) is a pore-forming toxin of biomedical interest that belongs to the actinoporin protein family. Sticholysins are currently under examination as an active immunomodulating component of a vaccinal platform against tumoral cells and as a key element of a nucleic acids delivery system to cell cytosol. These proteins form pores in the plasma membrane leading to ion imbalance and cell lysis. However, the intracellular mechanisms triggered by actinoporins upon binding to membranes and its consequences for cell death are barely understood. Here, we have examined the cytotoxicity and intracellular responses induced by StII upon binding to human B-cell lymphoma Raji in vitro. StII cytotoxicity involves a functional actin cytoskeleton, induces cellular swelling, lysis and the concomitant release of cytosol content. In addition, StII induces calcium release mainly from the Endoplasmic Reticulum, activates Mitogen-Activated Protein Kinase ERK and impairs mitochondrial membrane potential. Furthermore, StII stimulates the expression of receptor interacting protein kinase 1 (RIP1), normally related to different forms of regulated cell death such as apoptosis and necroptosis. In correspondence, necrostatin-1, an inhibitor of this kinase, reduces StII cytotoxicity. However, the mechanism of cell death activated by StII does not involve caspases activation, typical molecular features of apoptosis and pyroptosis. Our results suggest that, beyond pore-formation and cell lysis, StII-induced cytotoxicity could involve other regulated intracellular mechanisms connected to RIP1-MEK1/2 -ERK1/2- pathways. This opens new perspectives and challenges the general point of view that these toxins induce a completely unregulated mechanism of necrotic cell death. This study contributes to a better understanding of the molecular mechanisms involved in toxin-cell interaction and the implications for cell functioning, with connotation for the exploitations of these toxins in

VEGFR2-targeted fusion antibody improved NK cell-mediated immunosurveillance against K562 cells.

PubMed

Ren, Xueyan; Xie, Wei; Wang, Youfu; Xu, Menghuai; Liu, Fang; Tang, Mingying; Li, Chenchen; Wang, Min; Zhang, Juan

2016-08-01

MHC class I polypeptide-related sequence A (MICA), which is normally expressed on cancer cells, activates NK cells via NK group 2-member D pathway. However, some cancer cells escape NK-mediated immune surveillance by shedding membrane MICA causing immune suppression. To address this issue, we designed an antibody-MICA fusion targeting tumor-specific antigen (vascular endothelial growth factor receptor 2, VEGFR2) based on our patented antibody (mAb04) against VEGFR2. In vitro results demonstrate that the fusion antibody retains both the antineoplastic and the immunomodulatory activity of mAb04. Further, we revealed that it enhanced NK-mediated immunosurveillance against K562 cells through increasing degranulation and cytokine production of NK cells. The overall data suggest our new fusion protein provides a promising approach for cancer-targeted immunotherapy and has prospects for potential application of chronic myeloid leukemia.

Revealing strategies of quorum sensing in Azospirillum brasilense strains Ab-V5 and Ab-V6.

PubMed

Fukami, Josiane; Abrantes, Julia Laura Fernandes; Del Cerro, Pablo; Nogueira, Marco Antonio; Ollero, Francisco Javier; Megías, Manuel; Hungria, Mariangela

2018-01-01

Azospirillum brasilense is an important plant-growth promoting bacterium (PGPB) that requires several critical steps for root colonization, including biofilm and exopolysaccharide (EPS) synthesis and cell motility. In several bacteria these mechanisms are mediated by quorum sensing (QS) systems that regulate the expression of specific genes mediated by the autoinducers N-acyl-homoserine lactones (AHLs). We investigated QS mechanisms in strains Ab-V5 and Ab-V6 of A. brasilense, which are broadly used in commercial inoculants in Brazil. Neither of these strains carries a luxI gene, but there are several luxR solos that might perceive AHL molecules. By adding external AHLs we verified that biofilm and EPS production and cell motility (swimming and swarming) were regulated via QS in Ab-V5, but not in Ab-V6. Differences were observed not only between strains, but also in the specificity of LuxR-type receptors to AHL molecules. However, Ab-V6 was outstanding in indole acetic acid (IAA) synthesis and this molecule might mimic AHL signals. We also applied the quorum quenching (QQ) strategy, obtaining transconjugants of Ab-V5 and Ab-V6 carrying a plasmid with acyl-homoserine lactonase. When maize (Zea mays L.) was inoculated with the wild-type and transconjugant strains, plant growth was decreased with the transconjugant of Ab-V5-confirming the importance of an AHL-mediated QS system-but did not affect plant growth promotion by Ab-V6.

Arf-like GTPase Arl8b regulates lytic granule polarization and natural killer cell–mediated cytotoxicity

PubMed Central

Tuli, Amit; Thiery, Jerome; James, Ashley M.; Michelet, Xavier; Sharma, Mahak; Garg, Salil; Sanborn, Keri B.; Orange, Jordan S.; Lieberman, Judy; Brenner, Michael B.

2013-01-01

Natural killer (NK) lymphocytes contain lysosome-related organelles (LROs), known as lytic granules, which upon formation of immune synapse with the target cell, polarize toward the immune synapse to deliver their contents to the target cell membrane. Here, we identify a small GTP-binding protein, ADP-ribosylation factor-like 8b (Arl8b), as a critical factor required for NK cell–mediated cytotoxicity. Our findings indicate that Arl8b drives the polarization of lytic granules and microtubule-organizing centers (MTOCs) toward the immune synapse between effector NK lymphocytes and target cells. Using a glutathione S-transferase pull-down approach, we identify kinesin family member 5B (KIF5B; the heavy chain of kinesin-1) as an interaction partner of Arl8b from NK cell lysates. Previous studies showed that interaction between kinesin-1 and Arl8b is mediated by SifA and kinesin-interacting protein (SKIP) and the tripartite complex drives the anterograde movement of lysosomes. Silencing of both KIF5B and SKIP in NK cells, similar to Arl8b, led to failure of MTOC-lytic granule polarization to the immune synapse, suggesting that Arl8b and kinesin-1 together control this critical step in NK cell cytotoxicity. PMID:24088571

Rapid and preferential distribution of blood-borne αCD3εAb to the liver is followed by local stimulation of T cells and natural killer T cells

PubMed Central

Wingender, Gerhard; Schumak, Beatrix; Schurich, Anna; Gessner, J Engelbert; Endl, Elmar; Limmer, Andreas; Knolle, Percy A

