Application of Lectin Array Technology for Biobetter Characterization: Its Correlation with FcγRIII Binding and ADCC

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Markus Roucka

2016-12-01

Full Text Available Lectin microarray technology was applied to compare the glycosylation pattern of the monoclonal antibody MB311 expressed in SP2.0 cells to an antibody-dependent cellular cytotoxic effector function (ADCC-optimized variant (MB314. MB314 was generated by a plant expression system that uses genetically modified moss protoplasts (Physcomitrella patens to generate a de-fucosylated version of MB311. In contrast to MB311, no or very low interactions of MB314 with lectins Aspergillus oryzae l-fucose (AOL, Pisum sativum agglutinin (PSA, Lens culinaris agglutinin (LCA, and Aleuria aurantia lectin (AAL were observed. These lectins are specific for mono-/biantennary N-glycans containing a core fucose residue. Importantly, this fucose indicative lectin-binding pattern correlated with increased MB314 binding to CD16 (FcγRIII; receptor for the constant region of an antibody—whose affinity is mediated through core fucosylation—and stronger ADCC. In summary, these results demonstrate that lectin microarrays are useful orthogonal methods during antibody development and for characterization.

Modulating Cytotoxic Effector Functions by Fc Engineering to Improve Cancer Therapy.

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Kellner, Christian; Otte, Anna; Cappuzzello, Elisa; Klausz, Katja; Peipp, Matthias

2017-09-01

In the last two decades, monoclonal antibodies have revolutionized the therapy of cancer patients. Although antibody therapy has continuously been improved, still a significant number of patients do not benefit from antibody therapy. Therefore, rational optimization of the antibody molecule by Fc engineering represents a major area of translational research to further improve this potent therapeutic option. Monoclonal antibodies are able to trigger a variety of effector mechanisms. Especially Fc-mediated effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement- dependent cytotoxicity (CDC) are considered important in antibody therapy of cancer. Novel mechanistic insights into the action of monoclonal antibodies allowed the development of various Fc engineering approaches to modulate antibodies' effector functions. Strategies in modifying the Fc glycosylation profile (Fc glyco-engineering) or approaches in engineering the protein backbone (Fc protein engineering) have been intensively evaluated. In the current review, Fc engineering strategies resulting in improved ADCC, ADCP and CDC activity are summarized and discussed.

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

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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.

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.

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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.

Antitumor activity of chLpMab-2, a human-mouse chimeric cancer-specific antihuman podoplanin antibody, via antibody-dependent cellular cytotoxicity.

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Kaneko, Mika K; Yamada, Shinji; Nakamura, Takuro; Abe, Shinji; Nishioka, Yasuhiko; Kunita, Akiko; Fukayama, Masashi; Fujii, Yuki; Ogasawara, Satoshi; Kato, Yukinari

2017-04-01

Human podoplanin (hPDPN), a platelet aggregation-inducing transmembrane glycoprotein, is expressed in different types of tumors, and it binds to C-type lectin-like receptor 2 (CLEC-2). The overexpression of hPDPN is involved in invasion and metastasis. Anti-hPDPN monoclonal antibodies (mAbs) such as NZ-1 have shown antitumor and antimetastatic activities by binding to the platelet aggregation-stimulating (PLAG) domain of hPDPN. Recently, we developed a novel mouse anti-hPDPN mAb, LpMab-2, using the cancer-specific mAb (CasMab) technology. In this study we developed chLpMab-2, a human-mouse chimeric anti-hPDPN antibody, derived from LpMab-2. chLpMab-2 was produced using fucosyltransferase 8-knockout (KO) Chinese hamster ovary (CHO)-S cell lines. By flow cytometry, chLpMab-2 reacted with hPDPN-expressing cancer cell lines including glioblastomas, mesotheliomas, and lung cancers. However, it showed low reaction with normal cell lines such as lymphatic endothelial and renal epithelial cells. Moreover, chLpMab-2 exhibited high antibody-dependent cellular cytotoxicity (ADCC) against PDPN-expressing cells, despite its low complement-dependent cytotoxicity. Furthermore, treatment with chLpMab-2 abolished tumor growth in xenograft models of CHO/hPDPN, indicating that chLpMab-2 suppressed tumor development via ADCC. In conclusion, chLpMab-2 could be useful as a novel antibody-based therapy against hPDPN-expressing tumors. © 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

eCD4-Ig promotes ADCC activity of sera from HIV-1-infected patients.

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Meredith E Davis-Gardner

2017-12-01

Full Text Available Antibody-dependent cell-mediated cytotoxity (ADCC can eliminate HIV-1 infected cells, and may help reduce the reservoir of latent virus in infected patients. Sera of HIV-1 positive individuals include a number of antibodies that recognize epitopes usually occluded on HIV-1 envelope glycoprotein (Env trimers. We have recently described eCD4-Ig, a potent and exceptionally broad inhibitor of HIV-1 entry that can be used to protect rhesus macaques from multiple high-dose challenges with simian-human immunodeficiency virus AD8 (SHIV-AD8. Here we show that eCD4-Ig bearing an IgG1 Fc domain (eCD4-IgG1 can mediate efficient ADCC activity against HIV-1 isolates with differing tropisms, and that it does so at least 10-fold more efficiently than CD4-Ig, even when more CD4-Ig molecules bound cell surface-expressed Env. An ADCC-inactive IgG2 form of eCD4-Ig (eCD4-IgG2 exposes V3-loop and CD4-induced epitopes on cell-expressed trimers, and renders HIV-1-infected cells susceptible to ADCC mediated by antibodies of these classes. Moreover, eCD4-IgG2, but not IgG2 forms of the broadly neutralizing antibodies VRC01 and 10-1074, enhances the ADCC activities of serum antibodies from patients by 100-fold, and significantly enhanced killing of two latently infected T-cell lines reactivated by vorinostat or TNFα. Thus eCD4-Ig is qualitatively different from CD4-Ig or neutralizing antibodies in its ability to mediate ADCC, and it may be uniquely useful in treating HIV-1 infection or reducing the reservoir of latently infected cells.

Cytotoxicity of leukocytes from normal and Shigella-susceptible (opium-treated) guinea pigs against virulent Shigella sonnei.

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Morgan, D R; DuPont, H L; Wood, L V; Kohl, S

1984-01-01

Intraepithelial lymphocytes were collected from the ileum of adult Hartley strain guinea pigs and used as effector cells in a 60-min bactericidal assay with virulent Shigella sonnei as target cells. Natural killer cytotoxicity (NKC) and antibody-dependent cellular cytotoxicity (ADCC) were measured and correlated with the resistance of the animals to infection by S. sonnei. Normal guinea pig intraepithelial lymphocytes exhibited mean NKC and ADCC values of 22.8 +/- 5.0 and 34.1 +/- 13.6, respectively. These animals were resistant to oral challenge with virulent S. sonnei. Intraepithelial lymphocytes from guinea pigs which were fasted for 4 days demonstrated NKC and ADCC values similar to those of normal animals (31.0 +/- 8.1 and 41.7 +/- 6.7, respectively). These animals also were resistant to oral challenge. Intraepithelial lymphocytes from guinea pigs which were given 1 ml of deodorized tincture of opium 2 h before cell collection demonstrated deficient NKC (4.7 +/- 4.2) and ADCC (5.3 +/- 4.9) values but remained resistant to infection by S. sonnei. When guinea pigs were fasted for 4 days and given opium, deficient NKC (2.0 +/- 2.0) and ADCC (1.3 +/- 1.3) values were demonstrated; this group of animals was susceptible to infection by S. sonnei (P less than 0.04). These experiments demonstrated that opium treatment depresses one form of gut immunity. When combined with starvation, opium treatment may increase susceptibility to infection by shigellae by modulation of immunity in addition to the effects on gut motility and bacterial flora. PMID:6384044

Anti-Glycoprotein G Antibodies of Herpes Simplex Virus 2 Contribute to Complete Protection after Vaccination in Mice and Induce Antibody-Dependent Cellular Cytotoxicity and Complement-Mediated Cytolysis

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Staffan Görander

2014-11-01

Full Text Available We investigated the role of antibodies against the mature portion of glycoprotein G (mgG-2 of herpes simplex virus 2 (HSV-2 in protective immunity after vaccination. Mice were immunized intramuscularly with mgG-2 and oligodeoxynucleotides containing two CpG motifs plus alum as adjuvant. All C57BL/6 mice survived and presented no genital or systemic disease. High levels of immunoglobulin G subclass 1 (IgG1 and IgG2 antibodies were detected and re-stimulated splenic CD4+ T cells proliferated and produced IFN-γ. None of the sera from immunized mice exhibited neutralization, while all sera exerted antibody-dependent cellular cytotoxicity (ADCC and complement-mediated cytolysis (ACMC activity. Passive transfer of anti-mgG-2 monoclonal antibodies, or immune serum, to naive C57BL/6 mice did not limit disease progression. Immunized B‑cell KO mice presented lower survival rate and higher vaginal viral titers, as compared with vaccinated B-cell KO mice after passive transfer of immune serum and vaccinated C57BL/6 mice. Sera from mice that were vaccinated subcutaneously and intranasally with mgG-2 presented significantly lower titers of IgG antibodies and lower ADCC and ACMC activity. We conclude that anti-mgG-2 antibodies were of importance to limit genital HSV‑2 infection. ADCC and ACMC activity are potentially important mechanisms in protective immunity, and could tentatively be evaluated in future animal vaccine studies and in clinical trials.

Studies of cytotoxic antibodies against eye muscle antigens in patients with thyroid-associated ophthalmopathy

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Zhang, Z.-G.; Hiromatsu, Y.; Salvi, M.; Triller, H.; Bernard, N.; Wall, J.R.; Medeiros-Neto, G.; Iacona, A.; Lima, N.

1989-01-01

We have studied the prevalence and significance of cytotoxic antibodies against human eye muscle cells, as detected in antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-mediated antibody-dependent cytotoxicity (CMAC) in 51 Cr release assays, in patients with Graves' ophthalmopathy or Hashimoto's thyroiditis. A high prevalence of positive ADCC tests was found in all groups of patients with ophthalmopathy tested. Tests were positive in 64% of patients with Graves' ophthalmopathy from an area of severe iodine deficiency (Sao Paulo) and in 64% of such patients from an iodine replete area (Montreal). In patients with so-called ''euthyroid ophthalmopathy'', i.e. eye disease associated with thyroiditis, ADCC tests were positive in 75 and 38% of patients from the two areas, respectively, while tests were positive in 40 and 22%, respectively, of patients with Graves' hyperthyroidism without evident eye disease. In normal subjects, levels of 51 Cr release was always at background levels. In a group of patients from the high-iodine area, levels of antibodies in ADCC correlated positively with the intraocular pressure (mmHg) in primary position as a parameter of eye muscle dysfunction. In patients with ophthalmopathy, positive ADCC tests were assciated with antibodies to eye muscle membrane antigens of 55,65 and 95 kD as detected by immunoblotting, although the correlation was not close for any antigen. in contrast, CMAC tests were negative in all patients with ophthalmopathy. We also tested 9 mouse and 10 human monoclonal antibodies, reactive with orbital antigens in an enzyme-linked immunosorbent assay, for cytotoxic activity, in ADCC and CMAC, against eye muscle and thyroid cells. All monoclonal antibodies were of the IgM class and negative in ADCC assays. When tested in CMAC against eye muscle cells, one of 9 mouse and 5 of 8 human monoclonal antibodies showed significant activity while tests were positive in one of 9 and one of 10 monoclonal antibodies

Studies of cytotoxic antibodies against eye muscle antigens in patients with thyroid-associated ophthalmopathy

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Zhang, Z.-G.; Hiromatsu, Y.; Salvi, M.; Triller, H.; Bernard, N.; Wall, J.R. (Thyroid Research Unit, The Montreal General Hospital Research Institute, Montreal, Quebec (Canada)); Medeiros-Neto, G.; Iacona, A.; Lima, N. (Thyroid Clinic, Hospital das Clinicas, Sao Paulo (Brazil))

1989-01-01

We have studied the prevalence and significance of cytotoxic antibodies against human eye muscle cells, as detected in antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-mediated antibody-dependent cytotoxicity (CMAC) in {sup 51}Cr release assays, in patients with Graves' ophthalmopathy or Hashimoto's thyroiditis. A high prevalence of positive ADCC tests was found in all groups of patients with ophthalmopathy tested. Tests were positive in 64% of patients with Graves' ophthalmopathy from an area of severe iodine deficiency (Sao Paulo) and in 64% of such patients from an iodine replete area (Montreal). In patients with so-called ''euthyroid ophthalmopathy'', i.e. eye disease associated with thyroiditis, ADCC tests were positive in 75 and 38% of patients from the two areas, respectively, while tests were positive in 40 and 22%, respectively, of patients with Graves' hyperthyroidism without evident eye disease. In normal subjects, levels of {sup 51}Cr release was always at background levels. In a group of patients from the high-iodine area, levels of antibodies in ADCC correlated positively with the intraocular pressure (mmHg) in primary position as a parameter of eye muscle dysfunction. In patients with ophthalmopathy, positive ADCC tests were assciated with antibodies to eye muscle membrane antigens of 55,65 and 95 kD as detected by immunoblotting, although the correlation was not close for any antigen. in contrast, CMAC tests were negative in all patients with ophthalmopathy. We also tested 9 mouse and 10 human monoclonal antibodies, reactive with orbital antigens in an enzyme-linked immunosorbent assay, for cytotoxic activity, in ADCC and CMAC, against eye muscle and thyroid cells. All monoclonal antibodies were of the IgM class and negative in ADCC assays. (Abstract Truncated)

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

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Jiajun Fan

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

A novel antibody-dependent cellular cytotoxicity epitope in gp120 is identified by two monoclonal antibodies isolated from a long-term survivor of human immunodeficiency virus type 1 infection.

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Alsmadi, O; Herz, R; Murphy, E; Pinter, A; Tilley, S A

1997-01-01

Two monoclonal antibodies (MAbs), 42F and 43F, were isolated some 14 months apart from a single long-term survivor of human immunodeficiency virus type 1 (HIV-1) infection. These MAbs were found to be indistinguishable in terms of their isotypes, specificities, affinities, and biological activities. Both 42F and 43F directed substantial antibody-dependent cellular cytotoxicity (ADCC) against cells infected with four divergent lab-adapted strains of HIV-1, but no neutralizing activity against these strains was detectable. The ability of MAbs 42F and 43F, as well as that of MAbs against two other gp120 epitopes, to direct ADCC against uninfected CD4+ cells to which recombinant gp120SF2 had been adsorbed (i.e., "innocent bystanders") was demonstrated to be less efficient by at least an order of magnitude than their ability to direct ADCC against HIV-1-infected cells. Flow cytometry analyses showed that 42F and 43F also bind to native primary isolate Envs from clades B and E expressed on cell surfaces. By direct binding and competition assays, it was demonstrated that the 42F/43F epitope lies in a domain of gp120 outside the previously described CD4-binding site and V3 loop ADCC epitope clusters. Immunoblot analysis revealed that the 42F/43F epitope is not dependent on disulfide bonds or N-linked glycans in gp120. Epitope mapping of 42F and 43F by binding to linear peptides demonstrated specificity of these MAbs for a sequence of 10 amino acids in the C5 domain comprising residues 491 to 500 (Los Alamos National Laboratory numbering for the HXB2 strain). Thus, 42F and 43F define a new ADCC epitope in gp120. Because of the relative conservation of this epitope and the fact that it appears to have been significantly immunogenic in the individual from which these MAbs were derived, it may prove to be a useful component of HIV vaccines. Furthermore, these MAbs may be used as tools to probe the potential importance of ADCC as an antiviral activity in HIV-1 infection. PMID

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

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

2015-10-01

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

Evaluation of Different Parameters of Humoral and Cellular Immune Responses in HIV Serodiscordant Heterosexual Couples: Humoral Response Potentially Implicated in Modulating Transmission Rates

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María Julia Ruiz

2017-12-01

Full Text Available As the HIV/AIDS pandemic still progresses, understanding the mechanisms governing viral transmission as well as protection from HIV acquisition is fundamental. In this context, cohorts of HIV serodiscordant heterosexual couples (SDC represent a unique tool. The present study was aimed to evaluate specific parameters of innate, cellular and humoral immune responses in SDC. Specifically, plasma levels of cytokines and chemokines, HIV-specific T-cell responses, gp120-specific IgG and IgA antibodies, and HIV-specific antibody-dependent cellular cytotoxicity (ADCC activity were assessed in nine HIV-exposed seronegative individuals (ESN and their corresponding HIV seropositive partners (HIV+-P, in eighteen chronically infected HIV subjects (C, nine chronically infected subjects known to be HIV transmitters (CT and ten healthy HIV− donors (HD. Very low magnitude HIV-specific cellular responses were found in two out of six ESN. Interestingly, HIV+-P had the highest ADCC magnitude, the lowest IgA levels and the highest IgG/IgA ratio, all compared to CT. Positive correlations between CD4+ T-cell counts and both IgG/IgA ratios and %ADCC killing uniquely distinguished HIV+-P. Additionally, evidence of IgA interference with ADCC responses from HIV+-P and CT is provided. These data suggest for the first time a potential role of ADCC and/or gp120-specific IgG/IgA balance in modulating heterosexual transmission. In sum, this study provides key information to understand the host factors that influence viral transmission, which should be considered in both the development of prophylactic vaccines and novel immunotherapies for HIV-1 infection.

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

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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.

Cells of the J774 macrophage cell line are primed for antibody-dependent cell-mediated cytotoxicity following exposure to γ-irradiation

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Duerst, R.; Werberig, K.

1991-01-01

Activation of macrophages (M phi) for host defense against tumor cells follows a sequence of priming events followed by an initiating stimulus that results in production and release of cytotoxic molecules that mediate target cell killing. The authors have developed a model to study specific macrophage cytotoxicity in vitro utilizing a cultured murine M phi cell line, J774. Specific cytotoxicity of cultured human gastrointestinal tumor cells is achieved in the presence of murine IgG2a monoclonal antibody (mAb) 17-1-A. The ability of these cells to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) is greatly enhanced following gamma-irradiation. ADCC can be demonstrated at mAb 17-1-A concentrations greater than or equal to 1 microgram/ml and effector/target cell ratios greater than or equal to 2. Exposure to doses greater than or equal to 10 Gy of gamma-irradiation increases ADCC threefold. Varying the duration from J774 M phi exposure to γ-irradiation until addition of antibody-coated target cells showed that the primed state for ADCC is stable for at least 8 days but approximately 24 hr is required for complete development of the primed state. mAb-dependent target cell death begins 8 hr after addition of mAb and labeled target cells to primed effector cells and is complete by 24 hr. Incubation of unirradiated J774 M phi effector cells with recombinant murine interferon-γ (rmIFN-γ) also results in enhanced ADCC, but the extent of target cell killing achieved is less than that following priming by γ-irradiation. Concomitant priming of γ-irradiated J774 M phi with rmIFN-γ increases the extent of ADCC. Further study of irradiated J774 cells may elucidate the molecular pathways utilized by M phi for achieving and maintaining the primed state for ADCC

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

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Floyd Hassenrück

2018-01-01

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

Ex-vivo expanded human NK cells express activating receptors that mediate cytotoxicity of allogeneic and autologous cancer cell lines by direct recognition and antibody directed cellular cytotoxicity

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Campana Dario

2010-10-01

Full Text Available Abstract Background The possibility that autologous NK cells could serve as an effective treatment modality for solid tumors has long been considered. However, implementation is hampered by (i the small number of NK cells in peripheral blood, (ii the difficulties associated with large-scale production of GMP compliant cytolytic NK cells, (iii the need to activate the NK cells in order to induce NK cell mediated killing and (iv the constraints imposed by autologous inhibitory receptor-ligand interactions. To address these issues, we determined (i if large numbers of NK cells could be expanded from PBMC and GMP compliant cell fractions derived by elutriation, (ii their ability to kill allogeneic and autologous tumor targets by direct cytotoxitiy and by antibody-mediated cellular cytotoxicity and (iii defined NK cell specific receptor-ligand interactions that mediate tumor target cell killing. Methods Human NK cells were expanded during 14 days. Expansion efficiency, NK receptor repertoire before and after expansion, expression of NK specific ligands, cytolytic activity against allogeneic and autologous tumor targets, with and without the addition of chimeric EGFR monoclonal antibody, were investigated. Results Cell expansion shifted the NK cell receptor repertoire towards activation and resulted in cytotoxicity against various allogeneic tumor cell lines and autologous gastric cancer cells, while sparing normal PBMC. Blocking studies confirmed that autologous cytotoxicity is established through multiple activating receptor-ligand interactions. Importantly, expanded NK cells also mediated ADCC in an autologous and allogeneic setting by antibodies that are currently being used to treat patients with select solid tumors. Conclusion These data demonstrate that large numbers of cytolytic NK cells can be generated from PBMC and lymphocyte-enriched fractions obtained by GMP compliant counter current elutriation from PBMC, establishing the preclinical

Decreased Fc receptor expression on innate immune cells is associated with impaired antibody-mediated cellular phagocytic activity in chronically HIV-1 infected individuals.

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Dugast, Anne-Sophie; Tonelli, Andrew; Berger, Christoph T; Ackerman, Margaret E; Sciaranghella, Gaia; Liu, Qingquan; Sips, Magdalena; Toth, Ildiko; Piechocka-Trocha, Alicja; Ghebremichael, Musie; Alter, Galit

2011-07-05

In addition to neutralization, antibodies mediate other antiviral activities including antibody-dependent cellular phagocytosis (ADCP), antibody-dependent cellular cytotoxicity (ADCC), as well as complement deposition. While it is established that progressive HIV infection is associated with reduced ADCC and ADCP, the underlying mechanism for this loss of function is unknown. Here we report considerable changes in FcR expression over the course of HIV infection on both mDCs and monocytes, including elevated FcγRI expression in acute HIV infection and reduced expression of FcγRII and FcγRIIIa in chronic HIV infection. Furthermore, selective blockade of FcγRII alone was associated with a loss in ADCP activity, suggesting that FcγRII plays a central role in modulating ADCP. Overall, HIV infection is associated with a number of changes in FcR expression on phagocytic cells that are associated with changes in their ability to respond to antibody-opsonized targets, potentially contributing to a failure in viral clearance in progressive HIV-1 infection. Copyright © 2011 Elsevier Inc. All rights reserved.

Decreased Fc-Receptor expression on innate immune cells is associated with impaired antibody mediated cellular phagocytic activity in chronically HIV-1 infected individuals

Science.gov (United States)

Dugast, Anne-Sophie; Tonelli, Andrew; Berger, Christoph T.; Ackerman, Margaret E.; Sciaranghella, Gaia; Liu, Qingquan; Sips, Magdalena; Toth, Ildiko; Piechocka-Trocha, Alicja; Ghebremichael, Musie; Alter, Galit

2011-01-01

In addition to neutralization, antibodies mediate other antiviral activities including antibody-dependent cellular-phagocytosis (ADCP), antibody dependent cellular-cytotoxicity (ADCC), as well as complement deposition. While it is established that progressive HIV infection is associated with reduced ADCC and ADCP, the underlying mechanism for this loss of function is unknown. Here we report considerable changes in FcR expression over the course of HIV infection on both mDCs and monocytes, including elevated FcγRI expression in acute HIV infection and reduced expression of FcγRII and FcγRIIIa in chronic HIV infection. Furthermore, selective blockade of FcγRII alone was associated with a loss in ADCP activity, suggesting that FcγRII plays a central role in modulating ADCP. Overall, HIV infection is associated with a number of changes in FcR expression on phagocytic cells that are associated with changes in their ability to respond to antibody-opsonized targets, potentially contributing to a failure in viral clearance in progressive HIV-1 infection. PMID:21565376

Glycoengineered Monoclonal Antibodies with Homogeneous Glycan (M3, G0, G2, and A2) Using a Chemoenzymatic Approach Have Different Affinities for FcγRIIIa and Variable Antibody-Dependent Cellular Cytotoxicity Activities.

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Kurogochi, Masaki; Mori, Masako; Osumi, Kenji; Tojino, Mami; Sugawara, Shu-Ichi; Takashima, Shou; Hirose, Yuriko; Tsukimura, Wataru; Mizuno, Mamoru; Amano, Junko; Matsuda, Akio; Tomita, Masahiro; Takayanagi, Atsushi; Shoda, Shin-Ichiro; Shirai, Takashi

2015-01-01

Many therapeutic antibodies have been developed, and IgG antibodies have been extensively generated in various cell expression systems. IgG antibodies contain N-glycans at the constant region of the heavy chain (Fc domain), and their N-glycosylation patterns differ during various processes or among cell expression systems. The Fc N-glycan can modulate the effector functions of IgG antibodies, such as antibody-dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). To control Fc N-glycans, we performed a rearrangement of Fc N-glycans from a heterogeneous N-glycosylation pattern to homogeneous N-glycans using chemoenzymatic approaches with two types of endo-β-N-acetyl glucosaminidases (ENG'ases), one that works as a hydrolase to cleave all heterogeneous N-glycans, another that is used as a glycosynthase to generate homogeneous N-glycans. As starting materials, we used an anti-Her2 antibody produced in transgenic silkworm cocoon, which consists of non-fucosylated pauci-mannose type (Man2-3GlcNAc2), high-mannose type (Man4-9GlcNAc2), and complex type (Man3GlcNAc3-4) N-glycans. As a result of the cleavage of several ENG'ases (endoS, endoM, endoD, endoH, and endoLL), the heterogeneous glycans on antibodies were fully transformed into homogeneous-GlcNAc by a combination of endoS, endoD, and endoLL. Next, the desired N-glycans (M3; Man3GlcNAc1, G0; GlcNAc2Man3GlcNAc1, G2; Gal2GlcNAc2Man3GlcNAc1, A2; NeuAc2Gal2GlcNAc2Man3GlcNAc1) were transferred from the corresponding oxazolines to the GlcNAc residue on the intact anti-Her2 antibody with an ENG'ase mutant (endoS-D233Q), and the glycoengineered anti-Her2 antibody was obtained. The binding assay of anti-Her2 antibody with homogenous N-glycans with FcγRIIIa-V158 showed that the glycoform influenced the affinity for FcγRIIIa-V158. In addition, the ADCC assay for the glycoengineered anti-Her2 antibody (mAb-M3, mAb-G0, mAb-G2, and mAb-A2) was performed using SKBR-3 and BT-474 as target cells, and

Glycoengineered Monoclonal Antibodies with Homogeneous Glycan (M3, G0, G2, and A2 Using a Chemoenzymatic Approach Have Different Affinities for FcγRIIIa and Variable Antibody-Dependent Cellular Cytotoxicity Activities.

Directory of Open Access Journals (Sweden)

Masaki Kurogochi

Full Text Available Many therapeutic antibodies have been developed, and IgG antibodies have been extensively generated in various cell expression systems. IgG antibodies contain N-glycans at the constant region of the heavy chain (Fc domain, and their N-glycosylation patterns differ during various processes or among cell expression systems. The Fc N-glycan can modulate the effector functions of IgG antibodies, such as antibody-dependent cellular cytotoxicity (ADCC and complement dependent cytotoxicity (CDC. To control Fc N-glycans, we performed a rearrangement of Fc N-glycans from a heterogeneous N-glycosylation pattern to homogeneous N-glycans using chemoenzymatic approaches with two types of endo-β-N-acetyl glucosaminidases (ENG'ases, one that works as a hydrolase to cleave all heterogeneous N-glycans, another that is used as a glycosynthase to generate homogeneous N-glycans. As starting materials, we used an anti-Her2 antibody produced in transgenic silkworm cocoon, which consists of non-fucosylated pauci-mannose type (Man2-3GlcNAc2, high-mannose type (Man4-9GlcNAc2, and complex type (Man3GlcNAc3-4 N-glycans. As a result of the cleavage of several ENG'ases (endoS, endoM, endoD, endoH, and endoLL, the heterogeneous glycans on antibodies were fully transformed into homogeneous-GlcNAc by a combination of endoS, endoD, and endoLL. Next, the desired N-glycans (M3; Man3GlcNAc1, G0; GlcNAc2Man3GlcNAc1, G2; Gal2GlcNAc2Man3GlcNAc1, A2; NeuAc2Gal2GlcNAc2Man3GlcNAc1 were transferred from the corresponding oxazolines to the GlcNAc residue on the intact anti-Her2 antibody with an ENG'ase mutant (endoS-D233Q, and the glycoengineered anti-Her2 antibody was obtained. The binding assay of anti-Her2 antibody with homogenous N-glycans with FcγRIIIa-V158 showed that the glycoform influenced the affinity for FcγRIIIa-V158. In addition, the ADCC assay for the glycoengineered anti-Her2 antibody (mAb-M3, mAb-G0, mAb-G2, and mAb-A2 was performed using SKBR-3 and BT-474 as target

Tyrosine kinase inhibitors as modulators of trastuzumab-mediated antibody-dependent cell-mediated cytotoxicity in breast cancer cell lines.

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Collins, Denis M; Gately, Kathy; Hughes, Clare; Edwards, Connla; Davies, Anthony; Madden, Stephen F; O'Byrne, Kenneth J; O'Donovan, Norma; Crown, John

2017-09-01

Trastuzumab is an anti-HER2 monoclonal antibody (mAb) therapy capable of antibody-dependent cell-mediated cytotoxicity (ADCC) and used in the treatment of HER2+ breast cancer. Through interactions with FcƴR+ immune cell subsets, trastuzumab functions as a passive immunotherapy. The EGFR/HER2-targeting tyrosine kinase inhibitor (TKI) lapatinib and the next generation TKIs afatinib and neratinib, can alter HER2 levels, potentially modulating the ADCC response to trastuzumab. Using LDH-release assays, we investigated the impact of antigen modulation, assay duration and peripheral blood mononuclear cell (PBMC) activity on trastuzumab-mediated ADCC in breast cancer models of maximal (SKBR3) and minimal (MCF-7) target antigen expression to determine if modulating the ADCC response to trastuzumab using TKIs may be a viable approach for enhancing tumor immune reactivity. HER2 levels were determined in lapatinib, afatinib and neratinib-treated SKBR3 and MCF-7 using high content analysis (HCA). Trastuzumab-mediated ADCC was assessed following treatment with TKIs utilising a colorimetric LDH release-based protocol at 4 and 12h timepoints. PBMC activity was assessed against non-MHC-restricted K562 cells. A flow cytometry-based method (CFSE/7-AAD) was also used to measure trastuzumab-mediated ADCC in medium-treated SKBR3 and MCF-7. HER2 antigen levels were significantly altered by the three TKIs in both cell line models. The TKIs significantly reduced LDH levels directly in SKBR3 cells but not MCF-7. Lapatinib and neratinib augment trastuzumab-related ADCC in SKBR3 but the effect was not consistent with antigen expression levels and was dependent on volunteer PBMC activity (vs. K562). A 12h assay timepoint produced more consistent results. Trastuzumab-mediated ADCC (PBMC:target cell ratio of 10:1) was measured at 7.6±4.7% (T12) by LDH assay and 19±3.2 % (T12) using the flow cytometry-based method in the antigen-low model MCF-7. In the presence of effector cells with high

Avelumab: combining immune checkpoint inhibition and antibody-dependent cytotoxicity.

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Hamilton, Gerhard; Rath, Barbara

2017-04-01

Immune checkpoint inhibition holds great promise for selected tumors. The human monoclonal antibody (mAB) avelumab is directed to programmed death ligand-1 (PD-L1) and is supposed to inhibit the immunosuppressive PD-L1/PD-1 interaction and, furthermore, effect antibody-dependent cytotoxicity (ADCC) lysis of tumor cells. Areas covered: This article presents an overview of the current means to activate the antitumor immune defense by targeting PD-1 or PD-L1 with mABs and their possible role in ADCC-mediated tumor cell elimination. Expert opinion: Avelumab contains a Fc region which can bind cognate receptors on immune effector cells and induce ADCC-mediated tumor cell lysis, in contrast to other mABs directed to PD-1/PD-L1 which lack the ability to trigger ADCC due to belonging to the IgG4 subclass or possessing a mutated Fc region. Preclinical and clinical data indicate that avelumab can be safely administered to cancer patients with a toxicity profile comparable to other mABs and without lysis of PD-L1-positive activated immune cells. This antibody yielded durable responses in a phase II trial in advanced Merkel cell carcinoma patients. Tumor cell lysis by avelumab prevents cells from resorting to alternative checkpoints as shown by targeting PD-1 and the upregulation of TIM-3.

An Fc engineering approach that modulates antibody-dependent cytokine release without altering cell-killing functions.

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Kinder, Michelle; Greenplate, Allison R; Strohl, William R; Jordan, Robert E; Brezski, Randall J

2015-01-01

Cytotoxic therapeutic monoclonal antibodies (mAbs) often mediate target cell-killing by eliciting immune effector functions via Fc region interactions with cellular and humoral components of the immune system. Key functions include antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC). However, there has been increased appreciation that along with cell-killing functions, the induction of antibody-dependent cytokine release (ADCR) can also influence disease microenvironments and therapeutic outcomes. Historically, most Fc engineering approaches have been aimed toward modulating ADCC, ADCP, or CDC. In the present study, we describe an Fc engineering approach that, while not resulting in impaired ADCC or ADCP, profoundly affects ADCR. As such, when peripheral blood mononuclear cells are used as effector cells against mAb-opsonized tumor cells, the described mAb variants elicit a similar profile and quantity of cytokines as IgG1. In contrast, although the variants elicit similar levels of tumor cell-killing as IgG1 with macrophage effector cells, the variants do not elicit macrophage-mediated ADCR against mAb-opsonized tumor cells. This study demonstrates that Fc engineering approaches can be employed to uncouple macrophage-mediated phagocytic and subsequent cell-killing functions from cytokine release.

Antigenic modulation limits the effector cell mechanisms employed by type I anti-CD20 monoclonal antibodies.

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Tipton, Thomas R W; Roghanian, Ali; Oldham, Robert J; Carter, Matthew J; Cox, Kerry L; Mockridge, C Ian; French, Ruth R; Dahal, Lekh N; Duriez, Patrick J; Hargreaves, Philip G; Cragg, Mark S; Beers, Stephen A

2015-03-19

Following the success of rituximab, 2 other anti-CD20 monoclonal antibodies (mAbs), ofatumumab and obinutuzumab, have entered clinical use. Ofatumumab has enhanced capacity for complement-dependent cytotoxicity, whereas obinutuzumab, a type II mAb, lacks the ability to redistribute into lipid rafts and is glycoengineered for augmented antibody-dependent cellular cytotoxicity (ADCC). We previously showed that type I mAbs such as rituximab have a propensity to undergo enhanced antigenic modulation compared with type II. Here we assessed the key effector mechanisms affected, comparing type I and II antibodies of various isotypes in ADCC and antibody-dependent cellular-phagocytosis (ADCP) assays. Rituximab and ofatumumab depleted both normal and leukemic human CD20-expressing B cells in the mouse less effectively than glycoengineered and wild-type forms of obinutuzumab, particularly when human immunoglobulin G1 (hIgG1) mAbs were compared. In contrast to mouse IgG2a, hIgG1 mAbs were ineffective in ADCC assays with murine natural killer cells as effectors, whereas ADCP was equivalent for mouse IgG2a and hIgG1. However, rituximab's ability to elicit both ADCC and ADCP was reduced by antigenic modulation, whereas type II antibodies remained unaffected. These data demonstrate that ADCP and ADCC are impaired by antigenic modulation and that ADCP is the main effector function employed in vivo. © 2015 by The American Society of Hematology.

Combination of two anti-CD5 monoclonal antibodies synergistically induces complement-dependent cytotoxicity of chronic lymphocytic leukaemia cells.

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Klitgaard, Josephine L; Koefoed, Klaus; Geisler, Christian; Gadeberg, Ole V; Frank, David A; Petersen, Jørgen; Jurlander, Jesper; Pedersen, Mikkel W

2013-10-01

The treatment of chronic lymphocytic leukaemia (CLL) has been improved by introduction of monoclonal antibodies (mAbs) that exert their effect through secondary effector mechanisms. CLL cells are characterized by expression of CD5 and CD23 along with CD19 and CD20, hence anti-CD5 Abs that engage secondary effector functions represent an attractive opportunity for CLL treatment. Here, a repertoire of mAbs against human CD5 was generated and tested for ability to induce complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) both as single mAbs and combinations of two mAbs against non-overlapping epitopes on human CD5. The results demonstrated that combinations of two mAbs significantly increased the level of CDC compared to the single mAbs, while no enhancement of ADCC was seen with anti-CD5 mAb combinations. High levels of CDC and ADCC correlated with low levels of Ab-induced CD5 internalization and degradation. Importantly, an anti-CD5 mAb combination enhanced CDC of CLL cells when combined with the anti-CD20 mAbs rituximab and ofatumumab as well as with the anti-CD52 mAb alemtuzumab. These results suggest that an anti-CD5 mAb combination inducing CDC and ADCC may be effective alone, in combination with mAbs against other targets or combined with chemotherapy for CLL and other CD5-expressing haematological or lymphoid malignancies. © 2013 John Wiley & Sons Ltd.

Drifts in ADCC-related quality attributes of Herceptin®: Impact on development of a trastuzumab biosimilar.

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Kim, Seokkyun; Song, Jinsu; Park, Seungkyu; Ham, Sunyoung; Paek, Kyungyeol; Kang, Minjung; Chae, Yunjung; Seo, Heewon; Kim, Hyung-Chan; Flores, Michael

A biosimilar product needs to demonstrate biosimilarity to the originator reference product, and the quality profile of the latter should be monitored throughout the period of the biosimilar's development to match the quality attributes of the 2 products that relate to efficacy and safety. For the development of a biosimilar version of trastuzumab, the reference product, Herceptin®, was extensively characterized for the main physicochemical and biologic properties by standard or state-of-the-art analytical methods, using multiple lots expiring between March 2015 and December 2019. For lots with expiry dates up to July 2018, a high degree of consistency was observed for all the tested properties. However, among the lots expiring in August 2018 or later, a downward drift was observed in %afucose (G0+G1+G2). Furthermore, the upward drift of %high mannose (M5+M6) was observed in the lots with expiry dates from June 2019 to December 2019. As a result, the combination of %afucose and %high mannose showed 2 marked drifts in the lots with expiry dates from August 2018 to December 2019, which was supported by the similar trend of biologic data, such as FcγRIIIa binding and antibody-dependent cell-mediated cytotoxicity (ADCC) activity. Considering that ADCC is one of the clinically relevant mechanisms of action for trastuzumab, the levels of %afucose and %high mannose should be tightly monitored as critical quality attributes for biosimilar development of trastuzumab.

Cytotoxicity and cellular uptake of tri-block copolymer nanoparticles with different size and surface characteristics

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Bhattacharjee Sourav

2012-04-01

Full Text Available Abstract Background Polymer nanoparticles (PNP are becoming increasingly important in nanomedicine and food-based applications. Size and surface characteristics are often considered to be important factors in the cellular interactions of these PNP, although systematic investigations on the role of surface properties on cellular interactions and toxicity of PNP are scarce. Results Fluorescent, monodisperse tri-block copolymer nanoparticles with different sizes (45 and 90 nm and surface charges (positive and negative were synthesized, characterized and studied for uptake and cytotoxicity in NR8383 and Caco-2 cells. All types of PNP were taken up by the cells. The positive smaller PNP45 (45 nm showed a higher cytotoxicity compared to the positive bigger PNP90 (90 nm particles including reduction in mitochondrial membrane potential (ΔΨm, induction of reactive oxygen species (ROS production, ATP depletion and TNF-α release. The negative PNP did not show any cytotoxic effect. Reduction in mitochondrial membrane potential (ΔΨm, uncoupling of the electron transfer chain in mitochondria and the resulting ATP depletion, induction of ROS and oxidative stress may all play a role in the possible mode of action for the cytotoxicity of these PNP. The role of receptor-mediated endocytosis in the intracellular uptake of different PNP was studied by confocal laser scanning microscopy (CLSM. Involvement of size and charge in the cellular uptake of PNP by clathrin (for positive PNP, caveolin (for negative PNP and mannose receptors (for hydroxylated PNP were found with smaller PNP45 showing stronger interactions with the receptors than bigger PNP90. Conclusions The size and surface characteristics of polymer nanoparticles (PNP; 45 and 90 nm with different surface charges play a crucial role in cellular uptake. Specific interactions with cell membrane-bound receptors (clathrin, caveolin and mannose leading to cellular internalization were observed to depend on

Strong antitumor activities of IgG3 antibodies to a human melanoma-associated ganglioside

International Nuclear Information System (INIS)

Hellstroem, I.; Brankovan, V.; Hellstroem, K.E.

1985-01-01

Three mouse monoclonal IgG3 antibodies, 2B2, IF4, and MG-21, recognize a G/sub D3/ ganglioside antigen that is expressed at the cell surface of most human melanomas. All three antibodies mediate antibody-dependent cellular cytotoxicity (ADCC) in vitro when tested with human lymphocytes or effector cells in a 2-hr or 4-hr 51 Cr-release test, and one antibody, MG-21, also gives strong complement-dependent cytotoxicity with human serum. Antibody 2B2, which gives ADDC also in the presence of mouse lymphocytes, inhibited the outgrowth of a human melanoma in nude mice, but antibody IF4, which showed no ADCC with mouse lymphocyte effectors, did not

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

DEFF Research Database (Denmark)

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

2013-01-01

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

How immunoglobulin G antibodies kill target cells: revisiting an old paradigm.

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Biburger, Markus; Lux, Anja; Nimmerjahn, Falk

2014-01-01

The capacity of immunoglobulin G (IgG) antibodies to eliminate virtually any target cell has resulted in the widespread introduction of cytotoxic antibodies into the clinic in settings of cancer therapy, autoimmunity, and transplantation, for example. More recently, it has become apparent that also the protection from viral infection via IgG antibodies may require cytotoxic effector functions, suggesting that antibody-dependent cellular cytotoxicity (ADCC) directed against malignant or virally infected cells is one of the most essential effector mechanisms triggered by IgG antibodies to protect the host. A detailed understanding of the underlying molecular and cellular pathways is critical, therefore, to make full use of this antibody effector function. Several studies over the last years have provided novel insights into the effector pathways and innate immune effector cells responsible for ADCC reactions. One of the most notable outcomes of many of these reports is that cells of the mononuclear phagocytic system rather than natural killer cells are critical for removal of IgG opsonized target cells in vivo. © 2014 Elsevier Inc. All rights reserved.

Cellular cytotoxic response induced by highly purified multi-wall carbon nanotube in human lung cells.

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Tsukahara, Tamotsu; Haniu, Hisao

2011-06-01

Carbon nanotubes, a promising nanomaterial with unique characteristics, have applications in a variety of fields. The cytotoxic effects of carbon nanotubes are partially due to the induction of oxidative stress; however, the detailed mechanisms of nanotube cytotoxicity and their interaction with cells remain unclear. In this study, the authors focus on the acute toxicity of vapor-grown carbon fiber, HTT2800, which is one of the most highly purified multi-wall carbon nanotubes (MWCNT) by high-temperature thermal treatment. The authors exposed human bronchial epithelial cells (BEAS-2B) to HTT2800 and measured the cellular uptake, mitochondrial function, cellular LDH release, apoptotic signaling, reactive oxygen species (ROS) generation and pro-inflammatory cytokine release. The HTT2800-exposed cells showed cellular uptake of the carbon nanotube, increased cell death, enhanced DNA damage, and induced cytokine release. However, the exposed cells showed no obvious intracellular ROS generation. These cellular and molecular findings suggest that HTT2800 could cause a potentially adverse inflammatory response in BEAS-2B cells.

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

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Rajalingam, Raja

2016-01-01

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

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

Directory of Open Access Journals (Sweden)

Raja Rajalingam

2016-12-01

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

ADCC employing an NK cell line (haNK) expressing the high affinity CD16 allele with avelumab, an anti-PD-L1 antibody.

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Jochems, Caroline; Hodge, James W; Fantini, Massimo; Tsang, Kwong Y; Vandeveer, Amanda J; Gulley, James L; Schlom, Jeffrey

2017-08-01

NK-92 cells, and their derivative, designated aNK, were obtained from a patient with non-Hodgkin lymphoma. Prior clinical studies employing adoptively transferred irradiated aNK cells have provided evidence of clinical benefit and an acceptable safety profile. aNK cells have now been engineered to express IL-2 and the high affinity (ha) CD16 allele (designated haNK). Avelumab is a human IgG1 anti-PD-L1 monoclonal antibody, which has shown evidence of clinical activity in a range of human tumors. Prior in vitro studies have shown that avelumab has the ability to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) of human tumor cells when combined with NK cells. In the studies reported here, the ability of avelumab to enhance the lysis of a range of human carcinoma cells by irradiated haNK cells via the ADCC mechanism is demonstrated; this ADCC is shown to be inhibited by anti-CD16 blocking antibody and by concanamycin A, indicating the use of the granzyme/perforin pathway in tumor cell lysis. Studies also show that while NK cells have the ability to lyse aNK or haNK cells, the addition of NK cells to irradiated haNK cells does not inhibit haNK-mediated lysis of human tumor cells, with or without the addition of avelumab. Avelumab-mediated lysis of tumor cells by irradiated haNK cells is also shown to be similar to that of NK cells bearing the V/V Fc receptor high affinity allele. These studies thus provide the rationale for the clinical evaluation of the combined use of avelumab with that of irradiated adoptively transferred haNK cells. © 2017 UICC.

Cellular distribution of inorganic mercury and its relation to cytotoxicity in bovine kidney cell cultures

International Nuclear Information System (INIS)

Bracken, W.M.; Sharma, R.P.; Bourcier, D.R.

1984-01-01

A bovine kidney cell culture system was used to assess what relationship mercuric chloride (HgCl 2 ) uptake and subcellular distribution had to cytotoxicity. Twenty-four-hour incubations with 0.05-50 μM HgCl 2 elicited a concentration-related cytotoxicity. Cellular accumulation of 203 Hg was also concentration-related, with 1.0 nmol/10 6 cells at the IC50. Measurement of Hg uptake over the 24-h exposure period revealed a multiphasic process. Peak accumulation was attained by 1 h and was followed by extrusion and plateauing of intracellular Hg levels. Least-squares regression analysis of the cytotoxicity and cellular uptake data indicated a potential relationship between the Hg uptake and cytotoxicity. However, the subcellular distribution of Hg was not concentration-related. Mitochondria and soluble protein fractions accounted for greater than 65% of the cell-associated Hg at all concentrations. The remaining Hg was distributed between the microsomal (6-10%) and nuclear and cell debris (11-22%) fractions at all concentrations tested. Less than 20% of the total cell-associated Hg was bound with metallothionein-like protein. 31 references, 4 figures, 3 tables

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

Science.gov (United States)

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

2016-06-07

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

Cellular uptake and cytotoxicity of positively charged chitosan gold nanoparticles in human lung adenocarcinoma cells

Energy Technology Data Exchange (ETDEWEB)

Choi, Seon Young; Jang, Soo Hwa [Seoul National University, Laboratory of Veterinary Pharmacology, College of Veterinary Medicine and Institute for Veterinary Science (Korea, Republic of); Park, Jin; Jeong, Saeromi; Park, Jin Ho; Ock, Kwang Su [Soongsil University, Department of Chemistry (Korea, Republic of); Lee, Kangtaek [Yonsei University, Department of Chemical and Biomolecular Engineering (Korea, Republic of); Yang, Sung Ik [Kyung Hee University, College of Environment and Applied Chemistry (Korea, Republic of); Joo, Sang-Woo, E-mail: sjoo@ssu.ac.kr [Soongsil University, Department of Chemistry (Korea, Republic of); Ryu, Pan Dong; Lee, So Yeong, E-mail: leeso@snu.ac.kr [Seoul National University, Laboratory of Veterinary Pharmacology, College of Veterinary Medicine and Institute for Veterinary Science (Korea, Republic of)

2012-12-15

Cellular uptake, cytotoxicity, and mechanisms of cytotoxicity of the positively charged Au nanoparticles (NPs) were examined in A549 cells, which are one of the most characterized pulmonary cellular systems. Positively charged Au NPs were prepared by chemical reduction using chitosan. The dimension and surface charge of Au NPs were examined by transmission electron microscopy (TEM), dynamic light scattering, and zeta potential measurements. The uptake of Au NPs into A549 cells was also monitored using TEM and dark-field microscopy (DFM) and z-stack confocal microRaman spectroscopy. DFM live cell imaging was also performed to monitor the entry of chitosan Au NPs in real time. The cytotoxic assay, using both methylthiazol tetrazolium and lactate dehydrogenase assays revealed that positively charged Au NPs decreased cell viability. Flow cytometry, DNA fragmentation, real-time PCR, and western blot analysis suggest that positively charged chitosan Au NPs provoke cell damage through both apoptotic and necrotic pathways.