2006-01-01

Dissemination of soluble molecules or antigens via the blood stream is considered to lead to a uniform distribution in the various organs of the body, but organ-specific microarchitecture and vascularization may influence this. Following intravenous injection of αCD3ε antibody (αCD3εAb) we observed clear differences in antibody binding to Fcγ receptor (FcγR)+ antigen-presenting cells (APCs) or T lymphocytes in different organs. Significant binding of blood-borne αCD3εAb was only detected in the spleen and liver and not in the thymus or lymph node. In the spleen, only 10% of dendritic cells/macrophages and 40% of T-cell receptor (TCR)-β+ cells were positive for αCD3εAb, and, dependent on FcγR-mediated cross-linking of αCD3εAb, a similar percentage of splenic TCR-β+ cells were stimulated and became CD69+. Stimulation of TCR-β+ cells in the liver was at least as efficient as in the spleen, but almost all T cells and all scavenger liver sinusoidal endothelial cells bound αCD3εAb. In contrast to CD69 up-regulation, only CD4+ natural killer T (NKT) cells and CD11ahigh CD8+ T cells were activated by αCD3εAb and expressed interferon (IFN)-γ. Again, IFN-γ release from NKT/T cells was at least as efficient in the liver as in the spleen. Taken together, our results support the notion that the combination of extensive hepatic vascularization and very high scavenger activity allows the liver to fulfill its metabolic tasks and to promote stimulation of the large but widely distributed hepatic population of NKT/T cells. PMID:16423047

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

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

Johnson, Adam J.; Brown, Melissa N.; Black, Margaret E., E-mail: blackm@vetmed.wsu.edu

2011-12-09

Highlights: Black-Right-Pointing-Pointer Goal was to enhance dFdC cytotoxicity by the creation of a UCMK/dCK fusion enzyme. Black-Right-Pointing-Pointer The UCMK/dCK fusion enzyme possesses both native activities. Black-Right-Pointing-Pointer The fusion renders cells equally sensitive to dFdC relative to dCK expression alone. Black-Right-Pointing-Pointer Dual activities of fusion not sufficient to augment cell dFdC sensitivity in vitro. Black-Right-Pointing-Pointer Data may warrant the implementation of UCMK mutagenesis studies. -- Abstract: While gemcitabine (2 Prime -2 Prime -difluoro-2 Prime -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 enhancemore » 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.« less

Photocatalytic activity against azo dye and cytotoxicity on MCF-7 cell lines of zirconium oxide nanoparticle mediated using leaves of Lagerstroemia speciosa.

PubMed

Sai Saraswathi, V; Santhakumar, K

2017-04-01

Metal oxide nanoparticles are gaining interest in recent years. The present paper explains about the green synthesis of zirconium oxide nanoparticles (ZrO NPs) mediated from the leaves of Lagerstroemia speciosa. The prepared ZrO NPs were characterized by UV-vis spectroscopy, FT-IR, X-ray diffraction analysis (XRD), Transmission Electron Microscopy (TEM), Scanning Electron Microscopy (SEM), Energy Dispersive X-ray spectroscopy (EDX) and Thermogravimetric Analysis (TGA). The photocatalytic activity of ZrO NPs was studied for azo dye by exposing to sunlight. The azo dye was degraded up to 94.58%. Also the ZrO NPs were studied for in vitro cytotoxicity activity against breast cancer cell lines-MCF-7 and evaluated by MTT assay. The cell morphological changes were recorded by light microscope. The cells viability was seen at 500μg/mL when compared against control. Hence the research highlights, that the method was simple, eco-friendly towards environment by phytoremediation activity of the azo dye and cytotoxicity activity against MCF-7 cell lines. Hence the present paper may help to further explore the metal nanoparticle for its potential applications. Copyright © 2017 Elsevier B.V. All rights reserved.

Enhancement of the p27Kip1-mediated antiproliferative effect of trastuzumab (Herceptin) on HER2-overexpressing tumor cells.

PubMed

Marches, Radu; Uhr, Jonathan W

2004-11-10

The oncogenic activity of the overexpressed HER2 tyrosine kinase receptor requires its localization in the plasma membrane. The antitumor effect of anti-HER2 antibodies (Abs) is mainly dependent on receptor downregulation and comprises p27Kip1-mediated G1 cell cycle arrest. However, one major limitation of anti-HER2 therapy is the reversibility of tumor growth inhibition after discontinuation of treatment caused by the mitogenic signaling associated with cell surface receptor re-expression. We found that the level of p27Kip1 upregulation, inhibition of Cdk2 activity and magnitude of G1 arrest induced by the humanized Ab trastuzumab (Herceptin, HCT) on BT474 and SKBr3 HER2-overexpressing breast cancer cells correlates with the level of cell surface receptor. Thus, continuous exposure of cells to HCT for 72 hr results in downregulation of the cell surface receptor and a concurrent increase in the level of p27Kip1 protein. Discontinuation of Ab exposure after the first 8 hr results in failure to upregulate p27Kip1 and arrest of cell cycle progression. We show that the lysosomotropic amine chloroquine (CQ) augments receptor internalization in HER2-overexpressing cells either pretreated or continuously treated with HCT and leads to an increased and sustained inhibitory effect. The enhanced CQ-dependent loss of functional HER2 from the cell surface resulted in sustained inactivation of the serine/threonine kinase Akt, upregulation of p27Kip1 protein and inhibition of cyclin E/Cdk2 activity. Potentiation of the inhibitory effect of HCT by CQ was directly related to loss of HER2 from the plasma membrane since prevention of Ab-mediated receptor endocytosis by engagement of the receptor with immobilized HCT abrogated the effect of CQ.

Direct and indirect air particle cytotoxicity in human alveolar epithelial cells.

PubMed

Orona, N S; Astort, F; Maglione, G A; Saldiva, P H N; Yakisich, J S; Tasat, D R

2014-08-01

Air particulate matter has been associated with adverse impact on the respiratory system leading to cytotoxic and proinflammatory effects. The biological mechanisms behind these associations may be initiated by inhaled small size particles, particle components (soluble fraction) and/or mediators released by particle-exposed cells (conditioned media). The effect of Urban Air Particles from Buenos Aires (UAP-BA) and Residual Oil Fly Ash (ROFA) a surrogate of ambient air pollution, their Soluble Fractions (SF) and Conditioned Media (CM) on A549 lung epithelial cells was examined. After 24 h exposure to TP (10 and 100 μg/ml), SF or CM, several biological parameters were assayed on cultured A549 cells. We tested cell viability by MTT, superoxide anion (O₂(-)) generation by NBT and proinflammatory cytokine (TNFα, IL-6 and IL-8) production by ELISA. UAP-BA particles or its SF (direct effect) did not modify cell viability and generation of O₂(-) for any of the doses tested. On the contrary, UAP-BA CM (indirect effect) reduced cell viability and increased both generation of O₂(-) and IL-8 production. Exposure to ROFA particles, SF or ROFA CM reduced proliferation and O₂(-) but, stimulated IL-8. It is worth to note that UAP-BA and ROFA depicted distinct effects on particle-exposed A549 cells implicating morphochemical dependence. These in vitro findings support the hypothesis that particle-induced lung inflammation and disease may involve lung-derived mediators. Copyright © 2014 Elsevier Ltd. All rights reserved.

Clathrin-dependent entry and vesicle-mediated exocytosis define insulin transcytosis across microvascular endothelial cells

PubMed Central

Azizi, Paymon M.; Zyla, Roman E.; Guan, Sha; Wang, Changsen; Liu, Jun; Bolz, Steffen-Sebastian; Heit, Bryan; Klip, Amira; Lee, Warren L.

2015-01-01

Transport of insulin across the microvasculature is necessary to reach its target organs (e.g., adipose and muscle tissues) and is rate limiting in insulin action. Morphological evidence suggests that insulin enters endothelial cells of the microvasculature, and studies with large vessel–derived endothelial cells show insulin uptake; however, little is known about the actual transcytosis of insulin and how this occurs in the relevant microvascular endothelial cells. We report an approach to study insulin transcytosis across individual, primary human adipose microvascular endothelial cells (HAMECs), involving insulin uptake followed by vesicle-mediated exocytosis visualized by total internal reflection fluorescence microscopy. In this setting, fluorophore-conjugated insulin exocytosis depended on its initial binding and uptake, which was saturable and much greater than in muscle cells. Unlike its degradation within muscle cells, insulin was stable within HAMECs and escaped lysosomal colocalization. Insulin transcytosis required dynamin but was unaffected by caveolin-1 knockdown or cholesterol depletion. Instead, insulin transcytosis was significantly inhibited by the clathrin-mediated endocytosis inhibitor Pitstop 2 or siRNA-mediated clathrin depletion. Accordingly, insulin internalized for 1 min in HAMECs colocalized with clathrin far more than with caveolin-1. This study constitutes the first evidence of vesicle-mediated insulin transcytosis and highlights that its initial uptake is clathrin dependent and caveolae independent. PMID:25540431

RITA inhibits multiple myeloma cell growth through induction of p53-mediated caspase-dependent apoptosis and synergistically enhances nutlin-induced cytotoxic responses.

PubMed

Saha, Manujendra N; Jiang, Hua; Mukai, Asuka; Chang, Hong

2010-11-01

Mutations or deletions of p53 are relatively rare in multiple myeloma (MM), at least in newly diagnosed patients. Thus, restoration of p53 tumor suppressor function in MM by blocking the inhibitory role of murine double minute 2 (MDM2) is a promising and applicable therapeutic strategy. RITA and nutlin are two new classes of small molecule MDM2 inhibitors that prevent the p53-MDM2 interaction. Earlier reports showed p53-dependent activity of RITA in solid tumors as well as in leukemias. We and others recently described nutlin-induced apoptosis in MM cells, but it remains unclear whether RITA exerts antimyeloma activity. Here, we found that RITA activates the p53 pathway and induces apoptosis in MM cell lines and primary MM samples, preferentially killing myeloma cells. The activation of p53 induced by RITA was mediated through modulation of multiple apoptotic regulatory proteins, including upregulation of a proapoptotic protein (NOXA), downregulation of an antiapoptotic protein, Mcl-1, and activation of caspases through extrinsic pathways. Moreover, a number of key p53-mediated apoptotic target genes were identified by gene expression profiling and further validated by quantitative real-time PCR. Importantly, the combination of RITA with nutlin displayed a strong synergism on growth inhibition with the combination index ranging from 0.56 to 0.82 in MM cells. Our data support further clinical evaluation of RITA as a potential novel therapeutic intervention in MM. ©2010 AACR.

Oxidative stress mediated cytotoxicity of biologically synthesized silver nanoparticles in human lung epithelial adenocarcinoma cell line

NASA Astrophysics Data System (ADS)

Han, Jae Woong; Gurunathan, Sangiliyandi; Jeong, Jae-Kyo; Choi, Yun-Jung; Kwon, Deug-Nam; Park, Jin-Ki; Kim, Jin-Hoi

2014-09-01

The goal of the present study was to investigate the toxicity of biologically prepared small size of silver nanoparticles in human lung epithelial adenocarcinoma cells A549. Herein, we describe a facile method for the synthesis of silver nanoparticles by treating the supernatant from a culture of Escherichia coli with silver nitrate . The formation of silver nanoparticles was characterized using various analytical techniques. The results from UV-visible (UV-vis) spectroscopy and X-ray diffraction analysis show a characteristic strong resonance centered at 420 nm and a single crystalline nature, respectively. Fourier transform infrared spectroscopy confirmed the possible bio-molecules responsible for the reduction of silver from silver nitrate into nanoparticles. The particle size analyzer and transmission electron microscopy results suggest that silver nanoparticles are spherical in shape with an average diameter of 15 nm. The results derived from in vitro studies showed a concentration-dependent decrease in cell viability when A549 cells were exposed to silver nanoparticles. This decrease in cell viability corresponded to increased leakage of lactate dehydrogenase (LDH), increased intracellular reactive oxygen species generation (ROS), and decreased mitochondrial transmembrane potential (MTP). Furthermore, uptake and intracellular localization of silver nanoparticles were observed and were accompanied by accumulation of autophagosomes and autolysosomes in A549 cells. The results indicate that silver nanoparticles play a significant role in apoptosis. Interestingly, biologically synthesized silver nanoparticles showed more potent cytotoxicity at the concentrations tested compared to that shown by chemically synthesized silver nanoparticles. Therefore, our results demonstrated that human lung epithelial A549 cells could provide a valuable model to assess the cytotoxicity of silver nanoparticles.

Oxidative stress mediated cytotoxicity of biologically synthesized silver nanoparticles in human lung epithelial adenocarcinoma cell line

PubMed Central

2014-01-01

The goal of the present study was to investigate the toxicity of biologically prepared small size of silver nanoparticles in human lung epithelial adenocarcinoma cells A549. Herein, we describe a facile method for the synthesis of silver nanoparticles by treating the supernatant from a culture of Escherichia coli with silver nitrate. The formation of silver nanoparticles was characterized using various analytical techniques. The results from UV-visible (UV-vis) spectroscopy and X-ray diffraction analysis show a characteristic strong resonance centered at 420 nm and a single crystalline nature, respectively. Fourier transform infrared spectroscopy confirmed the possible bio-molecules responsible for the reduction of silver from silver nitrate into nanoparticles. The particle size analyzer and transmission electron microscopy results suggest that silver nanoparticles are spherical in shape with an average diameter of 15 nm. The results derived from in vitro studies showed a concentration-dependent decrease in cell viability when A549 cells were exposed to silver nanoparticles. This decrease in cell viability corresponded to increased leakage of lactate dehydrogenase (LDH), increased intracellular reactive oxygen species generation (ROS), and decreased mitochondrial transmembrane potential (MTP). Furthermore, uptake and intracellular localization of silver nanoparticles were observed and were accompanied by accumulation of autophagosomes and autolysosomes in A549 cells. The results indicate that silver nanoparticles play a significant role in apoptosis. Interestingly, biologically synthesized silver nanoparticles showed more potent cytotoxicity at the concentrations tested compared to that shown by chemically synthesized silver nanoparticles. Therefore, our results demonstrated that human lung epithelial A549 cells could provide a valuable model to assess the cytotoxicity of silver nanoparticles. PMID:25242904

Immunization with Clinical HIV-1 Env Proteins Induces Broad Antibody Dependent Cellular Cytotoxicity-Mediating Antibodies in a Rabbit Vaccination Model.