Cellular uptake and cytotoxicity of positively charged chitosan gold nanoparticles in human lung adenocarcinoma cells

International Nuclear Information System (INIS)

Choi, Seon Young; Jang, Soo Hwa; Park, Jin; Jeong, Saeromi; Park, Jin Ho; Ock, Kwang Su; Lee, Kangtaek; Yang, Sung Ik; Joo, Sang-Woo; Ryu, Pan Dong; Lee, So Yeong

2012-01-01

Cellular uptake, cytotoxicity, and mechanisms of cytotoxicity of the positively charged Au nanoparticles (NPs) were examined in A549 cells, which are one of the most characterized pulmonary cellular systems. Positively charged Au NPs were prepared by chemical reduction using chitosan. The dimension and surface charge of Au NPs were examined by transmission electron microscopy (TEM), dynamic light scattering, and zeta potential measurements. The uptake of Au NPs into A549 cells was also monitored using TEM and dark-field microscopy (DFM) and z-stack confocal microRaman spectroscopy. DFM live cell imaging was also performed to monitor the entry of chitosan Au NPs in real time. The cytotoxic assay, using both methylthiazol tetrazolium and lactate dehydrogenase assays revealed that positively charged Au NPs decreased cell viability. Flow cytometry, DNA fragmentation, real-time PCR, and western blot analysis suggest that positively charged chitosan Au NPs provoke cell damage through both apoptotic and necrotic pathways.

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

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

2013-12-12

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

Malignant mesothelioma effusions are infiltrated by CD3+ T cells highly expressing PD-L1 and the PD-L1+ tumor cells within these effusions are susceptible to ADCC by the anti-PD-L1 antibody avelumab

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Khanna, Swati; Thomas, Anish; Abate-Daga, Daniel; Zhang, Jingli; Morrow, Betsy; Steinberg, Seth M.; Orlandi, Augusto; Ferroni, Patrizia; Schlom, Jeffrey; Guadagni, Fiorella; Hassan, Raffit

2016-01-01

INTRODUCTION The functional aspects of programmed death 1 (PD-1) and PD ligand 1 (PD-L1) immune checkpoints in malignant mesothelioma have not been studied. METHODS Tumor samples from 65 patients with mesothelioma were evaluated for PD-L1 expression by immunohistochemistry and its prognostic significance. Malignant effusions from patients with pleural and peritoneal mesothelioma were evaluated for PD-1+ and PD-L1+ infiltrating lymphocytes and their role in inducing tumor cell PD-L1 expression. Antibody dependent cellular cytotoxicity (ADCC) of avelumab, a fully humanized IgG1 anti PD-L1 antibody towards primary mesothelioma cell lines was evaluated in presence of autologous and allogeneic NK cells. RESULTS Of 65 pleural and peritoneal mesothelioma tumors examined, 41 (63%) were PD-L1 positive, which was associated with slightly inferior overall survival compared to patients with PD-L1 negative tumors (median 23.0 vs. 33.3 months; p=0.35). The frequency of PD-L1 expression was similar in pleural and peritoneal mesothelioma patients with 62% and 64% of samples positive, respectively. Of nine mesothelioma effusion samples evaluated, the fraction of cells expressing PD-L1 ranged from 12 to 83%. Of 7 patients with paired malignant effusion and peripheral blood mononuclear cells (PBMC) samples, PD-L1 expression was significantly higher on CD3+ T cells present in malignant effusions as compared with PBMC (p=0.016). In addition, CD14+PD-1+ cells were elevated in malignant effusions compared with PBMC (p=0.031). The lymphocytes present in malignant effusions recognized autologous tumor cells and induced IFN-γ-mediated PD-L1 expression on the tumor cell surface. Of the three primary mesothelioma cell lines tested, two were susceptible to avelumab mediated ADCC in presence of autologous NK cells. CONCLUSION The majority of pleural as well as peritoneal mesothelioma express PD-L1. Malignant effusions in this disease are characterized by presence of tumor cells and CD3+ T

Poly-L-arginine: Enhancing Cytotoxicity and Cellular Uptake of Doxorubicin and Necrotic Cell Death.

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Movafegh, Bahareh; Jalal, Razieh; Mohammadi, Zobeideh; Aldaghi, Seyyede Araste

2018-04-11

Cell resistance to doxorubicin and its toxicity to healthy tissue reduce its efficiency. The use of cell penetrating peptides as drug delivery system along with doxorubicin is a strategy to reduce its side effects. In this study, the influence of poly-L-arginine on doxorubicin cytotoxicity, its cellular uptake and doxorubicin-induced apoptosis on human prostate cancer DU145 cells are assessed. The cytotoxicity of doxorubicin and poly-L-arginine, alone and in combination, in DU145 cells was evaluated at different exposure times using MTT assay. The influence of poly-L-arginine on doxorubicin delivery into cells was evaluated by fluorescence microscopy and ultraviolet spectroscopy. DAPI and ethidium bromide-acridine orange stainings, flow cytometry using annexin V/propidium iodide, western blot analysis with anti-p21 antibody and caspase-3 activity were used to examine the influence of poly-L-arginine on doxorubicin-induced cell death. Poly-L-arginine had no cytotoxicity at low concentrations and short exposure times. Poly-L-arginine increased the cytotoxic effect of doxorubicin in DU145 cells in a time-dependent manner. But no significant reduction was found in HFF cell viability. Poly-L-arginine seems to facilitate doxorubicin uptake and increase its intracellular concentration. 24 h combined treatment of cells with doxorubicin (0.5 μM) and poly-L-arginine (1 μg ml-1) caused a small increase in doxorubicin-induced apoptosis and significant elevated necrosis in DU145 cells as compared to each agent alone. Conlusion: Our results indicate that poly-L-arginine at lowest and highest concentrations act as proliferation-inducing and antiproliferative agents, respectively. Between these concentrations, poly-L-arginine increases the cellular uptake of doxorubicin and its cytotoxicity through induction of necrosis. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Malignant Mesothelioma Effusions Are Infiltrated by CD3+ T Cells Highly Expressing PD-L1 and the PD-L1+ Tumor Cells within These Effusions Are Susceptible to ADCC by the Anti-PD-L1 Antibody Avelumab.

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Khanna, Swati; Thomas, Anish; Abate-Daga, Daniel; Zhang, Jingli; Morrow, Betsy; Steinberg, Seth M; Orlandi, Augusto; Ferroni, Patrizia; Schlom, Jeffrey; Guadagni, Fiorella; Hassan, Raffit

2016-11-01

The functional aspects of programmed death 1 (PD-1) and PD ligand 1 (PD-L1) immune checkpoints in malignant mesothelioma have not been studied. Tumor samples from 65 patients with mesothelioma were evaluated for PD-L1 expression by immunohistochemistry, and its prognostic significance was examined. Malignant effusions from patients with pleural and peritoneal mesothelioma were evaluated for PD-1-positive and PD-L1-positive infiltrating lymphocytes and their role in inducing PD-L1 expression in tumor cells. Antibody-dependent cellular cytotoxicity (ADCC) of avelumab, a fully humanized immunoglobulin G1 anti PD-L1 antibody against primary mesothelioma cell lines, was evaluated in presence of autologous and allogeneic natural killer cells. Of 65 pleural and peritoneal mesothelioma tumors examined, 41 (63%) were PD-L1-positive, which was associated with slightly inferior overall survival compared to patients with PD-L1-negative tumors (median 23.0 versus 33.3 months, p = 0.35). The frequency of PD-L1 expression was similar in patients with pleural and peritoneal mesothelioma, with 62% and 64% of samples testing positive, respectively. In nine mesothelioma effusion samples evaluated, the fraction of cells expressing PD-L1 ranged from 12% to 83%. In seven patients with paired malignant effusion and peripheral blood mononuclear cell (PBMC) samples, PD-L1 expression was significantly higher on CD3-positive T cells present in malignant effusions as compared with PBMCs (p = 0.016). In addition, the numbers of CD14-positive PD-1-positive cells were increased in malignant effusions compared with PBMCs (p = 0.031). The lymphocytes present in malignant effusions recognized autologous tumor cells and induced interferon-γ-mediated PD-L1 expression on the tumor cell surface. Of the three primary mesothelioma cell lines tested, two were susceptible to avelumab-mediated ADCC in the presence of autologous natural killer cells. Most pleural as well as peritoneal mesotheliomas

Evaluation of the Cytotoxicity and Genotoxicity of Flavonolignans in Different Cellular Models

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Michal Bijak

2017-12-01

Full Text Available Flavonolignans are the main components of silymarin, which represents 1.5–3% of the dry fruit weight of Milk thistle (Silybum marianum L. Gaernt.. In ancient Greece and Romania, physicians and herbalists used the Silybum marianum to treat a range of liver diseases. Besides their hepatoprotective action, silymarin flavonolignans have many other healthy properties, such as anti-platelet and anti-inflammatory actions. The aim of this study was to evaluate the toxic effect of flavonolignans on blood platelets, peripheral blood mononuclear cells (PBMCs and human lung cancer cell line—A549—using different molecular techniques. We established that three major flavonolignans: silybin, silychristin and silydianin, in concentrations of up to 100 µM, have neither a cytotoxic nor genotoxic effect on blood platelets, PMBCs and A549. We also saw that silybin and silychristin have a protective effect on cellular mitochondria, observed as a reduction of spontaneous mitochondrial DNA (mtDNA damage in A549, measured as mtDNA copies, and mtDNA lesions in ND1 and ND5 genes. Additionally, we observed that flavonolignans increase the blood platelets’ mitochondrial membrane potential and reduce the generation of reactive oxygen species in blood platelets. Our current findings show for the first time that the three major flavonolignans, silybin, silychristin and silydianin, do not have any cytotoxicity and genotoxicity in various cellular models, and that they actually protect cellular mitochondria. This proves that the antiplatelet and anti-inflammatory effect of these compounds is part of our molecular health mechanisms.

The role of surface charge in cellular uptake and cytotoxicity of medical nanoparticles

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Fröhlich E

2012-11-01

Full Text Available Eleonore FröhlichCenter for Medical Research, Medical University of Graz, Graz, AustriaAbstract: Many types of nanoparticles (NPs are tested for use in medical products, particularly in imaging and gene and drug delivery. For these applications, cellular uptake is usually a prerequisite and is governed in addition to size by surface characteristics such as hydrophobicity and charge. Although positive charge appears to improve the efficacy of imaging, gene transfer, and drug delivery, a higher cytotoxicity of such constructs has been reported. This review summarizes findings on the role of surface charge on cytotoxicity in general, action on specific cellular targets, modes of toxic action, cellular uptake, and intracellular localization of NPs. Effects of serum and intercell type differences are addressed. Cationic NPs cause more pronounced disruption of plasma-membrane integrity, stronger mitochondrial and lysosomal damage, and a higher number of autophagosomes than anionic NPs. In general, nonphagocytic cells ingest cationic NPs to a higher extent, but charge density and hydrophobicity are equally important; phagocytic cells preferentially take up anionic NPs. Cells do not use different uptake routes for cationic and anionic NPs, but high uptake rates are usually linked to greater biological effects. The different uptake preferences of phagocytic and nonphagocytic cells for cationic and anionic NPs may influence the efficacy and selectivity of NPs for drug delivery and imaging.Keywords: endocytosis, plasma membrane, lysosomes, polystyrene particles, quantum dots, dendrimers

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

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

2001-04-15

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

Health and Cellular Impacts of Air Pollutants: From Cytoprotection to Cytotoxicity

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Karine Andreau

2012-01-01

Full Text Available Air pollution as one of the ravages of our modern societies is primarily linked to urban centers, industrial activities, or road traffic. These atmospheric pollutants have been incriminated in deleterious health effects by numerous epidemiological and in vitro studies. Environmental air pollutants are a heterogeneous mixture of particles suspended into a liquid and gaseous phase which trigger the disruption of redox homeostasis—known under the term of cellular oxidative stress—in relation with the establishment of inflammation and cell death via necrosis, apoptosis, or autophagy. Activation or repression of the apoptotic process as an adaptative response to xenobiotics might lead to either acute or chronic toxicity. The purpose of this paper is to highlight the central role of oxidative stress induced by air pollutants and to focus on the subsequent cellular impacts ranging from cytoprotection to cytotoxicity by decreasing or stimulating apoptosis, respectively.

Contribution of nonneutralizing vaccine-elicited antibody activities to improved protective efficacy in rhesus macaques immunized with Tat/Env compared with multigenic vaccines.

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Florese, Ruth H; Demberg, Thorsten; Xiao, Peng; Kuller, LaRene; Larsen, Kay; Summers, L Ebonita; Venzon, David; Cafaro, Aurelio; Ensoli, Barbara; Robert-Guroff, Marjorie

2009-03-15

Previously, chronic-phase protection against SHIV(89.6P) challenge was significantly greater in macaques primed with replicating adenovirus type 5 host range mutant (Ad5hr) recombinants encoding HIVtat and env and boosted with Tat and Env protein compared with macaques primed with multigenic adenovirus recombinants (HIVtat, HIVenv, SIVgag, SIVnef) and boosted with Tat, Env, and Nef proteins. The greater protection was correlated with Tat- and Env-binding Abs. Because the macaques lacked SHIV(89.6P)-neutralizing activity prechallenge, we investigated whether Ab-dependent cellular cytotoxicity (ADCC) and Ab-dependent cell-mediated viral inhibition (ADCVI) might exert a protective effect. We clearly show that Tat can serve as an ADCC target, although the Tat-specific activity elicited did not correlate with better protection. However, Env-specific ADCC activity was consistently higher in the Tat/Env group, with sustained cell killing postchallenge exhibited at higher levels (p vaccine regimens.

Antibody dependent cellular phagocytosis by macrophages is a novel mechanism of action of elotuzumab.

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Kurdi, Ahmed T; Glavey, Siobhan V; Bezman, Natalie A; Jhatakia, Amy; Guerriero, Jennifer L; Manier, Salomon; Moschetta, Michele; Mishima, Yuji; Roccaro, Aldo; Detappe, Alexandre; Liu, Chia-Jen; Sacco, Antonio; Huynh, Daisy; Tai, Yu-Tzu; Robbins, Michael D; Azzi, Jamil; Ghobrial, Irene M

2018-04-13

Elotuzumab, a recently approved antibody for the treatment of multiple myeloma (MM), has been shown to stimulate Fcγ receptor (FcγR)-mediated antibody-dependent cellular cytotoxicity (ADCC) by natural killer (NK) cells towards myeloma cells. The modulatory effects of elotuzumab on other effector cells in the tumor microenvironment, however, has not been fully explored. Antibody dependent cellular phagocytosis (ADCP) is a mechanism by which macrophages contribute to anti-tumor potency of monoclonal antibodies. Herein, we studied the NK cell independent effect of elotuzumab on tumor associated macrophages (TAMs) using a xenograft tumor model deficient in NK and adaptive immune cells. We demonstrate significant anti-tumor efficacy of single agent elotuzumab in immunocompromised xenograft models of multiple myeloma, which is in part mediated by Fc-FcγR interaction of elotuzumab with macrophages. Elotuzumab is shown in this study to induce phenotypic activation of macrophages in-vivo and mediates ADCP of myeloma cells though a FcγR dependent manner in-vitro. Together, these findings propose a novel immune mediated mechanism by which elotuzumab exerts anti-myeloma activity and helps to provide rationale for combination therapies that can enhance macrophage activity. Copyright ©2018, American Association for Cancer Research.

Correlation between NK function and response to trastuzumab in metastatic breast cancer patients

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Spadi Rosella

2008-05-01

Full Text Available Abstract Background Trastuzumab is a monoclonal antibody selectively directed against Her2 and approved for the treatment of Her2 overexpressing breast cancer patients. Its proposed mechanisms of action include mediation of antibody-dependent cellular cytotoxicity (ADCC by triggering FcγRIII on natural killer (NK cells. This study addresses the correlation between overall NK function and trastuzumab's clinical activity. Subjects and methods Clinical and immunological responses were assessed in 26 patients receiving trastuzumab monotherapy as maintenance management after chemotherapy (8 mg/kg load and then standard doses of 6 mg/kg every 3 weeks. Cytotoxic activity against the MHC class I-negative standard NK target K562 cell line and HER2-specific ADCC against a trastuzumab-coated Her2-positive SKBR3 cell line were assessed in peripheral blood mononuclear cells (PBMC harvested after the first standard dose. After six months, seventeen patients were scored as responders and nine as non-responders according to the RECIST criteria, while Progression-Free Survival (PFS was calculated during a 12 months follow-up. Results The responders had significantly higher levels of both NK and ADCC activities (p Conclusion One of the mechanisms of action of trastuzumab is NK cell-mediated ADCC lysis of the Her2-positve target cell. We show here that its potency is correlated with the short-term response to treatment, whereas longer protection against tumor expansion seems to be mediated by pure NK activity.

Cellular uptake and cytotoxic potential of respirable bentonite particles with different quartz contents and chemical modifications in human lung fibroblasts

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Geh, Stefan; Rettenmeier, Albert W.; Dopp, Elke [University Hospital, Institute of Hygiene and Occupational Medicine, Essen (Germany); Yuecel, Raif [University Hospital, Institute of Cell Biology (Cancer Research), Essen (Germany); Duffin, Rodger [Institute of Environmental Health Research (IUF), Duesseldorf (Germany); University of Edinburgh, ELEGI COLT Lab, Scotland (United Kingdom); Albrecht, Catrin; Borm, Paul J.A. [Institute of Environmental Health Research (IUF), Duesseldorf (Germany); Armbruster, Lorenz [Verein fuer Technische Sicherheit und Umweltschutz e.V., Gotha (Germany); Raulf-Heimsoth, Monika; Bruening, Thomas [Research Institute for Occupational Medicine of the Institutions for Statutory Accident Insurance and Prevention (BGFA), Bochum (Germany); Hoffmann, Eik [University of Rostock, Institute of Biology, Department of Cell Biology and Biosystems Technology, Rostock (Germany)

2006-02-01

Considering the biological reactivity of pure quartz in lung cells, there is a strong interest to clarify the cellular effects of respirable siliceous dusts, like bentonites. In the present study, we investigated the cellular uptake and the cytotoxic potential of bentonite particles (Oe< 10 {mu}m) with an {alpha}-quartz content of up to 6% and different chemical modifications (activation: alkaline, acidic, organic) in human lung fibroblasts (IMR90). Additionally, the ability of the particles to induce apoptosis in IMR90-cells and the hemolytic activity was tested. All bentonite samples were tested for endotoxins with the in vitro-Pyrogen test and were found to be negative. Cellular uptake of particles by IMR90-cells was studied by transmission electron microscopy (TEM). Cytotoxicity was analyzed in IMR90-cells by determination of viable cells using flow cytometry and by measuring of the cell respiratory activity. Induced apoptotic cells were detected by AnnexinV/Propidiumiodide-staining and gel electrophoresis. Our results demonstrate that activated bentonite particles are better taken up by IMR90-cells than untreated (native) bentonite particles. Also, activated bentonite particles with a quartz content of 5-6% were more cytotoxic than untreated bentonites or bentonites with a quartz content lower than 4%. The bentonite samples induced necrotic as well as apoptotic cell death. In general, bentonites showed a high membrane-damaging potential shown as hemolytic activity in human erythrocytes. We conclude that cellular effects of bentonite particles in human lung cells are enhanced after chemical treatment of the particles. The cytotoxic potential of the different bentonites is primarily characterized by a strong lysis of the cell membrane. (orig.)

Short communication: Anti-HIV-1 envelope immunoglobulin Gs in blood and cervicovaginal samples of Beninese commercial sex workers.

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Batraville, Laurie-Anne; Richard, Jonathan; Veillette, Maxime; Labbé, Annie-Claude; Alary, Michel; Guédou, Fernand; Kaufmann, Daniel E; Poudrier, Johanne; Finzi, Andrés; Roger, Michel

2014-11-01

Characterization of the immune correlates of protection against HIV infection is crucial for the development of preventive strategies. This study examined HIV-1 envelope (Env) glycoproteins, specifically immunoglobulin G (IgG), in systemic and mucosal compartments of female Beninese commercial sex workers (CSWs). Samples of 23 HIV-1-positive and 20 highly exposed HIV-1-seronegative (HESN) CSWs were studied. HIV-1 Env-specific IgG detection in sera and cervicovaginal lavages (CVLs) from the study population was done by cell-based ELISA. The HIV neutralizing activity was evaluated with a neutralization assay. The HIV-1-specific antibody-dependent cellular cytotoxicity (ADCC) response of the cohort was measured with a FACS-based assay evaluating the ADCC-mediated elimination of gp120-coated target cells. No anti-HIV-1 Env-specific IgG neutralizing or ADCC activities were detected in samples from HESN CSWs. Samples from HIV-1-infected CSWs presented ADCC activity in both sera and CVLs. Anti-Env IgG from sera and CVLs from HIV-1-infected CSWs preferentially recognized Env in its CD4-bound conformation. HIV-1-infected CSWs have ADCC-mediating IgG that preferentially recognizes Env in its CD4-bound conformation at the mucosal site.

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

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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

Monoclonal Antibodies, Derived from Humans Vaccinated with the RV144 HIV Vaccine Containing the HVEM Binding Domain of Herpes Simplex Virus (HSV) Glycoprotein D, Neutralize HSV Infection, Mediate Antibody-Dependent Cellular Cytotoxicity, and Protect Mice from Ocular Challenge with HSV-1.

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Wang, Kening; Tomaras, Georgia D; Jegaskanda, Sinthujan; Moody, M Anthony; Liao, Hua-Xin; Goodman, Kyle N; Berman, Phillip W; Rerks-Ngarm, Supachai; Pitisuttithum, Punnee; Nitayapan, Sorachai; Kaewkungwal, Jaranit; Haynes, Barton F; Cohen, Jeffrey I

2017-10-01

The RV144 HIV vaccine trial included a recombinant HIV glycoprotein 120 (gp120) construct fused to a small portion of herpes simplex virus 1 (HSV-1) glycoprotein D (gD) so that the first 40 amino acids of gp120 were replaced by the signal sequence and the first 27 amino acids of the mature form of gD. This region of gD contains most of the binding site for HVEM, an HSV receptor important for virus infection of epithelial cells and lymphocytes. RV144 induced antibodies to HIV that were partially protective against infection, as well as antibodies to HSV. We derived monoclonal antibodies (MAbs) from peripheral blood B cells of recipients of the RV144 HIV vaccine and showed that these antibodies neutralized HSV-1 infection in cells expressing HVEM, but not the other major virus receptor, nectin-1. The MAbs mediated antibody-dependent cellular cytotoxicity (ADCC), and mice that received the MAbs and were then challenged by corneal inoculation with HSV-1 had reduced eye disease, shedding, and latent infection. To our knowledge, this is the first description of MAbs derived from human recipients of a vaccine that specifically target the HVEM binding site of gD. In summary, we found that monoclonal antibodies derived from humans vaccinated with the HVEM binding domain of HSV-1 gD (i) neutralized HSV-1 infection in a cell receptor-specific manner, (ii) mediated ADCC, and (iii) reduced ocular disease in virus-infected mice. IMPORTANCE Herpes simplex virus 1 (HSV-1) causes cold sores and neonatal herpes and is a leading cause of blindness. Despite many trials, no HSV vaccine has been approved. Nectin-1 and HVEM are the two major cellular receptors for HSV. These receptors are expressed at different levels in various tissues, and the role of each receptor in HSV pathogenesis is not well understood. We derived human monoclonal antibodies from persons who received the HIV RV144 vaccine that contained the HVEM binding domain of HSV-1 gD fused to HIV gp120. These antibodies were

Cytotoxicity and cellular uptake of doxorubicin and its formamidine derivatives in HL60 sensitive and HL60/MX2 resistant cells.

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Kik, Krzysztof; Wasowska-Lukawska, Malgorzata; Oszczapowicz, Irena; Szmigiero, Leszek

2009-04-01

In this work a comparison was made of the cytotoxicity and cellular uptake of doxorubicin (DOX) and two of its derivatives containing a formamidino group (-N=CH-N<) at the 3' position with morpholine (DOXM) or hexamethyleneimine (DOXH) ring. All tests were performed in doxorubicin-sensitive HL60 and -resistant HL60/MX2 cells which are known for the presence of altered topoisomerase II. Cytotoxic activity of DOX toward HL60/MX2 cells was about 195 times lower when compared with the sensitive HL60 cell line. DOXM and DOXH were approximately 20 times more active in resistant cells than DOX. It was found that the uptake of DOX was lower in resistant cells by about 16%, while that of DOXM and DOXH was lower by about 36% and 19%, respectively. Thus the changes in the cellular uptake of anthracyclines are not associated with the fact that cytotoxicity of DOXM and DOXH exceed the cytotoxicity of DOX. Experiments in cell-free system containing human topoisomerase II showed that topoisomerase II is not inhibited by DOXM and DOXH. Formamidinoanthracyclines may be more useful than parent drugs in therapy against tumor cells with altered topoisomerase II activity.

Enhanced antibody-dependent cellular phagocytosis by chimeric monoclonal antibodies with tandemly repeated Fc domains.

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Nagashima, Hiroaki; Ootsubo, Michiko; Fukazawa, Mizuki; Motoi, Sotaro; Konakahara, Shu; Masuho, Yasuhiko

2011-04-01

We previously reported that chimeric monoclonal antibodies (mAbs) with tandemly repeated Fc domains, which were developed by introducing tandem repeats of Fc domains downstream of 2 Fab domains, augmented binding avidities for all Fcγ receptors, resulting in enhanced antibody (Ab)-dependent cellular cytotoxicity. Here we investigated regarding Ab-dependent cellular phagocytosis (ADCP) mediated by these chimeric mAbs, which is considered one of the most important mechanisms that kills tumor cells, using two-color flow cytometric methods. ADCP mediated by T3-Ab, a chimeric mAb with 3 tandemly repeated Fc domains, was 5 times more potent than that by native anti-CD20 M-Ab (M-Ab hereafter). Furthermore, T3-Ab-mediated ADCP was resistant to competitive inhibition by intravenous Ig (IVIG), although M-Ab-mediated ADCP decreased in the presence of IVIG. An Fcγ receptor-blocking study demonstrated that T3-Ab mediated ADCP via both FcγRIA and FcγRIIA, whereas M-Ab mediated ADCP exclusively via FcγRIA. These results suggest that chimeric mAbs with tandemly repeated Fc domains enhance ADCP as well as ADCC, and that Fc multimerization may significantly enhance the efficacy of therapeutic Abs. Copyright © 2010 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Daratumumab: a first-in-class CD38 monoclonal antibody for the treatment of multiple myeloma

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Larysa Sanchez

2016-06-01

Full Text Available Abstract Daratumumab is a human monoclonal antibody that targets CD38, a cell surface protein that is overexpressed on multiple myeloma (MM cells. Preclinical studies have shown that daratumumab induces MM cell death through several mechanisms, including complement-dependent cytotoxicity (CDC, antibody-dependent cell-mediated cytotoxicity (ADCC, antibody-dependent cellular phagocytosis (ADCP, and apoptosis. Given the encouraging efficacy and acceptable safety profile of daratumumab demonstrated in clinical trials, daratumumab has emerged as a novel treatment option for myeloma and became the first monoclonal antibody approved by the FDA for the treatment of MM.

Enhancement of antibody-dependent cell-mediated cytotoxicity by endowing IgG with FcαRI (CD89) binding.

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Borrok, M Jack; Luheshi, Nadia M; Beyaz, Nurten; Davies, Gareth C; Legg, James W; Wu, Herren; Dall'Acqua, William F; Tsui, Ping

2015-01-01

Fc effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP) are crucial to the efficacy of many antibody therapeutics. In addition to IgG, antibodies of the IgA isotype can also promote cell killing through engagement of myeloid lineage cells via interactions between the IgA-Fc and FcαRI (CD89). Herein, we describe a unique, tandem IgG1/IgA2 antibody format in the context of a trastuzumab variable domain that exhibits enhanced ADCC and ADCP capabilities. The IgG1/IgA2 tandem Fc format retains IgG1 FcγR binding as well as FcRn-mediated serum persistence, yet is augmented with myeloid cell-mediated effector functions via FcαRI/IgA Fc interactions. In this work, we demonstrate anti-human epidermal growth factor receptor-2 antibodies with the unique tandem IgG1/IgA2 Fc can better recruit and engage cytotoxic polymorphonuclear (PMN) cells than either the parental IgG1 or IgA2. Pharmacokinetics of IgG1/IgA2 in BALB/c mice are similar to the parental IgG, and far surpass the poor serum persistence of IgA2. The IgG1/IgA2 format is expressed at similar levels and with similar thermal stability to IgG1, and can be purified via standard protein A chromatography. The tandem IgG1/IgA2 format could potentially augment IgG-based immunotherapeutics with enhanced PMN-mediated cytotoxicity while avoiding many of the problems associated with developing IgAs.

Different cellular response mechanisms contribute to the length-dependent cytotoxicity of multi-walled carbon nanotubes

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Liu, Dun; Wang, Lijun; Wang, Zhigang; Cuschieri, Alfred

2012-07-01

To date, there has not been an agreement on the best methods for the characterisation of multi-walled carbon nanotube (MWCNT) toxicity. The length of MWCNTs has been identified as a factor in in vitro and in vivo studies, in addition to their purity and biocompatible coating. Another unresolved issue relates to the variable toxicity of MWCNTs on different cell types. The present study addressed the effects of MWCNTs' length on mammalian immune and epithelial cancer cells RAW264.7 and MCF-7, respectively. Our data confirm that MWCNTs induce cytotoxicity in a length- and cell type-dependent manner. Whereas, longer (3 to 14 μm) MWCNTs exert high toxicity, especially to RAW264.7 cells, shorter (1.5 μm) MWCNTs are significantly less cytotoxic. These findings confirm that the degree of biocompatibility of MWCNTs is closely related to their length and that immune cells appear to be more susceptible to damage by MWCNTs. Our study also indicates that MWCNT nanotoxicity should be analysed for various components of cellular response, and cytotoxicity data should be validated by the use of more than one assay system. Results from chromogenic-based assays should be confirmed by trypan blue exclusion.

Lack of protection following passive transfer of polyclonal highly functional low-dose non-neutralizing antibodies.

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Anne-Sophie Dugast

Full Text Available Recent immune correlates analysis from the RV144 vaccine trial has renewed interest in the role of non-neutralizing antibodies in mediating protection from infection. While neutralizing antibodies have proven difficult to induce through vaccination, extra-neutralizing antibodies, such as those that mediate antibody-dependent cellular cytotoxicity (ADCC, are associated with long-term control of infection. However, while several non-neutralizing monoclonal antibodies have been tested for their protective efficacy in vivo, no studies to date have tested the protective activity of naturally produced polyclonal antibodies from individuals harboring potent ADCC activity. Because ADCC-inducing antibodies are highly enriched in elite controllers (EC, we passively transferred highly functional non-neutralizing polyclonal antibodies, purified from an EC, to assess the potential impact of polyclonal non-neutralizing antibodies on a stringent SHIV-SF162P3 challenge in rhesus monkeys. Passive transfer of a low-dose of ADCC inducing antibodies did not protect from infection following SHIV-SF162P3 challenge. Passively administered antibody titers and gp120-specific, but not gp41-specific, ADCC and antibody induced phagocytosis (ADCP were detected in the majority of the monkeys, but did not correlate with post infection viral control. Thus these data raise the possibility that gp120-specific ADCC activity alone may not be sufficient to control viremia post infection but that other specificities or Fc-effector profiles, alone or in combination, may have an impact on viral control and should be tested in future passive transfer experiments.

Cellular pharmacodynamics of the cytotoxic guanidino-containing drug CHS 828. Comparison with methylglyoxal-bis(guanylhydrazone).

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Ekelund, S; Sjöholm, A; Nygren, P; Binderup, L; Larsson, R

2001-04-20

N-(6-(4-chlorophenoxy)hexyl)-N'-cyano-N"-4-pyridylguanidine (CHS 828) is a new guanidino-containing compound with antitumoral activity both in vitro and in vivo. Its activity profile differs from those of standard cytotoxic drugs but the mechanism of action is not yet fully understood. CHS 828 is presently in early phase I and II clinical trials. In the present study, the pharmacodynamic effects at the cellular level of CHS 828 was compared to another compound containing two guanidino groups, methylglyoxal-bis(guanylhydrazone) (MGBG). MGBG is known to inhibit the synthesis of polyamines, which are important in, e.g., proliferation and macromolecular synthesis. The concentration-response relationship of CHS 828 closely resembled that of MGBG and the drugs were similar with respect to inhibition of DNA and protein synthesis. On the other hand, CHS 828 induced a significant increase in cellular metabolism while MGBG did not. The cytotoxic effect of MGBG was reversed by the addition of exogenous polyamines, while that of CHS 828 was unaffected. Unlike MGBG, there was also no effect of CHS 828 on the levels of decarboxylating enzymes in the polyamine biosynthesis. In conclusion, CHS 828 does not appear to share any major mechanisms of action with the polyamine synthesis inhibitor MGBG. Further studies will be required to define the exact mechanism of action of CHS 828.

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

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

2016-01-01

Chordoma, a rare bone tumor derived from the notochord, has been shown to be resistant to conventional therapies. Checkpoint inhibition has shown great promise in immune-mediated therapy of diverse cancers. The anti-PD-L1 mAb avelumab is unique among checkpoint inhibitors in that it is a fully human IgG1 capable of mediating antibody-dependent cell-mediated cytotoxicity (ADCC) of PD-L1-expressing tumor cells. Here, we investigated avelumab as a potential therapy for chordoma. We examined 4 ch...

Antibody-dependent NK cell activation is associated with late kidney allograft dysfunction and the complement-independent alloreactive potential of donor-specific antibodies

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Tristan Legris

2016-08-01

Full Text Available Although kidney transplantation remains the best treatment for end-stage renal failure, it is limited by chronic humoral aggression of the graft vasculature by donor-specific antibodies (DSAs. The complement-independent mechanisms that lead to the antibody-mediated rejection (ABMR of kidney allografts remain poorly understood. Increasing lines of evidence have revealed the relevance of natural killer (NK cells as innate immune effectors of antibody-dependent cellular cytotoxicity, but few studies have investigated their alloreactive potential in the context of solid organ transplantation. Our study aimed to investigate the potential contribution of the antibody-dependent alloreactive function of NK cells to kidney graft dysfunction. We first conducted an observational study to investigate whether the cytotoxic function of NK cells is associated with chronic allograft dysfunction. The NK-Cellular Humoral Activation Test (NK-CHAT was designed to evaluate the recipient and antibody-dependent reactivity of NK cells against allogeneic target cells. The release of CD107a/Lamp1+ cytotoxic granules, resulting from the recognition of rituximab-coated B cells by NK cells, was analyzed in 148 kidney transplant recipients (KTRs, mean graft duration: 6.2 years. Enhanced ADCC responsiveness was associated with reduced graft function and identified as an independent risk factor predicting a decline in the estimated glomerular filtration rate (eGFR over a 1-year period (hazard ratio: 2.83. In a second approach, we used the NK-CHAT to reveal the cytotoxic potential of circulating alloantibodies in vitro. The level of CD16 engagement resulting from the in vitro recognition of serum-coated allogeneic B cells or splenic cells was further identified as a specific marker of DSA-induced ADCC. The NK-CHAT scoring of sera obtained from 40 patients at the time of transplant biopsy was associated with ABMR diagnosis. Our findings indicate that despite the administration

HIV-Specific ADCC Improves After Antiretroviral Therapy and Correlates With Normalization of the NK Cell Phenotype

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Jensen, Sanne S; Hartling, Hans J; Tingstedt, Jeanette L

2015-01-01

analyzed. RESULTS: The ability of NK cells to mediate ADCC was significantly increased after only 6 months of HAART and was not explained by a normalization of NK cell subsets (CD56 CD16 and CD56 CD16 NK cells) but rather by normalization in the frequency of NK cells expressing CCR7 and CD27...

Intratumoral delivery of CpG-conjugated anti-MUC1 antibody enhances NK cell anti-tumor activity.

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Schettini, Jorge; Kidiyoor, Amritha; Besmer, Dahlia M; Tinder, Teresa L; Roy, Lopamudra Das; Lustgarten, Joseph; Gendler, Sandra J; Mukherjee, Pinku

2012-11-01

Monoclonal antibodies (mAbs) against tumor-associated antigens are useful anticancer agents. Antibody-dependent cellular cytotoxicity (ADCC) is one of the major mechanisms responsible for initiating natural killer cell (NK)-mediated killing of tumors. However, the regulation of ADCC via NK cells is poorly understood. We have investigated the cytolytic activity of NK cells against pancreatic cancer cells that were coated with an antibody directed against the human tumor antigen, Mucin-1 designated HMFG-2, either alone or conjugated to CpG oligodeoxynucleotide (CpG ODN). Conjugated antibodies were tested for their ability to elicit ADCC in vitro and in vivo against pancreatic cancer cells. NK cells cultured in the presence of immobilized CpG ODN, HMFG-2 Ab, or CpG ODN-conjugated HMFG-2 Ab were able to up-regulate perforin similarly. Interestingly, a significant higher ADCC was observed when CpG ODN-conjugated HMFG-2-coated tumor cells were co-cultured with NK cells compared to unconjugated HMFG-2 Ab or CpG ODN alone. Moreover, MyD88-deficient NK cells can perform ADCC in vitro. Furthermore, intratumoral injections of CpG ODN-conjugated HMFG-2 induced a significant reduction in tumor burden in vivo in an established model of pancreatic tumor in nude mice compared to CpG ODN or the HMFG-2 alone. Depletion of macrophages or NK cells before treatment confirmed that both cells were required for the anti-tumor response in vivo. Results also suggest that CpG ODN and HMFG-2 Ab could be sensed by NK cells on the mAb-coated tumor cells triggering enhanced ADCC in vitro and in vivo.

Chimeric peptide containing both B and T cells epitope of tumor-associated antigen L6 enhances anti-tumor effects in HLA-A2 transgenic mice.

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Lin, Su-I; Huang, Ming-Hsi; Chang, Yu-Wen; Chen, I-Hua; Roffler, Steve; Chen, Bing-Mae; Sher, Yuh-Pyng; Liu, Shih-Jen

2016-07-28

Synthetic peptides are attractive for cancer immunotherapy because of their safety and flexibility. In this report, we identified a new B cell epitope of tumor-associated antigen L6 (TAL6) that could induce antibody-dependent cellular cytotoxicity (ADCC) in vivo. We incorporated the B cell epitope with a cytotoxic T lymphocyte (CTL) and a helper T (Th) epitope to form a chimeric long peptide. We formulated the chimeric peptide with different adjuvants to immunize HLA-A2 transgenic mice and evaluate their immunogenicity. The chimeric peptide formulated with an emulsion type nanoparticle (PELC) adjuvant and a toll-like receptor 9 agonist (CpG ODN) (PELC/CpG) induced the greatest ADCC and CTL responses. The induced anti-tumor immunity inhibited the growth of TAL6-positive cancer cells. Moreover, we observed that immunization with the chimeric peptide inhibited cancer cell migration in vitro and metastasis in vivo. These data suggest that a chimeric peptide containing both B and T cell epitopes of TAL6 formulated with PELC/CpG adjuvant is feasible for cancer immunotherapy. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Effects of daratumumab on natural killer cells and impact on clinical outcomes in relapsed or refractory multiple myeloma

DEFF Research Database (Denmark)

Casneuf, Tineke; Xu, Xu Steven; Adams, Homer C

2017-01-01

Daratumumab, a human CD38 imunoglobulin G 1κ monoclonal antibody, has demonstrated clinical activity and a manageable safety profile in monotherapy and combination therapy clinical trials in relapsed and/or refractory multiple myeloma. CD38 is expressed at high levels on myeloma cells and......, to a lesser extent, on immune effector cells, including natural killer (NK) cells, which are important for daratumumab-mediated antibody-dependent cellular cytotoxicity (ADCC). Here, the pharmacodynamic effects of daratumumab monotherapy on NK cells, and the effect of NK cell dynamics on daratumumab efficacy...

ZnO nanofluids for the improved cytotoxicity and cellular uptake of doxorubicin

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Safoura Soleymani

2018-01-01

Full Text Available Objective(s: Combination anticancer therapy holds promise for improving the therapeutic efficacy of chemotherapy drugs such as doxorubicin (DOX as well as decreasing their dose-limiting side effects. Overcoming the side effects of doxorubicin (DOX is a major challenge to the effective treatment of cancer. Zinc oxide nanoparticles (ZnO NPs are emerging as potent tools for a wide variety of biomedical applications. The aim of this study was to develop a combinatorial approach for enhancing the anticancer efficacy and cellular uptake of DOX. Materials and Methods: ZnO NPs were synthesized by the solvothermal method and were characterized by X-ray diffraction (XRD, dynamic light scattering (DLS and transmission electron microscopy (TEM. ZnO NPs were dispersed in 10% bovine serum albumin (BSA and the cytotoxic effect of the resulting ZnO nanofluids was evaluated alone and in combination with DOX on DU145 cells. The influence of ZnO nanofluids on the cellular uptake of DOX and DOX-induced catalase mRNA expression were investigated by fluorescence microscopy and semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR, respectively. Results: The MTT results revealed that ZnO nanofluids decreased the cell viability of DU145 cells in a timeand dose-dependent manner. Simultaneous combination treatment of DOX and ZnO nanofluid showed a significant increase in anticancer activity and the cellular uptake of DOX compared to DOX alone. Also, a time-dependent reduction of catalase mRNA expression was observed in the cells treated with ZnO nanofluids and DOX, alone and in combination with each other. Conclusion: These results indicate the role of ZnO nanofluid as a growth-inhibitory agent and a drug delivery system for DOX in DU145 cells. Thus, ZnO nanofluid could be a candidate for combination chemotherapy.

Identification of antibody glycosylation structures that predict monoclonal antibody Fc-effector function.

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Chung, Amy W; Crispin, Max; Pritchard, Laura; Robinson, Hannah; Gorny, Miroslaw K; Yu, Xiaojie; Bailey-Kellogg, Chris; Ackerman, Margaret E; Scanlan, Chris; Zolla-Pazner, Susan; Alter, Galit

2014-11-13

To determine monoclonal antibody (mAb) features that predict fragment crystalizable (Fc)-mediated effector functions against HIV. Monoclonal antibodies, derived from Chinese hamster ovary cells or Epstein-Barr virus-immortalized mouse heteromyelomas, with specificity to key regions of the HIV envelope including gp120-V2, gp120-V3 loop, gp120-CD4(+) binding site, and gp41-specific antibodies, were functionally profiled to determine the relative contribution of the variable and constant domain features of the antibodies in driving robust Fc-effector functions. Each mAb was assayed for antibody-binding affinity to gp140(SR162), antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and for the ability to bind to FcγRIIa, FcγRIIb and FcγRIIIa receptors. Antibody glycan profiles were determined by HPLC. Neither the specificity nor the affinity of the mAbs determined the potency of Fc-effector function. FcγRIIIa binding strongly predicted ADCC and decreased galactose content inversely correlated with ADCP, whereas N-glycolylneuraminic acid-containing structures exhibited enhanced ADCP. Additionally, the bi-antenary glycan arm onto which galactose was added predicted enhanced binding to FcγRIIIa and ADCC activity, independent of the specificity of the mAb. Our studies point to the specific Fc-glycan structures that can selectively promote Fc-effector functions independently of the antibody specificity. Furthermore, we demonstrated antibody glycan structures associated with enhanced ADCP activity, an emerging Fc-effector function that may aid in the control and clearance of HIV infection.

Combined use of anti-ErbB monoclonal antibodies and erlotinib enhances antibody-dependent cellular cytotoxicity of wild-type erlotinib-sensitive NSCLC cell lines

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Cavazzoni Andrea

2012-12-01

Full Text Available Abstract Background The epidermal growth factor receptor (EGFR is an established target for anti-cancer treatment in different tumour types. Two different strategies have been explored to inhibit this pivotal molecule in epithelial cancer development: small molecules TKIs and monoclonal antibodies. ErbB/HER-targeting by monoclonal antibodies such as cetuximab and trastuzumab or tyrosine-kinase inhibitors as gefitinib or erlotinib has been proven effective in the treatment of advanced NSCLC. Results In this study we explored the potential of combining either erlotinib with cetuximab or trastuzumab to improve the efficacy of EGFR targeted therapy in EGFR wild-type NSCLC cell lines. Erlotinib treatment was observed to increase EGFR and/or HER2 expression at the plasma membrane level only in NSCLC cell lines sensitive to the drug inducing protein stabilization. The combined treatment had marginal effect on cell proliferation but markedly increased antibody-dependent, NK mediated, cytotoxicity in vitro. Moreover, in the Calu-3 xenograft model, the combination significantly inhibited tumour growth when compared with erlotinib and cetuximab alone. Conclusion Our results indicate that erlotinib increases surface expression of EGFR and/or HER2 only in EGFR-TKI sensitive NSCLC cell lines and, in turns, leads to increased susceptibility to ADCC both in vitro and in a xenograft models. The combination of erlotinib with monoclonal antibodies represents a potential strategy to improve the treatment of wild-type EGFR NSCLC patients sensitive to erlotinib.

Antitumor effect of novel anti-podoplanin antibody NZ-12 against malignant pleural mesothelioma in an orthotopic xenograft model.

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Abe, Shinji; Kaneko, Mika Kato; Tsuchihashi, Yuki; Izumi, Toshihiro; Ogasawara, Satoshi; Okada, Naoto; Sato, Chiemi; Tobiume, Makoto; Otsuka, Kenji; Miyamoto, Licht; Tsuchiya, Koichiro; Kawazoe, Kazuyoshi; Kato, Yukinari; Nishioka, Yasuhiko

2016-09-01

Podoplanin (aggrus) is highly expressed in several types of cancers, including malignant pleural mesothelioma (MPM). Previously, we developed a rat anti-human podoplanin mAb, NZ-1, and a rat-human chimeric anti-human podoplanin antibody, NZ-8, derived from NZ-1, which induced antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity against podoplanin-positive MPM cell lines. In this study, we showed the antitumor effect of NZ-1, NZ-8, and NZ-12, a novel rat-human chimeric anti-human podoplanin antibody derived from NZ-1, in an MPM orthotopic xenograft SCID mouse model. Treatment with NZ-1 and rat NK (CD161a(+) ) cells inhibited the growth of tumors and the production of pleural effusion in NCI-H290/PDPN or NCI-H226 orthotopic xenograft mouse models. NZ-8 and human natural killer (NK) (CD56(+) ) cells also inhibited tumor growth and pleural effusion in MPM orthotopic xenograft mice. Furthermore, NZ-12 induced potent ADCC mediated by human MNC, compared with either NZ-1 or NZ-8. Antitumor effects were observed following treatment with NZ-12 and human NK (CD56(+) ) cells in MPM orthotopic xenograft mice. In addition, combined immunotherapy using the ADCC activity of NZ-12 mediated by human NK (CD56(+) ) cells with pemetrexed, led to enhanced antitumor effects in MPM orthotopic xenograft mice. These results strongly suggest that combination therapy with podoplanin-targeting immunotherapy using both NZ-12 and pemetrexed might provide an efficacious therapeutic strategy for the treatment of MPM. © 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Structure-cytotoxicity relationships for dietary flavonoids

DEFF Research Database (Denmark)

Breinholt, V.; Dragsted, L.O.