PubMed

Karlsson, Ingrid; Borggren, Marie; Jensen, Sanne Skov; Heyndrickx, Leo; Stewart-Jones, Guillaume; Scarlatti, Gabriella; Fomsgaard, Anders

2017-11-17

The induction of both neutralizing antibodies and non-neutralizing antibodies with effector functions, for example, antibody-dependent cellular cytotoxicity (ADCC), is desired in the search for effective vaccines against HIV-1. In the pursuit of novel immunogens capable of inducing an efficient antibody response, rabbits were immunized with selected antigens using different prime-boost strategies. We immunized 35 different groups of rabbits with Env antigens from clinical HIV-1 subtypes A and B, including immunization with DNA alone, protein alone, and DNA prime with protein boost. The rabbit sera were screened for ADCC activity using a GranToxiLux-based assay with human peripheral blood mononuclear cells as effector cells and CEM.NKR CCR5 cells coated with HIV-1 envelope as target cells. The groups with the highest ADCC activity were further characterized for cross-reactivity between HIV-1 subtypes. The immunogen inducing the most potent and broadest ADCC response was a trimeric gp140. The ADCC activity was highest against the HIV-1 subtype corresponding to the immunogen. The ADCC activity did not necessarily reflect neutralizing activity in the pseudovirus-TZMbl assay, but there was an overall correlation between the two antiviral activities. We present a rabbit vaccination model and an assay suitable for screening HIV-1 vaccine candidates for the induction of ADCC-mediating antibodies in addition to neutralizing antibodies. The antigens and/or immunization strategies capable of inducing antibodies with ADCC activity did not necessarily induce neutralizing activity and vice versa. Nevertheless, we identified vaccine candidates that were able to concurrently induce both types of responses and that had ADCC activity that was cross-reactive between different subtypes. When searching for an effective vaccine candidate, it is important to evaluate the antibody response using a model and an assay measuring the desired function.

Culture supernatants from V. cholerae O1 El Tor strains isolated from different geographic areas induce cell vacuolation and cytotoxicity.

PubMed

Vidal, Jorge E; Enríquez-Rincón, Fernando; Giono-Cerezo, Silvia; Ribas-Aparicio, Rosa María; Figueroa-Arredondo, Paula

2009-01-01

To investigate whether the HlyA-induced vacuolating effect is produced by V. cholerae O1 ElTor strains isolated from different geographic origins, including Mexico. Supernatant-induced haemolysis, vacuolating activity and cytotoxicity in Vero cells were recorded. PCR, RFLP analysis and molecular cloning were performed. All ElTor strains analyzed induced cellular vacuolation. Ribotype 2 strains isolates from the U.S. gulf coast yielded the highest titer of vacuolating activity. Eight of nine strains were haemolytic, while all strains were PCR positive for the hlyA gene. We cloned the hlyA gene from two ElTor strains, a toxigenic (2514-88, ctxAB+) and a non-toxigenic Mexican strain (CM 91-3, ctxAB-). Supernatant from those recombinant E. coli strains induced haemolysis, cell vacuolation and cytotoxicity. RFLP-PCR analysis revealed similarities in the hlyA gene from all strains tested. The HlyA-induced vacuolating effect is a widespread phenotype of epidemic V. cholerae O1 ElTor strains.

Suppressive effects of 3-methylcholanthrene on the in vitro antitumor activity of naturally cytotoxic cells

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

Lill, P.H.; Gangemi, D.

1986-01-01

Transient suppression of splenic natural killer (NK), natural cytotoxic (NC) and peritoneal macrophage cytotoxicity was observed following a single injection of 3-methylcholanthrene (3-MC) into C3H/HeN mice. Natural killer cell activity was depressed by 30-60% 4-6 d after injection of 1.0 mg 3-MC. Levels of NK reactivity returned to normal 8 d post 3-MC injection, and no suppression of natural killing was seen when tested 6 wk after 3-MC treatment. 3-MC did not affect propionibacterium acnes augmentation of NK cell activity when tested both 6 d and 6 wk after carcinogen injection. The results indicate that the observed suppression of naturallymore » cytotoxic cells may not be important in allowing 3-MC-induced tumors to grow, since suppression is not long-lasting. Therefore, any effect on tumor growth mediated by a suppression of naturally cytotoxic cells would have to be exerted at the earliest stages of tumor development.« less

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

PubMed

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.

The suppression of inflammatory macrophage-mediated cytotoxicity and proinflammatory cytokine production by transgenic expression of HLA-E.

PubMed

Maeda, Akira; Kawamura, Takuji; Ueno, Takehisa; Usui, Noriaki; Eguchi, Hiroshi; Miyagawa, Shuji

2013-12-01

Macrophages participate in xenogenic rejection and represent a major biological obstacle to successful xenotransplantation. The signal inhibitory regulatory protein α (SIRPα) receptor was reported to be a negative regulator of macrophage phagocytic activity via interaction with CD47, its ligand. Because a majority of human macrophages express the inhibitory receptor CD94/NKG2A, which binds specifically to the human leukocyte antigen (HLA)-E and contains immunoreceptor tyrosine-based inhibition motifs (ITIMs), the inhibitory function of HLA class I molecules, HLA-E, on macrophage-mediated cytolysis was examined. The suppressive effect against proinflammatory cytokine production by macrophages was also examined. Complementary DNA (cDNA) of HLA-E, and CD47 were prepared and transfected into swine endothelial cells (SEC). The expression of the modified genes was evaluated by flow cytometry and macrophage-mediated cytolysis was assessed using in vitro generated macrophages. Transgenic expression of HLA-E significantly suppressed the macrophage-mediated cytotoxicity. HLA-E transgenic expression demonstrated a significant suppression equivalent to CD47 transgenic expression. Furthermore, transgenic HLA-E suppressed the production of pro-inflammatory cytokines by inflammatory macrophages. These results indicate that generating transgenic HLA-E pigs might protect porcine grafts from, not only NK cytotoxicity, but also macrophage-mediated cytotoxicity. © 2013 Elsevier B.V. All rights reserved.

Tomentosin Induces Telomere Shortening and Caspase-Dependant Apoptosis in Cervical Cancer Cells.