1998-01-01

The cytotoxicity of a large series of dietary flavonoids was tested in a non-tumorigenic mouse and two human cancer cell lines, using the neutral red dye exclusion assay. All compounds tested exhibited a concentration-dependent cytotoxic action in the employed cell lines. The relative cytotoxicity...... of the flavonoids, however, Tvas found to vary greatly among the different cell Lines. With a few exceptions, the investigated flavonoids were more cytotoxic to the human cancer cell lines, than the mouse cell line. The differences in cytotoxicity were accounted for in part by differences in cellular uptake...... and metabolic capacity among the different cell types. In 3T3 cells fairly consistent structure-cytotoxicity relationships were found. The most cytotoxic structures tested in 3T3 cells were flavonoids with adjacent 3',4' hydroxy groups on the B-ring, such as luteolin, quercetin, myricetin, fisetin, eriodictyol...

Cytotoxicity and cellular uptake of pyrimidine nucleosides for imaging herpes simplex type-1 thymidine kinase (HSV-1 TK) expression in mammalian cells

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Morin, Kevin W.; Duan Weili; Xu Lihua; Zhou Aihua; Moharram, Sameh; Knaus, Edward E.; McEwan, Alexander J.B.; Wiebe, Leonard I. E-mail: leonard.wiebe@ualberta.ca

2004-07-01

In vivo transfer of the herpes simplex virus type-1 thymidine kinase (HSV-1 TK) gene, with subsequent administration of antiviral drugs such as ganciclovir, has emerged as a promising gene therapy protocol for treating proliferative disorders. The in vitro cytotoxicities (IC{sub 50}) for two series of 5-iodo- and (E)-5-(2-iodovinyl)-substituted 2'-deoxy- and 2'-deoxy-2'-fluoro-pyrimidine nucleosides ranged from millimolar to low nanomolar concentrations in mammalian tumor cell lines (KBALB; R-970-5; 143B; EMT-6) and their counterparts engineered to express HSV-1 TK (KBALB-STK; 143B-LTK). Their HSV-1 TK selectivity indices ranged from one (nonselective) to one million (highly selective) based on cytotoxicity, with FIRU being the least toxic to all cell lines, and FIAU being most toxic. HSV-1 TK selectivity, based on uptake, ranged from 10 to 140, with IVDU being most selective for HSV-1 TK expressing cells, followed by IVFRU, FIRU, FIAU, IVFAU and finally IUDR. Phosphorylation of [{sup 125}I]FIAU led to incorporation of the radiolabel into nucleic acids, whereas IVFRU and FIRU radioactivity was trapped primarily in the nucleotide pool. These data indicate that cytotoxicity does not depend on initial metabolic trapping (e.g., phosphorylation), but on elaboration of the mononucleotides to more cytotoxic anabolites. Lipophilicities and nucleoside transport rates of the six nucleosides tested were within narrow ranges. This supports the premise that cellular biochemistry, and not cellular bioavailability, is responsible for the observed broad range of cytotoxicity and trapping. In vivo biodistribution studies with 5-[{sup 125}I]iodo-2'-fluoro-2'-deoxyribouridine (FIRU), 5-[{sup 125}I]iodo-2'-fluoro-2'-deoxyarabinouridine (FIAU) and (E)-5-(2-[{sup 125}I]iodovinyl)-2'-fluoro-2'-deoxyuridine (IVFRU) demonstrate selective accumulation of all three radiotracers in HSV-1 TK-expressing KBABK-STK tumors, compared to their very low

Preclinical Characterization of a Novel Monoclonal Antibody NEO-201 for the Treatment of Human Carcinomas

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Massimo Fantini

2018-01-01

Full Text Available NEO-201 is a novel humanized IgG1 monoclonal antibody that was derived from an immunogenic preparation of tumor-associated antigens from pooled allogeneic colon tumor tissue extracts. It was found to react against a variety of cultured human carcinoma cell lines and was highly reactive against the majority of tumor tissues from many different carcinomas, including colon, pancreatic, stomach, lung, and breast cancers. NEO-201 also exhibited tumor specificity, as the majority of normal tissues were not recognized by this antibody. Functional assays revealed that treatment with NEO-201 is capable of mediating both antibody-dependent cellular cytotoxicity (ADCC and complement-dependent cytotoxicity (CDC against tumor cells. Furthermore, the growth of human pancreatic xenograft tumors in vivo was largely attenuated by treatment with NEO-201 both alone and in combination with human peripheral blood mononuclear cells as an effector cell source for ADCC. In vivo biodistribution studies in human tumor xenograft-bearing mice revealed that NEO-201 preferentially accumulates in the tumor but not organ tissue. Finally, a single-dose toxicity study in non-human primates demonstrated safety and tolerability of NEO-201, as a transient decrease in circulating neutrophils was the only related adverse effect observed. These findings indicate that NEO-201 warrants clinical testing as both a novel diagnostic and therapeutic agent for the treatment of a broad variety of carcinomas.

Environmentally persistent free radicals amplify ultrafine particle mediated cellular oxidative stress and cytotoxicity

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Balakrishna Shrilatha

2009-04-01

Full Text Available Abstract Background Combustion generated particulate matter is deposited in the respiratory tract and pose a hazard to the lungs through their potential to cause oxidative stress and inflammation. We have previously shown that combustion of fuels and chlorinated hydrocarbons produce semiquinone-type radicals that are stabilized on particle surfaces (i.e. environmentally persistent free radicals; EPFRs. Because the composition and properties of actual combustion-generated particles are complex, heterogeneous in origin, and vary from day-to-day, we have chosen to use surrogate particle systems. In particular, we have chosen to use the radical of 2-monochlorophenol (MCP230 as the EPFR because we have previously shown that it forms a EPFR on Cu(IIO surfaces and catalyzes formation of PCDD/F. To understand the physicochemical properties responsible for the adverse pulmonary effects of combustion by-products, we have exposed human bronchial epithelial cells (BEAS-2B to MCP230 or the CuO/silica substrate. Our general hypothesis was that the EPFR-containing particle would have greater toxicity than the substrate species. Results Exposure of BEAS-2B cells to our combustion generated particle systems significantly increased reactive oxygen species (ROS generation and decreased cellular antioxidants resulting in cell death. Resveratrol treatment reversed the decline in cellular glutathione (GSH, glutathione peroxidase (GPx, and superoxide dismutase (SOD levels for both types of combustion-generated particle systems. Conclusion The enhanced cytotoxicity upon exposure to MCP230 correlated with its ability to generate more cellular oxidative stress and concurrently reduce the antioxidant defenses of the epithelial cells (i.e. reduced GSH, SOD activity, and GPx. The EPFRs in MCP230 also seem to be of greater biological concern due to their ability to induce lipid peroxidation. These results are consistent with the oxidizing nature of the CuO/silica ultrafine

Cellular internalisation kinetics and cytotoxic properties of statistically designed and optimised neo-geometric copper nanocrystals.

Science.gov (United States)

Murugan, Karmani; Choonara, Yahya E; Kumar, Pradeep; du Toit, Lisa C; Pillay, Viness

2017-09-01

This study aimed to highlight a statistic design to precisely engineer homogenous geometric copper nanoparticles (CuNPs) for enhanced intracellular drug delivery as a function of geometrical structure. CuNPs with a dual functionality comprising geometric attributes for enhanced cell uptake and exerting cytotoxic activity on proliferating cells were synthesized as a novel drug delivery system. This paper investigated the defined concentrations of two key surfactants used in the reaction to mutually control and manipulate nano-shape and optimisation of the geometric nanosystems. A statistical experimental design comprising a full factorial model served as a refining factor to achieve homogenous geometric nanoparticles using a one-pot method for the systematic optimisation of the geometric CuNPs. Shapes of the nanoparticles were investigated to determine the result of the surfactant variation as the aim of the study and zeta potential was studied to ensure the stability of the system and establish a nanosystem of low aggregation potential. After optimisation of the nano-shapes, extensive cellular internalisation studies were conducted to elucidate the effect of geometric CuNPs on uptake rates, in addition to the vital toxicity assays to further understand the cellular effect of geometric CuNPs as a drug delivery system. In addition to geometry; volume, surface area, orientation to the cell membrane and colloidal stability is also addressed. The outcomes of the study demonstrated the success of homogenous geometric NP formation, in addition to a stable surface charge. The findings of the study can be utilized for the development of a drug delivery system for promoted cellular internalisation and effective drug delivery. Copyright © 2017 Elsevier B.V. All rights reserved.

A Novel Platform for the Potentiation of Therapeutic Antibodies Based on Antigen-Dependent Formation of IgG Hexamers at the Cell Surface

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de Jong, R. N.; Beurskens, F. J.; Verploegen, S.

2016-01-01

IgG antibodies can organize into ordered hexamers on cell surfaces after binding their antigen. These hexamers bind the first component of complement C1 inducing complement-dependent target cell killing. Here, we translated this natural concept into a novel technology platform (HexaBody technology......) for therapeutic antibody potentiation. We identified mutations that enhanced hexamer formation and complement activation by IgG1 antibodies against a range of targets on cells from hematological and solid tumor indications. IgG1 backbones with preferred mutations E345K or E430G conveyed a strong ability to induce...... conditional complement-dependent cytotoxicity (CDC) of cell lines and chronic lymphocytic leukemia (CLL) patient tumor cells, while retaining regular pharmacokinetics and biopharmaceutical developability. Both mutations potently enhanced CDC- and antibody-dependent cellular cytotoxicity (ADCC) of a type II CD...

Rationale for combination of therapeutic antibodies targeting tumor cells and immune checkpoint receptors: Harnessing innate and adaptive immunity through IgG1 isotype immune effector stimulation.

Science.gov (United States)

Ferris, Robert L; Lenz, Heinz-Josef; Trotta, Anna Maria; García-Foncillas, Jesús; Schulten, Jeltje; Audhuy, François; Merlano, Marco; Milano, Gerard

2018-02-01

Immunoglobulin (Ig) G1 antibodies stimulate antibody-dependent cell-mediated cytotoxicity (ADCC). Cetuximab, an IgG1 isotype monoclonal antibody, is a standard-of-care treatment for locally advanced and recurrent and/or metastatic squamous cell carcinoma of the head and neck (SCCHN) and metastatic colorectal cancer (CRC). Here we review evidence regarding the clinical relevance of cetuximab-mediated ADCC and other immune functions and provide a biological rationale concerning why this property positions cetuximab as an ideal partner for immune checkpoint inhibitors (ICIs) and other emerging immunotherapies. We performed a nonsystematic review of available preclinical and clinical data involving cetuximab-mediated immune activity and combination approaches of cetuximab with other immunotherapies, including ICIs, in SCCHN and CRC. Indeed, cetuximab mediates ADCC activity in the intratumoral space and primes adaptive and innate cellular immunity. However, counterregulatory mechanisms may lead to immunosuppressive feedback loops. Accordingly, there is a strong rationale for combining ICIs with cetuximab for the treatment of advanced tumors, as targeting CTLA-4, PD-1, and PD-L1 can ostensibly overcome these immunosuppressive counter-mechanisms in the tumor microenvironment. Moreover, combining ICIs (or other immunotherapies) with cetuximab is a promising strategy for boosting immune response and enhancing response rates and durability of response. Cetuximab immune activity-including, but not limited to, ADCC-provides a strong rationale for its combination with ICIs or other immunotherapies to synergistically and fully mobilize the adaptive and innate immunity against tumor cells. Ongoing prospective studies will evaluate the clinical effect of these combination regimens and their immune effect in CRC and SCCHN and in other indications. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

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

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

2015-06-11

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

Cytotoxicity of Phenol Red in Toxicity Assays for Carbon Nanoparticles

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Chunhai Fan

2012-09-01

Full Text Available To explore the novel properties of carbon nanoparticles (CNPs in nanotoxicity assays, the adsorption of phenol red (a pH indicator for culture medium by multi-walled carbon nanotubes (MWNTs and three kinds of carbon blacks (CBs with nanosize, and its effects on cytotoxicity were studied. Results indicated that the phenol red adsorbed and delivered into cells by CBs was responsible for the toxicity to Hela cells in the medium without serum. The cellular uptake of phenol red was verified using 125I-labeling techniques. The size-dependent cytotoxicity of CBs was found to closely correlate to adsorption of phenol red, cellular uptake of phenol red-CB complexes and the amount of phenol red delivered into the cells by CBs. Although the CBs were either nontoxic or slightly toxic, as vehicles of phenol red, they played an essential role in the cytotoxicity induced by phenol red. However, MWNTs showed an intrinsic cytotoxicity independent of phenol red. The implications associated with these findings are discussed.

Macrophage and NK-mediated killing of precursor-B acute lymphoblastic leukemia cells targeted with a-fucosylated anti-CD19 humanized antibodies.

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Matlawska-Wasowska, K; Ward, E; Stevens, S; Wang, Y; Herbst, R; Winter, S S; Wilson, B S

2013-06-01

This work reports the tumoricidal effects of a novel investigational humanized anti-CD19 monoclonal antibody (Medi-551). An a-fucosylated antibody with increased affinity for human FcγRIIIA, Medi-551 is shown to mediate both antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Medi-551/CD19 complexes internalize slowly (>5 h) and thus remain accessible to effector cells for prolonged periods. We evaluated in vitro ADCC and ADCP activities of primary human natural killer (NK) cells and macrophages against precursor-B (pre-B) acute lymphoblastic leukemia (ALL) cell lines and pediatric patient blasts. Fluorescent imaging studies document immunological synapses formed between anti-CD19-bound target leukemia cells and effector cells and capture the kinetics of both NK-mediated killing and macrophage phagocytosis. Genetic polymorphisms in FcγRIIIA-158F/V modulate in vitro activities of effector cells, with FcγRIIIA-158V homozygotes or heterozygotes showing the strongest activity. Medi-551 treatment of severe combined immunodeficiency (SCID) mice engrafted with human pre-B cells led to prolonged animal survival and markedly reduced disease burden in blood, liver and bone marrow. These data show that anti-CD19 antibodies effectively recruit immune cells to pre-B ALL cells and support a move forward to early phase trials in this disease.

Cytotoxicity of cadmium-containing quantum dots based on a study using a microfluidic chip

International Nuclear Information System (INIS)

Zheng Xiannuo; Weng Lixing; Tian Jing; Wang Lianhui; Wu Lei; Jin Qinghui; Zhao Jianlong

2012-01-01

There is a lack of reliable nanotoxicity assays available for monitoring and quantifying multiple cellular events in cultured cells. In this study, we used a microfluidic chip to systematically investigate the cytotoxicity of three kinds of well-characterized cadmium-containing quantum dots (QDs) with the same core but different shell structures, including CdTe core QDs, CdTe/CdS core–shell QDs, and CdTe/CdS/ZnS core–shell–shell QDs, in HEK293 cells. Using the microfluidic chip combined with fluorescence microscopy, multiple QD-induced cellular events including cell morphology, viability, proliferation, and QD uptake were simultaneously analysed. The three kinds of QDs showed significantly different cytotoxicities. The CdTe QDs, which are highly toxic to HEK293 cells, resulted in remarkable cellular and nuclear morphological changes, a dose-dependent decrease in cell viability, and strong inhibition of cell proliferation; the CdTe/CdS QDs were moderately toxic but did not significantly affect the proliferation of HEK293 cells; while the CdTe/CdS/ZnS QDs had no detectable influence on cytotoxicity with respect to cell morphology, viability, and proliferation. Our data indicated that QD cytotoxicity was closely related to their surface structures and specific physicochemical properties. This study also demonstrated that the microfluidic chip could serve as a powerful tool to systematically evaluate the cytotoxicity of nanoparticles in multiple cellular events. (paper)

Cytotoxicity of cadmium-containing quantum dots based on a study using a microfluidic chip

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Zheng, Xiannuo; Tian, Jing; Weng, Lixing; Wu, Lei; Jin, Qinghui; Zhao, Jianlong; Wang, Lianhui

2012-02-01

There is a lack of reliable nanotoxicity assays available for monitoring and quantifying multiple cellular events in cultured cells. In this study, we used a microfluidic chip to systematically investigate the cytotoxicity of three kinds of well-characterized cadmium-containing quantum dots (QDs) with the same core but different shell structures, including CdTe core QDs, CdTe/CdS core-shell QDs, and CdTe/CdS/ZnS core-shell-shell QDs, in HEK293 cells. Using the microfluidic chip combined with fluorescence microscopy, multiple QD-induced cellular events including cell morphology, viability, proliferation, and QD uptake were simultaneously analysed. The three kinds of QDs showed significantly different cytotoxicities. The CdTe QDs, which are highly toxic to HEK293 cells, resulted in remarkable cellular and nuclear morphological changes, a dose-dependent decrease in cell viability, and strong inhibition of cell proliferation; the CdTe/CdS QDs were moderately toxic but did not significantly affect the proliferation of HEK293 cells; while the CdTe/CdS/ZnS QDs had no detectable influence on cytotoxicity with respect to cell morphology, viability, and proliferation. Our data indicated that QD cytotoxicity was closely related to their surface structures and specific physicochemical properties. This study also demonstrated that the microfluidic chip could serve as a powerful tool to systematically evaluate the cytotoxicity of nanoparticles in multiple cellular events.

The cytotoxicity of polycationic iron oxide nanoparticles: Common endpoint assays and alternative approaches for improved understanding of cellular response mechanism

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Hoskins Clare

2012-04-01

Full Text Available Abstract Background Iron oxide magnetic nanoparticles (MNP's have an increasing number of biomedical applications. As such in vitro characterisation is essential to ensure the bio-safety of these particles. Little is known on the cellular interaction or effect on membrane integrity upon exposure to these MNPs. Here we synthesised Fe3O4 and surface coated with poly(ethylenimine (PEI and poly(ethylene glycol (PEG to achieve particles of varying surface positive charges and used them as model MNP's to evaluate the relative utility and limitations of cellular assays commonly applied for nanotoxicity assessment. An alternative approach, atomic force microscopy (AFM, was explored for the analysis of membrane structure and cell morphology upon interacting with the MNPs. The particles were tested in vitro on human SH-SY5Y, MCF-7 and U937 cell lines for reactive oxygen species (ROS production and lipid peroxidation (LPO, LDH leakage and their overall cytotoxic effect. These results were compared with AFM topography imaging carried out on fixed cell lines. Results Successful particle synthesis and coating were characterised using FTIR, PCS, TEM and ICP. The particle size from TEM was 30 nm (−16.9 mV which increased to 40 nm (+55.6 mV upon coating with PEI and subsequently 50 nm (+31.2 mV with PEG coating. Both particles showed excellent stability not only at neutral pH but also in acidic environment of pH 4.6 in the presence of sodium citrate. The higher surface charge MNP-PEI resulted in increased cytotoxic effect and ROS production on all cell lines compared with the MNP-PEI-PEG. In general the effect on the cell membrane integrity was observed only in SH-SY5Y and MCF-7 cells by MNP-PEI determined by LDH leakage and LPO production. AFM topography images showed consistently that both the highly charged MNP-PEI and the less charged MNP-PEI-PEG caused cell morphology changes possibly due to membrane disruption and cytoskeleton remodelling. Conclusions

Cytotoxicity of metal and semiconductor nanoparticles indicated by cellular micromotility.

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Tarantola, Marco; Schneider, David; Sunnick, Eva; Adam, Holger; Pierrat, Sebastien; Rosman, Christina; Breus, Vladimir; Sönnichsen, Carsten; Basché, Thomas; Wegener, Joachim; Janshoff, Andreas

2009-01-27

In the growing field of nanotechnology, there is an urgent need to sensitively determine the toxicity of nanoparticles since many technical and medical applications are based on controlled exposure to particles, that is, as contrast agents or for drug delivery. Before the in vivo implementation, in vitro cell experiments are required to achieve a detailed knowledge of toxicity and biodegradation as a function of the nanoparticles' physical and chemical properties. In this study, we show that the micromotility of animal cells as monitored by electrical cell-substrate impedance analysis (ECIS) is highly suitable to quantify in vitro cytotoxicity of semiconductor quantum dots and gold nanorods. The method is validated by conventional cytotoxicity testing and accompanied by fluorescence and dark-field microscopy to visualize changes in the cytoskeleton integrity and to determine the location of the particles within the cell.

HIV-1-Specific Antibody Response and Function after DNA Prime and Recombinant Adenovirus 5 Boost HIV Vaccine in HIV-Infected Subjects.

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Johannes S Gach

Full Text Available Little is known about the humoral immune response against DNA prime-recombinant adenovirus 5 (rAd5 boost HIV vaccine among HIV-infected patients on long-term suppressive antiretroviral therapy (ART. Previous studies emphasized cellular immune responses; however, current research suggests both cellular and humoral responses are likely required for a successful therapeutic vaccine. Thus, we aimed to understand antibody response and function induced by vaccination of ART-treated HIV-1-infected patients with immune recovery. All subjects participated in EraMune 02, an open-label randomized clinical trial of ART intensification followed by a six plasmid DNA prime (envA, envB, envC, gagB, polB, nefB and rAd5 boost HIV vaccine with matching inserts. Antibody binding levels were determined with a recently developed microarray approach. We also analyzed neutralization efficiency and antibody-dependent cellular cytotoxicity (ADCC. We found that the DNA prime-rAd5 boost vaccine induced a significant cross-clade HIV-specific antibody response, which correlated with antibody neutralization efficiency. However, despite the increase in antibody binding levels, the vaccine did not significantly stimulate neutralization or ADCC responses. This finding was also reflected by a lack of change in total CD4+ cell associated HIV DNA in those who received the vaccine. Our results have important implications for further therapeutic vaccine design and administration, especially in HIV-1 infected patients, as boosting of preexisting antibody responses are unlikely to lead to clearance of latent proviruses in the HIV reservoir.

Toxicology and cellular effect of manufactured nanomaterials

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Chen, Fanqing

2014-07-22

The increasing use of nanotechnology in consumer products and medical applications underlies the importance of understanding its potential toxic effects to people and the environment. Herein are described methods and assays to predict and evaluate the cellular effects of nanomaterial exposure. Exposing cells to nanomaterials at cytotoxic doses induces cell cycle arrest and increases apoptosis/necrosis, activates genes involved in cellular transport, metabolism, cell cycle regulation, and stress response. Certain nanomaterials induce genes indicative of a strong immune and inflammatory response within skin fibroblasts. Furthermore, the described multiwall carbon nanoonions (MWCNOs) can be used as a therapeutic in the treatment of cancer due to its cytotoxicity.

Association of Angiotensin-Converting Enzyme (ACE) Gene Polymorphism with Inflammation and Cellular Cytotoxicity in Vitiligo Patients.

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Rashed, Laila; Abdel Hay, Rania; Mahmoud, Rania; Hasan, Nermeen; Zahra, Amr; Fayez, Salwa

2015-01-01

Vitiligo is a disorder with profound heterogeneity in its aetio-pathophysiology. Angiotensin converting enzyme (ACE) plays an important role in the physiology of the vasculature, blood pressure and inflammation. An insertion/deletion (I/D) polymorphism of the ACE gene was reported be associated with the development of vitiligo. Our aim was to evaluate the ACE I/D polymorphism in vitiligo patients and controls. Our second aim was to find a possible association between ACE gene polymorphism and inflammatory mediators (as interleukin (IL)-6) and/or cellular cytotoxicity induced by serum nitrite (as a breakdown product of the cytotoxic nitric oxide) in vitiligo patients. This case-control study included 74 vitiligo patients and 75 apparently healthy controls. The distribution of ACE gene I/D genotype was investigated using PCR. Serum ACE, IL-6 and nitrite were measured by colorimetric method, ELISA and Griess assay respectively. The ACE allele frequency was significantly different between vitiligo patients and healthy controls (P = 0.026). However there was no significant difference between the ACE genotyping frequency in both groups (P = 0.115). There were statistically significant higher VIDA score (P = 0.007), and serum IL-6 (P ACE, IL-6 and nitrite in vitiligo patients were statistically significantly higher than those in controls. As a conclusion, ACE gene polymorphism might grant susceptibility to develop vitiligo. Serum IL-6 and nitrite levels might have an important role in the pathogenesis of vitiligo. Targeting these two factors might have an implication in the treatment of some resistant cases.

Biocompatibility of a fish scale-derived artificial cornea: Cytotoxicity, cellular adhesion and phenotype, and in vivo immunogenicity.

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van Essen, T H; van Zijl, L; Possemiers, T; Mulder, A A; Zwart, S J; Chou, C-H; Lin, C C; Lai, H J; Luyten, G P M; Tassignon, M J; Zakaria, N; El Ghalbzouri, A; Jager, M J

2016-03-01

To determine whether a fish scale-derived collagen matrix (FSCM) meets the basic criteria to serve as an artificial cornea, as determined with in vitro and in vivo tests. Primary corneal epithelial and stromal cells were obtained from human donor corneas and used to examine the (in)direct cytotoxicity effects of the scaffold. Cytotoxicity was assessed by an MTT assay, while cellular proliferation, corneal cell phenotype and adhesion markers were assessed using an EdU-assay and immunofluorescence. For in vivo-testing, FSCMs were implanted subcutaneously in rats. Ologen(®) Collagen Matrices were used as controls. A second implant was implanted as an immunological challenge. The FSCM was implanted in a corneal pocket of seven New Zealand White rabbits, and compared to sham surgery. The FSCM was used as a scaffold to grow corneal epithelial and stromal cells, and displayed no cytotoxicity to these cells. Corneal epithelial cells displayed their normal phenotypical markers (CK3/12 and E-cadherin), as well as cell-matrix adhesion molecules: integrin-α6 and β4, laminin 332, and hemi-desmosomes. Corneal stromal cells similarly expressed adhesion molecules (integrin-α6 and β1). A subcutaneous implant of the FSCM in rats did not induce inflammation or sensitization; the response was comparable to the response against the Ologen(®) Collagen Matrix. Implantation of the FSCM in a corneal stromal pocket in rabbits led to a transparent cornea, healthy epithelium, and, on histology, hardly any infiltrating immune cells. The FSCM allows excellent cell growth, is not immunogenic and is well-tolerated in the cornea, and thus meets the basic criteria to serve as a scaffold to reconstitute the cornea. Copyright © 2015 Elsevier Ltd. All rights reserved.

Cleavage of the interchain disulfide bonds in rituximab increases its affinity for FcγRIIIA.

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Suzuki, Mami; Yamanoi, Ayaka; Machino, Yusuke; Kobayashi, Eiji; Fukuchi, Kaori; Tsukimoto, Mitsutoshi; Kojima, Shuji; Kohroki, Junya; Akimoto, Kazunori; Masuho, Yasuhiko

2013-07-05

The Fc region of human IgG1 mediates effector function via binding to Fcγ receptors and complement activation. The H and L chains of IgG1 antibodies are joined by four interchain disulfide bonds. In this study, these bonds within the therapeutic IgG1 rituximab (RTX) were cleaved either by mild reduction followed by alkylation or by mild S-sulfonation; consequently, two modified RTXs - A-RTX (alkylated) and S-RTX (S-sulfonated) - were formed, and both were almost as potent as unmodified RTX when binding CD20 antigen. Unexpectedly, each modified RTX had a higher binding affinity for FcγRIIIA (CD16A) than did unmodified RTX. However, S-RTX and A-RTX were each less potent than RTX in an assay of antibody-dependent cellular cytotoxicity (ADCC). In this ADCC assay, each modified RTX showed decreased secretion of granzyme B, but no change in perforin secretion, from effector cells. These results provide significant information on the structures within IgG1 that are involved in binding FcγRIIIA, and they may be useful in the development of therapeutic antagonists for FcγRIIIA. Copyright © 2013 Elsevier Inc. All rights reserved.

In vitro assessment of cytotoxic activities of Lachesis muta muta snake venom.

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Stephanie Stransky

2018-04-01

Full Text Available Envenomation by the bushmaster snake Lachesis muta muta is considered severe, characterized by local effects including necrosis, the main cause of permanent disability. However, cellular mechanisms related to cell death and tissue destruction, triggered by snake venoms, are poorly explored. The purpose of this study was to investigate the cytotoxic effect caused by L. m. muta venom in normal human keratinocytes and to identify the cellular processes involved in in cellulo envenomation. In order to investigate venom effect on different cell types, Alamar Blue assay was performed to quantify levels of cellular metabolism as a readout of cell viability. Apoptosis, necrosis and changes in mitochondrial membrane potential were evaluated by flow cytometry, while induction of autophagy was assessed by expression of GFP-LC3 and analyzed using fluorescence microscopy. The cytotoxic potential of the venom is shown by reduced cell viability in a concentration-dependent manner. It was also observed the sequential appearance of cells undergoing autophagy (by 6 hours, apoptosis and necrosis (12 and 24 hours. Morphologically, incubation with L. m. muta venom led to a significant cellular retraction and formation of cellular aggregates. These results indicate that L. m. muta venom is cytotoxic to normal human keratinocytes and other cell lines, and this toxicity involves the integration of distinct modes of cell death. Autophagy as a cell death mechanism, in addition to apoptosis and necrosis, can help to unravel cellular pathways and mechanisms triggered by the venom. Understanding the mechanisms that underlie cellular damage and tissue destruction will be useful in the development of alternative therapies against snakebites.

A rapid fluorometric method for semiautomated determination of cytotoxicity and cellular proliferation of human tumor cell lines in microculture.

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Larsson, R; Nygren, P

1989-01-01

A fluorometric method for the determination of cellular growth and cytotoxicity of human tumor cell lines in 96-well microculture plates is described. The assay is based on the combined use of the DNA-binding dye Hoechst 33342 and the fluorogenic substrate fluorescein diacetate (FDA). Hoechst 33342 undergoes a dramatic enhancement of fluorescence when specifically intercalated with cellular DNA, whereas the FDA fluorescence is dependent on cellular hydrolysis of the non-fluorescent substrate into its fluorescent product. Fluorescence from both dyes was linearly related to the density of freshly seeded cells (6 x 10(3)-1 x 10(5)/well) and correlated well with physical cell count of cells under normal culture conditions as well as in response to the vinca alkaloid vincristine. However, the amount of FDA fluorescence produces and retained by the cultures was clearly dependent on the fraction of intact and viable cells, whereas the fluorescence reported by Hoechst 33342 was not. The assay was found to be simple, reliable and many samples could be analysed in a short period of time with minimal waste of cells and biological reagents. Apart from giving an estimate of cell density, the protocol described also provides a separate index of viability which in certain situations may be of importance for distinguishing between cytocidal and cytostatic drug actions. The method may be well suited for several applications, including the large scale screening for antitumor activity of compounds with potential cytocidal or cytostatic actions.

Functional Antibody Response Against V1V2 and V3 of HIV gp120 in the VAX003 and VAX004 Vaccine Trials.

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Balasubramanian, Preetha; Williams, Constance; Shapiro, Mariya B; Sinangil, Faruk; Higgins, Keith; Nádas, Arthur; Totrov, Maxim; Kong, Xiang-Peng; Fiore-Gartland, Andrew J; Haigwood, Nancy L; Zolla-Pazner, Susan; Hioe, Catarina E

2018-01-11

Immunization with HIV AIDSVAX gp120 vaccines in the phase III VAX003 and VAX004 trials did not confer protection. To understand the shortcomings in antibody (Ab) responses induced by these vaccines, we evaluated the kinetics of Ab responses to the V1V2 and V3 regions of gp120 and the induction of Ab-mediated antiviral functions during the course of 7 vaccinations over a 30.5-month period. Plasma samples from VAX003 and VAX004 vaccinees and placebo recipients were measured for ELISA-binding Abs and for virus neutralization, Ab-dependent cellular phagocytosis (ADCP), and Ab-dependent cellular cytotoxicity (ADCC). Ab responses to V1V2 and V3 peaked after 3 to 4 immunizations and declined after 5 to 7 immunizations. The deteriorating responses were most evident against epitopes in the underside of the V1V2 β-barrel and in the V3 crown. Correspondingly, vaccinees demonstrated higher neutralization against SF162 pseudovirus sensitive to anti-V1V2 and anti-V3 Abs after 3 or 4 immunizations than after 7 immunizations. Higher levels of ADCP and ADCC were also observed at early or mid-time points as compared with the final time point. Hence, VAX003 and VAX004 vaccinees generated V1V2- and V3-binding Abs and functional Abs after 3 to 4 immunizations, but subsequent boosts did not maintain these responses.

The cellular basis of skin injury after cytotoxic insult

International Nuclear Information System (INIS)

Potten, C.S.

1986-01-01

It is concluded that although the major target in terms of radiation damage is undoubtedly the epidermis, the skin is a complex tissue made up of many inter-dependent components each of which may constitute an important secondary target. Damage to each component has been considered at the cellular level. The precise inter-relationships and interdependencies remain somewhat obscure. Even within one site, the epidermis, a comprehensive cellular explanation of the various post-irradiation changes is difficult. Substantial bibliography. (UK)

Analyses of the peripheral immunome following multiple administrations of avelumab, a human IgG1 anti-PD-L1 monoclonal antibody

OpenAIRE

Donahue, Renee N.; Lepone, Lauren M.; Grenga, Italia; Jochems, Caroline; Fantini, Massimo; Madan, Ravi A.; Heery, Christopher R.; Gulley, James L.; Schlom, Jeffrey

2017-01-01

Background Multiple anti-PD-L1/PD-1 checkpoint monoclonal antibodies (MAb) have shown clear evidence of clinical benefit. All except one have been designed or engineered to omit the possibility to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) as a second potential mode of anti-tumor activity; the reason for this is the concern of lysis of PD-L1 positive immune cells. Avelumab is a fully human IgG1 MAb which has been shown in prior in vitro studies to mediate ADCC versus a range...

Fc engineering of anti-Nectin-2 antibody improved thrombocytopenic adverse event in monkey.

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Tsutomu Oshima

Full Text Available Nectin-2 is a transmembrane glycoprotein which is involved in the process of Ca2+-independent cell-cell adhesion. In our previous study, we have demonstrated that Nectin-2 is over-expressed in breast and ovarian cancer tissues by using gene expression analysis and immunohistochemistry. Furthermore, we discovered multiple anti-Nectin-2 fully human monoclonal antibodies which inhibited tumor growth in in vivo subcutaneous xenograft models with antibody-dependent cellular cytotoxicity (ADCC as the principal mechanism of action. In this report, we assessed the toxicity of Y-443, a fully human IgG1/kappa anti-Nectin-2 monoclonal antibody exhibiting strong in vitro ADCC and in vivo anti-tumor activity in cynomolgus monkeys (Macaca fascicularis (Cynos. Unexpectedly, upon administration, Y-443 induced strong thrombocytopenia through Nectin-2 expressed on Cyno platelets, presumably followed by phagocytosis in the mononuclear phagocytic system. To mitigate the adverse safety profile, we mutated the Fc region of Y-443 to reduce the Fc binding activity to Fcγ receptor I, which is the primary receptor for phagocytosis on macrophages. Moreover, we further engineered the Fc through defucosylation to maintain ADCC activity. The resultant Fc engineered antibody, termed Y-634, demonstrated diminished thrombocytopenia in Cyno toxicological studies and maintained anti-tumor activity in a mouse xenograft model. These findings suggest that Y-634 may have a therapeutic potential for the treatment of Nectin-2 positive cancers, and moreover, Fc engineering is a potential mitigation strategy to ameliorate safety liabilities in antibody induced thrombocytopenia while maintaining antibody potency.

Pyrosequencing for classification of human FcγRIIIA allotypes: a comparison with PCR-based techniques.

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Matlawska-Wasowska, Ksenia; Gale, James M; Nickl, Christian K; Khalili, Parisa; Shirley, Brian; Wilson, Bridget S; Vasef, Mohammad A; Winter, Stuart S

2014-12-01

Surface-specific antigens expressed by hematopoietic cells are attractive targets for antibody-mediated immunotherapy. Monoclonal antibodies (mAbs) involve various mechanisms to eliminate target cells, including antibody-dependent cellular cytotoxicity (ADCC)- and phagocytosis (ADCP)-mediated killing through natural killer (NK) and macrophage effector cells bearing FcγRIIIA (CD16). The clinical efficacy of ADCC is particularly impacted by a single nucleotide polymorphism (SNP) found in the gene encoding FcγRIIIA (FCGR3A), which generates a variable distribution of the 158 V/V, F/V or F/F CD16 allotypes (F = phenylalanine, V = valine) in the normal human population. Currently, most patients are not screened for CD16 allotypes, creating the potential to include in their treatment a mAb-based therapy that may have limited benefit. Therefore, it is important to identify CD16 allotypes when considering mAb therapies that require ADCC/ADCP. The objective of this study was to develop a reliable PCR-based assay for classification of human FcγRIIIA allotypes. We studied 42 normal human subjects for the incidence of FcγRIIIA-158 polymorphisms using comparative molecular approaches. The results of our study showed 100% accuracy in genotyping by pyrosequencing. In contrast, nested PCR-based allele-specific restriction assay and quantitative PCR techniques proved to be relatively less sensitive and less specific in distinguishing variant genotypes. Since the efficacy of the mAb-based targeted immunotherapy may be highly dependent upon the CD16 polymorphism in a given individual, we recommend pyrosequencing for CD16 allotype testing.

CA125 suppresses amatuximab immune-effector function and elevated serum levels are associated with reduced clinical response in first line mesothelioma patients.

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Nicolaides, Nicholas C; Schweizer, Charles; Somers, Elizabeth B; Wang, Wenquan; Fernando, Shawn; Ross, Erin N; Grasso, Luigi; Hassan, Raffit; Kline, J Bradford

2018-04-13

The tumor-shed antigen CA125 has recently been found to bind certain monoclonal antibodies (mAbs) and suppress immune-effector mediated killing through perturbation of the Fc domain with CD16a and CD32a Fc-γ activating receptors on immune-effector cells. Amatuximab is a mAb targeting mesothelin whose mechanism of action utilizes in part antibody-dependent cellular cytotoxicity (ADCC). It is being tested for its therapeutic activity in patients with mesothelioma in combination with first line standard-of-care. To determine if CA125 has immunosuppressive effects on amatuximab ADCC and associated clinical outcomes, post hoc subgroup analysis of patients from a Phase 2 study with primary diagnosed stage III/IV unresectable mesothelioma treated with amatuximab plus cisplatin and pemetrexed were conducted. Analysis found patients with baseline CA125 levels no greater than 57 U/m (∼3X the upper limit of normal) had a 2 month improvement in progression free survival (HR = 0.43, p = 0.0062) and a 7 month improvement in overall survival (HR = 0.40, p = 0.0022) as compared to those with CA125 above 57 U/mL. In vitro studies found that CA125 was able to bind amatuximab and perturb ADCC activity via decreased Fc-γ-receptor engagement. These data suggest that clinical trial designs of antibody-based drugs in cancers producing CA125, including mesothelioma, should consider stratifying patients on baseline CA125 levels for mAbs that are experimentally determined to be bound by CA125.

In vitro preliminary cytotoxicity testing of vegetal extracts, using colorimetric methods

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Claudia Patricia Cordero Camacho

2002-01-01

Full Text Available To advance in the study of the Colombian vegetal biodiversity, considered as a potential source of pharmacologically active products, the establishment of biological activity evaluation systems is necessary, which allow the detection of active products against pathologies with high social and economical impact, such as cancer. This work describes the implementation of a preliminary in vitro methodology for the determination of potential anticancer activity in vegetal extracts, by cytotoxicity testing upon human tumor cell lines, measuring the cellular mass indirectly with the colorimetric assays of MTT (methyl tetrazolium tiazole reduction and SRB (sulforhodamine Bstaining. HT-29, MCF-7, SiHa and HEp-2 cell lines cultures were adapted, MTT concentration, cellular density and treatment period parameters for the cytotoxicity assay were selected. Cell lines sensitivity to the chemotherapeutic agent Doxorubicin HCl was determined. Colombian vegetal species extracts cytotoxicity was tested and usefulness of the assay as a tool to bioguide the search of active products was evidenced.

In vitro preliminary cytotoxicity testing of vegetal extracts, using colorimetric methods

Directory of Open Access Journals (Sweden)

Claudia Patricia Cordero Camacho

2011-12-01

Full Text Available To advance in the study of the Colombian vegetal biodiversity, considered as a potential source of pharmacologically active products, the establishment of biological activity evaluation systems is necessary, which allow the detection of active products against pathologies with high social and economical impact, such as cancer. This work describes the implementation of a preliminary in vitro methodology for the determination of potential anticancer activity in vegetal extracts, by cytotoxicity testing upon human tumor cell lines, measuring the cellular mass indirectly with the colorimetric assays of MTT (methyl tetrazolium tiazole reduction and SRB (sulforhodamine Bstaining. HT-29, MCF-7, SiHa and HEp-2 cell lines cultures were adapted, MTT concentration, cellular density and treatment period parameters for the cytotoxicity assay were selected. Cell lines sensitivity to the chemotherapeutic agent Doxorubicin HCl was determined. Colombian vegetal species extracts cytotoxicity was tested and usefulness of the assay as a tool to bioguide the search of active products was evidenced.

Association of Angiotensin-Converting Enzyme (ACE Gene Polymorphism with Inflammation and Cellular Cytotoxicity in Vitiligo Patients.

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Laila Rashed

Full Text Available Vitiligo is a disorder with profound heterogeneity in its aetio-pathophysiology. Angiotensin converting enzyme (ACE plays an important role in the physiology of the vasculature, blood pressure and inflammation. An insertion/deletion (I/D polymorphism of the ACE gene was reported be associated with the development of vitiligo.Our aim was to evaluate the ACE I/D polymorphism in vitiligo patients and controls. Our second aim was to find a possible association between ACE gene polymorphism and inflammatory mediators (as interleukin (IL-6 and/or cellular cytotoxicity induced by serum nitrite (as a breakdown product of the cytotoxic nitric oxide in vitiligo patients.This case-control study included 74 vitiligo patients and 75 apparently healthy controls. The distribution of ACE gene I/D genotype was investigated using PCR. Serum ACE, IL-6 and nitrite were measured by colorimetric method, ELISA and Griess assay respectively.The ACE allele frequency was significantly different between vitiligo patients and healthy controls (P = 0.026. However there was no significant difference between the ACE genotyping frequency in both groups (P = 0.115. There were statistically significant higher VIDA score (P = 0.007, and serum IL-6 (P < 0.001 in patients with the DD genotype when compared to other genotypes. Serum nitrite in patients with the DD genotype was significantly higher (P = 0.007 when compared to patients with II genotype. Serum levels of ACE, IL-6 and nitrite in vitiligo patients were statistically significantly higher than those in controls.As a conclusion, ACE gene polymorphism might grant susceptibility to develop vitiligo. Serum IL-6 and nitrite levels might have an important role in the pathogenesis of vitiligo. Targeting these two factors might have an implication in the treatment of some resistant cases.

A cellular uptake and cytotoxicity properties study of gallic acid-loaded mesoporous silica nanoparticles on Caco-2 cells

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Rashidi, Ladan; Vasheghani-Farahani, Ebrahim; Soleimani, Masoud; Atashi, Amir; Rostami, Khosrow; Gangi, Fariba; Fallahpour, Masoud; Tahouri, Mohammad Taher

2014-03-01

In this study, the effects of intracellular delivery of various concentrations of gallic acid (GA) as a semistable antioxidant, gallic acid-loaded mesoporous silica nanoparticles (MSNs-GA), and cellular uptake of nanoparticles into Caco-2 cells were investigated. MSNs were synthesized and loaded with GA, then characterized using transmission electron microscopy (TEM), scanning electron microscopy (SEM), Fourier transform infrared spectroscopy, N2 adsorption isotherms, X-ray diffraction, and thermal gravimetric analysis. The cytotoxicity of MSNs and MSNs-GA at low and high concentrations were studied by means of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) test and flow cytometry. MSNs did not show significant toxicity in various concentrations (0-500 μg/ml) on Caco-2 cells. For MSNs-GA, cell viability was reduced as a function of incubation time and different concentrations of nanoparticles. The in vitro GA release from MSNs-GA exhibited the same antitumor properties as free GA on Caco-2 cells. Flow cytometry results confirmed those obtained using MTT assay. TEM and fluorescent microscopy confirmed the internalization of MSNs by Caco-2 cells through nonspecific cellular uptake. MSNs can easily internalize into Caco-2 cells without deleterious effects on cell viability. The cell viability of Caco-2 cells was affected during MSNs-GA uptake. MSNs could be designed as suitable nanocarriers for antioxidants delivery.

Reducing ZnO nanoparticle cytotoxicity by surface modification.

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Luo, Mingdeng; Shen, Cenchao; Feltis, Bryce N; Martin, Lisandra L; Hughes, Anthony E; Wright, Paul F A; Turney, Terence W

2014-06-07

Nanoparticulate zinc oxide (ZnO) is one of the most widely used engineered nanomaterials and its toxicology has gained considerable recent attention. A key aspect for controlling biological interactions at the nanoscale is understanding the relevant nanoparticle surface chemistry. In this study, we have determined the disposition of ZnO nanoparticles within human immune cells by measurement of total Zn, as well as the proportions of extra- and intracellular dissolved Zn as a function of dose and surface coating. From this mass balance, the intracellular soluble Zn levels showed little difference in regard to dose above a certain minimal level or to different surface coatings. PEGylation of ZnO NPs reduced their cytotoxicity as a result of decreased cellular uptake arising from a minimal protein corona. We conclude that the key role of the surface properties of ZnO NPs in controlling cytotoxicity is to regulate cellular nanoparticle uptake rather than altering either intracellular or extracellular Zn dissolution.

Cytotoxicity and intracellular dissolution of nickel nanowires

KAUST Repository

Perez, Jose E.

2015-12-22

The assessment of cytotoxicity of nanostructures is a fundamental step for their development as biomedical tools. As widely used nanostructures, nickel nanowires (Ni NWs) seem promising candidates for such applications. In this work, Ni NWs were synthesized and then characterized using vibrating sample magnetometry, energy dispersive X-Ray analysis and electron microscopy. After exposure to the NWs, cytotoxicity was evaluated in terms of cell viability, cell membrane damage and induced apoptosis/necrosis on the model human cell line HCT 116. The influence of NW to cell ratio (10:1 to 1000:1) and exposure times up to 72 hours was analyzed for Ni NWs of 5.4 µm in length, as well as for Ni ions. The results show that cytotoxicity markedly increases past 24 hours of incubation. Cellular uptake of NWs takes place through the phagocytosis pathway, with a fraction of the dose of NWs dissolved inside the cells. Cell death results from a combination of apoptosis and necrosis, where the latter is the outcome of the secondary necrosis pathway. The cytotoxicity of Ni ions and Ni NWs dissolution studies suggest a synergistic toxicity between NW aspect ratio and dissolved Ni, with the cytotoxic effects markedly increasing after 24 hours of incubation.

Cytotoxicity and intracellular dissolution of nickel nanowires.

Science.gov (United States)

Perez, Jose E; Contreras, Maria F; Vilanova, Enrique; Felix, Laura P; Margineanu, Michael B; Luongo, Giovanni; Porter, Alexandra E; Dunlop, Iain E; Ravasi, Timothy; Kosel, Jürgen

2016-09-01

The assessment of cytotoxicity of nanostructures is a fundamental step for their development as biomedical tools. As widely used nanostructures, nickel nanowires (Ni NWs) seem promising candidates for such applications. In this work, Ni NWs were synthesized and then characterized using vibrating sample magnetometry, energy dispersive X-Ray analysis, and electron microscopy. After exposure to the NWs, cytotoxicity was evaluated in terms of cell viability, cell membrane damage, and induced apoptosis/necrosis on the model human cell line HCT 116. The influence of NW to cell ratio (10:1 to 1000:1) and exposure times up to 72 hours was analyzed for Ni NWs of 5.4 μm in length, as well as for Ni ions. The results show that cytotoxicity markedly increases past 24 hours of incubation. Cellular uptake of NWs takes place through the phagocytosis pathway, with a fraction of the dose of NWs dissolved inside the cells. Cell death results from a combination of apoptosis and necrosis, where the latter is the outcome of the secondary necrosis pathway. The cytotoxicity of Ni ions and Ni NWs dissolution studies suggest a synergistic toxicity between NW aspect ratio and dissolved Ni, with the cytotoxic effects markedly increasing after 24 hours of incubation.

Cytotoxicity and intracellular dissolution of nickel nanowires

KAUST Repository

Perez, Jose E.; Contreras, Maria F.; Vidal, Enrique Vilanova; Felix Servin, Laura P.; Margineanu, Michael B.; Luongo, Giovanni; Porter, Alexandra E.; Dunlop, Iain E.; Ravasi, Timothy; Kosel, Jü rgen

2015-01-01

The assessment of cytotoxicity of nanostructures is a fundamental step for their development as biomedical tools. As widely used nanostructures, nickel nanowires (Ni NWs) seem promising candidates for such applications. In this work, Ni NWs were synthesized and then characterized using vibrating sample magnetometry, energy dispersive X-Ray analysis and electron microscopy. After exposure to the NWs, cytotoxicity was evaluated in terms of cell viability, cell membrane damage and induced apoptosis/necrosis on the model human cell line HCT 116. The influence of NW to cell ratio (10:1 to 1000:1) and exposure times up to 72 hours was analyzed for Ni NWs of 5.4 µm in length, as well as for Ni ions. The results show that cytotoxicity markedly increases past 24 hours of incubation. Cellular uptake of NWs takes place through the phagocytosis pathway, with a fraction of the dose of NWs dissolved inside the cells. Cell death results from a combination of apoptosis and necrosis, where the latter is the outcome of the secondary necrosis pathway. The cytotoxicity of Ni ions and Ni NWs dissolution studies suggest a synergistic toxicity between NW aspect ratio and dissolved Ni, with the cytotoxic effects markedly increasing after 24 hours of incubation.

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

Energy Technology Data Exchange (ETDEWEB)

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

2016-10-01

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

Early spatiotemporal-specific changes in intermediate signals are predictive of cytotoxic sensitivity to TNFα and co-treatments

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Loo, Lit-Hsin; Bougen-Zhukov, Nicola Michelle; Tan, Wei-Ling Cecilia

2017-03-01

Signaling pathways can generate different cellular responses to the same cytotoxic agents. Current quantitative models for predicting these differential responses are usually based on large numbers of intracellular gene products or signals at different levels of signaling cascades. Here, we report a study to predict cellular sensitivity to tumor necrosis factor alpha (TNFα) using high-throughput cellular imaging and machine-learning methods. We measured and compared 1170 protein phosphorylation events in a panel of human lung cancer cell lines based on different signals, subcellular regions, and time points within one hour of TNFα treatment. We found that two spatiotemporal-specific changes in an intermediate signaling protein, p90 ribosomal S6 kinase (RSK), are sufficient to predict the TNFα sensitivity of these cell lines. Our models could also predict the combined effects of TNFα and other kinase inhibitors, many of which are not known to target RSK directly. Therefore, early spatiotemporal-specific changes in intermediate signals are sufficient to represent the complex cellular responses to these perturbations. Our study provides a general framework for the development of rapid, signaling-based cytotoxicity screens that may be used to predict cellular sensitivity to a cytotoxic agent, or identify co-treatments that may sensitize or desensitize cells to the agent.

Fast intracellular dissolution and persistent cellular uptake of silver nanoparticles in CHO-K1 cells: implication for cytotoxicity

DEFF Research Database (Denmark)

Jiang, Xiumei; Miclaus, Teodora; Wang, Liming

2015-01-01

. Subsequent cytotoxicity studies show that Ag NPs decrease cell viability and increase ROS production. Pre-incubation with N-acetyl-l-cysteine, an efficient antioxidant and Ag+ chelator, diminished the cytotoxicity caused by Ag NPs or Ag+ exposure. Our study suggests that the cytotoxicity mechanism of Ag NPs...

Three-Year Durability of Immune Responses Induced by HIV-DNA and HIV-Modified Vaccinia Virus Ankara and Effect of a Late HIV-Modified Vaccinia Virus Ankara Boost in Tanzanian Volunteers.

Science.gov (United States)

Joachim, Agricola; Munseri, Patricia J; Nilsson, Charlotta; Bakari, Muhammad; Aboud, Said; Lyamuya, Eligius F; Tecleab, Teghesti; Liakina, Valentina; Scarlatti, Gabriella; Robb, Merlin L; Earl, Patricia L; Moss, Bernard; Wahren, Britta; Mhalu, Fred; Ferrari, Guido; Sandstrom, Eric; Biberfeld, Gunnel

2017-08-01

We explored the duration of immune responses and the effect of a late third HIV-modified vaccinia virus Ankara (MVA) boost in HIV-DNA primed and HIV-MVA boosted Tanzanian volunteers. Twenty volunteers who had previously received three HIV-DNA and two HIV-MVA immunizations were given a third HIV-MVA immunization 3 years after the second HIV-MVA boost. At the time of the third HIV-MVA, 90% of the vaccinees had antibodies to HIV-1 subtype C gp140 (median titer 200) and 85% to subtype B gp160 (median titer 100). The majority of vaccinees had detectable antibody-dependent cellular cytotoxicity (ADCC)-mediating antibodies, 70% against CRF01_AE virus-infected cells (median titer 239) and 84% against CRF01_AE gp120-coated cells (median titer 499). A high proportion (74%) of vaccinees had IFN-γ ELISpot responses, 63% to Gag and 42% to Env, 3 years after the second HIV-MVA boost. After the third HIV-MVA, there was an increase in Env-binding antibodies and ADCC-mediating antibodies relative to the response seen at the time of the third HIV-MVA vaccination, p < .0001 and p < .05, respectively. The frequency of IFN-γ ELISpot responses increased to 95% against Gag or Env and 90% to both Gag and Env, p = .064 and p = .002, respectively. In conclusion, the HIV-DNA prime/HIV-MVA boost regimen elicited potent antibody and cellular immune responses with remarkable durability, and a third HIV-MVA immunization significantly boosted both antibody and cellular immune responses relative to the levels detected at the time of the third HIV-MVA, but not to higher levels than after the second HIV-MVA.

Synergistic anti-tumor therapy by a comb-like multifunctional antibody nanoarray with exceptionally potent activity

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Li, Huafei; Sun, Yun; Chen, Di; Zhao, He; Zhao, Mengxin; Zhu, Xiandi; Ke, Changhong; Zhang, Ge; Jiang, Cheng; Zhang, Li; Zhang, Fulei; Wei, Huafeng; Li, Wei

2015-10-01

Simultaneously blocking multiple mediators offers new hope for the treatment of complex diseases. However, the curative potential of current combination therapy by chronological administration of separate monoclonal antibodies (mAbs) or multi-specific mAbs is still moderate due to inconvenient manipulation, low cooperative effectors, poor pharmacokinetics and insufficient tumor accumulation. Here, we describe a facile strategy that arms distinct mAbs with cooperative effectors onto a long chain to form a multicomponent comb-like nano mAb. Unlike dissociative parental mAbs, the multifunctional mAb nanoarray (PL-RB) constructed from type I/II anti-CD20 mAbs shows good pharmacokinetics. This PL-RB simultaneously targets distinct epitopes on a single antigen (Ag) and neighboring Ags on different lymphocytes. This unique intra- and intercellular Ag cross-linking endows the multifunctional mAb nanoarray with potent apoptosis activity. The exceptional apoptosis, complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC) that are synchronously evoked by the nano PL-RB are further synergistically promoted via enhanced permeability and retention (EPR), which resulted in high intratumor accumulation and excellent anti-lymphoma efficiency.

Shedding of Ebola Virus Surface Glycoprotein Is a Mechanism of Self-regulation of Cellular Cytotoxicity and Has a Direct Effect on Virus Infectivity.

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Dolnik, Olga; Volchkova, Valentina A; Escudero-Perez, Beatriz; Lawrence, Philip; Klenk, Hans-Dieter; Volchkov, Viktor E

2015-10-01

The surface glycoprotein (GP) is responsible for Ebola virus (EBOV) attachment and membrane fusion during virus entry. Surface expression of highly glycosylated GP causes marked cytotoxicity via masking of a wide range of cellular surface molecules, including integrins. Considerable amounts of surface GP are shed from virus-infected cells in a soluble truncated form by tumor necrosis factor α-converting enzyme. In this study, the role of GP shedding was investigated using a reverse genetics approach by comparing recombinant viruses possessing amino acid substitutions at the GP shedding site. Virus with an L635V substitution showed a substantial decrease in shedding, whereas a D637V substitution resulted in a striking increase in the release of shed GP. Variations in shedding efficacy correlated with observed differences in the amounts of shed GP in the medium, GP present in virus-infected cells, and GP present on virions. An increase in shedding appeared to be associated with a reduction in viral cytotoxicity, and, vice versa, the virus that shed less was more cytotoxic. An increase in shedding also resulted in a reduction in viral infectivity, whereas a decrease in shedding efficacy enhanced viral growth characteristics in vitro. Differences in shedding efficacy and, as a result, differences in the amount of mature GP available for incorporation into budding virions did not equate to differences in overall release of viral particles. Likewise, data suggest that the resulting differences in the amount of mature GP on the cell surface led to variations in the GP content of released particles and, as a consequence, in infectivity. In conclusion, fine-tuning of the levels of EBOV GP expressed at the surface of virus-infected cells via GP shedding plays an important role in EBOV replication by orchestrating the balance between optimal virion GP content and cytotoxicity caused by GP. © The Author 2015. Published by Oxford University Press on behalf of the Infectious

Synthesis, cytotoxicity, cellular uptake and influence on eicosanoid metabolism of cobalt-alkyne modified fructoses in comparison to auranofin and the cytotoxic COX inhibitor Co-ASS.

Science.gov (United States)

Ott, Ingo; Koch, Thao; Shorafa, Hashem; Bai, Zhenlin; Poeckel, Daniel; Steinhilber, Dieter; Gust, Ronald

2005-06-21

Propargylhexacarbonyldicobalt complexes with fructopyranose ligands were prepared and investigated for cytotoxicity in the MCF-7 human breast cancer cell line. The antiproliferative effects depended on the presence of isopropylidene protecting groups in the carbohydrate ligand and correlated with the cellular concentration of the complexes. IC(50) values of > 20 microM demonstrated that the fructose derivatives were only moderately active compared to the references auranofin and the aspirin (ASS) derivative [2-acetoxy(2-propynyl)benzoate]hexacarbonyldicobalt (Co-ASS). In continuation of our studies on the mode of action of cobalt-alkyne complexes we studied the influence of the compounds on the formation of 12-HHT (COX-1 product) and 12-HETE (12-LOX product) by human platelets as an indication of the interference in the eicosanoid metabolism, which is discussed as a target system of cytostatics. Co-ASS was an efficient COX-1 inhibitor without LOX inhibitory activity and auranofin inhibited both COX-1 and 12-LOX eicosanoid production. The missing activity of the fructopyranose complexes at the 12-LOX and the only moderate effects at COX-1 indicate that COX/LOX inhibition may be in part responsible for the pharmacological effects of auranofin and Co-ASS but not for those of the fructopyranose complexes.

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

Science.gov (United States)

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

2012-07-01

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

Evidence for the protective effect of maternal FcR-blocking IgG alloantibodies HLA-DR in Rh D-haemolytic disease of the newborn

NARCIS (Netherlands)

Dooren, M. C.; van Kamp, I. L.; Kanhai, H. H.; Gravenhorst, J. B.; von dem Borne, A. E.; Engelfriet, C. P.

1993-01-01

In cases of Rh D alloimmunization, strong results in the antibody-dependent cell-mediated cytotoxicity (ADCC) assay (> 80% lysis as compared to that of the standard anti-D serum) are indicative of severe hemolytic disease to occur in the newborn (HDN). However, discrepant cases were found in which

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

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Caroline B Madsen

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

Enzymatic Inactivation of Endogenous IgG by IdeS Enhances Therapeutic Antibody Efficacy.

Science.gov (United States)

Järnum, Sofia; Runström, Anna; Bockermann, Robert; Winstedt, Lena; Crispin, Max; Kjellman, Christian

2017-09-01

Endogenous plasma IgG sets an immunologic threshold that dictates the activity of tumor-directed therapeutic antibodies. Saturation of cellular antibody receptors by endogenous antibody limits antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Here, we show how enzymatic cleavage of IgG using the bacterial enzyme IdeS can be utilized to empty both high and low affinity Fcγ-receptors and clear the entire endogenous antibody pool. Using in vitro models, tumor animal models as well as ex vivo analysis of sera collected during a previous clinical trial with IdeS, we show how clearing of competing plasma antibody levels with IdeS unblocks cellular antibody receptors. We show that therapeutic antibodies against breast cancer (trastuzumab), colon cancer (cetuximab), and lymphomas (rituximab and alemtuzumab) can be potentiated when endogenous IgG is removed. Overall, IdeS is shown to be a potent tool to reboot the human antibody repertoire and to generate a window to preferentially load therapeutic antibodies onto effector cells and thereby create an armada of dedicated tumor-seeking immune cells. Mol Cancer Ther; 16(9); 1887-97. ©2017 AACR . ©2017 American Association for Cancer Research.

Polyfunctional HIV-Specific Antibody Responses Are Associated with Spontaneous HIV Control.

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Margaret E Ackerman

2016-01-01

Full Text Available Elite controllers (ECs represent a unique model of a functional cure for HIV-1 infection as these individuals develop HIV-specific immunity able to persistently suppress viremia. Because accumulating evidence suggests that HIV controllers generate antibodies with enhanced capacity to drive antibody-dependent cellular cytotoxicity (ADCC that may contribute to viral containment, we profiled an array of extra-neutralizing antibody effector functions across HIV-infected populations with varying degrees of viral control to define the characteristics of antibodies associated with spontaneous control. While neither the overall magnitude of antibody titer nor individual effector functions were increased in ECs, a more functionally coordinated innate immune-recruiting response was observed. Specifically, ECs demonstrated polyfunctional humoral immune responses able to coordinately recruit ADCC, other NK functions, monocyte and neutrophil phagocytosis, and complement. This functionally coordinated response was associated with qualitatively superior IgG3/IgG1 responses, whereas HIV-specific IgG2/IgG4 responses, prevalent among viremic subjects, were associated with poorer overall antibody activity. Rather than linking viral control to any single activity, this study highlights the critical nature of functionally coordinated antibodies in HIV control and associates this polyfunctionality with preferential induction of potent antibody subclasses, supporting coordinated antibody activity as a goal in strategies directed at an HIV-1 functional cure.

Anticancer agent CHS-828 inhibits cellular synthesis of NAD

DEFF Research Database (Denmark)

Olesen, U.H.; Christensen, M.K.; Bjorkling, F.

2008-01-01

Malignant cells display increased demands for energy production and DNA repair. Nicotinamide adenine dinucleotide (NAD) is required for both processes and is also continuously degraded by cellular enzymes. Nicotinamide phosphoribosyltransferase (Nampt) is a crucial factor in the resynthesis of NAD......, and thus in cancer cell survival. Here, we establish the cytotoxic mechanism of action of the small molecule inhibitor CHS-828 to result from impaired synthesis of NAD. Initially, we detected cross-resistance in cells between CHS-828 and a known inhibitor of Nampt, FK866, a compound of a structurally...... different class. We then showed that nicotinamide protects against CHS-828-mediated cytotoxicity. Finally, we observed that treatment with CHS-828 depletes cellular NAD levels in sensitive cancer cells. In conclusion, these results strongly suggest that, like FK866, CHS-828 kills cancer cells by depleting...

Analysis of cytotoxic effects of nickel on human blood lymphocytes.

Science.gov (United States)

Zarei, Mohammad Hadi; Hosseini Shirazi, Seyed Farshad; Aghvami, Marjan; Salimi, Ahmad; Pourahmad, Jalal

2018-02-01

Nickel compounds possess many applications in different industrial processes. Human beings are exposed to nickel commonly through occupational exposure and food. Although a few studies so far have investigated the effects of nickel compounds on human lymphocytes, the complete mechanism of cytotoxicity of this metal on human lymphocytes is yet to be determined. The intention of this paper was to determine the cytotoxicity mechanism of water soluble NiCl 2 toward human lymphocytes using the accelerated cytotoxicity mechanisms screening (ACMS) technique. Human lymphocytes were isolated from the blood of healthy subjects based on Ficoll-Paque PLUS standard method. For the assessment of cell viability, lymphocytes were incubated with 0.05-1 mM NiCl 2 for 12 h. Determination of mechanistic parameters was performed 2, 4 and 6 h after treatment of cells with ½ EC50 12h , EC50 12h and 2EC50 12h of NiCl 2 . Our results demonstrate that cytotoxicity of NiCl 2 on human lymphocytes is associated with increased ROS formation, mitochondrial membrane potential collapse, glutathione depletion, lysosomal membrane damage, cellular proteolysis and activation of caspase-3 before cytotoxicity ensued.

Complex immunological monitoring of breast cancer patients treated postoperatively by electron beam irradiation

International Nuclear Information System (INIS)

Horvath, M.; Horvath, A.; Fekete, B.; Toth, J.

1986-01-01

To monitor the electron beam therapy some immunological parameters of breast cancer patients previously undergone surgery were tested before, during and after irradiation. Immune complex levels measured by complement consumption technique were not altered by irradiation. Killer cell activity tested in so-called antibody dependent cellular cytotoxicity (ADCC) capacity assay showed a marked decrease in some cases. Based on the phagocytic capacity of the granulocytes the patients could be divided into two groups: one with declining activity and another with rising activity: The majority of the patients (22/45) were humoral leukocyte adherence inhibition (H-LAI) negative before and during irradiation. Those showed positive H-LAI indices before electron therapy had unchanged (7/45) or decreasing (9/45) tendencies during the observation period. Further study is needed to establish the clinical relevance of these in vitro assays used by us. (orig.) [de

Dynamics and mechanisms of quantum dot nanoparticle cellular uptake

Directory of Open Access Journals (Sweden)

Telford William G

2010-06-01

Full Text Available Abstract Background The rapid growth of the nanotechnology industry and the wide application of various nanomaterials have raised concerns over their impact on the environment and human health. Yet little is known about the mechanism of cellular uptake and cytotoxicity of nanoparticles. An array of nanomaterials has recently been introduced into cancer research promising for remarkable improvements in diagnosis and treatment of the disease. Among them, quantum dots (QDs distinguish themselves in offering many intrinsic photophysical properties that are desirable for targeted imaging and drug delivery. Results We explored the kinetics and mechanism of cellular uptake of QDs with different surface coatings in two human mammary cells. Using fluorescence microscopy and laser scanning cytometry (LSC, we found that both MCF-7 and MCF-10A cells internalized large amount of QD655-COOH, but the percentage of endocytosing cells is slightly higher in MCF-7 cell line than in MCF-10A cell line. Live cell fluorescent imaging showed that QD cellular uptake increases with time over 40 h of incubation. Staining cells with dyes specific to various intracellular organelles indicated that QDs were localized in lysosomes. Transmission electron microscopy (TEM images suggested a potential pathway for QD cellular uptake mechanism involving three major stages: endocytosis, sequestration in early endosomes, and translocation to later endosomes or lysosomes. No cytotoxicity was observed in cells incubated with 0.8 nM of QDs for a period of 72 h. Conclusions The findings presented here provide information on the mechanism of QD endocytosis that could be exploited to reduce non-specific targeting, thereby improving specific targeting of QDs in cancer diagnosis and treatment applications. These findings are also important in understanding the cytotoxicity of nanomaterials and in emphasizing the importance of strict environmental control of nanoparticles.

Cytotoxicity and inhibitory properties against topoisomerase II of doxorubicin and its formamidine derivatives.

Science.gov (United States)

Kik, Krzysztof; Studzian, Kazimierz; Wasowska-Łukawska, Małgorzata; Oszczapowicz, Irena; Szmigiero, Leszek

2009-01-01

This work was undertaken to compare cytotoxicity, DNA damaging properties and effect on DNA cleavage by topoisomerase II of the anthracycline drug doxorubicin (DOX) and its two derivatives with a formamidino group containing a cyclic amine moiety such as morpholine (DOXM) or hexamethyleneimine (DOXH). The tetrazolium dye colorimetric assay was used to determine the cytotoxic activity of anthracyclines toward L1210 leukemia cells. DNA damage was measured by alkaline elution technique. The effect of anthracyclines on DNA cleavage was studied in a cell-free system containing supercoiled pBR322 DNA and purified human topoisomerase II. The cytotoxicity data and the results of studies on the mechanism of DNA break formation by anthracyclines at the cellular level and in the cell-free system showed that the presence of the formamidino group in the doxorubicin molecule reduced its ability to stimulate DNA cleavage by DNA topoisomerase II. DNA topoisomerase II is not a primary cellular target for DOXM or DOXH. An advantageous feature of formamidinoanthracyclines is their mechanism of cytotoxic action which is not related to the inhibition of DNA topoisomerase II. Therefore this class of anthracyclines seems to be a good source for selection of an anticancer drug directed toward cancer cells with the developed multidrug resistance attributed to the presence of altered DNA topoisomerase II.

Cellular injury evidenced by impedance technology and infrared microspectroscopy

Science.gov (United States)

le Roux, K.; Prinsloo, L. C.; Meyer, D.

2015-03-01

Fourier Transform Infrared (FTIR) spectroscopy is finding increasing biological application, for example in the analysis of diseased tissues and cells, cell cycle studies and investigating the mechanisms of action of anticancer drugs. Cancer treatment studies routinely define the types of cell-drug responses as either total cell destruction by the drug (all cells die), moderate damage (cell deterioration where some cells survive) or reversible cell cycle arrest (cytostasis). In this study the loss of viability and related chemical stress experienced by cells treated with the medicinal plant, Plectranthus ciliatus, was investigated using real time cell electronic sensing (RT-CES) technology and FTIR microspectroscopy. The use of plants as medicines is well established and ethnobotany has proven that crude extracts can serve as treatments against various ailments. The aim of this study was to determine whether FTIR microspectroscopy would successfully distinguish between different types of cellular injury induced by a potentially anticancerous plant extract. Cervical adenocarcinoma (HeLa) cells were treated with a crude extract of Pciliatus and cells monitored using RT-CES to characterize the type of cellular responses induced. Cell populations were then investigated using FTIR microspectroscopy and statistically analysed using One-way Analysis of Variance (ANOVA) and Principal Component Analysis (PCA). The plant extract and a cancer drug control (actinomycin D) induced concentration dependent cellular responses ranging from nontoxic, cytostatic or cytotoxic. Thirteen spectral peaks (915 cm-1, 933 cm-1, 989 cm-1, 1192 cm-1, 1369 cm-1, 1437 cm-1, 1450 cm-1, 1546 cm-1, 1634 cm-1, 1679 cm-1 1772 cm-1, 2874 cm-1 and 2962 cm-1) associated with cytotoxicity were significantly (p value < 0.05, one way ANOVA, Tukey test, Bonferroni) altered, while two of the bands were also indicative of early stress related responses. In PCA, poor separation between nontoxic and cytostatic

Evaluation of Cytotoxic Effects of Different Concentrations of Porous Hollow Au Nanoparticles (PHAuNPs) on Cells

International Nuclear Information System (INIS)

Rao, S.; Tata, U.; Lin, V.K.; Chiao, J.C.; Huang, Ch.; Hao, Y.; Wu, P.; Arora, N.; Ahn, J.

2014-01-01

Nanoparticles (NPs) have been introduced as a suitable alternative in many in vivo bio applications. The risks of utilizing nanoparticles continue to be an ongoing research. Furthermore, the various chemicals used in their synthesis influence the cytotoxic effects of nanoparticles. We have investigated the cytotoxicity of Porous Hollow Au Nanoparticles (PHAuNPs) on cancer cell lines PC-3, PC-3ML, and MDA-MB-231 and the normal cell line PNT1A. Cell proliferation for the different cells in the presence of different concentrations of the PHAuNPs was assessed after 24 hours and 72 hours of incubation using MTT assay. The study also included the cytotoxic evaluation of pegylated PHAuNPs. Identical cell seeding densities, particle concentrations, and incubation times were employed for these two types of Au nanoparticles. Our results indicated that (1) impact on cell proliferation was concentration dependent and was different for the different cell types without cellular necrosis and (b) cellular proliferation might be impacted more based on the cell line.

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

Science.gov (United States)

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

1994-08-01

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

Nanosecond pulsed electric field (nsPEF) enhance cytotoxicity of cisplatin to hepatocellular cells by microdomain disruption on plasma membrane.

Science.gov (United States)

Yin, Shengyong; Chen, Xinhua; Xie, Haiyang; Zhou, Lin; Guo, Danjing; Xu, Yuning; Wu, Liming; Zheng, Shusen

2016-08-15

Previous studies showed nanosecond pulsed electric field (nsPEF) can ablate solid tumors including hepatocellular carcinoma (HCC) but its effect on cell membrane is not fully understood. We hypothesized nsPEF disrupt the microdomains on outer-cellular membrane with direct mechanical force and as a result the plasma membrane permeability increases to facilitate the small molecule intake. Three HCC cells were pulsed one pulse per minute, an interval longer than nanopore resealing time. The cationized ferritin was used to mark up the electronegative microdomains, propidium iodide (PI) for membrane permeabilization, energy dispersive X-ray spectroscopy (EDS) for the negative cell surface charge and cisplatin for inner-cellular cytotoxicity. We demonstrated that the ferritin marked-microdomain and negative cell surface charge were disrupted by nsPEF caused-mechanical force. The cell uptake of propidium and cytotoxicity of DNA-targeted cisplatin increased with a dose effect. Cisplatin gains its maximum inner-cellular cytotoxicity when combining with nsPEF stimulation. We conclude that nsPEF disrupt the microdomains on the outer cellular membrane directly and increase the membrane permeabilization for PI and cisplatin. The microdomain disruption and membrane infiltration changes are caused by the mechanical force from the changes of negative cell surface charge. Copyright © 2016 Elsevier Inc. All rights reserved.

Nanosecond pulsed electric field (nsPEF) enhance cytotoxicity of cisplatin to hepatocellular cells by microdomain disruption on plasma membrane

International Nuclear Information System (INIS)

Yin, Shengyong; Chen, Xinhua; Xie, Haiyang; Zhou, Lin; Guo, Danjing; Xu, Yuning; Wu, Liming; Zheng, Shusen

2016-01-01

Previous studies showed nanosecond pulsed electric field (nsPEF) can ablate solid tumors including hepatocellular carcinoma (HCC) but its effect on cell membrane is not fully understood. We hypothesized nsPEF disrupt the microdomains on outer-cellular membrane with direct mechanical force and as a result the plasma membrane permeability increases to facilitate the small molecule intake. Three HCC cells were pulsed one pulse per minute, an interval longer than nanopore resealing time. The cationized ferritin was used to mark up the electronegative microdomains, propidium iodide (PI) for membrane permeabilization, energy dispersive X-ray spectroscopy (EDS) for the negative cell surface charge and cisplatin for inner-cellular cytotoxicity. We demonstrated that the ferritin marked-microdomain and negative cell surface charge were disrupted by nsPEF caused-mechanical force. The cell uptake of propidium and cytotoxicity of DNA-targeted cisplatin increased with a dose effect. Cisplatin gains its maximum inner-cellular cytotoxicity when combining with nsPEF stimulation. We conclude that nsPEF disrupt the microdomains on the outer cellular membrane directly and increase the membrane permeabilization for PI and cisplatin. The microdomain disruption and membrane infiltration changes are caused by the mechanical force from the changes of negative cell surface charge.

Nanosecond pulsed electric field (nsPEF) enhance cytotoxicity of cisplatin to hepatocellular cells by microdomain disruption on plasma membrane

Energy Technology Data Exchange (ETDEWEB)

Yin, Shengyong; Chen, Xinhua; Xie, Haiyang; Zhou, Lin [Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Zhejiang University, 310003 Hangzhou (China); Key Laboratory of Combined Multi-organ Transplantation, Ministry of Public Health and Key Laboratory of Organ Transplantation of Zhejiang Province, The Department of Hepatobiliary and Pancreatic Surgery, The First Affiliated Hospital, Zhejiang University, Hangzhou 310003 (China); Guo, Danjing; Xu, Yuning [Key Laboratory of Combined Multi-organ Transplantation, Ministry of Public Health and Key Laboratory of Organ Transplantation of Zhejiang Province, The Department of Hepatobiliary and Pancreatic Surgery, The First Affiliated Hospital, Zhejiang University, Hangzhou 310003 (China); Wu, Liming, E-mail: wlm@zju.edu.cn [Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Zhejiang University, 310003 Hangzhou (China); Key Laboratory of Combined Multi-organ Transplantation, Ministry of Public Health and Key Laboratory of Organ Transplantation of Zhejiang Province, The Department of Hepatobiliary and Pancreatic Surgery, The First Affiliated Hospital, Zhejiang University, Hangzhou 310003 (China); Zheng, Shusen, E-mail: shusenzheng@zju.edu.cn [Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Zhejiang University, 310003 Hangzhou (China); Key Laboratory of Combined Multi-organ Transplantation, Ministry of Public Health and Key Laboratory of Organ Transplantation of Zhejiang Province, The Department of Hepatobiliary and Pancreatic Surgery, The First Affiliated Hospital, Zhejiang University, Hangzhou 310003 (China)

2016-08-15

Previous studies showed nanosecond pulsed electric field (nsPEF) can ablate solid tumors including hepatocellular carcinoma (HCC) but its effect on cell membrane is not fully understood. We hypothesized nsPEF disrupt the microdomains on outer-cellular membrane with direct mechanical force and as a result the plasma membrane permeability increases to facilitate the small molecule intake. Three HCC cells were pulsed one pulse per minute, an interval longer than nanopore resealing time. The cationized ferritin was used to mark up the electronegative microdomains, propidium iodide (PI) for membrane permeabilization, energy dispersive X-ray spectroscopy (EDS) for the negative cell surface charge and cisplatin for inner-cellular cytotoxicity. We demonstrated that the ferritin marked-microdomain and negative cell surface charge were disrupted by nsPEF caused-mechanical force. The cell uptake of propidium and cytotoxicity of DNA-targeted cisplatin increased with a dose effect. Cisplatin gains its maximum inner-cellular cytotoxicity when combining with nsPEF stimulation. We conclude that nsPEF disrupt the microdomains on the outer cellular membrane directly and increase the membrane permeabilization for PI and cisplatin. The microdomain disruption and membrane infiltration changes are caused by the mechanical force from the changes of negative cell surface charge.

Modification of the cytotoxic activity of mitomycin C

International Nuclear Information System (INIS)

Marshall, R.S.; Rauth, A.M.

1985-01-01

Utilizing a system in which oxygen levels could be altered and monitored during acute drug exposures, the authors have begun to characterize the cellular and molecular damage produced by MMC in CHO cells. The cytotoxic activity of MMC decreases sharply from 0 to 0.1% oxygen in solution, while from 0.1 to 20.0% there is little change. DNA crosslinking in cells was examined under these conditions by alkaline elution and found to be directly correlated with cell killing. While hypoxia increased crosslinking, significant levels were still observed under aerobic conditions. A cell-free assay for alkylation confirmed that overall levels increase in the absence of oxygen; however, negligible alkylation was observed under aerobic conditions. It was also noted that ascorbic acid present in the exposure medium (0.284 mM) increased the aerobic cytotoxicity without altering the hypoxic cytotoxicity. The present data suggest that MMC can be activated to an alkylating species by two mechanisms, one oxygen sensitive and one oxygen insensitive and that these two mechanisms may be independently modified

Industrial grade 2D molybdenum disulphide (MoS2): an in vitro exploration of the impact on cellular uptake, cytotoxicity, and inflammation

Science.gov (United States)

Moore, Caroline; Movia, Dania; Smith, Ronan J.; Hanlon, Damien; Lebre, Filipa; Lavelle, Ed C.; Byrne, Hugh J.; Coleman, Jonathan N.; Volkov, Yuri; McIntyre, Jennifer

2017-06-01

The recent surge in graphene research, since its liquid phase monolayer isolation and characterization in 2004, has led to advancements which are accelerating the exploration of alternative 2D materials such as molybdenum disulphide (MoS2), whose unique physico-chemical properties can be exploited in applications ranging from cutting edge electronic devices to nanomedicine. However, to assess any potential impact on human health and the environment, the need to understand the bio-interaction of MoS2 at a cellular and sub-cellular level is critical. Notably, it is important to assess such potential impacts of materials which are produced by large scale production techniques, rather than research grade materials. The aim of this study was to explore cytotoxicity, cellular uptake and inflammatory responses in established cell-lines that mimic different potential exposure routes (inhalation, A549; ingestion, AGS; monocyte, THP-1) following incubation with MoS2 flakes of varying sizes (50 nm, 117 nm and 177 nm), produced by liquid phase exfoliation. Using high content screening (HCS) and Live/Dead assays, it was established that 1 µg ml-1 (for the three different MoS2 sizes) did not induce toxic effects on any of the cell-lines. Confocal microscopy images revealed a normal cellular morphology in all cases. Transmission electron microscopy (TEM) confirmed the uptake of all MoS2 nanomaterials in all the cell-lines, the MoS2 ultimately locating in single membrane vesicles. At such sub-lethal doses, inflammatory responses are observed, however, associated, at least partially, with the presence of lipopolysaccharide endotoxin in nanomaterial suspensions and surfactant samples. Therefore, the inflammatory response of the cells to the MoS2 or endotoxin contamination was interrogated using a 10-plex ELISA which illustrates cytokine production. The experiments carried out using wild-type and endotoxin hyporesponsive bone marrow derived dendritic cells confirmed that the

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

African Journals Online (AJOL)

Background: Assessment of patients prior to cytotoxic chemotherapy usually includes absolute neutrophils count. Other cellular markers of susceptibility to infection as well as immunocompetence include the T Helper lymphocyte count. In cancer patients, decrease in these lymphocytes has been observed to be associated ...

Cellular glutathione prevents cytolethality of monomethylarsonic acid

International Nuclear Information System (INIS)

Sakurai, Teruaki; Kojima, Chikara; Ochiai, Masayuki; Ohta, Takami; Sakurai, Masumi H.; Waalkes, Michael P.; Fujiwara, Kitao

2004-01-01

Inorganic arsenicals are clearly toxicants and carcinogens in humans. In mammals, including humans, inorganic arsenic often undergoes methylation, forming compounds such as monomethylarsonic acid (MMAs V ) and dimethylarsinic acid (DMAs V ). However, much less information is available on the in vitro toxic potential or mechanisms of these methylated arsenicals, especially MMAs V . We studied the molecular mechanisms of in vitro cytolethality of MMAs V using a rat liver epithelial cell line (TRL 1215). MMAs V was not cytotoxic in TRL 1215 cells even at concentrations exceeding 10 mM, but it became weakly cytotoxic and induced both necrotic and apoptotic cell death when cellular reduced glutathione (GSH) was depleted with the glutathione synthase inhibitor, L-buthionine-[S,R]-sulfoximine (BSO), or the glutathione reductase inhibitor, carmustine. Similar results were observed in the other mammalian cells, such as human skin TIG-112 cells, chimpanzee skin CRT-1609 cells, and mouse metallothionein (MT) positive and MT negative embryonic cells. Ethacrynic acid (EA), an inhibitor of glutathione S-transferase (GST) that catalyses GSH-substrate conjugation, also enhanced the cytolethality of MMAs V , but aminooxyacetic acid (AOAA), an inhibitor of β-lyase that catalyses the final breakdown of GSH-substrate conjugates, had no effect. Both the cellular GSH levels and the cellular GST activity were increased by the exposure to MMAs V in TRL 1215 cells. On the other hand, the addition of exogenous extracellular GSH enhanced the cytolethality of MMAs V , although cellular GSH levels actually prevented the cytolethality of combined MMAs V and exogenous GSH. These findings indicate that human arsenic metabolite MMAs V is not a highly toxic compound in mammalian cells, and the level of cellular GSH is critical to its eventual toxic effects

Supplementary Material for: Cytotoxicity and intracellular dissolution of nickel nanowires

KAUST Repository

Perez, Jose E.; Contreras, Maria F.; Vidal, Enrique Vilanova; Felix Servin, Laura P.; Margineanu, Michael B.; Luongo, Giovanni; Porter, Alexandra E.; Dunlop, Iain E.; Ravasi, Timothy; Kosel, Jü rgen

2016-01-01

The assessment of cytotoxicity of nanostructures is a fundamental step for their development as biomedical tools. As widely used nanostructures, nickel nanowires (Ni NWs) seem promising candidates for such applications. In this work, Ni NWs were synthesized and then characterized using vibrating sample magnetometry, energy dispersive X-Ray analysis, and electron microscopy. After exposure to the NWs, cytotoxicity was evaluated in terms of cell viability, cell membrane damage, and induced apoptosis/necrosis on the model human cell line HCT 116. The influence of NW to cell ratio (10:1 to 1000:1) and exposure times up to 72 hours was analyzed for Ni NWs of 5.4 μm in length, as well as for Ni ions. The results show that cytotoxicity markedly increases past 24 hours of incubation. Cellular uptake of NWs takes place through the phagocytosis pathway, with a fraction of the dose of NWs dissolved inside the cells. Cell death results from a combination of apoptosis and necrosis, where the latter is the outcome of the secondary necrosis pathway. The cytotoxicity of Ni ions and Ni NWs dissolution studies suggest a synergistic toxicity between NW aspect ratio and dissolved Ni, with the cytotoxic effects markedly increasing after 24 hours of incubation.

Biocompatibility index of antiseptic agents by parallel assessment of antimicrobial activity and cellular cytotoxicity.

Science.gov (United States)

Müller, Gerald; Kramer, Axel

2008-06-01

To assess the suitability of an antiseptic agent, both the microbicidal activity and the cytotoxic effect must be taken into consideration to derive biocompatible antibacterial agents. We defined the biocompatibility index (BI) by measuring the antibacterial activity against the test organisms Escherichia coli and Staphylococcus aureus and, in parallel, the cytotoxicity on cultured murine fibroblasts. The antiseptic agents tested were benzalkonium chloride (BAC), cetylpyridinium chloride (CPC), chlorhexidine digluconate (CHX), mild silver protein (MSP), octenidine dihydrochloride (OCT), polyhexamethylene biguanide (PHMB), povidone iodine in solution [PVP-I(s)], povidone iodine in ointment [PVP-I(o)], silver nitrate (AgNO(3)), silver (I) sulfadiazine (SSD) and triclosan (TRI). Assays were carried out for 30 min of exposure at 37 degrees C in the presence of cell culture medium containing 10% fetal bovine serum. The resulting dimensionless BI was defined as the ratio of the concentration at which 50% of the murine fibroblasts are damaged and the microbicidal effect producing at least 3 log(10) (99.9%) reduction. The resulting rank ordering of BI for the ratio of fibroblast cytotoxicity to E. coli toxicity was OCT > PHMB > CHX > PVP-I(o) > PVP-I(s) > BAC > CPC > TRI > MSP and that to S. aureus was OCT > PHMB > CHX > CPC > PVP-I(o) > BAC > PVP(s) > TRI > MSP. OCT and PHMB were the most suitable agents with a BI greater than 1. The BI presented may be a useful tool to evaluate antiseptic agents for use in clinical practice.

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

DEFF Research Database (Denmark)

Caviglia, Claudia; Carminati, Marco; Heiskanen, Arto

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

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

Science.gov (United States)

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.

ROLE OF MEMBRANE LIPID-COMPOSITION IN THE CYTOTOXICITY OF THE SESQUITERPENE LACTONE EUPATORIOPICRIN

NARCIS (Netherlands)

VANDERLINDE, JCC; WOERDENBAG, HJ; MALINGRE, TM; KAMPINGA, HH; KONINGS, AWT

1993-01-01

The aim of the present study was to investigate a possible role of lipid peroxidation in the cytotoxicity of eupatoriopicrin, the principal sesquiterpene lactone from Eupatorum cannabinum L. Incorporation of arachidonic acid acyl chains in the phospholipids of cellular membranes of mouse fibroblast

Gold(III) bis(thiosemicarbazonate) compounds in breast cancer cells: Cytotoxicity and thioredoxin reductase targeting.

Science.gov (United States)

Rodríguez-Fanjul, Vanessa; López-Torres, Elena; Mendiola, M Antonia; Pizarro, Ana María

2018-03-25

Gold(III) compounds have received increasing attention in cancer research. Three gold complexes of general formula [Au III L]Cl, where L is benzil bis(thiosemicarbazonate), compound 1, benzil bis(4-methyl-3-thiosemicarbazonate), compound 2, or benzil bis(4-cyclohexyl-3-thiosemicarbazonate), compound 3, have been synthesized and fully characterized, including the X-ray crystal structure of compound 3, confirming square-planar geometry around the gold(III) centre. Compound 1 showed moderate cytotoxicity and accumulation in MCF7 breast cancer cells but did not inhibit thioredoxin reductase (TrxR) activity and did not induce reactive oxygen species (ROS) production. Compound 2, the least cytotoxic, was found to be capable of modestly inhibiting TrxR activity and produced low levels of ROS in the MCF7 cell line. The most cytotoxic compound, 3, had the highest cellular accumulation and its distribution pattern showed a clear preference for the cytosol and mitochondria of MCF7 cells. It readily hampered intracellular TrxR activity leading to a dramatic alteration of the cellular redox state and to the induction of cell death. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Cellular changes in motor neuron cell culture produced by cytotoxic cerebrospinal fluid from patients with amyotrophic lateral sclerosis.

Science.gov (United States)

Gomez-Pinedo, U; Yáñez, M; Matías-Guiu, J; Galán, L; Guerrero-Sola, A; Benito-Martin, M S; Vela, A; Arranz-Tagarro, J A; García, A G

2014-01-01

The neurotoxic effects of cerebrospinal fluid (CSF) from patients with amyotrophic lateral sclerosis (ALS) have been reported by various authors who have attributed this neurotoxicity to the glutamate in CSF-ALS. Cultures of rat embryonic cortical neurons were exposed to CSF from ALS patients during an incubation period of 24 hours. Optical microscopy was used to compare cellular changes to those elicited by exposure to 100μm glutamate, and confocal microscopy was used to evaluate immunohistochemistry for caspase-3, TNFα, and peripherin. In the culture exposed to CSF-ALS, we observed cells with nuclear fragmentation and scarce or null structural modifications to the cytoplasmic organelles or to plasma membrane maintenance. This did not occur in the culture exposed to glutamate. The culture exposed to CSF-ALS also demonstrated increases in caspase-3, TNFα, and in peripherin co-locating with caspase-3, but not with TNFα, suggesting that TNFα may play an early role in the process of apoptosis. CFS-ALS cytotoxicity is not related to glutamate. It initially affects the nucleus without altering the cytoplasmic membrane. It causes cytoplasmic apoptosis that involves an increase in caspase-3 co-located with peripherin, which is also overexpressed. Copyright © 2013 Sociedad Española de Neurología. Published by Elsevier Espana. All rights reserved.

Keynote address: cellular reduction of nitroimidazole drugs: potential for selective chemotherapy and diagnosis of hypoxic cells

International Nuclear Information System (INIS)

Chapman, J.D.; Lee, J.; Meeker, B.E.

1989-01-01

Nitroimidazole drugs were initially developed as selective radiosensitizers of hypoxic cells and, consequently, as adjuvants to improve the local control probabilities of current radiotherapies. Misonidazole (MISO), the prototype radiosensitizing drug, was found in Phase I clinical studies to cause dose-limiting neurotoxicities (mainly peripheral neuropathies). MISO was also found to be cytotoxic in the absence of radiation and to covalently bind to cellular molecules, both processes demonstrating rates much higher in hypoxic compared with oxygenated cells. It is likely that neurotoxicity, cellular cytotoxicity and adduct formation results from reactions between reduction intermediates of MISO and cellular target molecules. Spin-offs from radiosensitizer research include the synthesis and characterization of more potent hypoxic cytotoxins and the exploitation of sensitizer-adducts as probes for measuring cellular and tissue oxygen levels. Current developments in hypoxic cell cytotoxin and hypoxic cell marker research are reviewed with specific examples from studies which characterize the cellular reduction of TF-MISO, (1-(2-nitro-1-imidazolyl)-3[2,2,2-trifluoroethoxy]-2-propanol). 45 references

Viral control in chronic HIV-1 subtype C infection is associated with enrichment of p24 IgG1 with Fc effector activity.

Science.gov (United States)

Chung, Amy; Makuba, Jenniffer M; Ndlovu, Bongiwe; Licht, Anna; Robinson, Hannah; Ramlakhan, Yathisha; Ghebremichael, Musie; Reddy, Tarylee; Goulder, Philip; Walker, Bruce; Ndung'u, Thumbi; Alter, Galit

2018-04-03

Postinfection HIV viral control and immune correlates analysis of the RV144 vaccine trial indicate a potentially critical role for Fc receptor-mediated antibody functions. However, the influence of functional antibodies in clade C infection is largely unknown. Plasma samples from 361 chronic subtype C-infected, antiretroviral therapy-naïve participants were tested for their HIV-specific isotype and subclass distributions, along with their Fc receptor-mediated functional potential. Total IgG, IgG subclasses and IgA binding to p24 clade B/C and gp120 consensus C proteins were assayed by multiplex. Antibody-dependent uptake of antigen-coated beads and Fc receptor-mediated natural killer cell degranulation were evaluated as surrogates for antibody-dependent cellular phagocytosis (ADCP) and antibody-dependent cellular cytotoxicity (ADCC), respectively. p24 IgG1 was the only subclass associated with viral control (P = 0.01), with higher p24-specific ADCP and ADCC responses detected in individuals with high p24 IgG1. Although p24 IgG1 levels were enriched in patients with elevated Gag-specific T-cell responses, these levels remained an independent predictor of low-viral loads (P = 0.04) and high CD4 counts (P = 0.004) after adjusting for Gag-specific T-cell responses and for protective HLA class I alleles. p24 IgG1 levels independently predict viral control in HIV-1 clade C infection. Whether these responses contribute to direct antiviral control via the recruited killing of infected cells via the innate immune system or simply mark a qualitatively superior immune response to HIV, is uncertain, but highlights the role of p24-specific antibodies in control of clade C HIV-1 infection.

Immunoglobulin G1 Allotype Influences Antibody Subclass Distribution in Response to HIV gp140 Vaccination

Directory of Open Access Journals (Sweden)

Sven Kratochvil

2017-12-01

Full Text Available Antibody subclasses exhibit extensive polymorphisms (allotypes that could potentially impact the quality of HIV-vaccine induced B cell responses. Allotypes of immunoglobulin (Ig G1, the most abundant serum antibody, have been shown to display altered functional properties in regard to serum half-life, Fc-receptor binding and FcRn-mediated mucosal transcytosis. To investigate the potential link between allotypic IgG1-variants and vaccine-generated humoral responses in a cohort of 14 HIV vaccine recipients, we developed a novel protocol for rapid IgG1-allotyping. We combined PCR and ELISA assays in a dual approach to determine the IgG1 allotype identity (G1m3 and/or G1m1 of trial participants, using human plasma and RNA isolated from PBMC. The IgG1-allotype distribution of our participants mirrored previously reported results for caucasoid populations. We observed elevated levels of HIV gp140-specific IgG1 and decreased IgG2 levels associated with the G1m1-allele, in contrast to G1m3 carriers. These data suggest that vaccinees homozygous for G1m1 are predisposed to develop elevated Ag-specific IgG1:IgG2 ratios compared to G1m3-carriers. This elevated IgG1:IgG2 ratio was further associated with higher FcγR-dimer engagement, a surrogate for potential antibody-dependent cellular cytotoxicity (ADCC and antibody-dependent cellular phagocytosis (ADCP function. Although preliminary, these results suggest that IgG1 allotype may have a significant impact on IgG subclass distribution in response to vaccination and associated Fc-mediated effector functions. These results have important implications for ongoing HIV vaccine efficacy studies predicated on engagement of FcγR-mediated cellular functions including ADCC and ADCP, and warrant further investigation. Our novel allotyping protocol provides new tools to determine the potential impact of IgG1 allotypes on vaccine efficacy.