PubMed

Merghoub, Nawel; El Btaouri, Hassan; Benbacer, Laila; Gmouh, Saïd; Trentesaux, Chantal; Brassart, Bertrand; Attaleb, Mohammed; Madoulet, Claudie; Wenner, Thomas; Amzazi, Saaid; Morjani, Hamid; El Mzibri, Mohamed

2017-07-01

Tomentosin, a natural sesquiterpene lactone purified from of Inula viscosa L., was investigated for its anti-proliferative, telomere shortening, and apoptotic effects on human cervical cancer HeLa and SiHa cell lines. Tomentosin was found to inhibit the growth of SiHa and HeLa cell lines in dose and time-dependent manner (IC 50 values of 7.10 ± 0.78 μM and 5.87 ± 0.36 μM, respectively after 96 h of treatment). As evidenced by TTAGGG telomere length assay, tomentosin target specifically the telomeric overhang lengthening. This was confirmed by the evaluation of the cytotoxic effects of tomentosin in the foetal fibroblast Wi38 and JW10 cells which were derived from Wi38 and express hTERT, the telomerase catalytic subunit. We found that JW10 cells are 4.7-fold more sensitive to tomentosin which argues for telomere as its specific target. Furthermore, we found that tomentosin mediate this cytotoxic effect by inducing apoptosis and cell cycle arrest at G2/M phase. Morphological features of treated cells, as evidenced by Hoechst 33324 staining, revealed that the cytotoxic effect was due to induction of apoptosis. This was accompanied by pro-caspase-3 cleavage, an increase in caspase-3 activity and a cleavage of poly (ADP-ribose) polymerase (PARP). Moreover, tomentosin induced a decrease in mitochondrial membrane potential (ΔΨm) and an increase in reactive oxygen species (ROS), accompanied by a decrease in Bcl-2 expression. This indicates that tomentosin-induced apoptosis may involve a mitochondria-mediated signaling pathway. This study provides the first evidence that tomentosin targets telomere machinery and induces apoptosis in cervical cancer cells. The molecular mechanism underlying tomentosin-induced apoptosis may involve a mitochondria-mediated signaling pathway. J. Cell. Biochem. 118: 1689-1698, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

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

PubMed Central

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

2013-01-01

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. PMID:23430111

Diabody-based recombinant formats of humanized IgG-like bispecific antibody with effective retargeting of lymphocytes to tumor cells.

PubMed

Asano, Ryutaro; Kawaguchi, Hiroko; Watanabe, Yasuhiro; Nakanishi, Takeshi; Umetsu, Mitsuo; Hayashi, Hiroki; Katayose, Yu; Unno, Michiaki; Kudo, Toshio; Kumagai, Izumi

2008-10-01

Recently, recombinant antibodies have been dissected into antigen-binding regions and rebuilt into multivalent high-avidity formats. These new structural designs are expected to improve in vivo pharmacokinetics and efficacy in clinical use. Here, we designed effective recombinant bispecific antibody (BsAb) formats based on hEx3, a humanized bispecific diabody with epidermal growth factor receptor and CD3 retargeting. The bispecific and bivalent IgG-like antibodies engineered from hEx3 (or its single-chain form, hEx3-scDb) and the human Fc region showed stronger binding to each target cell than did monovalent diabody formats, and their affinity was identical to that of the corresponding parent IgG. The bivalent effect of the constructed IgG-like BsAbs resulted in cell cytotoxicity 10 times that of monovalent diabodies, and further, the fusion of Fc portion contributed intense cytotoxicity in peripheral blood mononuclear cells by the induction of the antibody-dependent cellular cytotoxicity. The growth-inhibition effects of IgG-like BsAbs were superior to those of the approved therapeutic antibody cetuximab, which recognizes the same epidermal growth factor receptor antigen, even when peripheral blood mononuclear cells were used as effector cells. We thus demonstrated a critical improvement in the effect of hEx3 by the bottom-up construction of IgG-like BsAbs; in adoptive immunotherapy, monotherapy without supplemental molecules may be able to induce antibody-dependent cellular cytotoxicity.

Betalains increase vitexin-2-O-xyloside cytotoxicity in CaCo-2 cancer cells.

PubMed

Farabegoli, F; Scarpa, E S; Frati, A; Serafini, G; Papi, A; Spisni, E; Antonini, E; Benedetti, S; Ninfali, P

2017-03-01

Vitexin-2-O-xyloside (XVX) from Beta vulgaris var. cicla L. (BVc) seeds, betaxanthin (R1) and betacyanin (R2) fractions from Beta vulgaris var. rubra L. (BVr) roots were combined and tested for cytotoxicity in CaCo-2 colon cancer cells. XVX was the most cytotoxic molecule, but the combination of XVX with R1 and R2 significantly prolonged its cytotoxicity. Cytotoxicity was mediated by the intrinsic apoptotic pathway, as shown by an increase in Bcl2-like protein 4, cleaved Poly ADP-Ribosyl Polymerase 1 and cleaved Caspase 3 levels with a parallel decrease in anti-apoptotic protein B-cell leukemia/lymphoma 2 levels. R1 and R2, used alone or in combination, reduced oxidative stress triggered by H 2 O 2 in CaCo-2 cells. Betalains dampened cyclooxygenase-2 and interleukin-8 mRNA expression after lipopolysaccharide induction in CaCo-2, showing an anti-inflammatory action. Our results support the use of a cocktail of R1, R2 and XVX as a chemopreventive tool against colon cancer. Copyright © 2016 Elsevier Ltd. All rights reserved.

Cetuximab-activated natural killer (NK) and dendritic cells (DC) collaborate to trigger tumor antigen-specific T cell immunity in head and neck cancer patients

PubMed Central

Srivastava, Raghvendra M.; Lee, Steve C.; Filho, Pedro A. Andrade; Lord, Christopher A.; Jie, Hyun-bae; Davidson, H. Carter; López-Albaitero, Andrés; Gibson, Sandra P.; Gooding, William E.; Ferrone, Soldano; Ferris, Robert L.

2013-01-01

Purpose Tumor antigen (TA)-specific monoclonal antibodies (mAb) block oncogenic signaling and induce Fcγ receptor (FcγR)-mediated cytotoxicity. However, the role of CD8+ cytotoxic T lymphocyte (CTL) and FcγR in initiating innate and adaptive immune responses in mAb-treated human cancer patients is still emerging. Experimental Design FcγRIIIa codon 158 polymorphism was correlated with survival in 107 cetuximab-treated head and neck cancer (HNC) patients. Flow cytometry was performed to quantify EGFR-specific T cells in cetuximab-treated HNC patients. The effect of cetuximab on NK cell, dendritic cell (DC), and T cell activation was measured using IFN-γ release assays and flow cytometry. Results FcγR IIIa polymorphism did not predict clinical outcome in cetuximab-treated HNC patients, however elevated circulating EGFR -specific CD8+ 853-861 T cells were found in cetuximab-treated HNC patients (p<0.005). Cetuximab promoted EGFR-specific cellular immunity through the interaction of EGFR+ tumor cells and FcγRIIIa on NK cells, but not on the polymorphism per se. Cetuximab-activated NK cells induced IFN-γ dependent expression of DC maturation markers, antigen presentation machinery (APM) components such as TAP-1/2, and Th1 chemokines through NKG2D/MICA binding. Cetuximab initiated adaptive immune responses via NK-cell induced DC maturation, which enhanced cross-presentation to CTL specific for EGFR as well as another TA, MAGE-3. Conclusion Cetuximab-activated NK cells promote DC maturation and CD8+ T cell priming, leading to TA spreading and Th1 cytokine release through ‘NK-DC cross-talk.’ FcγRIIIa polymorphism did not predict clinical response to cetuximab, but was necessary for NK-DC interaction and mAb induced cross-presentation. EGFR-specific T cells in cetuximab treated HNC patients may contribute to clinical response. PMID:23444227

Microchip Screening Platform for Single Cell Assessment of NK Cell Cytotoxicity

PubMed Central

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

Rebmab200, a humanized monoclonal antibody targeting the sodium phosphate transporter NaPi2b displays strong immune mediated cytotoxicity against cancer: a novel reagent for targeted antibody therapy of cancer.