Cytotoxicity of graphene: recent advances and future perspective.

Science.gov (United States)

Zhou, Ruhong; Gao, Huajian

2014-01-01

Graphene, a unique two-dimensional single-atom-thin nanomaterial with exceptional structural, mechanical, and electronic properties, has spurred an enormous interest in many fields, including biomedical applications, which at the same time ignites a growing concern on its biosafety and potential cytotoxicity to human and animal cells. In this review, we present a summary of some very recent studies on this important subject with both experimental and theoretical approaches. The molecular interactions of graphene with proteins, DNAs, and cell membranes (both bacteria and mammalian cells) are discussed in detail. Severe distortions in structures and functions of these biomacromolecules by graphene are identified and characterized. For example, the graphene is shown to disrupt bacteria cell membranes by insertion/cutting as well as destructive extraction of lipid molecules directly. More interestingly, this cytotoxicity has been shown to have implications in de novo design of nanomedicine, such as graphene-based band-aid, a potential 'green' antibiotics due to its strong physical-based (instead of chemical-based) antibacterial capability. These studies have provided a better understanding of graphene nanotoxicity at both cellular and molecular levels, and also suggested therapeutic potential by using graphene's cytotoxicity against bacteria cells. © 2014 Wiley Periodicals, Inc.

Effects of Iron-Oxide Nanoparticle Surface Chemistry on Uptake Kinetics and Cytotoxicity in CHO-K1 Cells

Directory of Open Access Journals (Sweden)

Camille C. Hanot

2015-12-01

Full Text Available Superparamagnetic iron-oxide nanoparticles (SPIONs show great promise for multiple applications in biomedicine. While a number of studies have examined their safety profile, the toxicity of these particles on reproductive organs remains uncertain. The goal of this study was to evaluate the cytotoxicity of starch-coated, aminated, and PEGylated SPIONs on a cell line derived from Chinese Hamster ovaries (CHO-K1 cells. We evaluated the effect of particle diameter (50 and 100 nm and polyethylene glycol (PEG chain length (2k, 5k and 20k Da on the cytotoxicity of SPIONs by investigating cell viability using the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT and sulforhodamine B (SRB assays. The kinetics and extent of SPION uptake by CHO-K1 cells was also studied, as well as the resulting generation of intracellular reactive oxygen species (ROS. Cell toxicity profiles of SPIONs correlated strongly with their cellular uptake kinetics, which was strongly dependent on surface properties of the particles. PEGylation caused a decrease in both uptake and cytotoxicity compared to aminated SPIONs. Interestingly, 2k Da PEG-modifed SPIONs displayed the lowest cellular uptake and cytotoxicity among all studied particles. These results emphasize the importance of surface coatings when engineering nanoparticles for biomedical applications.

Cellular Transport Mechanisms of Cytotoxic Metallodrugs: An Overview beyond Cisplatin

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Sarah Spreckelmeyer

2014-09-01

Full Text Available The field of medicinal inorganic chemistry has grown consistently during the past 50 years; however, metal-containing coordination compounds represent only a minor proportion of drugs currently on the market, indicating that research in this area has not yet been thoroughly realized. Although platinum-based drugs as cancer chemotherapeutic agents have been widely studied, exact knowledge of the mechanisms governing their accumulation in cells is still lacking. However, evidence suggests active uptake and efflux mechanisms are involved; this may be involved also in other experimental metal coordination and organometallic compounds with promising antitumor activities in vitro and in vivo, such as ruthenium and gold compounds. Such knowledge would be necessary to elucidate the balance between activity and toxicity profiles of metal compounds. In this review, we present an overview of the information available on the cellular accumulation of Pt compounds from in vitro, in vivo and clinical studies, as well as a summary of reports on the possible accumulation mechanisms for different families of experimental anticancer metal complexes (e.g., Ru Au and Ir. Finally, we discuss the need for rationalization of the investigational approaches available to study metallodrug cellular transport.

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

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

1980-01-01

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

Cytotoxicity and Initial Biocompatibility of Endodontic Biomaterials (MTA and Biodentine™) Used as Root-End Filling Materials.

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Escobar-García, Diana María; Aguirre-López, Eva; Méndez-González, Verónica; Pozos-Guillén, Amaury

2016-01-01

Objective. The aim of this study was to evaluate the cytotoxicity and cellular adhesion of Mineral Trioxide Aggregate (MTA) and Biodentine (BD) on periodontal ligament fibroblasts (PDL). Methods. PDL cells were obtained from nonerupted third molars and cultured; MTS cellular profusion test was carried out in two groups: MTA and BD, with respective controls at different time periods. Also, the LIVE/DEAD assay was performed at 24 h. For evaluation of cellular adhesion, immunocytochemistry was conducted to discern the expression of Integrin β1 and Vinculin at 12 h and 24 h. Statistical analysis was performed by the Kruskal-Wallis and Mann-Whitney U tests. Results. MTA and BD exhibited living cells up to 7 days. More expressions of Integrin β1 and Vinculin were demonstrated in the control group, followed by BD and MTA, which also showed cellular loss and morphological changes. There was a significant difference in the experimental groups cultured for 5 and 7 days compared with the control, but there was no significant statistical difference between both cements. Conclusions. Neither material was cytotoxic during the time evaluated. There was an increase of cell adhesion through the expression of focal contacts observed in the case of BD, followed by MTA, but not significantly.

Cytotoxicity and morphological effects induced by carvacrol and thymol on the human cell line Caco-2.

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Llana-Ruiz-Cabello, María; Gutiérrez-Praena, Daniel; Pichardo, Silvia; Moreno, F Javier; Bermúdez, José María; Aucejo, Susana; Cameán, Ana María

2014-02-01

Essential oils used as additives in the food industry due to its flavour, antimicrobial and antioxidant properties. Therefore, human can be exposed orally to these compounds through the ingestion of foods. In this sense, the present work aims to assess toxicological effects of oregano essential oil on the digestive tract. In concrete, the cytotoxic effects of two components of the oregano essential oils, carvacrol and thymol, and their mixture, on the intestinal cells line Caco-2 after 24 and 48 h of exposure are studied. The basal cytotoxicity endpoints assayed (total protein content, neutral red uptake and the tetrazolium salt reduction) and the annexin/propidium iodide staining indicated that carvacrol and the mixture carvacrol/thymol induced toxic effects. Moreover, a morphological study was performed in order to determine the ultrastructural cellular damages caused by these substances. The main morphological alterations were vacuolated cytoplasm, altered organelles and finally cell death. In addition, although no cytotoxic effects were recorded for thymol at any concentration and time of exposure, ultrastructural changes evidenced cellular damage such as lipid degeneration, mitochondrial damage, nucleolar segregation and apoptosis. Copyright © 2013 Elsevier Ltd. All rights reserved.

Dual antibody therapy to harness the innate anti-tumor immune response to enhance antibody targeting of tumors.

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Chester, Cariad; Marabelle, Aurelien; Houot, Roch; Kohrt, Holbrook E

2015-04-01

Cancer immunotherapy is a rapidly evolving field that offers a novel paradigm for cancer treatment: therapies focus on enhancing the immune system's innate and adaptive anti-tumor response. Early immunotherapeutics have achieved impressive clinical outcomes and monoclonal antibodies are now integral to therapeutic strategies in a variety of cancers. However, only recently have antibodies targeting innate immune cells entered clinical development. Innate immune effector cells play important roles in generating and maintaining antitumor immunity. Antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) are important innate immune mechanisms for tumor eradication. These cytolytic processes are initiated by the detection of a tumor-targeting antibody and can be augmented by activating co-stimulatory pathways or blocking inhibitory signals on innate immune cells. The combination of FDA-approved monoclonal antibodies with innate effector-targeting antibodies has demonstrated potent preclinical therapeutic synergy and early-phase combinatorial clinical trials are ongoing. Copyright © 2015 Elsevier Ltd. All rights reserved.

Sesquiterpene lactones: Mechanism of antineoplastic activity; relationship of cellular glutathione to cytotoxicity; and disposition

International Nuclear Information System (INIS)

Grippo, A.A.

1987-01-01

Helenalin, a sesquiterpene lactone, inhibited the growth of P388 lymphocytic and L1210 lymphoid leukemia, and Ehrlich ascites and KB carcinoma cells. The L1210 leukemia cells were most sensitive to the cytotoxic effects of helenalin. Helenalin's antineoplastic effects were due to inhibition of DNA synthesis by suppressing the activities of enzymes involved in this biosynthetic pathway; i.e., IMP dehydrogenase, ribonucleoside diphosphate reductase, thioredoxin complex, GSH disulfide oxidoreductase and DNA polymerase α activities. The relationship of reduced glutathione (GSH) to the cytotoxic effects of helanalin was evaluated. L1210 cells, which were more sensitive to helenalin's toxicity, contained lower basal concentrations of GSH. Helenalin decreased the concentration of reduced glutathione in both L1210 and P388 leukemia cells. Concurrent administration of helanalin with agents reported to raise GSH concentrations did not substantially effect GSH levels, nor were survival times of tumor-bearing mice enhanced. Following intraperitoneal administration of 3 H-plenolin, no radioactive drug and/or metabolite was sequestered in the organs of BDF 1 mice. Approximately 50% of 3 H-plenolin and/or its metabolites were eliminated via urine while lesser amounts of radioactive drug and/or metabolites were eliminated in the feces

Hypoxic cytotoxicity of chlorpromazine and the modification of radiation response in E. coli B/r

International Nuclear Information System (INIS)

Shenoy, M.A.; Singh, B.B.

1978-01-01

Chlorpromazine (0.1 mM) was cytotoxic to E. coli B/r cells under hypoxic but not euoxic conditions. Under nitrogen bubbling, there was no further enhancement in cellular lethality beyond 45 min contact time. The presence of the free drug seemed necessary for the cytocidal action to be demonstrated. Hypoxic cytotoxicity increased steadily with temperature between 30 and 37 0 C. Treatment of cells with N-ethyl maleimide (0.5 mM) completely abolished the subsequent hypoxic cytotoxicity of chlorpromazine (0.1 mM). Hypoxic gamma irradiation of cells pretreated for 45 min with chlorpromazine under nitrogen bubbling gave a DMF for survival of almost twice that produced by oxygen. Irradiation under aerobic conditions of cells subjected to the same pretreatment produced only the normal oxygen effect. The results indicate that the differential cytotoxicity of chlorpromazine is due to its effect on the changes induced in the membrane-associated biochemical state of the cells under euoxic and hypoxic conditions. (U.K.)

Klebsiella pneumoniae triggers a cytotoxic effect on airway epithelial cells

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Llobet-Brossa Enrique

2009-08-01

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

Evaluating Cytotoxicity and Cellular Uptake from the Presence of Variously Processed Ti02 Nanostructured Morphologies

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Chen, J.; Wong, S.; Zhou, H.; Santull, A.C.

2010-05-01

We evaluated the cytotoxicity of various morphological classes of TiO{sub 2} nanostructures (including 0-D nanoparticles, 1-D nanorods, and 3-D assemblies) toward living cells. These TiO{sub 2} nanostructures were modified with fluorescent dye molecules, mediated via a dopamine linkage, in order to facilitate a confocal study of their internalization. Specifically, we noted that both TiO{sub 2} 1-D nanorods and 0-D nanoparticles could internalize into cells after 24 h of incubation time. However, only incubation with TiO{sub 2} 1-D nanorods and 3-D micrometer-scale sea urchin-like assemblies at concentrations of up to 125 {mu}g/mL yielded data suggestive of cell viabilities of close to 100%. Moreover, upon irradiation with UV light for periods of a few minutes at energy densities of up to 1 J/cm{sub 2}, we observed up to 60% mortality rates, indicative of the cytotoxic potential of photoirradiated TiO{sub 2} nanostructures due to the generation of reactive oxygen species.

Analyses of functions of an anti-PD-L1/TGFβR2 bispecific fusion protein (M7824).

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Jochems, Caroline; Tritsch, Sarah R; Pellom, Samuel Troy; Su, Zhen; Soon-Shiong, Patrick; Wong, Hing C; Gulley, James L; Schlom, Jeffrey

2017-09-26

M7824 (MSB0011359C) is a novel first-in-class bifunctional fusion protein consisting of a fully human IgG1 anti-PD-L1 monoclonal antibody (with structural similarities to avelumab) linked to the extracellular domain of two TGFβ receptor 2 (TGFβR2) molecules serving as a TGFβ Trap. Avelumab has demonstrated clinical activity in a range of human cancers and has been approved by the Food and Drug Administration for the therapy of Merkel cell and bladder carcinomas. Preclinical studies have shown this anti-PD-L1 is capable of mediating antibody-dependent cell-mediated cytotoxicity (ADCC). In the studies reported here, it is shown that M7824 is also capable of mediating ADCC of a wide range of human carcinoma cells in vitro , employing natural killer (NK) cells as effectors, albeit not as potent as anti-PD-L1 employing some tumor cells as targets. The addition of the IL-15 superagonist fusion protein complex ALT-803 enhanced the ADCC capacity of both anti-PD-L1 and M7824, and to levels that both agents now demonstrated similar levels of ADCC of tumor cells. TGFβ is a known immunosuppressive entity. Studies reported here show TGFβ1 induced reduction of several NK activation markers as well as reduction of endogenous NK lytic activity and NK-mediated ADCC of tumor cells. These phenomena could be reduced or mitigated, however, by M7824, but not by anti-PD-L1. M7824, but not anti-PD-L1, was also shown to reduce the immunosuppressive activity of regulatory T cells on human CD4 + T-cell proliferation. These studies thus demonstrate the dual functionalities of M7824 and provide the rationale for its further clinical development.

Noscapinoids bearing silver nanocrystals augmented drug delivery, cytotoxicity, apoptosis and cellular uptake in B16F1, mouse melanoma skin cancer cells.

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Soni, Naina; Jyoti, Kiran; Jain, Upendra Kumar; Katyal, Anju; Chandra, Ramesh; Madan, Jitender

2017-06-01

Noscapine (Nos) and reduced brominated analogue of noscapine (Red-Br-Nos) prevent cellular proliferation and induce apoptosis in cancer cells either alone or in combination with other chemotherapeutic drugs. However, owing to poor physicochemical properties, Nos and Red-Br-Nos have demonstrated their anticancer activity at higher and multiple doses. Therefore, in present investigation, silver nanocrystals of noscapinoids (Nos-Ag 2+ nanocrystals and Red-Br-Nos-Ag 2+ nanocrystals) were customized to augment drug delivery, cytotoxicity, apoptosis and cellular uptake in B16F1 mouse melanoma cancer cells. Nos-Ag 2+ nanocrystals and Red-Br-Nos-Ag 2+ nanocrystals were prepared separately by precipitation method. The mean particle size of Nos-Ag 2+ nanocrystals was measured to be 25.33±3.52nm, insignificantly (P>0.05) different from 27.43±4.51nm of Red-Br-Nos-Ag 2+ nanocrystals. Furthermore, zeta-potential of Nos-Ag 2+ nanocrystals was determined to be -25.3±3.11mV significantly (Pcellular uptake. The Nos-Ag 2+ nanocrystals and Red-Br-Nos-Ag 2+ nanocrystals exhibited an IC 50 of 16.6μM and 6.5μM, significantly (Pcellular morphological alterations in B16F1 cells upon internalization of Nos-Ag 2+ nanocrystals and Red-Br-Nos-Ag 2+ nanocrystals provided the evidences for accumulation within membrane-bound cytoplasmic vacuoles and in enlarged lysosomes and thus triggered mitochondria mediated apoptosis via caspase activation. Preliminary investigations substantiated that Nos-Ag 2+ nanocrystals and Red-Br-Nos-Ag 2+ nanocrystals must be further explored and utilized for the delivery of noscapinoids to melanoma cancer cells. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Rhodium metalloinsertor binding generates a lesion with selective cytotoxicity for mismatch repair-deficient cells.

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Bailis, Julie M; Weidmann, Alyson G; Mariano, Natalie F; Barton, Jacqueline K

2017-07-03

The DNA mismatch repair (MMR) pathway recognizes and repairs errors in base pairing and acts to maintain genome stability. Cancers that have lost MMR function are common and comprise an important clinical subtype that is resistant to many standard of care chemotherapeutics such as cisplatin. We have identified a family of rhodium metalloinsertors that bind DNA mismatches with high specificity and are preferentially cytotoxic to MMR-deficient cells. Here, we characterize the cellular mechanism of action of the most potent and selective complex in this family, [Rh(chrysi)(phen)(PPO)] 2+ (Rh-PPO). We find that Rh-PPO binding induces a lesion that triggers the DNA damage response (DDR). DDR activation results in cell-cycle blockade and inhibition of DNA replication and transcription. Significantly, the lesion induced by Rh-PPO is not repaired in MMR-deficient cells, resulting in selective cytotoxicity. The Rh-PPO mechanism is reminiscent of DNA repair enzymes that displace mismatched bases, and is differentiated from other DNA-targeted chemotherapeutics such as cisplatin by its potency, cellular mechanism, and selectivity for MMR-deficient cells.

Effect of vitamin E on cytotoxicity, DNA single strand breaks, chromosomal aberrations, and mutation in Chinese hamster V-79 cells exposed to ultraviolet-B light

International Nuclear Information System (INIS)

Sugiyama, M.; Tsuzuki, K.; Matsumoto, K.; Ogura, R.

1992-01-01

The effect of pretreatment with vitamin E on cytotoxicity, DNA single strand breaks, and chromosomal aberrations as well as on mutation induced by ultraviolet-B light (UV-B) was investigated in Chinese hamster V-79 cells. Cellular pretreatment with non-toxic levels of 25 μM α-tocopherol succinate (vitamin E) for 24h prior to exposure resulted in a 10-fold increase in cellular levels of α-tocopherol. Using a colony-forming assay, this pretreatment decreased the cytotoxicity of UV-B light. However, alkaline elution assays demonstrated that pretreatment with vitamin E did not affect the number of DNA single strand breaks caused by UV-B light. UV-B exposure produced a dose-dependent induction of chromosomal aberrations and mutations at the HGPRT locus, and neither of these actions of UV-B was influenced by pretreatment with the vitamin. These results suggest that vitamin E protects cells from UV-B-induced cytotoxicity, possibly through its ability to scavenge free radicals. The genotoxicity induced by UV-B light may not correlate directly with the cytotoxic action of this wavelength region in sunlight. (author)

Detection of tumor-specific cytotoxic drug activity in vitro using the fluorometric microculture cytotoxicity assay and primary cultures of tumor cells from patients.

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Nygren, P; Fridborg, H; Csoka, K; Sundström, C; de la Torre, M; Kristensen, J; Bergh, J; Hagberg, H; Glimelius, B; Rastad, J

1994-03-01

The semi-automated fluorometric microculture cytotoxicity assay (FMCA), based on the measurement of fluorescence generated from cellular hydrolysis of fluorescein diacetate (FDA) by viable cells, was employed for cytotoxic drug sensitivity testing of tumor cells from patients with hematological or solid tumors. In total, 390 samples from 20 diagnoses were tested with up to 12 standard cytotoxic drugs. The technical success rate for different tumor types ranged from 67 to 95%. Fluorescence was linearly related to cell number but variably steep depending on tumor type. Samples from most solid tumors thus showed higher signal-to-noise ratios than hematological samples. A wide spectrum of in vitro drug activity was obtained, with acute leukemias and non-Hodgkin's lymphomas being sensitive to almost all tested drugs, whereas renal and adrenocortical carcinomas were essentially totally resistant. Between these extremes were samples of breast and ovarian carcinomas and sarcomas. When in vitro response was compared with known clinical response patterns, a good correspondence was observed. The results indicate that the FMCA is a rapid and efficient method for in vitro measurement of tumor-specific drug activity both in hematological and in solid tumors. The assay may be suitable for new drug development and direction of phase-2 trials to suitable patients.

Cytotoxicity and cellular uptake of ZnS:Mn nanocrystals biofunctionalized with chitosan and aminoacids

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Augustine, M.S.; Anas, A.; Das, A.V.; Sreekanth, S.; Jayalekshmi, S.

into solution and by generating free radical species. In animal experiments, QDs preferentially enter the liver and spleen follow-ing intravascular injection which undergo minimal excretion if lar-ger than 6 nm, and appear to be safe to the animal. The present... recently on toxicity studies of quantum dots (QDs) in cells and animals [49,50]. Cell culture experiments have shown that QDs undergo design- dependent intracellular localiza-tion which can cause cytotoxicity by releasing free toxic materials...

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

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

2013-05-15

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

The role of autophagy inhibition in the enhanced cytotoxicity of temozolomide on melanoma cell lines

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O. O. Ryabaya

2017-01-01

Full Text Available Background. Despite advantages in treatment of metastatic melanoma it remains resistant to current therapy. Recent evidence indicates that tumor cells could overcome death through autophagy, a process that degrades cellular proteins and organelles to maintain cellular biosynthesis during nutrient deprivation or lack of energy. Objective: to investigate the involvement of autophagy inhibitors chloroquine (CQ and LY-294.002 (LY in temozolomide (TMZ cytotoxicity in human melanoma cell lines.Materials and methods. The study was performed on patient-derived melanoma cell lines Mel Z, Mel IL and Mel MTP. The antiproliferative activity of combined TMZ and autophagy inhibitors treatment was determined by MTT assay and colony-forming assay. Cell cycle analysis, apoptosis activation and expression analysis of key autophagy markers under combined treatment was evaluated.Results. CQ and LY enhanced the cytotoxicity of TMZ and reduced colony formation in 3 melanoma cell lines, moreover both inhibitors increased cell population in G0 / G1 phase of cell cycle in Mel Z, Mel IL cell lines, but not in Mel MTP. CQ and LY synergistically activated apoptosis in all cell lines. The matrix RNA expression analysis of key autophagy genes showed autophagy involvement in enhanced cytotoxicity.Conclusions. Thus, autophagy inhibition on different stages of this process could overcome resistance to TMZ and be applicable as potent target in metastatic melanoma treatment.

Assessing cellular toxicities in fibroblasts upon exposure to lipid-based nanoparticles: a high content analysis approach

International Nuclear Information System (INIS)

Solmesky, Leonardo J; Weil, Miguel; Shuman, Michal; Goldsmith, Meir; Peer, Dan

2011-01-01

Lipid-based nanoparticles (LNPs) are widely used for the delivery of drugs and nucleic acids. Although most of them are considered safe, there is confusing evidence in the literature regarding their potential cellular toxicities. Moreover, little is known about the recovery process cells undergo after a cytotoxic insult. We have previously studied the systemic effects of common LNPs with different surface charge (cationic, anionic, neutral) and revealed that positively charged LNPs ((+)LNPs) activate pro-inflammatory cytokines and induce interferon response by acting as an agonist of Toll-like receptor 4 on immune cells. In this study, we focused on the response of human fibroblasts exposed to LNPs and their cellular recovery process. To this end, we used image-based high content analysis (HCA). Using this strategy, we were able to show simultaneously, in several intracellular parameters, that fibroblasts can recover from the cytotoxic effects of (+)LNPs. The use of HCA opens new avenues in understanding cellular response and nanotoxicity and may become a valuable tool for screening safe materials for drug delivery and tissue engineering.

Assessing cellular toxicities in fibroblasts upon exposure to lipid-based nanoparticles: a high content analysis approach

Science.gov (United States)

Solmesky, Leonardo J.; Shuman, Michal; Goldsmith, Meir; Weil, Miguel; Peer, Dan

2011-12-01

Lipid-based nanoparticles (LNPs) are widely used for the delivery of drugs and nucleic acids. Although most of them are considered safe, there is confusing evidence in the literature regarding their potential cellular toxicities. Moreover, little is known about the recovery process cells undergo after a cytotoxic insult. We have previously studied the systemic effects of common LNPs with different surface charge (cationic, anionic, neutral) and revealed that positively charged LNPs ((+)LNPs) activate pro-inflammatory cytokines and induce interferon response by acting as an agonist of Toll-like receptor 4 on immune cells. In this study, we focused on the response of human fibroblasts exposed to LNPs and their cellular recovery process. To this end, we used image-based high content analysis (HCA). Using this strategy, we were able to show simultaneously, in several intracellular parameters, that fibroblasts can recover from the cytotoxic effects of (+)LNPs. The use of HCA opens new avenues in understanding cellular response and nanotoxicity and may become a valuable tool for screening safe materials for drug delivery and tissue engineering.

Cytotoxicity of Cerastes cerastes snake venom: Involvement of imbalanced redox status.

Science.gov (United States)

Kebir-Chelghoum, Hayet; Laraba-Djebari, Fatima

2017-09-01

Envenomation caused by Cerastes cerastes snake venom is characterized by a local and a systemic tissue damage due to myonecrosis, hemorrhage, edema and acute muscle damage. The present study aimed to evaluate the relationship between the pro/anti-oxidants status and the cytotoxicity of C. cerastes snake venom. The in vivo cytotoxicity analysis was undertaken by the injection of C. cerastes venom (48μg/20g body weight) by i.p. route, mice were then sacrificed at 3, 24 and 48h post injection, organs were collected for further analysis. In vitro cytotoxicity analysis was investigated on cultured PBMC, hepatocytes and isolated liver. The obtained results showed a significant cell infiltration characterized by a significant increase of myeloperoxidase (MPO) and eosinoperoxidase (EPO) activities. These results showed also a potent oxidative activity of C. cerastes venom characterized by increased levels of residual nitrites and lipid peroxidation associated with a significant decrease of glutathione and catalase activity in sera and tissues (heart, lungs, liver and kidneys). The in vitro cytotoxicity of C. cerastes venom on PBMC seems to be dose-dependent (IC50 of 21μg/ml/10 6 cells) and correlated with an imbalanced redox status at high doses of venom. However, in the case of cultured hepatocytes, the LDH release and oxidative stress were observed only at high doses of the venom. The obtained results of in vivo study were confirmed by the culture of isolated liver. Therefore, these results suggest that the venom induces a direct cytotoxic effect which alters the membrane integrity causing a leakage of the cellular contents. This cytotoxic effect can lead indirectly to inflammatory response and oxidative stress. These data suggest that an early anti-inflammatory and antioxidant treatment could be useful in the management of envenomed victims. Copyright © 2017. Published by Elsevier B.V.

Curcumin-cyclodextrin encapsulated chitosan nanoconjugates with enhanced solubility and cell cytotoxicity.

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Popat, Amirali; Karmakar, Surajit; Jambhrunkar, Siddharth; Xu, Chun; Yu, Chengzhong

2014-05-01

Curcumin (CUR), a naturally derived anti-cancer cocktail is arguably the most widely studied neutraceutical. Despite a lot of promises, it is yet to reach the market as an active anti-cancer formulation. In the present study, we have prepared highly soluble (3 mg/ml) CUR-γ-hydroxypropyl cyclodextrin (CUR-CD) hollow spheres. CUR-CD hollow spheres were prepared by a novel and scalable spray drying method. CUR-CD was then encapsulated into positively charged biodegradable chitosan (CUR-CD-CS) nanoparticles. The CUR-CD-CS nanoparticles were characterised by TEM, SEM, DLS, drug loading and in vitro release. We tested the efficacy of these CUR-CD-CS nanoparticles in SCC25 cell lines using MTT assay and investigated its cellular uptake mechanism. We also studied Oligo DNA loading in CUR-CD-CS nanoparticles and its delivery via confocal imaging and FACS analysis. Our results demonstrated that CUR-CD-CS nanoparticles showed superior in vitro release performance and higher cytotoxicity in SCC25 cell line amongst all tested formulations. The cytotoxicity results were corroborated by cell cycle analysis and apoptosis test, showing nearly 100% apoptotic cell death in the case of CUR-CD-CS nanoparticles. Compared to CS nanoparticles, CS-CD nanoformulation showed higher cellular delivery of Cy3-Oligo DNA which was tested quantitatively using flowcytometry analysis, indicating that CD not only enhanced CUR solubility but also boosted the cellular uptake. Our study shows that rationally designed bio-degradable natural biomaterials have great potential as next generation nano-carriers for hydrophobic drug delivery such as CUR with potential of dual drug-gene delivery. Copyright © 2014 Elsevier B.V. All rights reserved.

Effects of folic acid deficiency and MTHFRC677T polymorphisms on cytotoxicity in human peripheral blood lymphocytes

International Nuclear Information System (INIS)

Wu Xiayu; Liang Ziqing; Zou Tianning; Wang Xu

2009-01-01

Apoptosis (APO) and necrosis (NEC) are two different types of cell death occurring in response to cellular stress factors. Cells with DNA damage may undergo APO or NEC. Folate is an essential micronutrient associated with DNA synthesis, repair and methylation. Methylenetetrahydrofolate reductase (MTHFR) regulates intracellular folate metabolism. Folate deficiency and MTHFR C677T polymorphisms have been shown to be related to DNA damage. To verify the cytotoxic effects of folate deficiency on cells with different MTHFR C677T genotypes, 15 human peripheral lymphocyte cases with different MTHFR C677T genotypes were cultured in folic acid (FA)-deficient and -sufficient media for 9 days. Cytotoxicity was quantified using the frequencies of APO and NEC as endpoints, the nuclear division index (NDI), and the number of viable cells (NVC). These results showed that FA is an important factor in reducing cytotoxicity and increasing cell proliferation. Lymphocytes with the TT genotype proliferated easily under stress and exhibited different responses to FA deficiency than lymphocytes with the CC and CT genotypes. A TT individual may accumulate more cytotoxicity under cytotoxic stress, suggesting that the effects of FA deficiency on cytotoxicity are greater than the effects in individuals with the other MTHFR C677T variants.

DNA binding, antioxidant, cytotoxicity (MTT, lactate dehydrogenase, NO), and cellular uptake studies of structurally different nickel(II) thiosemicarbazone complexes: synthesis, spectroscopy, electrochemistry, and X-ray crystallography.

Science.gov (United States)

Prabhakaran, R; Kalaivani, P; Huang, R; Poornima, P; Vijaya Padma, V; Dallemer, F; Natarajan, K

2013-02-01

Three new nickel(II) thiosemicarbazone complexes have been synthesized and characterized by analytical, spectral, and single-crystal X-ray diffraction studies. In complex 1, the ligand 2-hydroxy-1-naphthaldehydethiosemicarbazone coordinated as a monobasic tridentate donor, whereas in complexes 2 and 3, the ligands salicylaldehyde-4(N)-ethylthiosemicarbazone and 2-hydroxy-1-naphthaldehyde-4(N)-ethylthiosemicarbazone coordinated as a dibasic tridentate donor. The DNA binding ability of the complexes in calf thymus DNA was explored by absorption and emission titration experiments. The antioxidant property of the new complexes was evaluated to test their free-radical scavenging ability. In vitro cytotoxicity assays were performed for the new complexes in A549 and HepG2 cell lines. The new compounds overcome cisplatin resistance in the A549 cell line and they were also active in the HepG2 cell line. The cellular uptake study showed the accumulation of the complexes in tumor cells depended on the nature of the ligand attached to the nickel ion.

Gold Nanocluster-Mediated Cellular Death under Electromagnetic Radiation.

Science.gov (United States)

Cifuentes-Rius, Anna; Ivask, Angela; Das, Shreya; Penya-Auladell, Nuria; Fabregas, Laura; Fletcher, Nicholas L; Houston, Zachary H; Thurecht, Kristofer J; Voelcker, Nicolas H

2017-11-29

Gold nanoclusters (Au NCs) have become a promising nanomaterial for cancer therapy because of their biocompatibility and fluorescent properties. In this study, the effect of ultrasmall protein-stabilized 2 nm Au NCs on six types of mammalian cells (fibroblasts, B-lymphocytes, glioblastoma, neuroblastoma, and two types of prostate cancer cells) under electromagnetic radiation is investigated. Cellular association of Au NCs in vitro is concentration-dependent, and Au NCs have low intrinsic toxicity. However, when Au NC-incubated cells are exposed to a 1 GHz electromagnetic field (microwave radiation), cell viability significantly decreases, thus demonstrating that Au NCs exhibit specific microwave-dependent cytotoxicity, likely resulting from localized heating. Upon i.v. injection in mice, Au NCs are still present at 24 h post administration. Considering the specific microwave-dependent cytotoxicity and low intrinsic toxicity, our work suggests the potential of Au NCs as effective and safe nanomedicines for cancer therapy.

Cytotoxicity evaluation of a copaiba oil-based root canal sealer compared to three commonly used sealers in endodontics

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Angela Delfina Bittencourt Garrido

2015-01-01

Full Text Available Background: The constant development of new root canal sealers has allowed the solution of a large number of clinical cases in endodontics, however, cytotoxicity of such sealers must be tested before their validation as filling materials. The aim of this study was to evaluate the cytotoxic effect of a new Copaiba oil-based root canal sealer (Biosealer [BS] on osteoblast-like Osteo-1 cells. Materials and Methods: The experimental groups were formed according to the culture medium conditioned with the tested sealers, as follows: Control group (CG (culture medium without conditioning; Sealer 26 (S26 - culture medium + S26; Endofill (EF - culture medium + EF; AH Plus (AHP - culture medium + AHP; and BS - culture medium + BS (Copaiba oil-based sealer. The conditioned culture medium was placed in contact with 2 × 10 4 cells cultivated on 60 mm diameter Petri dishes for 24 h. Then, hemocytometer count was performed to evaluate cellular viability, using Trypan Blue assay. The normal distribution of data was tested by the Kolmogorov-Smirnov test and the values obtained for cellular viability were statistically analyzed (1-way ANOVA, Tukey′s test - P 0.05. Conclusion: The Copaiba oil-based root canal sealer presented promising results in terms of cytotoxicity which indicated its usefulness as a root canal sealer.

Cytotoxicity evaluation of a copaiba oil-based root canal sealer compared to three commonly used sealers in endodontics

Science.gov (United States)

Garrido, Angela Delfina Bittencourt; de Cara, Sueli Patricia Harumi Miyagi; Marques, Marcia Martins; Sponchiado, Emílio Carlos; Garcia, Lucas da Fonseca Roberti; de Sousa-Neto, Manoel Damião

2015-01-01

Background: The constant development of new root canal sealers has allowed the solution of a large number of clinical cases in endodontics, however, cytotoxicity of such sealers must be tested before their validation as filling materials. The aim of this study was to evaluate the cytotoxic effect of a new Copaiba oil-based root canal sealer (Biosealer [BS]) on osteoblast-like Osteo-1 cells. Materials and Methods: The experimental groups were formed according to the culture medium conditioned with the tested sealers, as follows: Control group (CG) (culture medium without conditioning); Sealer 26 (S26) - culture medium + S26; Endofill (EF) - culture medium + EF; AH Plus (AHP) - culture medium + AHP; and BS - culture medium + BS (Copaiba oil-based sealer). The conditioned culture medium was placed in contact with 2 × 104 cells cultivated on 60 mm diameter Petri dishes for 24 h. Then, hemocytometer count was performed to evaluate cellular viability, using Trypan Blue assay. The normal distribution of data was tested by the Kolmogorov-Smirnov test and the values obtained for cellular viability were statistically analyzed (1-way ANOVA, Tukey's test - P 0.05). Conclusion: The Copaiba oil-based root canal sealer presented promising results in terms of cytotoxicity which indicated its usefulness as a root canal sealer. PMID:25878676

Impact of cell adhesion and migration on nanoparticle uptake and cellular toxicity.

Science.gov (United States)

Pitchaimani, Arunkumar; Nguyen, Tuyen Duong Thanh; Koirala, Mukund; Zhang, Yuntao; Aryal, Santosh

2017-09-01

In vitro cell-nanoparticle (NP) studies involve exposure of NPs onto the monolayer cells growing at the bottom of a culture plate, and assumed that the NPs evenly distributed for a dose-responsive effect. However, only a few proportion of the administered dose reaches the cells depending on their size, shape, surface, and density. Often the amount incubated (administered dose) is misled as a responsive dose. Herein, we proposed a cell adhesion-migration (CAM) strategy, where cells incubated with the NP coated cell culture substrate to maximize the cell-NP interaction and investigated the physiological properties of the cells. In the present study, cell adhesion and migration pattern of human breast cancer cell (MCF-7) and mouse melanoma cell (B16-F10) on cell culture substrate decorated with toxic (cetyltrimethylammonium bromide, CTAB) and biocompatible (poly (sodium 4-styrenesulphonate), PSS) gold nanoparticles (AuNPs) of different sizes (5 and 40nm) were investigated and evaluated for cellular uptake efficiency, proliferation, and toxicity. Results showed enhanced cell adhesion, migration, and nanoparticle uptake only on biocompatible PSS coated AuNP, irrespective of its size. Whereas, cytotoxic NP shows retard proliferation with reduced cellular uptake efficiency. Considering the importance of cell adhesion and migration on cellular uptake and cytotoxicity assessment of nanoparticle, CAM strategy would hold great promises in cell-NP interaction studies. Copyright © 2017. Published by Elsevier Ltd.

Luminescent cyclometalated iridium(III) polypyridine indole complexes--synthesis, photophysics, electrochemistry, protein-binding properties, cytotoxicity, and cellular uptake.

Science.gov (United States)

Lau, Jason Shing-Yip; Lee, Pui-Kei; Tsang, Keith Hing-Kit; Ng, Cyrus Ho-Cheong; Lam, Yun-Wah; Cheng, Shuk-Han; Lo, Kenneth Kam-Wing

2009-01-19

A series of luminescent cyclometalated iridium(III) polypyridine indole complexes, [Ir(N--C)(2)(N--N)](PF(6)) (HN--C = 2-phenylpyridine (Hppy), N--N = 4-((2-(indol-3-yl)ethyl)aminocarbonyl)-4'-methyl-2,2'-bipyridine (bpy-ind) (1a), N--N = 4-((5-((2-(indol-3-yl)ethyl)aminocarbonyl)pentyl)aminocarbonyl)-4'-methyl-2,2'-bipyridine (bpy-C6-ind) (1b); HN--C = 7,8-benzoquinoline (Hbzq), N--N = bpy-ind (2a), N--N = bpy-C6-ind (2b); and HN--C = 2-phenylquinoline (Hpq), N--N = bpy-ind (3a), N--N = bpy-C6-ind (3b)), have been synthesized, characterized, and their photophysical and electrochemical properties and lipophilicity investigated. Photoexcitation of the complexes in fluid solutions at 298 K and in alcohol glass at 77 K resulted in intense and long-lived luminescence (lambda(em) = 540-616 nm, tau(o) = 0.13-5.15 mus). The emission of the complexes has been assigned to a triplet metal-to-ligand charge-transfer ((3)MLCT) (dpi(Ir) --> pi*(N--N)) excited state, probably with some mixing of triplet intraligand ((3)IL) (pi --> pi*) (pq) character for complexes 3a,b. Electrochemical measurements revealed that all the complexes showed an irreversible indole oxidation wave at ca. +1.1 V versus SCE, a quasi-reversible iridium(IV/III) couple at ca. +1.3 V, and a reversible diimine reduction couple at ca. -1.3 V. The interactions of these complexes with an indole-binding protein, bovine serum albumin (BSA), have been studied by emission titrations, and the K(a) values are on the order of 10(4) M(-1). Additionally, the cytotoxicity of the complexes toward human cervix epithelioid carcinoma (HeLa) cells has been examined by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay. The IC(50) values of the complexes ranged from 1.1 to 6.3 microM, which are significantly smaller than that of cisplatin (30.7 microM) under the same experimental conditions. Furthermore, the cellular uptake of the complexes has been investigated by flow cytometry and laser

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.

Science.gov (United States)

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

Acrolein cytotoxicity in hepatocytes involves endoplasmic reticulum stress, mitochondrial dysfunction and oxidative stress

OpenAIRE

Mohammad, Mohammad K; Avila, Diana; Zhang, Jingwen; Barve, Shirish; Arteel, Gavin; McClain, Craig; Joshi-Barve, Swati

2012-01-01

Acrolein is a common environmental, food and water pollutant and a major component of cigarette smoke. Also, it is produced endogenously via lipid peroxidation and cellular metabolism of certain amino acids and drugs. Acrolein is cytotoxic to many cell types including hepatocytes; however the mechanisms are not fully understood. We examined the molecular mechanisms underlying acrolein hepatotoxicity in primary human hepatocytes and hepatoma cells. Acrolein, at pathophysiological concentration...

Nitric oxide-releasing prodrug triggers cancer cell death through deregulation of cellular redox balance

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Anna E. Maciag

2013-01-01

Full Text Available JS-K is a nitric oxide (NO-releasing prodrug of the O2-arylated diazeniumdiolate family that has demonstrated pronounced cytotoxicity and antitumor properties in a variety of cancer models both in vitro and in vivo. The current study of the metabolic actions of JS-K was undertaken to investigate mechanisms of its cytotoxicity. Consistent with model chemical reactions, the activating step in the metabolism of JS-K in the cell is the dearylation of the diazeniumdiolate by glutathione (GSH via a nucleophilic aromatic substitution reaction. The resulting product (CEP/NO anion spontaneously hydrolyzes, releasing two equivalents of NO. The GSH/GSSG redox couple is considered to be the major redox buffer of the cell, helping maintain a reducing environment under basal conditions. We have quantified the effects of JS-K on cellular GSH content, and show that JS-K markedly depletes GSH, due to JS-K's rapid uptake and cascading release of NO and reactive nitrogen species. The depletion of GSH results in alterations in the redox potential of the cellular environment, initiating MAPK stress signaling pathways, and inducing apoptosis. Microarray analysis confirmed signaling gene changes at the transcriptional level and revealed alteration in the expression of several genes crucial for maintenance of cellular redox homeostasis, as well as cell proliferation and survival, including MYC. Pre-treating cells with the known GSH precursor and nucleophilic reducing agent N-acetylcysteine prevented the signaling events that lead to apoptosis. These data indicate that multiplicative depletion of the reduced glutathione pool and deregulation of intracellular redox balance are important initial steps in the mechanism of JS-K's cytotoxic action.

The cellular magnetic response and biocompatibility of biogenic zinc- and cobalt-doped magnetite nanoparticles

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Moise, Sandhya; Céspedes, Eva; Soukup, Dalibor; Byrne, James M.; El Haj, Alicia J.; Telling, Neil D.

2017-01-01

The magnetic moment and anisotropy of magnetite nanoparticles can be optimised by doping with transition metal cations, enabling their properties to be tuned for different biomedical applications. In this study, we assessed the suitability of bacterially synthesized zinc- and cobalt-doped magnetite nanoparticles for biomedical applications. To do this we measured cellular viability and activity in primary human bone marrow-derived mesenchymal stem cells and human osteosarcoma-derived cells. Using AC susceptibility we studied doping induced changes in the magnetic response of the nanoparticles both as stable aqueous suspensions and when associated with cells. Our findings show that the magnetic response of the particles was altered after cellular interaction with a reduction in their mobility. In particular, the strongest AC susceptibility signal measured in vitro was from cells containing high-moment zinc-doped particles, whilst no signal was observed in cells containing the high-anisotropy cobalt-doped particles. For both particle types we found that the moderate dopant levels required for optimum magnetic properties did not alter their cytotoxicity or affect osteogenic differentiation of the stem cells. Thus, despite the known cytotoxicity of cobalt and zinc ions, these results suggest that iron oxide nanoparticles can be doped to sufficiently tailor their magnetic properties without compromising cellular biocompatibility.

Structure-property relationship in cytotoxicity and cell uptake of poly(2-oxazoline) amphiphiles

KAUST Repository

Luxenhofer, Robert

2011-07-01

The family of poly(2-oxazoline)s (POx) is being increasingly investigated in the context of biomedical applications. We tested the relative cytotoxicity of POx and were able to confirm that these polymers are typically not cytotoxic even at high concentrations. Furthermore, we report structure-uptake relationships of a series of amphiphilic POx block copolymers that have different architectures, molar mass and chain termini. The rate of endocytosis can be fine-tuned over a broad range by changing the polymer structure. The cellular uptake increases with the hydrophobic character of the polymers and is observed even at nanomolar concentrations. Considering the structural versatility of this class of polymers, the relative ease of preparation and their stability underlines the potential of POx as a promising platform candidate for the preparation of next-generation polymer therapeutics.

Modulation in vitro and in vivo of cytotoxicity but not cellular levels of doxorubicin by the calmodulin inhibitor trifluoperazine is dependent on the level of resistance.

Science.gov (United States)

Ganapathi, R.; Schmidt, H.; Grabowski, D.; Melia, M.; Ratliff, N.

1988-01-01

The role of the calmodulin inhibitor trifluoperazine (TFP) in modulating the cellular levels and cytotoxicity in vitro and antitumour effects in vivo of doxorubicin (DOX), was evaluated in progressively DOX-resistant (5- to 40-fold) sublines of B16-BL6 mouse melanoma. In parental-sensitive B16-BL6 cells treated for 3 h, the IC50 of DOX was 0.1 microgram ml-1, and a less than 2-fold enhancement in DOX cell kill in the presence of a noncytotoxic concentration of 5 microM TFP was observed. However, in the DOX-resistant sublines, the IC50 was 0.7 to 5.0 micrograms ml-1 DOX in the absence of 5 microM TFP and 0.3 to 0.7 microgram ml-1 DOX in the presence of 5 microM TFP. The 2- to 7.5-fold decrease in the IC50 of DOX in the presence of 5 microM TFP, was dependent on the level of DOX-resistance in the various sublines. Compared to parental-sensitive cells, a 2-fold decrease in DOX-accumulation was evident only in the 40-fold DOX-resistant subline. Further, maximal enhancement (50%) of cellular DOX accumulation in the presence of 5 microM TFP was observed only in the 40-fold resistant cells treated with 5.0 micrograms ml-1 DOX. Retention of DOX in the 40-fold resistant subline was only 20% lower than similarly treated sensitive cells, and the inclusion of TFP increased DOX retention less than 10-15%. Antitumour studies in mice with experimental pulmonary metastases revealed that although DOX and DOX plus TFP had similar antitumour activity with the parental sensitive B16-BL6 cells, the combination of DOX plus TFP was significantly more effective than DOX alone with the DOX-resistant sublines. No overt toxicity was observed in normal mice treated with doses of TFP, DOX or DOX plus TFP used for in vivo chemotherapy studies. Results from this study suggest that gross cellular DOX levels do not appear to correlate with the magnitude of resistance, and the effects of TFP in modulating DOX resistance is possibly due to mechanisms other than mere alterations in cellular drug

Identification of cytotoxic drugs that selectively target tumor cells with MYC overexpression.

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Anna Frenzel

Full Text Available Expression of MYC is deregulated in a wide range of human cancers, and is often associated with aggressive disease and poorly differentiated tumor cells. Identification of compounds with selectivity for cells overexpressing MYC would hence be beneficial for the treatment of these tumors. For this purpose we used cell lines with conditional MYCN or c-MYC expression, to screen a library of 80 conventional cytotoxic compounds for their ability to reduce tumor cell viability and/or growth in a MYC dependent way. We found that 25% of the studied compounds induced apoptosis and/or inhibited proliferation in a MYC-specific manner. The activities of the majority of these were enhanced both by c-MYC or MYCN over-expression. Interestingly, these compounds were acting on distinct cellular targets, including microtubules (paclitaxel, podophyllotoxin, vinblastine and topoisomerases (10-hydroxycamptothecin, camptothecin, daunorubicin, doxorubicin, etoposide as well as DNA, RNA and protein synthesis and turnover (anisomycin, aphidicholin, gliotoxin, MG132, methotrexate, mitomycin C. Our data indicate that MYC overexpression sensitizes cells to disruption of specific pathways and that in most cases c-MYC and MYCN overexpression have similar effects on the responses to cytotoxic compounds. Treatment of the cells with topoisomerase I inhibitors led to down-regulation of MYC protein levels, while doxorubicin and the small molecule MYRA-A was found to disrupt MYC-Max interaction. We conclude that the MYC pathway is only targeted by a subset of conventional cytotoxic drugs currently used in the clinic. Elucidating the mechanisms underlying their specificity towards MYC may be of importance for optimizing treatment of tumors with MYC deregulation. Our data also underscores that MYC is an attractive target for novel therapies and that cellular screenings of chemical libraries can be a powerful tool for identifying compounds with a desired biological activity.

Comparative Cytotoxicity and Genotoxicity of Particulate and Soluble Hexavalent Chromium in Human and Sperm Whale (Physeter macrocephalus) Skin Cells

Science.gov (United States)

Li Chen, Tânia; LaCerte, Carolyne; Wise, Sandra S.; Holmes, Amie; Martino, Julieta; Wise, John Pierce; Thompson, W. Douglas; Wise, John Pierce

2014-01-01

Chromium (Cr) is a global marine pollutant, present in marine mammal tissues. Hexavalent chromium [Cr(VI)] is a known human carcinogen. In this study we compare the cytotoxic and clastogenic effects of Cr(VI) in human (Homo sapiens) and sperm whale (Physeter macrocephalus) skin fibroblasts. Our data show that increasing concentrations of both particulate and soluble Cr(VI) induce increasing amounts of cytotoxicity and clastogenicity in human and sperm whale skin cells. Furthermore, the data show that sperm whale cells are resistant to these effects exhibiting less cytotoxicity and genotoxicity than the human cells. Differences in Cr uptake accounted for some but not all of the differences in particulate and soluble Cr(VI) genotoxicity, although it did explain the differences in particulate Cr(VI) cytotoxicity. Altogether the data indicate that Cr(VI) is a genotoxic threat to whales, but also suggest that whales have evolved cellular mechanisms to protect them against the genotoxicity of environmental agents such as Cr(VI). PMID:21466859

Activation of immune functions via induction of glutathione of lymphocytes by low-dose, whole-body irradiation with gamma-rays

International Nuclear Information System (INIS)

Shuji Kojima; Hisatake Hayase; Mareyuki Takahashi

2007-01-01

Complete text of publication follows. We have recently found that low doses of radiation, unlike higher doses, do not always cause a decrease of cellular glutathione, but they can increase it, leading to an elevation of Con A-induced proliferation of splenocytes. In this study, we first examined whether the increase of glutathione level induced by low-dose gamma-ray irradiation is involved in the appearance of enhanced natural killer (NK) activity and antibody-dependent cellular cytotoxicity (ADCC), leading to delayed tumor growth in Ehrlich solid tumor (EST)-bearing mice. NK activity in ICR mouse splenocytes was significantly increased from 4 h to 6 h after a single whole-body gamma-ray irradiation at 0.5 Gy, and thereafter decreased almost to the zero-time level by 24 h post-irradiation. ADCC was also increased significantly in a similar way. Reduced glutathione exogenously added to splenocytes obtained from normal mice enhanced both NK activity and ADCC in a dose-dependent manner. The inhibitory effect of the radiation on tumor growth was then examined in EST-bearing mice. Repeated low-dose irradiation (0.5 Gy, four times, before and within an early time after the inoculation) significantly delayed the tumor growth. Finally, the effect of single low-dose (0.5 Gy), whole-body gamma-ray irradiation on immune balance (Th1/Th2) was examined in order to elucidate the mechanism underlying the anti-tumor immunity. Recent studies indicate that Th1/Th2 balance plays an important role in the immune responses involved in anti-tumor immunity. The activity of NK is hallmarks of cell-mediated immunity, and play key roles in anti-tumor immunity. The percentage of B cells in blood lymphocytes was selectively decreased after the radiation, concomitantly with an increase in that of helper T cell population, favoring Th1 polarization. The IFN-gamma level in splenocyte culture prepared from EST-bearing mice was significantly increased 48 h after the radiation, though the level of

The entomopathogenic fungus Nomuraea rileyi impairs cellular immunity of its host Helicoverpa armigera.

Science.gov (United States)

Zhong, Ke; Liu, Zhan-Chi; Wang, Jia-Lin; Liu, Xu-Sheng

2017-09-01

In this study, we investigated the effect of the entomopathogenic fungus Nomuraea rileyi on Helicoverpa armigera cellular immune responses. Nomuraea rileyi infection had no effect on total hemocyte count (THC), but impaired hemocyte-mediated phagocytosis, nodulation, and encapsulation responses. Nomuraea rileyi infection led to a significant reduction in hemocyte spreading. An in vitro assay revealed that plasma from N. rileyi infected H. armigera larvae suppressed the spreading ability of hemocytes from naïve larvae. We infer that N. rileyi suppresses the cellular immune response of its host, possibly by secreting exogenous, cytotoxic compounds into the host's hemolymph. © 2017 Wiley Periodicals, Inc.

Morphologic categorization of cell death induced by mild hyperthermia and comparison with death induced by ionizing radiation and cytotoxic drugs

International Nuclear Information System (INIS)

Allan, D.J.; Harmon, B.V.

1986-01-01

This paper presents a summary of the morphological categorization of cell death, results of two in vivo studies on the cell death induced by mild hyperthermia in rat small intestine and mouse mastocytoma, and a comparison of the cell death induced by hyperthermia, radiation and cytotoxic drugs. Two distinct forms of cell death, apoptosis and necrosis, can be recognized on morphologic grounds. Apoptosis appears to be a process of active cellular self-destruction to which a biologically meaningful role can usually be attributed, whereas necrosis is a passive degenerative phenomenon that results from irreversible cellular injury. Light and transmission electron microscopic studies showed that lower body hyperthermia (43 degrees C for 30 min) induced only apoptosis of intestinal epithelial cells, and of lymphocytes, plasma cells, and eosinophils. In the mastocytoma, hyperthermia (43 degrees C for 15 min) produced widespread tumor necrosis and also enhanced apoptosis of tumor cells. Ionizing radiation and cytotoxic drugs are also known to induce apoptosis in a variety of tissues. It is attractive to speculate that DNA damage by each agent is the common event which triggers the same process of active cellular self-destruction that characteristically effects selective cell deletion in normal tissue homeostasis

Study of Galfenol direct cytotoxicity and remote microactuation in cells.

Science.gov (United States)

Vargas-Estevez, Carolina; Blanquer, Andreu; Dulal, Prabesh; Pérez Del Real, Rafael; Duch, Marta; Ibáñez, Elena; Barrios, Leonardo; Murillo, Gonzalo; Torras, Núria; Nogués, Carme; Stadler, Bethanie J H; Plaza, José A; Esteve, Jaume

2017-09-01

Remote microactuators are of great interest in biology and medicine as minimally-invasive tools for cellular stimulation. Remote actuation can be achieved by active magnetostrictive transducers which are capable of changing shape in response to external magnetic fields thereby creating controlled displacements. Among the magnetostrictive materials, Galfenol, the multifaceted iron-based smart material, offers high magnetostriction with robust mechanical properties. In order to explore these capabilities for biomedical applications, it is necessary to study the feasibility of material miniaturization in standard fabrication processes as well as evaluate the biocompatibility. Here we develop a technology to fabricate, release, and suspend Galfenol-based microparticles, without affecting the integrity of the material. The morphology, composition and magnetic properties of the material itself are characterized. The direct cytotoxicity of Galfenol is evaluated in vitro using human macrophages, osteoblast and osteosarcoma cells. In addition, cytotoxicity and actuation of Galfenol microparticles in suspension are evaluated using human macrophages. The biological parameters analyzed indicate that Galfenol is not cytotoxic, even after internalization of some of the particles by macrophages. The microparticles were remotely actuated forming intra- and extracellular chains that did not impact the integrity of the cells. The results propose Galfenol as a suitable material to develop remote microactuators for cell biology studies and intracellular applications. Copyright © 2017 Elsevier Ltd. All rights reserved.

Imaging and measuring the biophysical properties of Fc gamma receptors on single macrophages using atomic force microscopy

Energy Technology Data Exchange (ETDEWEB)

Li, Mi [State Key Laboratory of Robotics, Shenyang Institute of Automation, Chinese Academy of Sciences, Shenyang 110016 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Liu, Lianqing, E-mail: lqliu@sia.cn [State Key Laboratory of Robotics, Shenyang Institute of Automation, Chinese Academy of Sciences, Shenyang 110016 (China); Xi, Ning [Department of Mechanical and Biomedical Engineering, City University of Hong Kong, Hong Kong (China); Wang, Yuechao [State Key Laboratory of Robotics, Shenyang Institute of Automation, Chinese Academy of Sciences, Shenyang 110016 (China); Xiao, Xiubin [Department of Lymphoma, Affiliated Hospital of Military Medical Academy of Sciences, Beijing 100071 (China); Zhang, Weijing, E-mail: zhangwj3072@163.com [Department of Lymphoma, Affiliated Hospital of Military Medical Academy of Sciences, Beijing 100071 (China)

2013-09-06

Highlights: •Nanoscale cellular ultra-structures of macrophages were observed. •The binding affinities of FcγRs were measured directly on macrophages. •The nanoscale distributions of FcγRs were mapped on macrophages. -- Abstract: Fc gamma receptors (FcγR), widely expressed on effector cells (e.g., NK cells, macrophages), play an important role in clinical cancer immunotherapy. The binding of FcγRs to the Fc portions of antibodies that are attached to the target cells can activate the antibody-dependent cell-mediated cytotoxicity (ADCC) killing mechanism which leads to the lysis of target cells. In this work, we used atomic force microscopy (AFM) to observe the cellular ultra-structures and measure the biophysical properties (affinity and distribution) of FcγRs on single macrophages in aqueous environments. AFM imaging was used to obtain the topographies of macrophages, revealing the nanoscale cellular fine structures. For molecular interaction recognition, antibody molecules were attached onto AFM tips via a heterobifunctional polyethylene glycol (PEG) crosslinker. With AFM single-molecule force spectroscopy, the binding affinities of FcγRs were quantitatively measured on single macrophages. Adhesion force mapping method was used to localize the FcγRs, revealing the nanoscale distribution of FcγRs on local areas of macrophages. The experimental results can improve our understanding of FcγRs on macrophages; the established approach will facilitate further research on physiological activities involved in antibody-based immunotherapy.

HIV-specific Fc effector function early in infection predicts the development of broadly neutralizing antibodies.

Science.gov (United States)

Richardson, Simone I; Chung, Amy W; Natarajan, Harini; Mabvakure, Batsirai; Mkhize, Nonhlanhla N; Garrett, Nigel; Abdool Karim, Salim; Moore, Penny L; Ackerman, Margaret E; Alter, Galit; Morris, Lynn

2018-04-01

While the induction of broadly neutralizing antibodies (bNAbs) is a major goal of HIV vaccination strategies, there is mounting evidence to suggest that antibodies with Fc effector function also contribute to protection against HIV infection. Here we investigated Fc effector functionality of HIV-specific IgG plasma antibodies over 3 years of infection in 23 individuals, 13 of whom developed bNAbs. Antibody-dependent cellular phagocytosis (ADCP), complement deposition (ADCD), cellular cytotoxicity (ADCC) and cellular trogocytosis (ADCT) were detected in almost all individuals with levels of activity increasing over time. At 6 months post-infection, individuals with bNAbs had significantly higher levels of ADCD and ADCT that correlated with antibody binding to C1q and FcγRIIa respectively. In addition, antibodies from individuals with bNAbs showed more IgG subclass diversity to multiple HIV antigens which also correlated with Fc polyfunctionality. Germinal center activity represented by CXCL13 levels and expression of activation-induced cytidine deaminase (AID) was found to be associated with neutralization breadth, Fc polyfunctionality and IgG subclass diversity. Overall, multivariate analysis by random forest classification was able to group bNAb individuals with 85% sensitivity and 80% specificity based on the properties of their antibody Fc early in HIV infection. Thus, the Fc effector function profile predicted the development of neutralization breadth in this cohort, suggesting that intrinsic immune factors within the germinal center provide a mechanistic link between the Fc and Fab of HIV-specific antibodies.

Augmentation of natural cytotoxicity by chronic low-dose ionizing radiation in murine natural killer cells primed by IL-2

International Nuclear Information System (INIS)

Sonn, Chung-Hee; Choi, Jong-Rip; Kim, Tae-Jin

2012-01-01

The possible beneficial effects of chronic low-dose irradiation (LDR) and its mechanism of action in a variety of pathophysiological processes such as cancer are a subject of intense investigation. While animal studies involving long-term exposure to LDR have yielded encouraging results, the influence of LDR at the cellular level has been less well defined. We reasoned that since natural killer (NK) cells constitute an early responder to exogenous stress, NK cells may reveal sentinel alterations in function upon exposure to LDR. When purified NK cells received LDR at 4.2 mGy/h for a total of 0.2 Gy in vitro, no significant difference in cell viability was observed. Likewise, no functional changes were detected in LDR-exposed NK cells, demonstrating that LDR alone was insufficient to generate changes at the cellular level. Nonetheless, significant augmentation of cytotoxic, but not proliferative, function was detected when NK cells were stimulated with low-dose IL-2 prior to irradiation. This enhancement of NK cytotoxicity was not due to alterations in NK-activating receptors, NK1.1, NKG2D, CD69 and 2B4, or changes in the rate of early or late apoptosis. Therefore, LDR, in the presence of suboptimal cytokine levels, can facilitate anti-tumor cytotoxicity of NK cells without influencing cellular proliferation or apoptosis. Whether these results translate to in vivo consequences remains to be seen; however, our data provide initial evidence that exposure to LDR can lead to subtle immune-enhancing effects on NK cells and may explain, in part, the functional basis underlying, diverse beneficial effects seen in the animals chronically exposed to LDR. (author)

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

Directory of Open Access Journals (Sweden)

St Germain Carly

2010-09-01

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

Determination of spectral markers of cytotoxicity and genotoxicity using in vitro Raman microspectroscopy: cellular responses to polyamidoamine dendrimer exposure.

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Efeoglu, Esen; Casey, Alan; Byrne, Hugh J

2017-10-09

Although consumer exposure to nanomaterials is ever increasing, with potential increased applications in areas such as drug and/or gene delivery, contrast agents and diagnosis, the determination of the cyto- and geno-toxic effects of nanomaterials on human health and the environment still remains challenging. Although many techniques have been established and adapted to determine the cytotoxicity and genotoxicity of nano-sized materials, these techniques remain limited by the number of assays required, total cost, and use of labels and they struggle to explain the underlying interaction mechanisms. In this study, Raman microspectroscopy is employed as an in vitro label-free, high content screening technique to observe toxicological changes within the cell in a multi-parametric fashion. The evolution of spectral markers as a function of time and applied dose has been used to elucidate the mechanism of action of polyamidoamine (PAMAM) dendrimers associated with cytotoxicity and their impact on nuclear biochemistry. PAMAM dendrimers are chosen as a model nanomaterial due to their widely studied cytotoxic and genotoxic properties and commercial availability. Point spectra were acquired from the cytoplasm to monitor the cascade of toxic events occurring in the cytoplasm upon nanoparticle exposure, whereas the spectra acquired from the nucleus and the nucleolus were used to explore PAMAM-nuclear material interaction as well as genotoxic responses.

Acinetobacter baumannii and A. pittii clinical isolates lack adherence and cytotoxicity to lung epithelial cells in vitro.

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Lázaro-Díez, María; Navascués-Lejarza, Teresa; Remuzgo-Martínez, Sara; Navas, Jesús; Icardo, José Manuel; Acosta, Felix; Martínez-Martínez, Luis; Ramos-Vivas, José

2016-09-01

The molecular and genetic basis of Acinetobacter baumannii and Acinetobacter pittii virulence remains poorly understood, and there is still lack of knowledge in host cell response to these bacteria. In this study, we have used eleven clinical Acinetobacter strains (A. baumannii n = 5; A. pittii n = 6) to unravel bacterial adherence, invasion and cytotoxicity to human lung epithelial cells. Our results showed that adherence to epithelial cells by Acinetobacter strains is scarce and cellular invasion was not truly detected. In addition, all Acinetobacter strains failed to induce any cytotoxic effect on A549 cells. Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Molecular mechanisms of resistance to Rituximab and pharmacologic strategies for its circumvention.

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Stolz, Claudia; Schuler, Martin

2009-06-01

The introduction of Rituximab has greatly improved therapeutic options for patients with B-cell non-Hodgkin lymphoma (B-NHL). However, a substantial fraction of patients with aggressive B-NHL fails first-line therapy, and most patients with relapsing indolent B-NHL eventually acquire Rituximab resistance. Molecular understanding of the underlying mechanisms facilitates the development of pharmacologic strategies to overcome resistance. Rituximab exerts its activity on CD20-expressing B-cells by indirect and direct effector mechanisms. Indirect mechanisms are complement-dependent cytotoxicity (CDC), and antibody-dependent cell-mediated cytotoxicity (ADCC). Direct activities, such as growth inhibition, induction of apoptosis and chemosensitisation, have been reported, but are less defined. Moreover, the relative contribution of CDC, ADCC and direct mechanisms to the activity of Rituximab in vivo is unclear. Down-regulation of CD20 and expression of complement inhibitors have been described as escape mechanisms in B-NHL. Recent reports suggest that deregulated phosphoinositide-3-kinase (PI3K)/Akt, mitogen-activated kinases (MAPK) and nuclear-factor kappaB (NF-kappaB), as well as up-regulation of anti-apoptotic proteins may determine the efficacy of Rituximab to kill B-NHL cells in vitro and in vivo. The latter signalling pathways are attractive targets for pharmacologic modulation of resistance to Rituximab. With the advent of new inhibitors and antibodies, rationally designed clinical trials addressing Rituximab resistance are feasible.

Characterization of non-dimer DNA lesions and cellular damages caused by ultraviolet light

International Nuclear Information System (INIS)

Nakao, Kumi

1989-01-01

To understand the mechanisms of carcinogenicity and cytotoxicity induced by ultraviolet (UV) light, non-dimer DNA damages produced by near UV light (wave-length: 290∼320 nm) were examined by alkaline elution using Chinese hamster V-79 cells. UV exposure produced a dose-dependent induction of DNA single strand breaks and DNA-protein crosslinks. However, neither of these DNA lesions were repaired within a 24 hr incubation of the cells following UV exposure. Rather the number of these lesions increased. Also, UV exposure inhibited DNA and RNA synthesis. In addition, UV induced both cytotoxicity and chromosomal aberration. Electron spin resornance (ESR) studies showed that the exposure of cells to UV light resulted in the appearance of an ESR signal at -120degC. The roles of glutathione, vitamin E and vitamin B 2 , which were celluar antioxidant, on the induction of cytotoxicity by UV exposure were also examined. Pretreatment with vitamin E reduced the cytotoxicty caused by UV, whereas neither preteatment with vitamin B 2 nor the alteration of cellular gluthaione content affected the cytotoxicity. These results suggest that non-dimer DNA damages, such as DNA single strand breaks and DNA-protein crosslinks play an important role in inducing UV-carcinogenicity and UV-cytotoxicity, and that the mechanisms of these damages may be associated with the generation of free radicals. (author)

Cytotoxic and biological effects of bulk fill composites on rat cortical neuron cells.

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Kamalak, Hakan; Kamalak, Aliye; Taghizadehghalehjoughi, Ali; Hacımüftüoğlu, Ahmet; Nalcı, Kemal Alp

2018-03-28

The aim of this study was to investigate potential cellular responses and biological effects of new generation dental composites on cortical neuron cells in two different exposure times. The study group included five different bulk-fill flow able composites; Surefil SDR Flow, X-tra Base Flow, Venus Bulk Flow, Filtek Bulk Flow and Tetric-Evo Flow. They were filled in Teflon molds (Height: 4 mm, Width: 6 mm) and irradiated for 20 s. Cortical neuron cells were inoculated into 24-well plates. After 80% of the wells were coated, the 3 µm membrane was inserted and dental filling materials were added. The experiment was continued for 24 and 72 h. Cell viability measured by MTT assay test, total antioxidant and total oxidant status were examined using real assay diagnostic kits. The patterns of cell death (apoptosis) were analyzed using annexin V-FITC staining with flow cytometry. Β-defensins were quantitatively assessed by RT-PCR. IL-6, IL-8 and IL-10 cytokines were measured from the supernatants. All composites significantly affected analyses parameters during the exposure durations. Our data provide evidence that all dental materials tested are cytotoxic in acute phase and these effects are induced cellular death after different exposure periods. Significant cytotoxicity was detected in TE, XB, SS, FBF and VBF groups at 24 and 72 h, respectively.

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

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

2018-04-01

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

Potentiation of luteolin cytotoxicity by flavonols fisetin and quercetin in human chronic lymphocytic leukemia cell lines.

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Sak, Katrin; Kasemaa, Kristi; Everaus, Hele

2016-09-14

Despite numerous studies chronic lymphocytic leukemia (CLL) still remains an incurable disease. Therefore, all new compounds and novel strategies which are able to eradicate CLL cells should be considered as valuable clues for a potential future remedy against this malignancy. In the present study, the cytotoxic profiles of natural flavonoids were described in two human CLL cell lines, HG-3 and EHEB, indicating the flavone luteolin as the most potent flavonoid with half-maximal inhibitory constants (IC50) of 37 μM and 26 μM, respectively. Luteolin significantly increased the apoptotic cell population in both cell lines by increasing the activities of caspases-3 and -9 and triggering the intrinsic apoptotic pathway. Two flavonols, fisetin and quercetin, were somewhat less efficient in suppressing cellular viability, whereas baicalein, chrysin, (+)-catechin and hesperetin exerted only a small or no response at doses as high as 100 μM. Both fisetin and quercetin were able to augment the cytotoxic activity of luteolin in both cell lines by reducing the IC50 values up to four fold. As a result of this, luteolin displayed cytotoxicity activity already at low micromolar concentrations that could potentially be physiologically achievable through oral ingestion. No other tested flavonoids were capable of sensitizing CLL cells to luteolin pointing to a specific binding of fisetin and quercetin to the cellular targets which interfere with the signaling pathways induced by luteolin. Although further molecular studies to unravel this potentiating mechanism are certainly needed, this phenomenon could contribute to future remedies for prevention and treatment of chronic lymphocytic leukemia.

Cytotoxic effects of nickel nanowires in human fibroblasts

KAUST Repository

Felix Servin, Laura P.

2016-03-09

The increasing interest in the use of magnetic nanostructures for biomedical applications necessitates rigorous studies to be carried out in order to determine their potential toxicity. This work attempts to elucidate the cytotoxic effects of nickel nanowires (NWs) in human fibroblasts WI-38 by a colorimetric assay (MTT) under two different parameters: NW concentration and exposure time. This was complemented with TEM and confocal images to assess the NWs internalization and to identify any changes in the cell morphology. Ni NWs were fabricated by electrodeposition using porous alumina templates. Energy dispersive X-Ray analysis, scanning electron microscopy and transmission electron microscopy imaging were used for NW characterization. The results showed decreased cell metabolic activity for incubation times longer than 24 hours and no negative effects for exposure times shorter than that. The cytotoxicity effects for human fibroblasts were then compared with those reported for HCT 116 cells, and the findings point out that it is relevant to consider the cellular size. In addition, the present study compares the toxic effects of equivalent amounts of nickel in the form of its salt to those of NWs and shows that the NWs are more toxic than the salts. Internalized NWs were found in vesicles inside of the cells where their presence induced inflammation of the endoplasmic reticulum.

Increased cellular uptake of lauryl gallate loaded in superparamagnetic poly(methyl methacrylate) nanoparticles due to surface modification with folic acid.

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Feuser, Paulo Emilio; Arévalo, Juan Marcelo Carpio; Junior, Enio Lima; Rossi, Gustavo Rodrigues; da Silva Trindade, Edvaldo; Rocha, Maria Eliane Merlin; Jacques, Amanda Virtuoso; Ricci-Júnior, Eduardo; Santos-Silva, Maria Claudia; Sayer, Claudia; de Araújo, Pedro H Hermes

2016-12-01

Lauryl gallate loaded in superparamagnetic poly(methyl methacrylate) nanoparticles surface modified with folic acid were synthesized by miniemulsion polymerization in just one step. In vitro biocompatibility and cytotoxicity assays on L929 (murine fibroblast), human red blood, and HeLa (uterine colon cancer) cells were performed. The effect of folic acid at the nanoparticles surface was evaluated through cellular uptake assays in HeLa cells. Results showed that the presence of folic acid did not affect substantially the polymer particle size (~120 nm), the superparamagnetic behavior, the encapsulation efficiency of lauryl gallate (~87 %), the Zeta potential (~38 mV) of the polymeric nanoparticles or the release profile of lauryl gallate. The release profile of lauryl gallate from superparamagnetic poly(methyl methacrylate) nanoparticles presented an initial burst effect (0-1 h) followed by a slow and sustained release, indicating a biphasic release system. Lauryl gallate loaded in superparamagnetic poly(methyl methacrylate) nanoparticles with folic acid did not present cytotoxicity effects on L929 and human red blood cells. However, free lauryl gallate presented significant cytotoxic effects on L929 and human red blood cells at all tested concentrations. The presence of folic acid increased the cytotoxicity of lauryl gallate loaded in nanoparticles on HeLa cells due to a higher cellular uptake when HeLa cells were incubated at 37 °C. On the other hand, when the nanoparticles were incubated at low temperature (4 °C) cellular uptake was not observed, suggesting that the uptake occurred by folate receptor mediated energy-dependent endocytosis. Based on presented results our work suggests that this carrier system can be an excellent alternative in targeted drug delivery by folate receptor.

Cytotoxicity and Effects on Cell Viability of Nickel Nanowires

KAUST Repository

Rodriguez, Jose E.

2013-05-01

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

Synthesis of mono Mannich bases of 2-(4-hydroxybenzylidene)-2,3-dihydroinden-1-one and evaluation of their cytotoxicities.

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Tugrak, Mehtap; Yamali, Cem; Sakagami, Hiroshi; Gul, Halise Inci

2016-10-01

Chalcones and Mannich bases are a group of compounds known for their cytotoxicities. In this study restricted chalcone analogue, compound 2-(4-hydroxybenzylidene)-2,3-dihydroinden-1-one MT1, was used as a starting compound to synthesize new mono Mannich bases since Mannich bases may induce more cytotoxicity than chalcone analogue that they are derived from by producing additional alkylating center for cellular thiols. In this study, cyclic and acyclic amines were used to synthesize Mannich bases. All compounds were tested against Ca9-22 (gingival carcinoma), HSC-2, HSC-3 and HSC-4 (oral squamous cell carcinoma) as tumour cell lines and HGF (gingival fibroblasts), HPC (pulp cells) and HPLF (periodontal ligament fibroblasts) human normal oral cells as non tumour cell lines. Cytotoxicity, selectivity index (SI) values and potency selectivity expression (PSE) values expressed as a percentage were determined for the compounds. According to data obtained, the compound MT8 with the highest PSE value bearing N-methylpiperazine moiety seems to be a good candidate to develop new cytotoxic compounds and is suited for further investigation.

Alpha-ketoglutarate and N-acetyl cysteine protect PC12 cells from cyanide-induced cytotoxicity and altered energy metabolism.

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Satpute, R M; Hariharakrishnan, J; Bhattacharya, R

2008-01-01

Cyanide is a rapidly acting neurotoxin that inhibits cellular respiration and energy metabolism leading to histotoxic hypoxia. This results in the dissipation of mitochondrial membrane potential (MMP) accompanied by decreased cellular ATP content which in turn is responsible for increased levels of intracellular calcium ions ([Ca(2+)](i)) and total lactic acid content of the cells. Rat pheochromocytoma (PC12) cells possess much of the biochemical machinery associated with synaptic neurons. In the present study, we evaluated the cytoprotective effects of alpha-ketoglutarate (A-KG) and N-acetylcysteine (NAC) against cyanide-induced cytotoxicity and altered energy metabolism in PC12 cells. Cyanide-antagonism by A-KG is attributed to cyanohydrin formation whereas NAC is known for its antioxidant properties. Data on leakage of intracellular lactate dehydrogenase and mitochondrial function (MTT assay) revealed that simultaneous treatment of A-KG (0.5 mM) and NAC (0.25 mM) significantly prevented the cytotoxicity of cyanide. Also, cellular ATP content was found to improve, followed by restoration of MMP, intracellular calcium [Ca(2+)](i) and lactic acid levels. Treatment with A-KG and NAC also attenuated the levels of peroxides generated by cyanide. The study indicates that combined administration of A-KG and NAC protected the cyanide-challenged PC12 cells by resolving the altered energy metabolism. The results have implications in the development of new treatment regimen for cyanide poisoning.

Artifacts by marker enzyme adsorption on nanomaterials in cytotoxicity assays with tissue cultures

International Nuclear Information System (INIS)

Wohlleben, Wendel; Kolle, Susanne N; Hasenkamp, Laura-Carolin; Boeser, Alexander; Vogel, Sandra; Vacano, Bernhard von; Ravenzwaay, Ben van; Landsiedel, Robert

2011-01-01

We used precision cut lung slices (PCLS) to study the cytotoxicity of cobalt ferrite nanomaterials with and without bovine serum albumin (BSA) stabilization. Using mitochondrial activity as an indicator of cytotoxicity (WST-1 assay) increasing concentrations of cobalt ferrite nanomaterial caused increasing levels of cytotoxicity in PCLS irrespective of BSA stabilization. However, there was no increase in released lactate dehydrogenase (LDH) levels caused by BSA stabilized nanomaterial indicating concentration depended cytotoxictiy. Moreover, non-stabilized nanomaterial caused a decrease of background LDH levels in the PCLS culture supernatant confirmed by complementary methods. Direct characterization of the protein corona of extracted nanomaterial shows that the LDH decrease is due to adsorption of LDH onto the surface of the non-stabilized nanomaterial, correlated with strong agglomeration. Preincubation with serum protein blocks the adsorption of LDH and stabilizes the nanomaterial at low agglomeration. We have thus demonstrated the cytotoxicity of nanomaterials in PCLS does not correlate with disrupted membrane integrity followed by LDH release. Furthermore, we found that intracellular enzymes such as the marker enzyme LDH are able to bind onto surfaces of nanomaterial and thereby adulterate the detection of toxic effects. A replacement of BSA by LDH or a secondary LDH-on-BSA-corona were not observed, confirming earlier indications that the protein corona exchange rate are slow or vanishing on inorganic nanomaterial. Thus, the method(s) to assess nanomaterial-mediated effects have to be carefully chosen based on the cellular effect and possible nano-specific artifacts.

Chlorpromazine inhibits tumour necrosis factor synthesis and cytotoxicity in vitro.

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Zinetti, M; Galli, G; Demitri, M T; Fantuzzi, G; Minto, M; Ghezzi, P; Alzani, R; Cozzi, E; Fratelli, M

1995-11-01

Chlorpromazine (CPZ) has been previously shown to protect against endotoxin [lipopolysaccharide (LPS)] lethality and inhibit the release of tumour necrosis factor in vivo. We investigated at the cellular level whether this was due to direct inhibition of tumour necrosis factor-alpha (TNF-alpha) synthesis, using LPS-stimulated THP-1 human monocytic leukemia cells. We also studied the effect of CPZ on human TNF-alpha action by assessing TNF-alpha cytotoxicity on mouse fibrosarcoma L929 cells. CPZ (1-100 microM) inhibited TNF-alpha production in THP-1 cells in a dose dependent manner by a maximum of 80%. This effect was comparable to that of two well-known inhibitory drugs, dexamethasone and cyclicAMP. Inhibition was also evident at the mRNA level. On the other hand CPZ (10-25 microM) also inhibited TNF-alpha activity: in fact it reduced the cytotoxicity of TNF-alpha on L929 cells (EC50 was increased four times) and could provide protection even as a post-treatment. CPZ inhibited TNF-induced apoptosis in L929 cells, as detected by analysis of nuclear morphology. However, since we showed that apoptosis was very limited, and was not the main mode of cell death in our conditions, this could not explain the overall protection. Since CPZ did not interfere with either the oligomerization state of TNF-alpha or its receptor binding, our data suggest that it reduced cytotoxicity by inhibiting some steps in the TNF-alpha signalling pathways.

Evaluation of Cytotoxicity and Genotoxicity of Acacia aroma Leaf Extracts

Directory of Open Access Journals (Sweden)

C. M. Mattana

2014-01-01

Full Text Available Acacia aroma, native plant from San Luis, Argentina, is commonly used as antiseptic and for healing of wounds. The present study was conducted to investigate the in vitro cytotoxicity and genotoxicity of hot aqueous extract (HAE and ethanolic extract (EE of A. aroma. The cytotoxic activity was assayed by neutral red uptake assay on Vero cell. Cell treatment with a range from 100 to 5000 μg/mL of HAE and EE showed that 500 μg/mL and 100 μg/mL were the maximum noncytotoxic concentrations, respectively. The CC50 was 658 μg/mL for EE and 1020 μg/mL for HAE. The genotoxicity was tested by the single-cell gel electrophoresis comet assay. The results obtained in the evaluation of DNA cellular damage exposed to varied concentrations of the HAE showed no significant genotoxic effect at range of 1–20 mg/mL. The EE at 20 mg/mL showed moderate genotoxic effect related to the increase of the DNA percentage contained in tail of the comet; DNA was classified in category 2. At concentrations below 5 mg/mL, the results of cytotoxicity and genotoxicity of aqueous and ethanolic extracts of Acacia aroma guarantee the safety at cell and genomic level. However further studies are needed for longer periods including animal models to confirm the findings.

Putative contribution of CD56 positive cells in cetuximab treatment efficacy in first-line metastatic colorectal cancer patients

International Nuclear Information System (INIS)

Maréchal, Raphaël; De Schutter, Jef; Nagy, Nathalie; Demetter, Pieter; Lemmers, Arnaud; Devière, Jacques; Salmon, Isabelle; Tejpar, Sabine; Van Laethem, Jean-Luc

2010-01-01

Activity of cetuximab, a chimeric monoclonal antibody targeting the epidermal growth factor receptor, is largely attributed to its direct antiproliferative and proapoptotic effects. Antibody-dependent cell-mediated cytotoxicity (ADCC) could be another possible mechanism of cetuximab antitumor effects and its specific contribution on the clinical activity of cetuximab is unknown. We assessed immune cells infiltrate (CD56, CD68, CD3, CD4, CD8, Foxp3) in the primary tumor of metastatic colorectal cancer (mCRC) patients treated with a first-line cetuximab-based chemotherapy in the framework of prospective trials (treatment group) and in a matched group of mCRC patients who received the same chemotherapy regimen without cetuximab (control group). The relationship between intra-tumoral immune effector cells, the K-ras status and the efficacy of the treatment were investigated. We also evaluated in vitro, the ADCC activity in healthy donors and chemonaive mCRC patients and the specific contribution of CD56 + cells. ADCC activity against DLD1 CRC cell line is maintained in cancer patients and significantly declined after CD56 + cells depletion. In multivariate analysis, K-ras wild-type (HR: 4.7 (95% CI 1.8-12.3), p = 0.001) and tumor infiltrating CD56 + cells (HR: 2.6, (95%CI:1.14-6.0), p = 0.019) were independent favourable prognostic factors for PFS and response only in the cetuximab treatment group. By contrast CD56 + cells failed to predict PFS and response in the control group. CD56 + cells, mainly NK cells, may be the major effector of ADCC related-cetuximab activity. Assessment of CD56 + cells infiltrate in primary colorectal adenocarcinoma may provide additional information to K-ras status in predicting response and PFS in mCRC patients treated with first-line cetuximab-based chemotherapy

FX knockout CHO hosts can express desired ratios of fucosylated or afucosylated antibodies with high titers and comparable product quality.

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Louie, Salina; Haley, Benjamin; Marshall, Brett; Heidersbach, Amy; Yim, Mandy; Brozynski, Martina; Tang, Danming; Lam, Cynthia; Petryniak, Bronislawa; Shaw, David; Shim, Jeongsup; Miller, Aaron; Lowe, John B; Snedecor, Brad; Misaghi, Shahram

2017-03-01

During antibody dependent cell cytotoxicity (ADCC) the target cells are killed by monocytes and natural killer cells. ADCC is enhanced when the antibody heavy chain's core N-linked glycan lacks the fucose molecule(s). Several strategies have been utilized to generate fully afucosylated antibodies. A commonly used and efficient approach has been knocking out the FUT8 gene of the Chinese hamster ovary (CHO) host cells, which results in expression of antibody molecules with fully afucosylated glycans. However, a major drawback of the FUT8-KO host is the requirement for undertaking two separate cell line development (CLD) efforts in order to obtain both primarily fucosylated and fully afucosylated antibody species for comparative studies in vitro and in vivo. Even more challenging is obtaining primarily fucosylated and FUT8-KO clones with similar enough product quality attributes to ensure that any observed ADCC advantage(s) can be strictly attributed to afucosylation. Here, we report generation and use of a FX knockout (FXKO) CHO host cell line that is capable of expressing antibody molecules with either primarily fucosylated or fully afucosylated glycan profiles with otherwise similar product quality attributes, depending on addition of fucose to the cell culture media. Hence, the FXKO host not only obviates the requirement for undertaking two separate CLD efforts, but it also averts the need for screening many colonies to identify clones with comparable product qualities. Finally, FXKO clones can express antibodies with the desired ratio of primarily fucosylated to afucosylated glycans when fucose is titrated into the production media, to allow achieving intended levels of FcγRIII-binding and ADCC for an antibody. Biotechnol. Bioeng. 2017;114: 632-644. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

A perillyl alcohol-conjugated analog of 3-bromopyruvate without cellular uptake dependency on monocarboxylate transporter 1 and with activity in 3-BP-resistant tumor cells.

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Chen, Thomas C; Yu, Jiali; Nouri Nigjeh, Eslam; Wang, Weijun; Myint, Phyo Thazin; Zandi, Ebrahim; Hofman, Florence M; Schönthal, Axel H

2017-08-01

The anticancer agent 3-bromopyruvate (3-BP) is viewed as a glycolytic inhibitor that preferentially kills glycolytic cancer cells through energy depletion. However, its cytotoxic activity is dependent on cellular drug import through transmembrane monocarboxylate transporter 1 (MCT-1), which restricts its anticancer potential to MCT-1-positive tumor cells. We created and characterized an MCT-1-independent analog of 3-BP, called NEO218. NEO218 was synthesized by covalently conjugating 3-BP to perillyl alcohol (POH), a natural monoterpene. The responses of various tumor cell lines to treatment with either compound were characterized in the presence or absence of supplemental pyruvate or antioxidants N-acetyl-cysteine (NAC) and glutathione (GSH). Drug effects on glyceraldehyde 3-phosphate dehydrogenase (GAPDH) enzyme activity were investigated by mass spectrometric analysis. The development of 3-BP resistance was investigated in MCT-1-positive HCT116 colon carcinoma cells in vitro. Our results show that NEO218: (i) pyruvylated GAPDH on all 4 of its cysteine residues and shut down enzymatic activity; (ii) severely lowered cellular ATP content below life-sustaining levels, and (iii) triggered rapid necrosis. Intriguingly, supplemental antioxidants effectively prevented cytotoxic activity of NEO218 as well as 3-BP, but supplemental pyruvate powerfully protected cells only from 3-BP, not from NEO218. Unlike 3-BP, NEO218 exerted its potent cytotoxic activity irrespective of cellular MCT-1 status. Treatment of HCT116 cells with 3-BP resulted in prompt development of resistance, based on the emergence of MCT-1-negative cells. This was not the case with NEO218, and highly 3-BP-resistant cells remained exquisitely sensitive to NEO218. Thus, our study identifies a mechanism by which tumor cells develop rapid resistance to 3-BP, and presents NEO218 as a superior agent not subject to this cellular defense. Furthermore, our results offer alternative interpretations of previously

Submicron and nano formulations of titanium dioxide and zinc oxide stimulate unique cellular toxicological responses in the green microalga Chlamydomonas reinhardtii

Energy Technology Data Exchange (ETDEWEB)

Gunawan, Cindy, E-mail: c.gunawan@unsw.edu.au [ARC Centre of Excellence for Functional Nanomaterials, School of Chemical Engineering, The University of New South Wales, Sydney, NSW (Australia); Sirimanoonphan, Aunchisa [ARC Centre of Excellence for Functional Nanomaterials, School of Chemical Engineering, The University of New South Wales, Sydney, NSW (Australia); Teoh, Wey Yang [Clean Energy and Nanotechnology (CLEAN) Laboratory, School of Energy and Environment, City University of Hong Kong, Kowloon, Hong Kong Special Administrative Region (Hong Kong); Marquis, Christopher P., E-mail: c.marquis@unsw.edu.au [School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, NSW (Australia); Amal, Rose [ARC Centre of Excellence for Functional Nanomaterials, School of Chemical Engineering, The University of New South Wales, Sydney, NSW (Australia)

2013-09-15

Highlights: • Uptake of TiO{sub 2} solids by C. reinhardtii generates ROS as an early stress response. • Submicron and nanoTiO{sub 2} exhibit benign effect on cell proliferation. • Uptake of ZnO solids and leached zinc by C. reinhardtii inhibit the alga growth. • No cellular oxidative stress is detected with submicron and nano ZnO exposure. • The toxicity of particles is not necessarily mediated by cellular oxidative stress. -- Abstract: The work investigates the eco-cytoxicity of submicron and nano TiO{sub 2} and ZnO, arising from the unique interactions of freshwater microalga Chlamydomonas reinhardtii to soluble and undissolved components of the metal oxides. In a freshwater medium, submicron and nano TiO{sub 2} exist as suspended aggregates with no-observable leaching. Submicron and nano ZnO undergo comparable concentration-dependent fractional leaching, and exist as dissolved zinc and aggregates of undissolved ZnO. Cellular internalisation of solid TiO{sub 2} stimulates cellular ROS generation as an early stress response. The cellular redox imbalance was observed for both submicron and nano TiO{sub 2} exposure, despite exhibiting benign effects on the alga proliferation (8-day EC50 > 100 mg TiO{sub 2}/L). Parallel exposure of C. reinhardtii to submicron and nano ZnO saw cellular uptake of both the leached zinc and solid ZnO and resulting in inhibition of the alga growth (8-day EC50 ≥ 0.01 mg ZnO/L). Despite the sensitivity, no zinc-induced cellular ROS generation was detected, even at 100 mg ZnO/L exposure. Taken together, the observations confront the generally accepted paradigm of cellular oxidative stress-mediated cytotoxicity of particles. The knowledge of speciation of particles and the corresponding stimulation of unique cellular responses and cytotoxicity is vital for assessment of the environmental implications of these materials.

Submicron and nano formulations of titanium dioxide and zinc oxide stimulate unique cellular toxicological responses in the green microalga Chlamydomonas reinhardtii

International Nuclear Information System (INIS)

Gunawan, Cindy; Sirimanoonphan, Aunchisa; Teoh, Wey Yang; Marquis, Christopher P.; Amal, Rose

2013-01-01

Highlights: • Uptake of TiO 2 solids by C. reinhardtii generates ROS as an early stress response. • Submicron and nanoTiO 2 exhibit benign effect on cell proliferation. • Uptake of ZnO solids and leached zinc by C. reinhardtii inhibit the alga growth. • No cellular oxidative stress is detected with submicron and nano ZnO exposure. • The toxicity of particles is not necessarily mediated by cellular oxidative stress. -- Abstract: The work investigates the eco-cytoxicity of submicron and nano TiO 2 and ZnO, arising from the unique interactions of freshwater microalga Chlamydomonas reinhardtii to soluble and undissolved components of the metal oxides. In a freshwater medium, submicron and nano TiO 2 exist as suspended aggregates with no-observable leaching. Submicron and nano ZnO undergo comparable concentration-dependent fractional leaching, and exist as dissolved zinc and aggregates of undissolved ZnO. Cellular internalisation of solid TiO 2 stimulates cellular ROS generation as an early stress response. The cellular redox imbalance was observed for both submicron and nano TiO 2 exposure, despite exhibiting benign effects on the alga proliferation (8-day EC50 > 100 mg TiO 2 /L). Parallel exposure of C. reinhardtii to submicron and nano ZnO saw cellular uptake of both the leached zinc and solid ZnO and resulting in inhibition of the alga growth (8-day EC50 ≥ 0.01 mg ZnO/L). Despite the sensitivity, no zinc-induced cellular ROS generation was detected, even at 100 mg ZnO/L exposure. Taken together, the observations confront the generally accepted paradigm of cellular oxidative stress-mediated cytotoxicity of particles. The knowledge of speciation of particles and the corresponding stimulation of unique cellular responses and cytotoxicity is vital for assessment of the environmental implications of these materials

Oxygen concentration modulates cellular senescence and autophagy in human trophoblast cells.

Science.gov (United States)

Seno, Kotomi; Tanikawa, Nao; Takahashi, Hironori; Ohkuchi, Akihide; Suzuki, Hirotada; Matsubara, Shigeki; Iwata, Hisataka; Kuwayama, Takehito; Shirasuna, Koumei

2018-02-15

We investigated the effect of oxygen concentrations on cellular senescence and autophagy and examined the role of autophagy in human trophoblast cells. Human first-trimester trophoblast cells (Sw.71) were incubated under 21%, 5%, or 1% O 2 concentrations for 24 hours. We examined the extent of senescence caused using senescence-associated β-galactosidase (SA-β-Gal) and senescence-associated secretory phenotype (SASP) as markers. Moreover, we examined the role of autophagy in causing cellular senescence using an autophagy inhibitor (3-methyladenine, 3MA). Physiological normoxia (5% O 2 ) decreased SA-β-Gal-positive cells and SASP including interleukin-6 (IL-6) and IL-8 compared with cultured cells in 21% O 2 . Pathophysiological hypoxia (1% O 2 ) caused cytotoxicity, including extracellular release of ATP and lactate dehydrogenase, and decreased senescence phenotypes. 3MA-treated trophoblast cells significantly suppressed senescence markers (SA-β-Gal-positive cells and SASP secretion) in O 2 -independent manner. We conclude that O 2 concentration modulates cellular senescence phenotypes regulating autophagy in the human trophoblast cells. Moreover, inhibiting autophagy suppresses cellular senescence, suggesting that autophagy contributes to oxygen stress-induced cellular senescence. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

HIV-1 Env DNA vaccine plus protein boost delivered by EP expands B- and T-cell responses and neutralizing phenotype in vivo.

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Kar Muthumani

Full Text Available An effective HIV vaccine will most likely require the induction of strong T-cell responses, broadly neutralizing antibodies (bNAbs, and the elicitation of antibody-dependent cellular cytotoxicity (ADCC. Previously, we demonstrated the induction of strong HIV/SIV cellular immune responses in macaques and humans using synthetic consensus DNA immunogens delivered via adaptive electroporation (EP. However, the ability of this improved DNA approach to prime for relevant antibody responses has not been previously studied. Here, we investigate the immunogenicity of consensus DNA constructs encoding gp140 sequences from HIV-1 subtypes A, B, C and D in a DNA prime-protein boost vaccine regimen. Mice and guinea pigs were primed with single- and multi-clade DNA via EP and boosted with recombinant gp120 protein. Sera were analyzed for gp120 binding and induction of neutralizing antibody activity. Immunization with recombinant Env protein alone induced low-titer binding antibodies with limited neutralization breath. In contrast, the synthetic DNA prime-protein boost protocol induced significantly higher antibody binding titers. Furthermore, sera from DNA prime-protein boost groups were able to neutralize a broader range of viruses in a panel of tier 1 clade B viruses as well as multiple tier 1 clade A and clade C viruses. Further investigation of synthetic DNA prime plus adaptive EP plus protein boost appears warranted.