PubMed

Lopes dos Santos, Mariana; Yeda, Fernanda Perez; Tsuruta, Lilian Rumi; Horta, Bruno Brasil; Pimenta, Alécio A; Degaki, Theri Leica; Soares, Ibere C; Tuma, Maria Carolina; Okamoto, Oswaldo Keith; Alves, Venancio A F; Old, Lloyd J; Ritter, Gerd; Moro, Ana Maria

2013-01-01

NaPi2b, a sodium-dependent phosphate transporter, is highly expressed in ovarian carcinomas and is recognized by the murine monoclonal antibody MX35. The antibody had shown excellent targeting to ovarian cancer in several early phase clinical trials but being murine the antibody's full therapeutic potential could not be explored. To overcome this impediment we developed a humanized antibody version named Rebmab200, expressed in human PER.C6® cells and cloned by limiting dilution. In order to select a clone with high therapeutic potential clones were characterized using a series of physicochemical assays, flow cytometry, real-time surface plasmon resonance, glycosylation analyses, immunohistochemistry, antibody-dependent cell-mediated cytotoxicity, complement-dependent-cytotoxicity assays and quantitative PCR. Comparative analyses of Rebmab200 and MX35 monoclonal antibodies demonstrated that the two antibodies had similar specificity for NaPi2b by flow cytometry with a panel of 30 cell lines and maintained similar kinetic parameters. Robust and high producer cell clones potentially suitable for use in manufacturing were obtained. Rebmab200 antibodies were assessed by immunohistochemistry using a large panel of tissues including human carcinomas of ovarian, lung, kidney and breast origin. An assessment of its binding towards 33 normal human organs was performed as well. Rebmab200 showed selected strong reactivity with the tested tumor types but little or no reactivity with the normal tissues tested confirming its potential for targeted therapeutics strategies. The remarkable cytotoxicity shown by Rebmab200 in OVCAR-3 cells is a significant addition to the traits of stability and productivity displayed by the top clones of Rebmab200. Antibody-dependent cell-mediated toxicity functionality was confirmed in repeated assays using cancer cell lines derived from ovary, kidney and lung as targets. To explore use of this antibody in clinical trials, GMP production of Rebmab

Rebmab200, a Humanized Monoclonal Antibody Targeting the Sodium Phosphate Transporter NaPi2b Displays Strong Immune Mediated Cytotoxicity against Cancer: A Novel Reagent for Targeted Antibody Therapy of Cancer

PubMed Central

dos Santos, Mariana Lopes; Yeda, Fernanda Perez; Tsuruta, Lilian Rumi; Horta, Bruno Brasil; Pimenta, Alécio A.; Degaki, Theri Leica; Soares, Ibere C.; Tuma, Maria Carolina; Okamoto, Oswaldo Keith; Alves, Venancio A. F.; Ritter, Gerd; Moro, Ana Maria

2013-01-01

NaPi2b, a sodium-dependent phosphate transporter, is highly expressed in ovarian carcinomas and is recognized by the murine monoclonal antibody MX35. The antibody had shown excellent targeting to ovarian cancer in several early phase clinical trials but being murine the antibody's full therapeutic potential could not be explored. To overcome this impediment we developed a humanized antibody version named Rebmab200, expressed in human PER.C6® cells and cloned by limiting dilution. In order to select a clone with high therapeutic potential clones were characterized using a series of physicochemical assays, flow cytometry, real-time surface plasmon resonance, glycosylation analyses, immunohistochemistry, antibody-dependent cell-mediated cytotoxicity, complement-dependent-cytotoxicity assays and quantitative PCR. Comparative analyses of Rebmab200 and MX35 monoclonal antibodies demonstrated that the two antibodies had similar specificity for NaPi2b by flow cytometry with a panel of 30 cell lines and maintained similar kinetic parameters. Robust and high producer cell clones potentially suitable for use in manufacturing were obtained. Rebmab200 antibodies were assessed by immunohistochemistry using a large panel of tissues including human carcinomas of ovarian, lung, kidney and breast origin. An assessment of its binding towards 33 normal human organs was performed as well. Rebmab200 showed selected strong reactivity with the tested tumor types but little or no reactivity with the normal tissues tested confirming its potential for targeted therapeutics strategies. The remarkable cytotoxicity shown by Rebmab200 in OVCAR-3 cells is a significant addition to the traits of stability and productivity displayed by the top clones of Rebmab200. Antibody-dependent cell-mediated toxicity functionality was confirmed in repeated assays using cancer cell lines derived from ovary, kidney and lung as targets. To explore use of this antibody in clinical trials, GMP production of Rebmab

4SC-202 activates ASK1-dependent mitochondrial apoptosis pathway to inhibit hepatocellular carcinoma cells

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

Fu, Meili, E-mail: fumeilidrlinyi@tom.com; Wan, Fuqiang; Li, Zhengling

The aim of the present study is to investigate the potential anti-hepatocellular carcinoma (HCC) cell activity by 4SC-202, a novel class I HDAC inhibitor (HDACi). The associated signaling mechanisms were also analyzed. We showed that 4SC-202 treatment induced potent cytotoxic and proliferation–inhibitory activities against established HCC cell lines (HepG2, HepB3, SMMC-7721) and patient-derived primary HCC cells. Further, adding 4SC-202 in HCC cells activated mitochondrial apoptosis pathway, which was evidenced by mitochondrial permeability transition pore (mPTP) opening, cytochrome C cytosol release and caspase-3/-9 activation. Inhibition of this apoptosis pathway, by caspase-3/-9 inhibitors, mPTP blockers, or by shRNA-mediated knockdown of cyclophilin-D (Cyp-D,more » a key component of mPTP), significantly attenuated 4SC-202-induced HCC cell death and apoptosis. Reversely, over-expression of Cyp-D enhanced 4SC-202's sensitivity in HCC cells. Further studies showed that 4SC-202 induced apoptosis signal-regulating kinase 1 (ASK1) activation, causing it translocation to mitochondria and physical association with Cyp-D. This mitochondrial ASK1-Cyp-D complexation appeared required for mediating 4SC-202-induced apoptosis activation. ASK1 stable knockdown by targeted-shRNAs largely inhibited 4SC-202-induced mPTP opening, cytochrome C release, and following HCC cell apoptotic death. Together, we suggest that 4SC-202 activates ASK1-dependent mitochondrial apoptosis pathway to potently inhibit human HCC cells. - Highlights: • 4SC-202 exerts potent anti-proliferative and cytotoxic activity against established/primary HCC cells. • SC-202-induced anti-HCC cell activity relies on caspase-dependent apoptosis activation. • 4SC-202 activates Cyp-D-dependent mitochondrial apoptosis pathway in HCC cells. • 4SC-202 activates ASK1 in HCC cells, causing it translocation to mitochondria. • Mitochondrial ASK1-Cyp-D complexation mediates 4SC-202's activity in HCC cells.« less

Size- and coating-dependent cytotoxicity and genotoxicity of silver nanoparticles evaluated using in vitro standard assays.