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

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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.

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

Science.gov (United States)

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

2010-10-15

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

Mulberry Fruit Extract Affords Protection against Ethyl Carbamate-Induced Cytotoxicity and Oxidative Stress

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Wei Chen

2017-01-01

Full Text Available Ethyl carbamate (EC is a food and environmental toxicant and is a cause of concern for human exposure. Several studies indicated that EC-induced toxicity was associated with oxidative stress. Mulberry fruits are reported to have a wide range of bioactive compounds and pharmacological activities. The present study was therefore aimed to investigate the protective property of mulberry fruit extract (MFE on EC-induced cytotoxicity and oxidative stress. Chemical composition analysis showed that total phenolic content and total flavonoid content in MFE were 502.43 ± 5.10 and 219.12 ± 4.45 mg QE/100 g FW. Cyanidin-3-O-glucoside and cyanidin-3-O-rutinoside were the major anthocyanins in MFE. In vitro antioxidant studies (DPPH, ABTS, and FRAP assays jointly exhibited the potent antioxidant capacity of MFE. Further study indicated that MFE protected human liver HepG2 cells from EC-induced cytotoxicity by scavenging overproduced cellular ROS. EC treatment promoted intracellular glutathione (GSH depletion and caused mitochondrial membrane potential (MMP collapse, as well as mitochondrial membrane lipid peroxidation, whereas MFE pretreatment significantly inhibited GSH depletion and restored the mitochondrial membrane function. Overall, our study suggested that polyphenolic-rich MFE could afford a potent protection against EC-induced cytotoxicity and oxidative stress.

A novel bicistronic gene design couples stable cell line selection with a fucose switch in a designer CHO host to produce native and afucosylated glycoform antibodies.

Science.gov (United States)

Roy, Gargi; Martin, Tom; Barnes, Arnita; Wang, Jihong; Jimenez, Rod Brian; Rice, Megan; Li, Lina; Feng, Hui; Zhang, Shu; Chaerkady, Raghothama; Wu, Herren; Marelli, Marcello; Hatton, Diane; Zhu, Jie; Bowen, Michael A

2018-04-01

The conserved glycosylation site Asn 297 of a monoclonal antibody (mAb) can be decorated with a variety of sugars that can alter mAb pharmacokinetics and recruitment of effector proteins. Antibodies lacking the core fucose at Asn 297 (afucosylated mAbs) show enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) and increased efficacy. Here, we describe the development of a robust platform for the manufacture of afucosylated therapeutic mAbs by engineering a Chinese hamster ovary (CHO) host cell line to co-express a mAb with GDP-6-deoxy-D-lyxo-4-hexulose reductase (RMD), a prokaryotic enzyme that deflects an intermediate in the de novo synthesis of fucose to a dead-end product, resulting in the production of afucosylated mAb (GlymaxX™ Technology, ProBioGen). Expression of the mAb and RMD genes was coordinated by co-transfection of separate mAb and RMD vectors or use of an internal ribosome entry site (IRES) element to link the translation of RMD with either the glutamine synthase selection marker or the mAb light chain. The GS-IRES-RMD vector format was more suitable for the rapid generation of high yielding cell lines, secreting afucosylated mAb with titers exceeding 6.0 g/L. These cell lines maintained production of afucosylated mAb over 60 generations, ensuring their suitability for use in large-scale manufacturing. The afucosylated mAbs purified from these RMD-engineered cell lines showed increased binding in a CD16 cellular assay, demonstrating enhancement of ADCC compared to fucosylated control mAb. Furthermore, the afucosylation in these mAbs could be controlled by simple addition of L-fucose in the culture medium, thereby allowing the use of a single cell line for production of the same mAb in fucosylated and afucosylated formats for multiple therapeutic indications.

High-content, high-throughput screening for the identification of cytotoxic compounds based on cell morphology and cell proliferation markers.

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Heather L Martin

Full Text Available Toxicity is a major cause of failure in drug discovery and development, and whilst robust toxicological testing occurs, efficiency could be improved if compounds with cytotoxic characteristics were identified during primary compound screening. The use of high-content imaging in primary screening is becoming more widespread, and by utilising phenotypic approaches it should be possible to incorporate cytotoxicity counter-screens into primary screens. Here we present a novel phenotypic assay that can be used as a counter-screen to identify compounds with adverse cellular effects. This assay has been developed using U2OS cells, the PerkinElmer Operetta high-content/high-throughput imaging system and Columbus image analysis software. In Columbus, algorithms were devised to identify changes in nuclear morphology, cell shape and proliferation using DAPI, TOTO-3 and phosphohistone H3 staining, respectively. The algorithms were developed and tested on cells treated with doxorubicin, taxol and nocodazole. The assay was then used to screen a novel, chemical library, rich in natural product-like molecules of over 300 compounds, 13.6% of which were identified as having adverse cellular effects. This assay provides a relatively cheap and rapid approach for identifying compounds with adverse cellular effects during screening assays, potentially reducing compound rejection due to toxicity in subsequent in vitro and in vivo assays.

Do Dental X-Rays Induce Genotoxicity and Cytotoxicity in Oral Mucosa Cells? A Critical Review.

Science.gov (United States)

Angelieri, Fernanda; Yujra, Veronica Quispe; Oshima, Celina Tizuko Fujiyama; Ribeiro, Daniel Araki

2017-10-01

Dental X-rays are widely used in clinical practice, since the technique is an important approach for diagnosing diseases in dental and periodontal tissues. However, it is widely known that radiation is capable of causing damage to cellular systems, such as genotoxicity or cytotoxicity. Thus, the aim of this review was to present a critical analysis regarding the studies published on genotoxicity and cytotoxicity induced by dental X-rays in oral mucosa cells. Such studies have revealed that some oral cell types are more sensitive than others following exposure to dental X-rays. Certainly, this review will contribute to a better understanding of this matter as well as to highlighting perspectives for further studies. Ultimately, such data will promote better safety for both patients and dental professionals. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

The enhanced cytotoxicity of misonidazole in the thiol depleted state - An oxygen dependent mechanism

International Nuclear Information System (INIS)

Tuttle, S.W.; Varnes, M.E.; Donahue, L.; Biaglow, J.E.

1985-01-01

Incubating A549 cells in the presence of L-buthionine-S, R-sulfoximine and misonidazole under aerobic conditions results in lowered rates of cell growth and greater cytotoxicity than is seen with either drug alone. The authors previously demonstrated the accumulation of hydrogen peroxide from cells treated with misonidazole following the inhibition of GSH-peroxidase with thiol depleting agents. They hypothesize that the enhancement of misonidazole toxicity by L-BSO results from the increased exposure to hydrogen peroxide, and the possible formation of the highly reactive hydroxyl radical in the presence of trace metals via Fenton chemistry. Support for this hypothesis comes from their observations that addition of radical scavengers (such as SOD and catalase) and nutritional antioxidants (vitamin E) to the culture medium will partially inhibit the cytotoxic effects. Further work is being done to measure the products of reaction of toxic oxygen species with cellular macromolecules, i.e. lipids

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

DEFF Research Database (Denmark)

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

2013-01-01

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

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

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Thurber Aaron

2009-01-01

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

In vitro cytotoxity of silver: implication for clinical wound care.

Science.gov (United States)

Poon, Vincent K M; Burd, Andrew

2004-03-01

In this study, we look at the cytotoxic effects of silver on keratinocytes and fibroblasts. We have assessed the viability of monolayer cultures using the MTT and BrdU assays. The composition of the culture medium and also the culture technique were modified to assess the effects of culture 'environment' on the susceptibility of the cells to the toxic action of silver. Further in vitro, experiments were performed using tissue culture models to allow cellular behavior in three dimensional planes which more closely simulated in vivo behavior. The silver source was both silver released from silver nitrate solution but also nanocrystalline silver released from a commercially available dressing. The results show that silver is highly toxic to both keratinocytes and fibroblasts in monolayer culture. When using optimized and individualized culture the fibroblasts appear to be more sensitive to silver than keratinocytes. However, when both cell types were grown in the same medium their viability was the same. Using tissue culture models again indicated an 'environmental effect' with decreased sensitivity of the cells to the cytotoxic effects of the silver. Nevertheless in these studies the toxic dose of skin cells ranging from 7 x 10(-4) to 55 x 10(-4)% was similar to that of bacteria. These results suggest that consideration of the cytotoxic effects of silver and silver-based products should be taken when deciding on dressings for specific wound care strategies. This is important when using keratinocyte culture, in situ, which is playing an increasing role in contemporary wound and burn care.

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

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Ryan J Mailloux

2010-10-01

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

Synthesis, Characterization, and Cytotoxicity of Iron Oxide Nanoparticles

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S. Kanagesan

2013-01-01

Full Text Available In order to study the response of human breast cancer cells' exposure to nanoparticle, iron oxide (α-Fe2O3 nanoparticles were synthesized by a simple low temperature combustion method using Fe(NO33·9H2O as raw material. X-ray diffraction studies confirmed that the resultant powders are pure α-Fe2O3. Transmission electron microscopy study revealed the spherical shape of the primary particles, and the size of the iron oxide nanoparticles is in the range of 19 nm. The magnetic hysteresis loops demonstrated that the sample exposed ferromagnetic behaviors with a relatively low coercivity. The cytotoxicity of α-Fe2O3 nanoparticle was also evaluated on human breast cancer cells to address the current deficient knowledge of cellular response to nanoparticle exposure.

Ganoderma lucidum stimulates NK cell cytotoxicity by inducing NKG2D/NCR activation and secretion of perforin and granulysin.

Science.gov (United States)

Chang, Chih-Jung; Chen, Yi-Yuan M; Lu, Chia-Chen; Lin, Chuan-Sheng; Martel, Jan; Tsai, Sheng-Hui; Ko, Yun-Fei; Huang, Tsung-Teng; Ojcius, David M; Young, John D; Lai, Hsin-Chih

2014-04-01

Ganoderma lucidum (G. lucidum) is a medicinal mushroom long used in Asia as a folk remedy to promote health and longevity. Recent studies indicate that G. lucidum activates NK cells, but the molecular mechanism underlying this effect has not been studied so far. To address this question, we prepared a water extract of G. lucidum and examined its effect on NK cells. We observed that G. lucidum treatment increases NK cell cytotoxicity by stimulating secretion of perforin and granulysin. The mechanism of activation involves an increased expression of NKG2D and natural cytotoxicity receptors (NCRs), as well as increased phosphorylation of intracellular MAPKs. Our results indicate that G. lucidum induces NK cell cytotoxicity against various cancer cell lines by activating NKG2D/NCR receptors and MAPK signaling pathways, which together culminate in exocytosis of perforin and granulysin. These observations provide a cellular and molecular mechanism to account for the reported anticancer effects of G. lucidum extracts in humans.

Resistance to Degradation and Cellular Distribution are Important Features for the Antitumor Activity of Gomesin

Science.gov (United States)

Buri, Marcus V.; Domingues, Tatiana M.; Paredes-Gamero, Edgar J.; Casaes-Rodrigues, Rafael L.; Rodrigues, Elaine Guadelupe; Miranda, Antonio

2013-01-01

Many reports have shown that antimicrobial peptides exhibit anticancer abilities. Gomesin (Gm) exhibits potent cytotoxic activity against cancer cells by a membrane pore formation induced after well-orchestrated intracellular mechanisms. In this report, the replacements of the Cys by Ser or Thr, and the use D-amino acids in the Gm structure were done to investigate the importance of the resistance to degradation of the molecule with its cytotoxicity. [Thr2,6,11,15]-Gm, and [Ser2,6,11,15]-Gm exhibits low cytotoxicity, and low resistance to degradation, and after 24 h are present in localized area near to the membrane. Conversely, the use of D-amino acids in the analogue [D-Thr2,6,11,15]-D-Gm confers resistance to degradation, increases its potency, and maintained this peptide spread in the cytosol similarly to what happens with Gm. Replacements of Cys by Thr and Gln by L- or D-Pro ([D-Thr2,6,11,15, Pro9]-D-Gm, and [Thr2,6,11,15, D-Pro9]-Gm), which induced a similar β-hairpin conformation, also increase their resistance to degradation, and cytotoxicity, but after 24 h they are not present spread in the cytosol, exhibiting lower cytotoxicity in comparison to Gm. Additionally, chloroquine, a lysosomal enzyme inhibitor potentiated the effect of the peptides. Furthermore, the binding and internalization of peptides was determined, but a direct correlation among these factors was not observed. However, cholesterol ablation, which increase fluidity of cellular membrane, also increase cytotoxicity and internalization of peptides. β-hairpin spatial conformation, and intracellular localization/target, and the capability of entry are important properties of gomesin cytotoxicity. PMID:24312251

Resistance to degradation and cellular distribution are important features for the antitumor activity of gomesin.

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Marcus V Buri

Full Text Available Many reports have shown that antimicrobial peptides exhibit anticancer abilities. Gomesin (Gm exhibits potent cytotoxic activity against cancer cells by a membrane pore formation induced after well-orchestrated intracellular mechanisms. In this report, the replacements of the Cys by Ser or Thr, and the use D-amino acids in the Gm structure were done to investigate the importance of the resistance to degradation of the molecule with its cytotoxicity. [Thr(2,6,11,15]-Gm, and [Ser(2,6,11,15]-Gm exhibits low cytotoxicity, and low resistance to degradation, and after 24 h are present in localized area near to the membrane. Conversely, the use of D-amino acids in the analogue [D-Thr(2,6,11,15]-D-Gm confers resistance to degradation, increases its potency, and maintained this peptide spread in the cytosol similarly to what happens with Gm. Replacements of Cys by Thr and Gln by L- or D-Pro ([D-Thr(2,6,11,15, Pro(9]-D-Gm, and [Thr(2,6,11,15, D-Pro(9]-Gm, which induced a similar β-hairpin conformation, also increase their resistance to degradation, and cytotoxicity, but after 24 h they are not present spread in the cytosol, exhibiting lower cytotoxicity in comparison to Gm. Additionally, chloroquine, a lysosomal enzyme inhibitor potentiated the effect of the peptides. Furthermore, the binding and internalization of peptides was determined, but a direct correlation among these factors was not observed. However, cholesterol ablation, which increase fluidity of cellular membrane, also increase cytotoxicity and internalization of peptides. β-hairpin spatial conformation, and intracellular localization/target, and the capability of entry are important properties of gomesin cytotoxicity.

Resistance to degradation and cellular distribution are important features for the antitumor activity of gomesin.

Science.gov (United States)

Buri, Marcus V; Domingues, Tatiana M; Paredes-Gamero, Edgar J; Casaes-Rodrigues, Rafael L; Rodrigues, Elaine Guadelupe; Miranda, Antonio

2013-01-01

Many reports have shown that antimicrobial peptides exhibit anticancer abilities. Gomesin (Gm) exhibits potent cytotoxic activity against cancer cells by a membrane pore formation induced after well-orchestrated intracellular mechanisms. In this report, the replacements of the Cys by Ser or Thr, and the use D-amino acids in the Gm structure were done to investigate the importance of the resistance to degradation of the molecule with its cytotoxicity. [Thr(2,6,11,15)]-Gm, and [Ser(2,6,11,15)]-Gm exhibits low cytotoxicity, and low resistance to degradation, and after 24 h are present in localized area near to the membrane. Conversely, the use of D-amino acids in the analogue [D-Thr(2,6,11,15)]-D-Gm confers resistance to degradation, increases its potency, and maintained this peptide spread in the cytosol similarly to what happens with Gm. Replacements of Cys by Thr and Gln by L- or D-Pro ([D-Thr(2,6,11,15), Pro(9)]-D-Gm, and [Thr(2,6,11,15), D-Pro(9)]-Gm), which induced a similar β-hairpin conformation, also increase their resistance to degradation, and cytotoxicity, but after 24 h they are not present spread in the cytosol, exhibiting lower cytotoxicity in comparison to Gm. Additionally, chloroquine, a lysosomal enzyme inhibitor potentiated the effect of the peptides. Furthermore, the binding and internalization of peptides was determined, but a direct correlation among these factors was not observed. However, cholesterol ablation, which increase fluidity of cellular membrane, also increase cytotoxicity and internalization of peptides. β-hairpin spatial conformation, and intracellular localization/target, and the capability of entry are important properties of gomesin cytotoxicity.

Cytotoxicity of Cheese and Cheddar Cheese food flavorings on Allim cepa L root meristems

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A. G. Moura

Full Text Available Abstract Despite their great importance for the food industry, flavorings, in general, raise a number of questions regarding their cytotoxicity, mutagenicity and carcinogenicity, since, in the literature, there are few studies found evaluating the toxicity on the systemic and cellular level, of these chemical compounds. The root meristems of Allium cepa (onion are widely used for the assessment of toxicity of chemical compounds of interest. Thus, this study aimed to evaluate, in A. cepa meristematic cells, individually and in combination at the cellular level, the toxicity of synthetic Cheese and Cheddar Cheese food flavorings, identical to the natural, at doses of 1.0 and 2.0 mL, at exposure times of 24 and 48 hours. In combination we used 0.5 mL of Cheese flavor associated with 0.5 mL of Cheddar flavor; and 1.0 mL of Cheese flavor associated with 1.0 mL of Cheddar flavor, at exposure times of 24 and 48 hours. For these evaluations, we used groups of five onion bulbs, which were first embedded in distilled water and then transferred to their respective doses. The root tips were collected and fixed in acetic acid (3:1 for 24 hours. The slides were prepared by crushing and were stained with 2% acetic orcein. Cells were analyzed throughout the cell cycle, totaling 5,000 for each control and exposure time. The mitotic indices calculated and cellular aberrations observed were subjected to statistical analysis using the chi-square test (p <0.05. No chromosomal abnormalities nor those of mitotic spindle were observed for the treatments performed. The results, both individually and in combination, showed that the flavorings under study significantly reduced the cell division rate of the test system cells used. Therefore, under the conditions studied, the two flavorings were cytotoxic.

HIV-specific Fc effector function early in infection predicts the development of broadly neutralizing antibodies.

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Simone I Richardson

2018-04-01

Full Text Available While the induction of broadly neutralizing antibodies (bNAbs is a major goal of HIV vaccination strategies, there is mounting evidence to suggest that antibodies with Fc effector function also contribute to protection against HIV infection. Here we investigated Fc effector functionality of HIV-specific IgG plasma antibodies over 3 years of infection in 23 individuals, 13 of whom developed bNAbs. Antibody-dependent cellular phagocytosis (ADCP, complement deposition (ADCD, cellular cytotoxicity (ADCC and cellular trogocytosis (ADCT were detected in almost all individuals with levels of activity increasing over time. At 6 months post-infection, individuals with bNAbs had significantly higher levels of ADCD and ADCT that correlated with antibody binding to C1q and FcγRIIa respectively. In addition, antibodies from individuals with bNAbs showed more IgG subclass diversity to multiple HIV antigens which also correlated with Fc polyfunctionality. Germinal center activity represented by CXCL13 levels and expression of activation-induced cytidine deaminase (AID was found to be associated with neutralization breadth, Fc polyfunctionality and IgG subclass diversity. Overall, multivariate analysis by random forest classification was able to group bNAb individuals with 85% sensitivity and 80% specificity based on the properties of their antibody Fc early in HIV infection. Thus, the Fc effector function profile predicted the development of neutralization breadth in this cohort, suggesting that intrinsic immune factors within the germinal center provide a mechanistic link between the Fc and Fab of HIV-specific antibodies.

Multifunctional non-viral gene vectors with enhanced stability, improved cellular and nuclear uptake capability, and increased transfection efficiency

Science.gov (United States)

Yang, Zhe; Jiang, Zhaozhong; Cao, Zhong; Zhang, Chao; Gao, Di; Luo, Xingen; Zhang, Xiaofang; Luo, Huiyan; Jiang, Qing; Liu, Jie

2014-08-01

We have developed a new multifunctional, non-viral gene delivery platform consisting of cationic poly(amine-co-ester) (PPMS) for DNA condensation, PEG shell for nanoparticle stabilization, poly(γ-glutamic acid) (γ-PGA) and mTAT (a cell-penetrating peptide) for accelerated cellular uptake, and a nuclear localization signal peptide (NLS) for enhanced intracellular transport of DNA to the nucleus. In vitro study showed that coating of the binary PPMS/DNA polyplex with γ-PGA promotes cellular uptake of the polyplex particles, particularly by γ-glutamyl transpeptidase (GGT)-positive cells through the GGT-mediated endocytosis pathway. Conjugating PEG to the γ-PGA led to the formation of a ternary PPMS/DNA/PGA-g-PEG polyplex with decreased positive charges on the surface of the polyplex particles and substantially higher stability in serum-containing aqueous medium. The cellular uptake rate was further improved by incorporating mTAT into the ternary polyplex system. Addition of the NLS peptide was designed to facilitate intracellular delivery of the plasmid to the nucleus--a rate-limiting step in the gene transfection process. As a result, compared with the binary PPMS/LucDNA polyplex, the new mTAT-quaternary PPMS/LucDNA/NLS/PGA-g-PEG-mTAT system exhibited reduced cytotoxicity, remarkably faster cellular uptake rate, and enhanced transport of DNA to the nucleus. All these advantageous functionalities contribute to the remarkable gene transfection efficiency of the mTAT-quaternary polyplex both in vitro and in vivo, which exceeds that of the binary polyplex and commercial Lipofectamine™ 2000/DNA lipoplex. The multifunctional mTAT-quaternary polyplex system with improved efficiency and reduced cytotoxicity represents a new type of promising non-viral vectors for the delivery of therapeutic genes to treat tumors.We have developed a new multifunctional, non-viral gene delivery platform consisting of cationic poly(amine-co-ester) (PPMS) for DNA condensation, PEG shell

Surface-dependent cytotoxicity on bacteria as a model for environmental stress of halloysite nanotubes

Energy Technology Data Exchange (ETDEWEB)

Choi, Hyo-Jick, E-mail: hyojickchoi@gmail.com [National Institute for Nanotechnology, Nanotechnology Accelerator and Department of Chemical and Materials Engineering, University of Alberta (Canada); Stazak, Theodore J. [School of Energy, Environmental, Biological and Medical Engineering, University of Cincinnati (United States); Montemagno, Carlo D., E-mail: montemag@ualberta.ca [National Institute for Nanotechnology, Nanotechnology Accelerator and Department of Chemical and Materials Engineering, University of Alberta (Canada)

2013-10-15

This study examined the cytotoxicity of halloysite nanotubes (HNTs) by investigating physiological responses of Escherichia coli, from cell growth to protein expression. Surfaces of HNTs were modified by amine functionalization (NH{sub 2}-HNTs) or bovine serum albumin (BSA) coating and their cytotoxicity levels were compared with that of non-modified HNTs (Bare-HNTs). Bare- and NH{sub 2}-HNTs exhibited accelerated cell death rates at {>=}0.5 mg/ml of HNTs. It was also found that concentration as low as 0.01 mg/ml of HNTs exerted significant toxic effects on the bacterial cells. Cellular viability, metabolic activity, and DNA replication all decreased with increasing concentrations of Bare- and NH{sub 2}-HNTs. In contrast, 0.01 mg/ml of BSA-coated HNTs (BSA-HNTs) coated showed no evidence of cytotoxicity. Even at concentrations {<=}0.1 mg/ml, the cytocompatibility of BSA-HNTs was significantly better than those of Bare- and NH{sub 2}-HNTs, which was confirmed by the observation of (i) the same or similar levels of cell proliferation and cell viability to the control, and (ii) higher levels of metabolic activity and plasmid DNA replication than those of Bare- and NH{sub 2}-HNTs. In addition, higher ranaspumin-2 protein yield was observed from bacterial culture supplemented with BSA-HNTs (100, 83, and 80 % of yield at 0.01, 0.05, and 0.1 mg/ml, respectively, relative to the control). This work showed that the increase of bacterial cytotoxicity of HNTs correlated well with elevating HNT concentration and that surface modification of HNTs with amine functional group and BSA coating was an effective strategy to reduce cytotoxicity up to 0.1 mg/ml of HNTs.

Comparison of Cytotoxic Activity in Leukemic Lineages Reveals Important Features of β-Hairpin Antimicrobial Peptides.

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Buri, Marcus V; Torquato, Heron F Vieira; Barros, Carlos Castilho; Ide, Jaime S; Miranda, Antonio; Paredes-Gamero, Edgar J

2017-07-01

Several reports described different modes of cell death triggered by antimicrobial peptides (AMPs) due to direct effects on membrane disruption, and more recently by apoptosis and necrosis-like patterns. Cytotoxic curves of four β-hairpin AMPs (gomesin, protegrin, tachyplesin, and polyphemusin) were obtained from several human leukemic lineages and normal monocytes and Two cell lines were then selected based on their cytotoxic sensitivity. One was sensitive to AMPs (K562) and the other resistant (KG-1) and their effect compared between these lineages. Thus, these lineages were chosen to further investigate biological features related with their cytotoxicities to AMPs. Stimulation with AMPs produced cell death, with activation of caspase-3, in K562 lineage. Increase on the fluidity of plasmatic membrane by reducing cholesterol potentiated cytotoxicity of AMPs in both lineages. Quantification of internal and external gomesin binding to the cellular membrane of both K562 and KG-1 cells showed that more peptide is accumulated inside of K562 cells. Additionally, evaluation of multi-drug resistant pumps activity showed that KG-1 has more activity than K562 lineage. A comparison of intrinsic gene patterns showed great differences between K562 and KG-1, but stimulation with gomesin promoted few changes in gene expression patterns. Differences in internalization process through the plasma membrane, multidrug resistance pumps activity, and gene expression pattern are important features to AMPs regulated cell death. J. Cell. Biochem. 118: 1764-1773, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

ABP 501 for the treatment of rheumatoid arthritis.

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Pelechas, Eleftherios; Voulgari, Paraskevi V; Drosos, Alexandros A

2018-03-01

Rheumatoid arthritis (RA) is an autoimmune disease, which has a negative impact on the ability to perform activities daily. Tumour necrosis factor α (TNF) is a cytokine with diverse cellular effects, and a key regulator of the inflammatory response. ABP 501 is a biosimilar to adalimumab, a TNF inhibitor. Areas covered: In this review, we examined ABP 501, as a biosimilar candidate to adalimumab in the treatment of RA focusing on the available data. Current data indicate that ABP 501 is a highly similar alternative to adalimumab in terms of safety, efficacy, tolerability and immunogenicity. ABP 501 has already been approved by health authorities in Europe and the United States of America, as a subcutaneous (s.c.) therapy option for the treatment of patients with RA, but also for the full spectrum approved for its bio-originator adalimumab. Expert opinion: Current body of evidence suggests that all biologic activities have been demonstrated to be equivalent between ABP 501 and the originator, including binding rates and affinity to TNF, and also the effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC). Therefore, it is fully expected to have same efficacy and safety in all indications.

Recombinant tumor necrosis factor alpha inhibits growth of methylcholanthrene-induced sarcoma and enhances natural killer activity of tumor-infiltrating lymphocytes in aging rats

International Nuclear Information System (INIS)

Ziolkowska, Maria; Nowak Joanna, J.; Janiak, Marek; Ryzewska, Alicja

1994-01-01

The effect of recombinant human tumor necrosis factors alpha (rHuTNF-α) on the growth of immunogenic, methylcholanthrene-induced sarcoma (MC-Sa) and natural killer (NK) cell activity of tumor-infiltrating lymphocytes (TIL) in adult and aging rats was investigated. In both groups of animals the growth of transplantable MC-Sa was markedly and similarly inhibited by multiple intratumoral (i.t.) injections of rHuTF-α. This effect was accompanied by stimulation of NK activity of tumor-infiltrating lymphocytes in adult as well as in aging rats. Studies ''in vitro'' demonstrated additionally that rHuTNF-α was a potent stimulator of NK but not of ADCC (antibody-dependent cellular cytotoxicity) activity of spleen lymphocytes from healthy animals. Our results indicate that the antitumor effect of TNF-α is comparable in adult and in aging rats bearing immunogenic MC-Sa. The inhibition of MC-Sa growth may be attributed not only to the TNF-α-induced necrosis of the neoplastic tissue but also to the ''in vivo'' stimulatory effect of this cytokine upon the NK-type function of lymphocytes infiltrating the tumor mass. (author). 31 refs, 5 figs, 2 tabs

Effective combination treatment of GD2-expressing neuroblastoma and Ewing's sarcoma using anti-GD2 ch14.18/CHO antibody with Vγ9Vδ2+ γδT cells.

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Fisher, Jonathan P H; Flutter, Barry; Wesemann, Florian; Frosch, Jennifer; Rossig, Claudia; Gustafsson, Kenth; Anderson, John

Gamma delta T lymphocytes (γδT cells) have pleiotropic properties including innate cytotoxicity, which make them attractive effectors for cancer immunotherapy. Combination treatment with zoledronic acid and IL-2 can activate and expand the most common subset of blood γδT, which express the Vγ9Vδ2 T cell receptor (TCR) (Vδ2 T cells). Vγ9Vδ2 T cells are equipped for antibody-dependent cell-mediated cytotoxicity (ADCC) through expression of the low-affinity FcγR CD16. GD2 is a highly ranked tumor associated antigen for immunotherapy due to bright expression on the cell surface, absent expression on normal tissues and availability of therapeutic antibodies with known efficacy in neuroblastoma. To explore the hypothesis that zoledronic acid, IL-2 and anti-GD2 antibodies will synergize in a therapeutic combination, we evaluated in vitro cytotoxicity and tumor growth inhibition in the GD2 expressing cancers neuroblastoma and Ewing's sarcoma. Vδ2 T cells exert ADCC against GD2-expressing Ewing's sarcoma and neuroblastoma cell lines, an effect which correlates with the brightness of GD2 expression. In an immunodeficient mouse model of small established GD2-expressing Ewing's sarcoma or neuroblastoma tumors, the combination of adoptively transferred Vδ2+ T cells, expanded in vitro with zoledronic acid and IL-2, with anti-GD2 antibody ch14.18/CHO, and with systemic zoledronic acid, significantly suppressed tumor growth compared to antibody or γδT cell-free controls. Combination treatment using ch14.18/CHO, zoledronic acid and IL-2 is more effective than their use in isolation. The already-established safety profiles of these agents make testing of the combination in GD2 positive cancers such as neuroblastoma or Ewing's sarcoma both rational and feasible.

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

OpenAIRE

sprotocols

2015-01-01

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

Involvement of enniatins-induced cytotoxicity in human HepG2 cells.

Science.gov (United States)

Juan-García, Ana; Manyes, Lara; Ruiz, María-José; Font, Guillermina

2013-04-12

Enniatins (ENNs) are mycotoxins found in Fusarium fungi and they appear in nature as mixtures of cyclic depsipeptides. The ability to form ionophores in the cell membrane is related to their cytotoxicity. Changes in ion distribution between inner and outer phases of the mitochondria affect to their metabolism, proton gradient, and chemiosmotic coupling, so a mitochondrial toxicity analysis of enniatins is highly recommended because they host the homeostasis required for cellular survival. Two ENNs, ENN A and ENN B on hepatocarcinoma cells (HepG2) at 1.5 and 3 μM and three exposure times (24, 48 and 72 h) were studied. Flow cytometry was used to examine their effects on cell proliferation, to characterize at which phase of the cell cycle progression the cells were blocked and to study the role of the mitochondrial in ENNs-induced apoptosis. In conclusion, apoptosis induction on HepG2 cells allowed to compare cytotoxic effects caused by both ENNs, A and B. It is reported the possible mechanism observed in MMP changes, cell cycle analysis and apoptosis/necrosis, identifying ENN B more toxic than ENN A. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Cytotoxicity and cellular mechanisms involved in the toxicity of CdS quantum dots in hemocytes and gill cells of the mussel Mytilus galloprovincialis

Energy Technology Data Exchange (ETDEWEB)

Katsumiti, A. [CBET Research Group, Dept. Zoology and Animal Cell Biology, Faculty of Science and Technology and Research Centre for Experimental Marine Biology and Biotechnology PIE, University of the Basque Country UPV/EHU, Basque Country (Spain); Gilliland, D. [EU Commission–Joint Research Centre, Institute of Health and Consumer Protection, NSB Unit, Ispra (Italy); Arostegui, I. [Department of Applied Mathematics, Statistics and Operations Research, Faculty of Science and Technology, University of the Basque Country UPV/EHU, Leioa (Spain); Cajaraville, M.P., E-mail: mirenp.cajaraville@ehu.es [CBET Research Group, Dept. Zoology and Animal Cell Biology, Faculty of Science and Technology and Research Centre for Experimental Marine Biology and Biotechnology PIE, University of the Basque Country UPV/EHU, Basque Country (Spain)

2014-08-15

Highlights: • CdS QDs were cytotoxic for mussel hemocytes and gill cells in vitro. • Ionic Cd was the most toxic form, followed by CdS QDs and bulk CdS. • CdS QDs altered oxidative balance and caused DNA damage in mussel cells. • CdS QDs caused a particle-specific immunostimulation on phagocytosis of hemocytes. • Conceptual models for cellular handling and toxicity of CdS QDs are proposed. - Abstract: CdS quantum dots (QDs) show a great promise for treatment and diagnosis of cancer and for targeted drug delivery, due to their size-tunable fluorescence and ease of functionalization for tissue targeting. In spite of their advantages it is important to determine if CdS QDs can exert toxicity on biological systems. In the present work, cytotoxicity of CdS QDs (5 nm) at a wide range of concentrations (0.001–100 mg Cd/L) was screened using neutral red (NR) and thiazolyl blue tetrazolium bromide (MTT) assays in isolated hemocytes and gill cells of mussels (Mytilus galloprovincialis). The mechanisms of action of CdS QDs were assessed at sublethal concentrations (0.31–5 mg Cd/L) in the same cell types through a series of functional in vitro assays: production of reactive oxygen species (ROS), catalase (CAT) activity, DNA damage, lysosomal acid phosphatase (AcP) activity, multixenobiotic resistance (MXR) transport activity, Na-K-ATPase activity (only in gill cells) and phagocytic activity and damage to actin cytoskeleton (only in hemocytes). Exposures to CdS QDs lasted for 24 h and were performed in parallel with exposures to bulk CdS and ionic Cd. Ionic Cd was the most toxic form to both cell types, followed by CdS QDs and bulk CdS. ROS production, DNA damage, AcP activity and MXR transport were significantly increased in both cell types exposed to the 3 forms of Cd. CAT activity increased in hemocytes exposed to the three forms of Cd while in gill cells only in those exposed to ionic Cd. No effects were found on hemocytes cytoskeleton integrity. Effects on

Antioxidant Activity of Lawsonia inermis Extracts Inhibits Chromium(VI-Induced Cellular and DNA Toxicity

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Gunjan Guha

2011-01-01

Full Text Available Hexavalent chromium Cr(VI is a very strong oxidant which consequently causes high cytotoxicity through oxidative stress. Prevention of Cr(VI-induced cellular damage has been sought in this study in aqueous and methanolic extracts of Lawsonia inermis Linn. (Lythraceae, commonly known as Henna. The extracts showed significant (P < .05 potential in scavenging free radicals (DPPH• and ABTS•+ and Fe3+, and in inhibiting lipid peroxidation. DNA damage caused by exposure of pBR322 to Cr(VI-UV is markedly inhibited by both extracts in varying degrees. A distinct decline in Cr(VI-induced cytotoxicity was noticed in MDA-MB-435S (human breast carcinoma cells with an increase in dosage of both extracts individually. Furthermore, both extracts proved to contain a high content of phenolic compounds which were found to have a strong and significant (P < .05 positive correlation to the radical scavenging potential, lipid peroxidation inhibition capacity and cyto-protective efficiency against Cr(VI-induced oxidative cellular damage. HPLC analysis identified some of the major phenolic compounds in both extracts, which might be responsible for the antioxidant potential and the properties of DNA and cyto-protection. This study contributes to the search for natural resources that might yield potent therapeutic drugs against Cr(VI-induced oxidative cell damage.

Broad-spectrum antiviral properties of andrographolide.

Science.gov (United States)

Gupta, Swati; Mishra, K P; Ganju, Lilly

2017-03-01

Andrographolide, a diterpenoid, is known for its anti-inflammatory effects. It can be isolated from various plants of the genus Andrographis, commonly known as 'creat'. This purified compound has been tested for its anti-inflammatory effects in various stressful conditions, such as ischemia, pyrogenesis, arthritis, hepatic or neural toxicity, carcinoma, and oxidative stress, Apart from its anti-inflammatory effects, andrographolide also exhibits immunomodulatory effects by effectively enhancing cytotoxic T cells, natural killer (NK) cells, phagocytosis, and antibody-dependent cell-mediated cytotoxicity (ADCC). All these properties of andrographolide form the foundation for the use of this miraculous compound to restrain virus replication and virus-induced pathogenesis. The present article covers antiviral properties of andrographolide in variety of viral infections, with the hope of developing of a new highly potent antiviral drug with multiple effects.

Oxidative Mechanisms of Monocyte-Mediated Cytotoxicity

Science.gov (United States)

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

1980-01-01

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

Surface modification of amorphous nanosilica particles suppresses nanosilica-induced cytotoxicity, ROS generation, and DNA damage in various mammalian cells

International Nuclear Information System (INIS)

Yoshida, Tokuyuki; Yoshioka, Yasuo; Matsuyama, Keigo; Nakazato, Yasutaro; Tochigi, Saeko; Hirai, Toshiro; Kondoh, Sayuri; Nagano, Kazuya; Abe, Yasuhiro; Kamada, Haruhiko; Tsunoda, Shin-ichi; Nabeshi, Hiromi; Yoshikawa, Tomoaki; Tsutsumi, Yasuo

2012-01-01

Highlights: ► There is increasing concern regarding the potential health risks of nanomaterials. ► We evaluated the effect of surface properties of nanomaterials on cellular responses. ► We showed that the surface properties play an important in determining its safety. ► These data provide useful information for producing safer nanomaterials. -- Abstract: Recently, nanomaterials have been utilized in various fields. In particular, amorphous nanosilica particles are increasingly being used in a range of applications, including cosmetics, food technology, and medical diagnostics. However, there is concern that the unique characteristics of nanomaterials might induce undesirable effects. The roles played by the physical characteristics of nanomaterials in cellular responses have not yet been elucidated precisely. Here, by using nanosilica particles (nSPs) with a diameter of 70 nm whose surface was either unmodified (nSP70) or modified with amine (nSP70-N) or carboxyl groups (nSP70-C), we examined the relationship between the surface properties of nSPs and cellular responses such as cytotoxicity, reactive oxygen species (ROS) generation, and DNA damage. To compare the cytotoxicity of nSP70, nSP70-N, or nSP70-C, we examined in vitro cell viability after nSP treatment. Although the susceptibility of each cell line to the nSPs was different, nSP70-C and nSP70-N showed lower cytotoxicity than nSP70 in all cell lines. Furthermore, the generation of ROS and induction of DNA damage in nSP70-C- and nSP70-N-treated cells were lower than those in nSP70-treated cells. These results suggest that the surface properties of nSP70 play an important role in determining its safety, and surface modification of nSP70 with amine or carboxyl groups may be useful for the development of safer nSPs. We hope that our results will contribute to the development of safer nanomaterials.

Cytotoxicity, Intestinal Transport, and Bioavailability of Dispersible Iron and Zinc Supplements

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Jae-Min Oh

2017-04-01

Full Text Available Iron or zinc deficiency is one of the most important nutritional disorders which causes health problem. However, food fortification with minerals often induces unacceptable organoleptic changes during preparation process and storage, has low bioavailability and solubility, and is expensive. Nanotechnology surface modification to obtain novel characteristics can be a useful tool to overcome these problems. In this study, the efficacy and potential toxicity of dispersible Fe or Zn supplement coated in dextrin and glycerides (SunActive FeTM and SunActive ZnTM were evaluated in terms of cytotoxicity, intestinal transport, and bioavailability, as compared with each counterpart without coating, ferric pyrophosphate (FePP and zinc oxide (ZnO nanoparticles (NPs, respectively. The results demonstrate that the cytotoxicity of FePP was not significantly affected by surface modification (SunActive FeTM, while SunActive ZnTM was more cytotoxic than ZnO-NPs. Cellular uptake and intestinal transport efficiency of SunActive FeTM were significantly higher than those of its counterpart material, which was in good agreement with enhanced oral absorption efficacy after a single-dose oral administration to rats. These results seem to be related to dissolution, particle dispersibility, and coating stability of materials depending on suspending media. Both SunActiveTM products and their counterpart materials were determined to be primarily transported by microfold (M cells through the intestinal epithelium. It was, therefore, concluded that surface modification of food fortification will be a useful strategy to enhance oral absorption efficiency at safe levels.

DNA damage and cellular death in oral mucosa cells of children who have undergone panoramic dental radiography

Energy Technology Data Exchange (ETDEWEB)

Angelieri, Fernanda; Oliveira, Gabriela R. de [Sao Paulo Metodista University (UMESP), Department of Orthodontics, Sao Bernardo do Campo, Sao Paulo (Brazil); Sannomiya, Eduardo K. [Sao Paulo Metodista University (UMESP), Department of Dento-Maxillofacial Radiology, Sao Bernardo do Campo, Sao Paulo (Brazil); Ribeiro, Daniel A. [Federal University of Sao Paulo (UNIFESP), Department of Health Sciences, Santos, Sao Paulo (Brazil); Universidade Federal de Sao Paulo (UNIFESP), Departamento de Ciencias da Saude, Santos, Sao Paulo (Brazil)

2007-06-15

Despite wide use as a diagnostic tool in medical and dental practice, radiography can induce cytotoxic effects and genetic damage. To evaluate DNA damage (micronucleus) and cellular death (pyknosis, karyolysis and karyorrhexis) in exfoliated buccal mucosa cells taken from healthy children following exposure to radiation during dental radiography. A total of 17 children who had undergone panoramic dental radiography were included. We found no statistically significant differences (P > 0.05) between micronucleated oral mucosa cells in children before and after exposure to radiation. On the other hand, radiation did cause other nuclear alterations closely related to cytotoxicity including karyorrhexis, pyknosis and karyolysis. Taken together, these results indicate that panoramic dental radiography might not induce chromosomal damage, but may be cytotoxic. Overall, the results reinforce the importance of evaluating the health side effects of radiography and contribute to the micronucleus database, which will improve our understanding and practice of this methodology in children. (orig.)

DNA damage and cellular death in oral mucosa cells of children who have undergone panoramic dental radiography

International Nuclear Information System (INIS)

Angelieri, Fernanda; Oliveira, Gabriela R. de; Sannomiya, Eduardo K.; Ribeiro, Daniel A.

2007-01-01

Despite wide use as a diagnostic tool in medical and dental practice, radiography can induce cytotoxic effects and genetic damage. To evaluate DNA damage (micronucleus) and cellular death (pyknosis, karyolysis and karyorrhexis) in exfoliated buccal mucosa cells taken from healthy children following exposure to radiation during dental radiography. A total of 17 children who had undergone panoramic dental radiography were included. We found no statistically significant differences (P > 0.05) between micronucleated oral mucosa cells in children before and after exposure to radiation. On the other hand, radiation did cause other nuclear alterations closely related to cytotoxicity including karyorrhexis, pyknosis and karyolysis. Taken together, these results indicate that panoramic dental radiography might not induce chromosomal damage, but may be cytotoxic. Overall, the results reinforce the importance of evaluating the health side effects of radiography and contribute to the micronucleus database, which will improve our understanding and practice of this methodology in children. (orig.)

A Mathematical Model for Cisplatin Cellular Pharmacodynamics

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Ardith W. El-Kareh

2003-03-01

Full Text Available A simple theoretical model for the cellular pharmacodynamics of cisplatin is presented. The model, which takes into account the kinetics of cisplatin uptake by cells and the intracellular binding of the drug, can be used to predict the dependence of survival (relative to controls on the time course of extracellular exposure. Cellular pharmacokinetic parameters are derived from uptake data for human ovarian and head and neck cancer cell lines. Survival relative to controls is assumed to depend on the peak concentration of DNA-bound intracellular platinum. Model predictions agree well with published data on cisplatin cytotoxicity for three different cancer cell lines, over a wide range of exposure times. In comparison with previously published mathematical models for anticancer drug pharmacodynamics, the present model provides a better fit to experimental data sets including long exposure times (∼100 hours. The model provides a possible explanation for the fact that cell kill correlates well with area under the extracellular concentration-time curve in some data sets, but not in others. The model may be useful for optimizing delivery schedules and for the dosing of cisplatin for cancer therapy.

Uptake and cytotoxic effects of multi-walled carbon nanotubes in human bronchial epithelial cells

International Nuclear Information System (INIS)

Hirano, Seishiro; Fujitani, Yuji; Furuyama, Akiko; Kanno, Sanae

2010-01-01

Carbon nanotubes (CNT) are cytotoxic to several cell types. However, the mechanism of CNT toxicity has not been fully studied, and dosimetric analyses of CNT in the cell culture system are lacking. Here, we describe a novel, high throughput method to measure cellular uptake of CNT using turbimetry. BEAS-2B, a human bronchial epithelial cell line, was used to investigate cellular uptake, cytotoxicity, and inflammatory effects of multi-walled CNT (MWCNT). The cytotoxicity of MWCNT was higher than that of crocidolite asbestos in BEAS-2B cells. The IC 50 of MWCNT was 12 μg/ml, whereas that of asbestos (crocidolite) was 678 μg/ml. Over the course of 5 to 8 h, BEAS-2B cells took up 17-18% of the MWCNT when they were added to the culture medium at a concentration of 10 μg/ml. BEAS-2B cells were exposed to 2, 5, or 10 μg/ml of MWCNT, and total RNA was extracted for cytokine cDNA primer array assays. The culture supernatant was collected for cytokine antibody array assays. Cytokines IL-6 and IL-8 increased in a dose dependent manner at both the mRNA and protein levels. Migration inhibitory factor (MIF) also increased in the culture supernatant in response to MWCNT. A phosphokinase array study using lysates from BEAS-2B cells exposed to MWCNT indicated that phosphorylation of p38, ERK1, and HSP27 increased significantly in response to MWCNT. Results from a reporter gene assays using the NF-κB or AP-1 promoter linked to the luciferase gene in transiently transfected CHO-KI cells revealed that NF-κB was activated following MWCNT exposure, while AP-1 was not changed. Collectively, MWCNT activated NF-κB, enhanced phosphorylation of MAP kinase pathway components, and increased production of proinflammatory cytokines in human bronchial epithelial cells.

Evaluating Cytotoxicity of Hyaluronate Targeted Solid Lipid Nanoparticles of Etoposide on SK-OV-3 Cells

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Parviz Mohammadi Ghalaei

2014-01-01

Full Text Available The epithelial ovarian carcinoma is one of the most fatal gynecological cancers. Etoposide is used in treating platinum-resistant ovarian cancer. Sodium hyaluronate is a substance that binds to the CD44 receptors overexpressed in SK-OV-3 cells of epithelial ovarian carcinoma. The aim of the present work was to study the cytotoxicity effect of hyaluronate targeted solid lipid nanoparticles (SLNs of etoposide on SK-OV-3 cells. The cytotoxicity of the targeted and nontargeted SLNs of etoposide was compared to free drug on the SK-OV-3 cells by MTT assay method. The cellular uptake of the targeted and nontargeted nanoparticles containing sodium fluorescein was also studied. The difference of cell vitality between nontargeted nanoparticles and also targeted nanoparticles with free drug was significant. Targeted nanoparticles also caused more toxicity than nontargeted nanoparticles (P<0.05. After 4 hours of incubating, the fluorescence was remarkably higher in the cells treated by targeted SLNs rather than nontargeted ones, and there was no observable fluorescence in cells incubated with pure sodium fluorescein. Hyaluronate targeted SLNs containing etoposide increased the cytotoxicity of etoposide on SK-OV-3 cells which may be a worthwhile potential method for reducing the prescribed dose and systemic side effects of this drug in epithelial ovarian carcinoma.