PubMed

Guo, Xiaoqing; Li, Yan; Yan, Jian; Ingle, Taylor; Jones, Margie Yvonne; Mei, Nan; Boudreau, Mary D; Cunningham, Candice K; Abbas, Mazhar; Paredes, Angel M; Zhou, Tong; Moore, Martha M; Howard, Paul C; Chen, Tao

2016-11-01

The physicochemical characteristics of silver nanoparticles (AgNPs) may greatly alter their toxicological potential. To explore the effects of size and coating on the cytotoxicity and genotoxicity of AgNPs, six different types of AgNPs, having three different sizes and two different coatings, were investigated using the Ames test, mouse lymphoma assay (MLA) and in vitro micronucleus assay. The genotoxicities of silver acetate and silver nitrate were evaluated to compare the genotoxicity of nanosilver to that of ionic silver. The Ames test produced inconclusive results for all types of the silver materials due to the high toxicity of silver to the test bacteria and the lack of entry of the nanoparticles into the cells. Treatment of L5718Y cells with AgNPs and ionic silver resulted in concentration-dependent cytotoxicity, mutagenicity in the Tk gene and the induction of micronuclei from exposure to nearly every type of the silver materials. Treatment of TK6 cells with these silver materials also resulted in concentration-dependent cytotoxicity and significantly increased micronucleus frequency. With both the MLA and micronucleus assays, the smaller the AgNPs, the greater the cytotoxicity and genotoxicity. The coatings had less effect on the relative genotoxicity of AgNPs than the particle size. Loss of heterozygosity analysis of the induced Tk mutants indicated that the types of mutations induced by AgNPs were different from those of ionic silver. These results suggest that AgNPs induce cytotoxicity and genotoxicity in a size- and coating-dependent manner. Furthermore, while the MLA and in vitro micronucleus assay (in both types of cells) are useful to quantitatively measure the genotoxic potencies of AgNPs, the Ames test cannot.

Interleukin 2 and interleukin 10 function synergistically to promote CD8+ T cell cytotoxicity, which is suppressed by regulatory T cells in breast cancer.

PubMed

Li, Xiaogang; Lu, Ping; Li, Bo; Zhang, Wanfu; Yang, Rong; Chu, Yan; Luo, Kaiyuan

2017-06-01

The precise role of interleukin (IL)-10 in breast cancer is not clear. Previous studies suggested a tumor-promoting role of IL-10 in breast cancer, whereas recent discoveries that IL-10 activated and expanded tumor-resident CD8 + T cells challenged the traditional view. Here, we investigated the role of IL-10 in HLA-A2-positive breast cancer patients with Grade III, Stage IIA or IIB in-situ and invasive ductal carcinoma, and compared it with that of IL-2, the canonical CD8 + T cell growth factor. We first observed that breast cancer patients presented higher serum levels of IL-2 and IL-10 than healthy controls. Upon prolonged TCR stimulation, peripheral blood CD8 + T cells from breast cancer patients tended to undergo apoptosis, which could be prevented by the addition of IL-2 and/or IL-10. The cytotoxicity of TCR-activated CD8 + T cells was also enhanced by exogenous IL-2 and/or IL-10. Interestingly, IL-2 and IL-10 demonstrated synergistic effects, since the enhancement in CD8 + T cell function when both cytokines were added was greater than the sum of the improvements mediated by each individual cytokine. IL-10 by itself could not promote the proliferation of CD8 + T cells but could significantly enhance IL-2-mediated promotion of CD8 + T cell proliferation. In addition, the cytotoxicity of tumor-infiltrating CD8 + T cells in breast tumor was elevated when both IL-2 and IL-10 were present but not when either one was absent. This synergistic effect was stopped by CD4 + CD25 + regulatory T cells (Treg), which depleted IL-2 in a cell number-dependent manner. Together, these results demonstrated that IL-2 and IL-10 could work synergistically to improve the survival, proliferation, and cytotoxicity of activated CD8 + T cells, an effect suppressible by CD4 + CD25 + Treg cells. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

PubMed

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.

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.

PubMed

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.

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

PubMed Central

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

2018-01-01

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. PMID:29541243

The Voltage-dependent Anion Channel 1 Mediates Amyloid β Toxicity and Represents a Potential Target for Alzheimer Disease Therapy.

PubMed

Smilansky, Angela; Dangoor, Liron; Nakdimon, Itay; Ben-Hail, Danya; Mizrachi, Dario; Shoshan-Barmatz, Varda

2015-12-25

The voltage-dependent anion channel 1 (VDAC1), found in the mitochondrial outer membrane, forms the main interface between mitochondrial and cellular metabolisms, mediates the passage of a variety of molecules across the mitochondrial outer membrane, and is central to mitochondria-mediated apoptosis. VDAC1 is overexpressed in post-mortem brains of Alzheimer disease (AD) patients. The development and progress of AD are associated with mitochondrial dysfunction resulting from the cytotoxic effects of accumulated amyloid β (Aβ). In this study we demonstrate the involvement of VDAC1 and a VDAC1 N-terminal peptide (VDAC1-N-Ter) in Aβ cell penetration and cell death induction. Aβ directly interacted with VDAC1 and VDAC1-N-Ter, as monitored by VDAC1 channel conductance, surface plasmon resonance, and microscale thermophoresis. Preincubated Aβ interacted with bilayer-reconstituted VDAC1 and increased its conductance ∼ 2-fold. Incubation of cells with Aβ resulted in mitochondria-mediated apoptotic cell death. However, the presence of non-cell-penetrating VDAC1-N-Ter peptide prevented Aβ cellular entry and Aβ-induced mitochondria-mediated apoptosis. Likewise, silencing VDAC1 expression by specific siRNA prevented Aβ entry into the cytosol as well as Aβ-induced toxicity. Finally, the mode of Aβ-mediated action involves detachment of mitochondria-bound hexokinase, induction of VDAC1 oligomerization, and cytochrome c release, a sequence of events leading to apoptosis. As such, we suggest that Aβ-mediated toxicity involves mitochondrial and plasma membrane VDAC1, leading to mitochondrial dysfunction and apoptosis induction. The VDAC1-N-Ter peptide targeting Aβ cytotoxicity is thus a potential new therapeutic strategy for AD treatment. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Long noncoding RNA AB074169 inhibits cell proliferation via modulation of KHSRP-mediated p21 expression in papillary thyroid carcinoma.

PubMed

Gou, Qiheng; Gao, Linbo; Nie, Xinwen; Pu, Wenchen; Zhu, Jingqiang; Wang, Yichao; Liu, Xuesha; Tan, Shuangyan; Zhou, Jian-Kang; Gong, Yanqiu; He, Juan; Wu, Ke; Xie, Yuxin; Zhao, Wanjun; Dai, Lunzhi; Liu, Lunxu; Xiang, Rong; Wei, Yu-Quan; Zhang, Lin; Peng, Yong

2018-05-07

Long noncoding RNAs (lncRNAs) are emerging as a novel class of regulators in gene expression associated with tumorigenesis. However, the role of lncRNAs in papillary thyroid carcinoma (PTC) is poorly understood. Here we conducted global lncRNA profiling and identified lncRNA AB074169 (lncAB) as significantly downregulated in PTC. Decreased expression of lncAB in PTC was caused by CpG hypermethylation within its gene promoter. Functional studies showed that lncAB overexpression led to cell cycle arrest and tumor growth inhibition in vitro and in vivo, whereas lncAB knockdown promoted cell proliferation. Mechanistic analyses revealed that lncAB bound KH-type splicing regulatory protein (KHSRP) and also decreased expression of KHSRP, thus increasing CDKN1a (p21) expression and decreasing CDK2 expression to repress cell proliferation. Taken together, these findings demonstrate that lncAB functions as a tumor suppressor during PTC tumorigenesis. Copyright ©2018, American Association for Cancer Research.

Human Follicular Lymphoma CD39+-Infiltrating T Cells Contribute to Adenosine-Mediated T Cell Hyporesponsiveness1

PubMed Central

Hilchey, Shannon P.; Kobie, James J.; Cochran, Mathew R.; Secor-Socha, Shelley; Wang, Jyh-Chiang E.; Hyrien, Ollivier; Burack, W. Richard; Mosmann, Tim R.; Quataert, Sally A.; Bernstein, Steven H.

2010-01-01

Our previous work has demonstrated that human follicular lymphoma (FL) infiltrating T cells are anergic, in part due to suppression by regulatory T cells. In this study, we identify pericellular adenosine, interacting with T cell-associated G protein-coupled A2A/B adenosine receptors (AR), as contributing to FL T cell hyporesponsiveness. In a subset of FL patient samples, treatment of lymph node mononuclear cells (LNMC) with specific A2A/B AR antagonists results in an increase in IFN-γ or IL-2 secretion upon anti-CD3/CD28 Ab stimulation, as compared with that seen without inhibitors. In contrast, treatment with an A1 AR antagonist had no effect on cytokine secretion. As the rate limiting step for adenosine generation from pericellular ATP is the ecto-ATPase CD39, we next show that inhibition of CD39 activity using the inhibitor ARL 67156 partially overcomes T cell hyporesponsiveness in a subset of patient samples. Phenotypic characterization of LNMC demonstrates populations of CD39-expressing CD4+ and CD8+ T cells, which are overrepresented in FL as compared with that seen in normal or reactive nodes, or normal peripheral blood. Thirty percent of the FL CD4+CD39+ T cells coexpress CD25high and FOXP3 (consistent with regulatory T cells). Finally, FL or normal LNMC hydrolyze ATP in vitro, in a dose- and time-dependent fashion, with the rate of ATP consumption being associated with the degree of CD39+ T cell infiltration. Together, these results support the finding that the ATP-ectonucleotidase-adenosine system mediates T cell anergy in a human tumor. In addition, these studies suggest that the A2A/B AR as well as CD39 are novel pharmacological targets for augmenting cancer immunotherapy. PMID:19864600

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

PubMed

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 <10%. Novel treatment strategies are therefore urgently required, particularly for patients bearing BRAF/NRAS wild-type tumors. Targeting autophagy is a means to promote cancer cell death in chemotherapy-resistant tumors, and the aim of this study was to test the hypothesis that cannabinoids promote autophagy-dependent apoptosis in melanoma. Treatment with Δ(9)-Tetrahydrocannabinol (THC) resulted in the activation of autophagy, loss of cell 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.

HDAC6 regulates the dynamics of lytic granules in cytotoxic T lymphocytes

PubMed Central

Núñez-Andrade, Norman; Iborra, Salvador; Trullo, Antonio; Moreno-Gonzalo, Olga; Calvo, Enrique; Catalán, Elena; Menasche, Gaël; Sancho, David; Vázquez, Jesús; Yao, Tso-Pang

2016-01-01

HDAC6 is a tubulin deacetylase involved in many cellular functions related to cytoskeleton dynamics including cell migration and autophagy. In addition, HDAC6 affects antigen-dependent CD4+ T cell activation. In this study, we show that HDAC6 contributes to the cytotoxic function of CD8+ T cells. Immunization studies revealed defective cytotoxic activity in vivo in the absence of HDAC6. Adoptive transfer of wild-type or Hdac6-/- CD8+ T cells to Rag1-/- mice demonstrated specific impairment in CD8+ T cell responses against vaccinia infection. Mechanistically, HDAC6-deficient cytotoxic T lymphocytes (CTLs) showed defective in vitro cytolytic activity related to altered dynamics of lytic granules, inhibited kinesin 1 – dynactin mediated terminal transport of lytic granules to the immune synapse and deficient exocytosis, but not to target cell recognition, T cell receptor (TCR) activation or interferon (IFNγ) production. Our results establish HDAC6 as an effector of the immune cytotoxic response that acts by affecting the dynamics, transport and secretion of lytic granules by CTLs. PMID:26869226

INTEGRIN-MEDIATED CELL ATTACHMENT SHOWS TIME-DEPENDENT UPREGULATION OF GAP JUNCTION COMMUNICATION.

EPA Science Inventory

Integrin-mediated Cell Attachment Shows Time-Dependent Upregulation of Gap Junction
Communication

Rachel Grindstaff and Carl Blackman, National Health & Environmental Effects Research
Laboratory, Office of Research and Development, US EPA, Research Triang...

HPV vaccine stimulates cytotoxic activity of killer dendritic cells and natural killer cells against HPV-positive tumour cells

PubMed Central

Van den Bergh, Johan M J; Guerti, Khadija; Willemen, Yannick; Lion, Eva; Cools, Nathalie; Goossens, Herman; Vorsters, Alex; Van Tendeloo, Viggo F I; Anguille, Sébastien; Van Damme, Pierre; Smits, Evelien L J M

2014-01-01

Cervarix™ is approved as a preventive vaccine against infection with the human papillomavirus (HPV) strains 16 and 18, which are causally related to the development of cervical cancer. We are the first to investigate in vitro the effects of this HPV vaccine on interleukin (IL)-15 dendritic cells (DC) as proxy of a naturally occurring subset of blood DC, and natural killer (NK) cells, two innate immune cell types that play an important role in antitumour immunity. Our results show that exposure of IL-15 DC to the HPV vaccine results in increased expression of phenotypic maturation markers, pro-inflammatory cytokine production and cytotoxic activity against HPV-positive tumour cells. These effects are mediated by the vaccine adjuvant, partly through Toll-like receptor 4 activation. Next, we demonstrate that vaccine-exposed IL-15 DC in turn induce phenotypic activation of NK cells, resulting in a synergistic cytotoxic action against HPV-infected tumour cells. Our study thus identifies a novel mode of action of the HPV vaccine in boosting innate immunity, including killing of HPV-infected cells by DC and NK cells. PMID:24979331