Endogenous glutathione protects human skin fibroblasts against the cytotoxic action of UVB, UVA and near-visible radiations

International Nuclear Information System (INIS)

Tyrell, R.M.; Pidoux, Mireille

1986-01-01

Both the UVB (290-320 nm) and UVA (320-380 nm) regions of sunlight damage human skin cells but, particularly at the longer wavelengths, information is scant concerning the mechanism(s) of damage induction and the roles of cellular defense mechanisms. Following extensive glutathione depletion of cultured human skin fibroblasts, the cells become strongly sensitized to the cytotoxic action of near-visible (405 nm), UVA (334 nm, 365 nm) and UVB (313 nm) but not UVC (254 nm) radiations. In the critical UVB region, the magnitude of the protection afforded by endogenous glutathione approaches that of the protection provided by excision repair. The results suggest that a significant fraction of even UVB damage can be mediated by free radical attack and that a major role of glutathione in human skin cells is to protect them from the cytotoxic action of sunlight. (author)

Downregulation of SWI/SNF chromatin remodeling factor subunits modulates cisplatin cytotoxicity

International Nuclear Information System (INIS)

Kothandapani, Anbarasi; Gopalakrishnan, Kathirvel; Kahali, Bhaskar; Reisman, David; Patrick, Steve M.

2012-01-01

Chromatin remodeling complex SWI/SNF plays important roles in many cellular processes including transcription, proliferation, differentiation and DNA repair. In this report, we investigated the role of SWI/SNF catalytic subunits Brg1 and Brm in the cellular response to cisplatin in lung cancer and head/neck cancer cells. Stable knockdown of Brg1 and Brm enhanced cellular sensitivity to cisplatin. Repair kinetics of cisplatin DNA adducts revealed that downregulation of Brg1 and Brm impeded the repair of both intrastrand adducts and interstrand crosslinks (ICLs). Cisplatin ICL-induced DNA double strand break repair was also decreased in Brg1 and Brm depleted cells. Altered checkpoint activation with enhanced apoptosis as well as impaired chromatin relaxation was observed in Brg1 and Brm deficient cells. Downregulation of Brg1 and Brm did not affect the recruitment of DNA damage recognition factor XPC to cisplatin DNA lesions, but affected ERCC1 recruitment, which is involved in the later stages of DNA repair. Based on these results, we propose that SWI/SNF chromatin remodeling complex modulates cisplatin cytotoxicity by facilitating efficient repair of the cisplatin DNA lesions. -- Highlights: ► Stable knockdown of Brg1 and Brm enhances cellular sensitivity to cisplatin. ► Downregulation of Brg1 and Brm impedes the repair of cisplatin intrastrand adducts and interstrand crosslinks. ► Brg1 and Brm deficiency results in impaired chromatin relaxation, altered checkpoint activation as well as enhanced apoptosis. ► Downregulation of Brg1 and Brm affects recruitment of ERCC1, but not XPC to cisplatin DNA lesions.

Cytotoxicity of Silver Nanoparticles in Human Embryonic Stem Cell-Derived Fibroblasts and an L-929 Cell Line

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Hui Peng

2012-01-01

Full Text Available Consensus about the toxicity of silver nanoparticles (Ag-NPs has not been reached, even though extensive attention has been paid to this issue. This confusion may be due to physicochemical factors of Ag-NPs and the cell model used for biological safety evaluation. In the present study, human embryonic stem cell-derived fibroblasts (EBFs, which have been considered a closer representative of the in vivo response, were used as a novel cell model to assess the cytotoxicity of Ag-NPs (~20 nm and ~100 nm in comparison with L-929 fibroblast cell line. Cell proliferation, cell cycle, apoptosis, p53 expression, and cellular uptake were examined. Results showed that Ag-NPs presented higher cytotoxicity to EBF than to L-929. EBF demonstrated a stronger capacity to ingest Ag-NPs, a higher G2/M arrest, and more upgraduated p53 expression after exposed to Ag-NPs for 48 h when compared with L-929. It could be concluded that EBF exhibited a more sensitive response to Ag-NPs compared with L-929 cells, indicating that EBF may be a valid candidate for cytotoxicity screening assays of nanoparticles.

Curcumin stably interacts with DNA hairpin through minor groove binding and demonstrates enhanced cytotoxicity in combination with FdU nucleotides.

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Ghosh, Supratim; Mallick, Sumana; Das, Upasana; Verma, Ajay; Pal, Uttam; Chatterjee, Sabyasachi; Nandy, Abhishek; Saha, Krishna D; Maiti, Nakul Chandra; Baishya, Bikash; Suresh Kumar, G; Gmeiner, William H

2018-03-01

We report, based on biophysical studies and molecular mechanical calculations that curcumin binds DNA hairpin in the minor groove adjacent to the loop region forming a stable complex. UV-Vis and fluorescence spectroscopy indicated interaction of curcumin with DNA hairpin. In this novel binding motif, two ɣ H of curcumin heptadiene chain are closely positioned to the A 16 -H8 and A 17 -H8, while G 12 -H8 is located in the close proximity of curcumin α H. Molecular dynamics (MD) simulations suggest, the complex is stabilized by noncovalent forces including; π-π stacking, H-bonding and hydrophobic interactions. Nuclear magnetic resonance (NMR) spectroscopy in combination with molecular dynamics simulations indicated curcumin is bound in the minor groove, while circular dichroism (CD) spectra suggested minute enhancement in base stacking and a little change in DNA helicity, without significant conformational change of DNA hairpin structure. The DNA:curcumin complex formed with FdU nucleotides rather than Thymidine, demonstrated enhanced cytotoxicity towards oral cancer cells relative to the only FdU substituted hairpin. Fluorescence co-localization demonstrated stability of the complex in biologically relevant conditions, including its cellular uptake. Acridine orange/EtBr staining further confirmed the enhanced cytotoxic effects of the complex, suggesting apoptosis as mode of cell death. Thus, curcumin can be noncovalently complexed to small DNA hairpin for cellular delivery and the complex showed increased cytotoxicity in combination with FdU nucleotides, demonstrating its potential for advanced cancer therapy. Copyright © 2017 Elsevier B.V. All rights reserved.

Anticancer Activity of Chloroform Extract and Sub-fractions of Nepeta deflersiana on Human Breast and Lung Cancer Cells: An In vitro Cytotoxicity Assessment.

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Al-Oqail, Mai M; Al-Sheddi, Ebtesam S; Siddiqui, Maqsood A; Musarrat, Javed; Al-Khedhairy, Abdulaziz A; Farshori, Nida N

2015-10-01

Cancer is one of the major causes of death worldwide. The plant-derived natural products have received considerable attention in recent years due to their diverse pharmacological properties including anticancer effects. Nepeta deflersiana (ND) is used in the folk medicine as antiseptic, carminative, antimicrobial, antioxidant, and for treating rheumatic disorders. However, the anticancer activity of ND chloroform extract has not been explored so far. The present study was aimed to investigate the anticancer activities of chloroform Nepeta deflersiana extract and various sub-fractions (ND-1-ND-15) of ND against human breast cancer cells (MCF-7) and human lung cancer cells (A-549). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and neutral red uptake assays, and cellular morphological alterations using phase contrast light microscope were studied. Cells were exposed with 10-1000 μg/ml of sub-fractions of ND for 24 h. Results showed that selected sub-fractions of the chloroform extract significantly reduced the cell viability of MCF-7 and A-549 cells, and altered the cellular morphology in a concentration-dependent manner. Among the sub-fractions, ND-10 fraction showed relatively higher cytotoxicity compared to other fractions whereas, ND-1 did not cause any cytotoxicity even at higher concentrations. The A-549 cells were found to be more sensitive to growth inhibition by all the extracts as compared to the MCF-7 cells. The present study provides preliminary screening of anticancer activities of chloroform extract and sub-fractions of ND, which can be further used for the development of a potential therapeutic anticancer agent. Nepeta deflersiana extract exhibit cytotoxicity and altered the cellular morphology. Sub-fractions of the chloroform extract of Nepeta deflersiana reduced the cell viability of MCF-7 and A-549 cells. Among the sub-fractions, ND-10 fraction showed relatively higher cytotoxicity. The A-549 cells were found to be more sensitive

Pingyangmycin and Bleomycin Share the Same Cytotoxicity Pathway

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Yanli He

2016-06-01

Full Text Available Pingyangmycin is an anticancer drug known as bleomycin A5 (A5, discovered in the Pingyang County of Zhejiang Province of China. Bleomycin (BLM is a mixture of mainly two compounds (A2 and B2, which is on the World Health Organization’s list of essential medicines. Both BLM and A5 are hydrophilic molecules that depend on transporters or endocytosis receptors to get inside of cells. Once inside, the anticancer activities rely on their abilities to produce DNA breaks, thus leading to cell death. Interestingly, the half maximal inhibitory concentration (IC50 of BLMs in different cancer cell lines varies from nM to μM ranges. Different cellular uptake, DNA repair rate, and/or increased drug detoxification might be some of the reasons; however, the molecules and signaling pathways responsible for these processes are largely unknown. In the current study, we purified the A2 and B2 from the BLM and tested the cytotoxicities and the molecular mechanisms of each individual compound or in combination with six different cell lines, including a Chinese hamster ovary (CHO cell line defective in glycosaminoglycan biosynthesis. Our data suggested that glycosaminoglycans might be involved in the cellular uptake of BLMs. Moreover, both BLM and A5 shared similar signaling pathways and are involved in cell cycle and apoptosis in different cancer cell lines.

Poly (ɛ-caprolactone) nanoparticles of carboplatin: Preparation, characterization and in vitro cytotoxicity evaluation in U-87 MG cell lines.

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Karanam, Vamshikrishna; Marslin, Gregory; Krishnamoorthy, Balakumar; Chellan, Vijayaraghavan; Siram, Karthik; Natarajan, Tamilselvan; Bhaskar, Balaji; Franklin, Gregory

2015-06-01

Carboplatin is a platinum based drug used in the treatment of several malignancies. Due to poor cellular uptake, generally, a larger dose of drug is administered to achieve therapeutic levels, causing harmful side-effects such as hematologic toxicity. In order to enhance the cellular uptake of carboplatin, we have developed carboplatin loaded nanoparticles using the biodegradable polymer poly (ɛ-caprolactone) (PCL). Nanoparticles ranging from the size of 23.77±1.37 to 96.73±2.79 nm with positive zeta potential and moderate entrapment efficiency (54.21±0.98%) were obtained. Transmission electron microscopy (TEM) and atomic force microscopy (AFM) confirmed the spherical morphology and smooth surface of all nanoformulations. The concentrations of PCL and the stabilizer (DMAB) are found to play a role in determining the size and the entrapment efficiency of the nanoparticles. Drug release from nanoparticles followed a biphasic pattern with an initial burst release followed by a sustained release for 10h. Results of in vitro cellular uptake and cytotoxicity studies revealed that carboplatin in the form of PCL-nanoparticles were efficiently up taken and displayed profound cytotoxicity to U-87 MG (human glioma) cells than the free drug. Importantly, unlike the free carboplatin, carboplatin in the form of PCL nanoparticles did not present any haemolytic activity in rat erythrocytes, a major side effect of this chemotherapeutic drug. This suggests that poly (ɛ-caprolactone) nanoencapsulation of carboplatin might be an efficient approach to treat cancer, while reducing carboplatin induced haemolysis. Copyright © 2015 Elsevier B.V. All rights reserved.

Colossolactone H, a new Ganoderma triterpenoid exhibits cytotoxicity and potentiates drug efficacy of gefitinib in lung cancer.

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Chen, Su-Yu; Chang, Chao-Lin; Chen, Teng-Hai; Chang, Ya-Wen; Lin, Shwu-Bin

2016-10-01

Three pentacyclic triterpene dilactones were isolated from the fruiting bodies of Ganoderma colossum, a medicinal mushroom. Colossolactone H (colo H) as a new compound and the most cytotoxic among the isolates was studied for its anticancer mechanism and the potential use in cancer therapy. Gene expression profiling analysis indicated that treatment of lung cancer cells with colo H caused upregulation of 252 genes and downregulation of 398 genes. Gene ontology enrichment analysis indicated that the downregulated genes were the most significantly enriched in cell cycle progression, and the upregulated genes were significantly enriched in metabolic process, cellular response to stimulus, and oxidation reduction. Accordingly, colo H was found to halt cell growth and induce cell apoptosis via the elevation of cellular reactive oxygen species to cause DNA damage and the increase of tumor suppressor p53 protein. These events facilitate additive cytotoxicity of colo H and gefitinib for gefitinib-resistant H1650 lung cancer cells. Furthermore, combination of colo H and gefitinib effectively inhibited the growth of tumor xenografts in athymic mice. In addition to the efficacy in adjunctive cancer therapy, we have also demonstrated the isolation of colo H from cultivated G. colossum. Thus it is feasible to use colo H or Ganoderma colossum for cancer therapy. Copyright © 2016. Published by Elsevier B.V.

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

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Shuichi Kitayama

2016-02-01

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

In-vitro cytotoxicity and cellular uptake studies of luminescent functionalized core-shell nanospheres

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Anees A. Ansari

2017-09-01

Full Text Available Monodispersed luminescent functionalized core-shell nanospheres (LFCSNs were successfully synthesized and investigated for their cyto-toxic effect on human liver hepatocellular carcinoma cell line (HepG2 cells by adopting MTT, DNA Ladder, TUNEL assay and qPCR based gene expressions through mRNA quantifications. The TUNEL and DNA ladder assays suggested an insignificant apoptosis in HepG2 cells due to the LFCSNs treatment. Further, the qPCR results also show that the mRNA expressions of cell cycle checkpoint gene p53 and apoptosis related gene (caspase-9 was up-regulated, while the antiapoptotic gene BCl-2 and apoptosis related genes FADD and CAS-3 (apoptosis effecter gene were down-regulated in the LFCSNs treated cells. The nanospheres that were loaded into the cells confirm their intracellular uptake by light and fluorescent spectro-photometry and microscopy imaging analysis. The loaded nanospheres demonstrate an absolute resistance to photo-bleaching, which were applied for dynamic imaging to real-time tracking in-vitro cell migratory activity for continuous 24 and 48 h durations using a time-lapsed fluorescent microscope. These properties of LFCSNs could therefore promote applications in the area of fluorescent protein biolabeling and drug-delivery.

Nanodiamonds act as Trojan horse for intracellular delivery of metal ions to trigger cytotoxicity.

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Zhu, Ying; Zhang, Yu; Shi, Guosheng; Yang, Jinrong; Zhang, Jichao; Li, Wenxin; Li, Aiguo; Tai, Renzhong; Fang, Haiping; Fan, Chunhai; Huang, Qing

2015-02-05

Nanomaterials hold great promise for applications in the delivery of various molecules with poor cell penetration, yet its potential for delivery of metal ions is rarely considered. Particularly, there is limited insight about the cytotoxicity triggered by nanoparticle-ion interactions. Oxidative stress is one of the major toxicological mechanisms for nanomaterials, and we propose that it may also contribute to nanoparticle-ion complexes induced cytotoxicity. To explore the potential of nanodiamonds (NDs) as vehicles for metal ion delivery, we used a broad range of experimental techniques that aimed at getting a comprehensive assessment of cell responses after exposure of NDs, metal ions, or ND-ion mixture: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, Trypan blue exclusion text, optical microscope observation, synchrotron-based scanning transmission X-ray microscopy (STXM) and micro X-ray fluorescence (μXRF) microscopy, inductively coupled plasma-mass spectrometry (ICP-MS), reactive oxygen species (ROS) assay and transmission electron microscopy (TEM) observation. In addition, theoretical calculation and molecular dynamics (MD) computation were used to illustrate the adsorption properties of different metal ion on NDs as well as release profile of ion from ND-ion complexes at different pH values. The adsorption capacity of NDs for different metal ions was different, and the adsorption for Cu2+ was the most strong among divalent metal ions. These different ND-ion complexes then had different cytotoxicity by influencing the subsequent cellular responses. Detailed investigation of ND-Cu2+ interaction showed that the amount of released Cu2+ from ND-Cu2+ complexes at acidic lysosomal conditions was much higher than that at neutral conditions, leading to the elevation of intracellular ROS level, which triggered cytotoxicity. By theoretical approaches, we demonstrated that the functional carbon surface and cluster structures of NDs made them

Preventive effects of fructose and N-acetyl-L-cysteine against cytotoxicity induced by the psychoactive compounds N-methyl-5-(2-aminopropyl)benzofuran and 3,4-methylenedioxy-N-methamphetamine in isolated rat hepatocytes.

Science.gov (United States)

Nakagawa, Yoshio; Suzuki, Toshinari; Inomata, Akiko

2018-02-01

Psychoactive compounds, N-methyl-5-(2-aminopropyl)benzofuran (5-MAPB) and 3,4-methylenedioxy-N-methamphetamine (MDMA), are known to be hepatotoxic in humans and/or experimental animals. As previous studies suggested that these compounds elicited cytotoxicity via mitochondrial dysfunction and/or oxidative stress in rat hepatocytes, the protective effects of fructose and N-acetyl-l-cysteine (NAC) on 5-MAPB- and MDMA-induced toxicity were studied in rat hepatocytes. These drugs caused not only concentration-dependent (0-4 mm) and time-dependent (0-3 hours) cell death accompanied by the depletion of cellular levels of adenosine triphosphate (ATP) and glutathione (reduced form; GSH) but also an increase in the oxidized form of GSH. The toxic effects of 5-MAPB were greater than those of MDMA. Pretreatment of hepatocytes with either fructose at a concentration of 10 mm or NAC at a concentration of 2.5 mm prevented 5-MAPB-/MDMA-induced cytotoxicity. In addition, the exposure of hepatocytes to 5-MAPB/MDMA caused the loss of mitochondrial membrane potential, although the preventive effect of fructose was weaker than that of NAC. These results suggest that: (1) 5-MAPB-/MDMA-induced cytotoxicity is linked to mitochondrial failure and depletion of cellular GSH; (2) insufficient cellular ATP levels derived from mitochondrial dysfunction were ameliorated, at least in part, by the addition of fructose; and (3) GSH loss via oxidative stress was prevented by NAC. Taken collectively, these results indicate that the onset of toxic effects caused by 5-MAPB/MDMA may be partially attributable to cellular energy stress as well as oxidative stress. Copyright © 2017 John Wiley & Sons, Ltd.

Idarubicin induces mTOR-dependent cytotoxic autophagy in leukemic cells

International Nuclear Information System (INIS)

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

2014-01-01

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

Idarubicin induces mTOR-dependent cytotoxic autophagy in leukemic cells

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

2014-08-01

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

Evaluation of the cytotoxic and antimutagenic effects of biflorin, an antitumor 1,4 o-naphthoquinone isolated from Capraria biflora L.

Science.gov (United States)

Vasconcellos, Marne C; Moura, Dinara J; Rosa, Renato M; Machado, Miriana S; Guecheva, Temenouga N; Villela, Izabel; Immich, Bruna F; Montenegro, Raquel C; Fonseca, Aluísio M; Lemos, Telma L G; Moraes, Maria Elisabete A; Saffi, Jenifer; Costa-Lotufo, Letícia V; Moraes, Manoel O; Henriques, João A P

2010-10-01

Biflorin is a natural quinone isolated from Capraria biflora L. Previous studies demonstrated that biflorin inhibits in vitro and in vivo tumor cell growth and presents potent antioxidant activity. In this paper, we report concentration-dependent cytotoxic, genotoxic, antimutagenic, and protective effects of biflorin on Salmonella tiphymurium, yeast Saccharomyces cerevisiae, and V79 mammalian cells, using different approaches. In the Salmonella/microsome assay, biflorin was not mutagenic to TA97a TA98, TA100, and TA102 strains. However, biflorin was able to induce cytotoxicity in haploid S. cerevisiae cells in stationary and exponential phase growth. In diploid yeast cells, biflorin did not induce significant mutagenic and recombinogenic effects at the employed concentration range. In addition, the pre-treatment with biflorin prevented the mutagenic and recombinogenic events induced by hydrogen peroxide (H(2)O(2)) in S. cerevisiae. In V79 mammalian cells, biflorin was cytotoxic at higher concentrations. Moreover, at low concentrations biflorin pre-treatment protected against H(2)O(2)-induced oxidative damage by reducing lipid peroxidation and DNA damage as evaluated by normal and modified comet assay using DNA glycosylases. Our results suggest that biflorin cellular effects are concentration dependent. At lower concentrations, biflorin has significant antioxidant and protective effects against the cytotoxicity, genotoxicity, mutagenicity, and intracellular lipid peroxidation induced by H(2)O(2) in yeast and mammalian cells, which can be attributed to its hydroxyl radical-scavenging property. However, at higher concentrations, biflorin is cytotoxic and genotoxic.

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

Energy Technology Data Exchange (ETDEWEB)

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

2013-03-15

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

Antitumor activity of anti-C-ERC/mesothelin monoclonal antibody in vivo.

Science.gov (United States)

Inami, Koichi; Abe, Masaaki; Takeda, Kazuyoshi; Hagiwara, Yoshiaki; Maeda, Masahiro; Segawa, Tatsuya; Suyama, Masafumi; Watanabe, Sumio; Hino, Okio

2010-04-01

Mesothelioma is an aggressive cancer often caused by chronic asbestos exposure, and its prognosis is very poor despite the therapies currently used. Due to the long latency period between asbestos exposure and tumor development, the worldwide incidence will increase substantially in the next decades. Thus, novel effective therapies are warranted to improve the prognosis. The ERC/mesothelin gene (MSLN) is expressed in wide variety of human cancers, including mesotheliomas, and encodes a precursor protein cleaved by proteases to generate C-ERC/mesothelin and N-ERC/mesothelin. In this study, we investigated the antitumor activity of C-ERC/mesothelin-specific mouse monoclonal antibody, 22A31, against tumors derived from a human mesothelioma cell line, ACC-MESO-4, in a xenograft experimental model using female BALB/c athymic nude mice. Treatment with 22A31 did not inhibit cell proliferation of ACC-MESO-4 in vitro; however, therapeutic treatment with 22A31 drastically inhibited tumor growth in vivo. 22A31 induced antibody-dependent cell-mediated cytotoxicity by natural killer (NK) cells, but not macrophages, in vitro. Consistently, the F(ab')(2) fragment of 22A31 did not inhibit tumor growth in vivo, nor did it induce antibody-dependent cell mediated cytotoxicity (ADCC) in vitro. Moreover, NK cell depletion diminished the antitumor effect of 22A31. Thus, 22A31 induced NK cell-mediated ADCC and exerted antitumor activity in vivo. 22A31 could have potential as a therapeutic tool to treat C-ERC/mesothelin-expressing cancers including mesothelioma.

Enantioselective Cytotoxicity Profile of o,p’-DDT in PC 12 Cells

Science.gov (United States)

Zhang, Chunlong; Wen, Yuezhong; Liu, Weiping

2012-01-01

Background The continued uses of dichlordiphenyltrichloroethane (DDT) for indoor vector control in some developing countries have recently fueled intensive debates toward the global ban of this persistent legacy contaminant. Current approaches for ecological and health risk assessment has ignored the chiral nature of DDT. In this study by employing an array of cytotoxicity related endpoints, we investigated the enantioselective cytotoxicity of o,p’-DDT. Principal Findings we demonstrated for the first time that R-(−)-o,p’-DDT caused more neuron cell death by inducing more severe oxidative stress, which selectively imbalanced the transcription of stress-related genes (SOD1, SOD2, HSP70) and enzyme (superoxide dismutase and lactate dehydrogenase) activities, and greater cellular apoptosis compared to its enantiomer S-(+)-o,p’-DDT at the level comparable to malaria area exposure (parts per million). We further elucidated enantioselective modes of action using microarray combined with enzyme-linked immunosorbent assay. The enantioselective apoptosis might involve three signaling pathways via caspase 3, tumor protein 53 (p53) and NFkB. Conclusions Based on DDT stereochemistry and results reported for other chiral pesticides, our results pointed to the same directional enantioselectivity of chiral DDT toward mammalian cells. We proposed that risk assessment on DDT should consider the enantiomer ratio and enantioselectivities. PMID:22937105

Membrane adsorption and binding, cellular uptake and cytotoxicity of cell-penetrating peptidomimetics with α-peptide/β-peptoid backbone

DEFF Research Database (Denmark)

Jing, Xiaona; Yang, Mingjun; Kasimova, Marina Robertovna

2012-01-01

to evaluate the effect of α-chirality in the β-peptoid residues and the presence of guanidinium groups in the α-amino acid residues on membrane interaction. The molecular properties of the peptidomimetics in solution (surface and intramolecular hydrogen bonding, aqueous diffusion rate and molecular size) were...... studied along with their adsorption to lipid bilayers, cellular uptake, and toxicity. The surface hydrogen bonding ability of the peptidomimetics reflected their adsorbed amounts onto lipid bilayers as well as with their cellular uptake, indicating the importance of hydrogen bonding for their membrane...

Processed fruit juice ready to drink: screening acute toxicity at the cellular level

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Erick Leal da Silva

2017-06-01

Full Text Available The present study evaluated the acute toxicity at the cellular level of processed juice ready for consumption Orange and Grape flavors, produced by five companies with significant influence on the food market of South American countries, especially in Brazil. This evaluation was performed in root meristem cells of Allium cepa L., at the exposure times of 24 and 48 hours, directly with marketed liquid preparations. Based on the results, it was found that fruit juices, of all companies considered, promoted significant antiproliferative effect to root meristems at the exposure time of 24 hours and resulted in at both exposure times, statistically significant number of mitotic spindle changes and chromosomal breaks. Therefore, under the study conditions, all juice samples analyzed were cytotoxic, genotoxic and mutagenic to root meristem cells. These results indicate that such beverages have relevant potential to cause cellular disorders and, thus, need to be evaluated more fully in more complex test systems, as those in rodents, and then establish specific toxicity at the cellular level of these juices and ensure the well-being of those who consume them.

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

Energy Technology Data Exchange (ETDEWEB)

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

2012-06-01

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

Molecular cytotoxic mechanisms of anticancer hydroxychalcones.

Science.gov (United States)

Sabzevari, Omid; Galati, Giuseppe; Moridani, Majid Y; Siraki, Arno; O'Brien, Peter J

2004-06-30

Chalcones are being considered as anticancer agents as they are natural compounds that are particularly cytotoxic towards K562 leukemia or melanoma cells. In this study, we have investigated phloretin, isoliquiritigenin, and 10 other hydroxylated chalcones for their cytotoxic mechanisms towards isolated rat hepatocytes. All hydroxychalcones partly depleted hepatocyte GSH and oxidized GSH to GSSG. These chalcones also caused a collapse of mitochondrial membrane potential and increased oxygen uptake. Furthermore, glycolytic or citric acid cycle substrates prevented cytotoxicity and mitochondrial membrane potential collapse. The highest pKa chalcones were the most effective at collapsing the mitochondrial membrane potential which suggests that the cytotoxic activity of hydroxychalcones are likely because of their ability to uncouple mitochondria.

Cellular uptake and transport of zein nanoparticles: effects of sodium caseinate.

Science.gov (United States)

Luo, Yangchao; Teng, Zi; Wang, Thomas T Y; Wang, Qin

2013-08-07

Cellular evaluation of zein nanoparticles has not been studied systematically due to their poor redispersibility. Caseinate (CAS)-stabilized zein nanoparticles have been recently developed with better redispersibility in salt solutions. In this study, zein-CAS nanoparticles were prepared with different zein/CAS mass ratios. The prepared nanoparticles demonstrated good stabilities to maintain particle size (120-140 nm) in cell culture medium and HBSS buffer at 37 °C. The nanoparticles showed no cytotoxicity for Caco-2 cells for 72 h. CAS not only significantly enhanced cell uptake of zein nanoparticles in a concentration- and time-dependent manner but also remarkably improved epithelial transport through Caco-2 cell monolayer. The cell uptake of zein-CAS nanoparticles indicated an energy-dependent endocytosis process as evidenced by cell uptake under blocking conditions, that is, 4 °C, sodium azide, and colchicine. Fluorescent microscopy clearly showed the internalization of zein-CAS nanoparticles. This study may shed some light on the cellular evaluations of hydrophobic protein nanoparticles.

The anti-canine distemper virus activities of ex vivo-expanded canine natural killer cells.

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Park, Ji-Yun; Shin, Dong-Jun; Lee, Soo-Hyeon; Lee, Je-Jung; Suh, Guk-Hyun; Cho, Duck; Kim, Sang-Ki

2015-04-17

Natural killer (NK) cells play critical roles in induction of antiviral effects against various viruses of humans and animals. However, few data on NK cell activities during canine distemper virus (CDV) infections are available. Recently, we established a culture system allowing activation and expansion of canine non-B, non-T, large granular NK lymphocytes from PBMCs of normal dogs. In the present study, we explored the ability of such expanded NK cells to inhibit CDV infection in vitro. Cultured CD3-CD5-CD21- NK cells produced large amounts of IFN-γ, exhibited highly upregulated expression of mRNAs encoding NK-cell-associated receptors, and demonstrated strong natural killing activity against canine tumor cells. Although the expanded NK cells were dose-dependently cytotoxic to both normal and CDV-infected Vero cells, CDV infection rendered Vero cells more susceptible to NK cells. Pretreatment with anti-CDV serum from hyperimmunized dogs enhanced the antibody-dependent cellular cytotoxicity (ADCC) of NK cells against CDV-infected Vero cells. The culture supernatants of NK cells, added before or after infection, dose-dependently inhibited both CDV replication and development of CDV-induced cytopathic effects (CPEs) in Vero cells. Anti-IFN-γ antibody neutralized the inhibitory effects of NK cell culture supernatants on CDV replication and CPE induction in Vero cells. Such results emphasize the potential significance of NK cells in controlling CDV infection, and indicate that NK cells may play roles both during CDV infection and in combating such infections, under certain conditions. Copyright © 2015 Elsevier B.V. All rights reserved.

Advances in therapeutic Fc engineering - modulation of IgG associated effector functions and serum half-life

Directory of Open Access Journals (Sweden)

Abhishek Saxena

2016-12-01

Full Text Available Today monoclonal immunoglobulin gamma (IgG antibodies have become a major option in cancer therapy especially for the patients with advanced or metastatic cancers. Efficacy of monoclonal antibodies (mAbs are achieved through both its antigen binding fragment (Fab and crystallizable fragment (Fc. Fab can specifically recognize tumor associated antigen (TAA and thus modulate TAA-linked downstream signaling pathways that may lead to inhibition of tumor growth, induction of tumor apoptosis and differentiation. The Fc region can further improve mAbs’ efficacy by mediating effector functions such as antibody-dependent cellular cytotoxicity (ADCC, complement-dependent cytotoxicity (CDC and antibody dependent cell-mediated phagocytosis (ADCP. Moreover, Fc is the region interacting with the neonatal Fc receptor (FcRn in a pH-dependent manner that can slow down IgG’s degradation and extend its serum half-life. Loss of the antibody Fc region dramatically shortens its serum half-life and weakens its anti-cancer effects. Given the essential roles that the Fc region plays in the modulation of the efficacy of mAb in cancer treatment, Fc engineering has been extensively studied in the past years. This review focuses on the recent advances in therapeutic Fc engineering that modulates its related effector functions and serum half-life. We also discuss the progress made in aglycosylated mAb development that may substantially reduce cost of manufacture but maintain similar efficacies as conventional glycosylated mAb. Finally, we highlight several Fc engineering based mAbs under clinical trials.

Cellular determinants involving mitochondrial dysfunction, oxidative stress and apoptosis correlate with the synergic cytotoxicity of epigallocatechin-3-gallate and menadione in human leukemia Jurkat T cells.

Science.gov (United States)

Tofolean, Ioana Teodora; Ganea, Constanta; Ionescu, Diana; Filippi, Alexandru; Garaiman, Alexandru; Goicea, Alexandru; Gaman, Mihnea-Alexandru; Dimancea, Alexandru; Baran, Irina

2016-01-01

We have investigated the growth-suppressive action of epigallocatechin-3-gallate (EGCG) on human leukemia Jurkat T cells. Results show a strong correlation between the dose-dependent reduction of clonogenic survival following acute EGCG treatments and the EGCG-induced decline of the mitochondrial level of Ca(2+). The cell killing ability of EGCG was synergistically enhanced by menadione. In addition, the cytotoxic effect of EGCG applied alone or in combination with menadione was accompanied by apoptosis induction. We also observed that in acute treatments EGCG displays strong antioxidant properties in the intracellular milieu, but concurrently triggers some oxidative stress generating mechanisms that can fully develop on a longer timescale. In parallel, EGCG dose-dependently induced mitochondrial depolarization during exposure, but this condition was subsequently reversed to a persistent hyperpolarized mitochondrial state that was dependent on the activity of respiratory Complex I. Fluorimetric measurements suggest that EGCG is a mitochondrial Complex III inhibitor and indicate that EGCG evokes a specific cellular fluorescence with emission at 400nm and two main excitation bands (at 330nm and 350nm) that may originate from a mitochondrial supercomplex containing dimeric Complex III and dimeric ATP-synthase, and therefore could provide a valuable means to characterize the functional properties of the respiratory chain. Copyright © 2015 Elsevier Ltd. All rights reserved.

Anti-Melanogenic Activity and Cytotoxicity of Pistacia vera Hull on Human Melanoma SKMEL-3 Cells.

Science.gov (United States)

Sarkhail, Parisa; Salimi, Mona; Sarkheil, Pantea; Mostafapour Kandelous, Hirsa

2017-07-01

Pistacia vera seed is a common food and medicinal seed in Iran. It's hull (outer skin) as a significant byproduct of pistachio, is traditionally used as tonic, sedative and antidiarrheal and has been shown to be a rich source of antioxidants. The aim of the present study is to evaluate the anti-melanogenic activity of the pistachio hulls in order to discover a new alternative herbal agent to treat skin hyperpigmentation disorders. In this work, antioxidant and anti-tyrosinase activity of MeOH extract from Pistacia vera hull (MPH) were evaluated in vitro, respectively, by DPPH radical scavenging and mushroom tyrosinase activity assays. Then the effect of MPH on the melanin content, cellular tyrosinase activity and cytotoxicity (MTT assay) on human melanoma SKMEL-3 cell were determined followed by 72 h incubation. The results indicated that MPH had valuable DPPH radical scavenging effect and weak anti-tyrosinase activity when compared to the well-known antioxidant (BHT) and tyrosinase inhibitor (kojic acid), respectively. MPH, at a high dose (0.5 mg/mL), showed significant cytotoxic activity (~63%) and strong anti-melanogenic effect (~57%) on SKMEL-3 cells. The effect of MPH in the reduction of melanin content may be related to its cytotoxicity. The results obtained suggest that MPH can be used as an effective agent in the treatment of some skin hyperpigmentation disorders such as melanoma.

Cytotoxicity assessment of functionalized CdSe, CdTe and InP quantum dots in two human cancer cell models

International Nuclear Information System (INIS)

Liu, Jing; Hu, Rui; Liu, Jianwei; Zhang, Butian; Wang, Yucheng; Liu, Xin; Law, Wing-Cheung; Liu, Liwei; Ye, Ling; Yong, Ken-Tye

2015-01-01

The toxicity of quantum dots (QDs) has been extensively studied over the past decade. Some common factors that originate the QD toxicity include releasing of heavy metal ions from degraded QDs and the generation of reactive oxygen species on the QD surface. In addition to these factors, we should also carefully examine other potential QD toxicity causes that will play crucial roles in impacting the overall biological system. In this contribution, we have performed cytotoxicity assessment of four types of QD formulations in two different human cancer cell models. The four types of QD formulations, namely, mercaptopropionic acid modified CdSe/CdS/ZnS QDs (CdSe-MPA), PEGylated phospholipid encapsulated CdSe/CdS/ZnS QDs (CdSe-Phos), PEGylated phospholipid encapsulated InP/ZnS QDs (InP-Phos) and Pluronic F127 encapsulated CdTe/ZnS QDs (CdTe-F127), are representatives for the commonly used QD formulations in biomedical applications. Both the core materials and the surface modifications have been taken into consideration as the key factors for the cytotoxicity assessment. Through side-by-side comparison and careful evaluations, we have found that the toxicity of QDs does not solely depend on a single factor in initiating the toxicity in biological system but rather it depends on a combination of elements from the particle formulations. More importantly, our toxicity assessment shows different cytotoxicity trend for all the prepared formulations tested on gastric adenocarcinoma (BGC-823) and neuroblastoma (SH-SY5Y) cell lines. We have further proposed that the cellular uptake of these nanocrystals plays an important role in determining the final faith of the toxicity impact of the formulation. The result here suggests that the toxicity of QDs is rather complex and it cannot be generalized under a few assumptions reported previously. We suggest that one have to evaluate the QD toxicity on a case to case basis and this indicates that standard procedures and comprehensive

Cytotoxicity assessment of functionalized CdSe, CdTe and InP quantum dots in two human cancer cell models

Energy Technology Data Exchange (ETDEWEB)

Liu, Jing [Institute of Gerontology and Geriatrics & Beijing Key Lab of Aging and Geriatrics, Chinese PLA General Hospital, Beijing 100853 (China); Hu, Rui [School of Electrical and Electronic Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798 (Singapore); Liu, Jianwei [Institute of Gerontology and Geriatrics & Beijing Key Lab of Aging and Geriatrics, Chinese PLA General Hospital, Beijing 100853 (China); Zhang, Butian; Wang, Yucheng [School of Electrical and Electronic Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798 (Singapore); Liu, Xin [Lawrence Berkeley National Laboratory, Berkeley, CA (United States); Law, Wing-Cheung [Department of Industrial and System Engineering, The Hang Kong Polytechnic University, Hung Hom (Hong Kong); Liu, Liwei [School of Science, Changchun University of Science and Technology, Changchun 130022 (China); Ye, Ling, E-mail: lye_301@163.com [Institute of Gerontology and Geriatrics & Beijing Key Lab of Aging and Geriatrics, Chinese PLA General Hospital, Beijing 100853 (China); Yong, Ken-Tye, E-mail: ktyong@ntu.edu.sg [School of Electrical and Electronic Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798 (Singapore)

2015-12-01

The toxicity of quantum dots (QDs) has been extensively studied over the past decade. Some common factors that originate the QD toxicity include releasing of heavy metal ions from degraded QDs and the generation of reactive oxygen species on the QD surface. In addition to these factors, we should also carefully examine other potential QD toxicity causes that will play crucial roles in impacting the overall biological system. In this contribution, we have performed cytotoxicity assessment of four types of QD formulations in two different human cancer cell models. The four types of QD formulations, namely, mercaptopropionic acid modified CdSe/CdS/ZnS QDs (CdSe-MPA), PEGylated phospholipid encapsulated CdSe/CdS/ZnS QDs (CdSe-Phos), PEGylated phospholipid encapsulated InP/ZnS QDs (InP-Phos) and Pluronic F127 encapsulated CdTe/ZnS QDs (CdTe-F127), are representatives for the commonly used QD formulations in biomedical applications. Both the core materials and the surface modifications have been taken into consideration as the key factors for the cytotoxicity assessment. Through side-by-side comparison and careful evaluations, we have found that the toxicity of QDs does not solely depend on a single factor in initiating the toxicity in biological system but rather it depends on a combination of elements from the particle formulations. More importantly, our toxicity assessment shows different cytotoxicity trend for all the prepared formulations tested on gastric adenocarcinoma (BGC-823) and neuroblastoma (SH-SY5Y) cell lines. We have further proposed that the cellular uptake of these nanocrystals plays an important role in determining the final faith of the toxicity impact of the formulation. The result here suggests that the toxicity of QDs is rather complex and it cannot be generalized under a few assumptions reported previously. We suggest that one have to evaluate the QD toxicity on a case to case basis and this indicates that standard procedures and comprehensive

An exploratory, open-label, randomized, multicenter study to investigate the pharmacodynamics of a glycoengineered antibody (imgatuzumab) and cetuximab in patients with operable head and neck squamous cell carcinoma.

Science.gov (United States)

Temam, S; Spicer, J; Farzaneh, F; Soria, J C; Oppenheim, D; McGurk, M; Hollebecque, A; Sarini, J; Hussain, K; Soehrman Brossard, S; Manenti, L; Evers, S; Delmar, P; Di Scala, L; Mancao, C; Feuerhake, F; Andries, L; Ott, M G; Passioukov, A; Delord, J P

2017-11-01

In addition to inhibiting epidermal growth factor receptor (EGFR) signaling, anti-EGFR antibodies of the IgG1 'subtype' can induce a complementary therapeutic effect through the induction of antibody-dependent cell-mediated cytotoxicity (ADCC). Glycoengineering of therapeutic antibodies increases the affinity for the Fc-gamma receptor, thereby enhancing ADCC. We investigated the changes in immune effector cells and EGFR pathway biomarkers in 44 patients with operable, advanced stage head and neck squamous cell carcinoma treated with two preoperative doses of either glycoengineered imgatuzumab (GA201; 700 or 1400 mg) or cetuximab (standard dosing) in a neoadjuvant setting with paired pre- and post-treatment tumor biopsies. Significant antitumor activity was observed with both antibodies after just two infusions. Metabolic responses were seen in 23 (59.0%) patients overall. One imgatuzumab-treated patient (700 mg) achieved a 'pathological' complete response. An immediate and sustained decrease in peripheral natural killer cells was consistently observed with the first imgatuzumab infusion but not with cetuximab. The functionality of the remaining peripheral natural killer cells was maintained. Similarly, a pronounced increase in circulating cytokines was seen following the first infusion of imgatuzumab but not cetuximab. Overall, tumor-infiltrating CD3+ cell counts increased following treatment with both antibodies. A significant increase from baseline in CD3+/perforin+ cytotoxic T cells occurred only in the 700-mg imgatuzumab group (median 95% increase, P < 0.05). The most prominent decrease of EGFR-expressing cells was recorded after treatment with imgatuzumab (700 mg, -34.6%; 1400 mg, -41.8%). The post-treatment inflammatory tumor microenvironment was strongly related to baseline tumor-infiltrating immune cell density, and baseline levels of EGFR and pERK in tumor cells most strongly predicted therapeutic response. These pharmacodynamic

Cellular effects of the microtubule-targeting agent peloruside A in hypoxia-conditioned colorectal carcinoma cells.

Science.gov (United States)

Řehulka, Jiří; Annadurai, Narendran; Frydrych, Ivo; Znojek, Pawel; Džubák, Petr; Northcote, Peter; Miller, John H; Hajdúch, Marián; Das, Viswanath

2017-07-01

Hypoxia is a prominent feature of solid tumors, dramatically remodeling microtubule structures and cellular pathways and contributing to paclitaxel resistance. Peloruside A (PLA), a microtubule-targeting agent, has shown promising anti-tumor effects in preclinical studies. Although it has a similar mode of action to paclitaxel, it binds to a distinct site on β-tubulin that differs from the classical taxane site. In this study, we examined the unexplored effects of PLA in hypoxia-conditioned colorectal HCT116 cancer cells. Cytotoxicity of PLA was determined by cell proliferation assay. The effects of a pre-exposure to hypoxia on PLA-induced cell cycle alterations and apoptosis were examined by flow cytometry, time-lapse imaging, and western blot analysis of selected markers. The hypoxia effect on stabilization of microtubules by PLA was monitored by an intracellular tubulin polymerization assay. Our findings show that the cytotoxicity of PLA is not altered in hypoxia-conditioned cells compared to paclitaxel and vincristine. Furthermore, hypoxia does not alter PLA-induced microtubule stabilization nor the multinucleation of cells. PLA causes cyclin B1 and G2/M accumulation followed by apoptosis. The cellular and molecular effects of PLA have been determined in normoxic conditions, but there are no reports of PLA effects in hypoxic cells. Our findings reveal that hypoxia preconditioning does not alter the sensitivity of HCT116 to PLA. These data report on the cellular and molecular effects of PLA in hypoxia-conditioned cells for the first time, and will encourage further exploration of PLA as a promising anti-tumor agent. Copyright © 2017 Elsevier B.V. All rights reserved.

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

International Nuclear Information System (INIS)

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

2014-01-01

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

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

Energy Technology Data Exchange (ETDEWEB)

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

2014-11-15

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

Membrane and Nuclear Permeabilization by Polymeric pDNA Vehicles: Efficient Method for Gene Delivery or Mechanism of Cytotoxicity?

Science.gov (United States)

Grandinetti, Giovanna; Smith, Adam E.; Reineke, Theresa M.

2012-01-01

The aim of this study is to compare the cytotoxicity mechanisms of linear PEI to two analogous polymers synthesized by our group: a hydroxyl-containing poly(L-tartaramidoamine) (T4) and a version containing an alkyl chain spacer poly(adipamidopentaethylenetetramine) (A4) by studying the cellular responses to polymer transfection. We have also synthesized analogues of T4 with different molecular weights (degrees of polymerization of 6, 12, and 43) to examine the role of molecular weight on the cytotoxicity mechanisms. Several mechanisms of polymer-induced cytotoxicity are investigated, including plasma membrane permeabilization, the formation of potentially harmful polymer degradation products during transfection including reactive oxygen species, and nuclear membrane permeabilization. We hypothesized that since cationic polymers are capable of disrupting the plasma membrane, they may also be capable of disrupting the nuclear envelope, which could be a potential mechanism of how the pDNA is delivered into the nucleus (other than nuclear envelope breakdown during mitosis). Using flow cytometry and confocal microscopy, we show that the polycations with the highest amount of protein expression and toxicity, PEI and T443, are capable of inducing nuclear membrane permeability. This finding is important for the field of nucleic acid delivery in that not only could direct nucleus permeabilization be a mechanism for pDNA nuclear import but also a potential mechanism of cytotoxicity and cell death. We also show that the production of reactive oxygen species is not a main mechanism of cytotoxicity, and that the presence or absence of hydroxyl groups as well as polymer length plays a role in polyplex size and charge in addition to protein expression efficiency and toxicity. PMID:22175236

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

Science.gov (United States)

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

Human cytomegalovirus antigens in malignant gliomas as targets for adoptive cellular therapy

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Daniel eLandi

2014-11-01

Full Text Available Malignant gliomas are the most common primary brain tumor in adults, with over 12,000 new cases diagnosed in the United States each year. Over the last decade, investigators have reliably identified human cytomegalovirus (HCMV proteins, nucleic acids, and virions in most high-grade gliomas, including glioblastoma (GBM. This discovery is significant because human cytomegalovirus gene products can be targeted by immune-based therapies.In this review, we describe the current level of understanding regarding the presence and role in pathogenesis of HCMV in GBM. We describe our success detecting and expanding HCMV-specific cytotoxic T lymphocytes to kill GBM cells and explain how these cells can be used as a platform for enhanced cellular therapies. We discuss alternative approaches that capitalize on HCMV infection to treat patients with HCMV-positive tumors. Adoptive cellular therapy for HCMV-positive GBM has been tried in a small number of patients with some benefit, but we reason why, to date, these approaches generally fail to generate long-term remission or cure. We conjecture how cellular therapy for GBM can be improved and describe the barriers that must be overcome to cure these patients.

ADCC-Mediated CD56DIM NK Cell Responses Are Associated with Early HBsAg Clearance in Acute HBV Infection.

Science.gov (United States)

Yu, Wen-Han; Cosgrove, Cormac; Berger, Christoph T; Cheney, Patrick C; Krykbaeva, Marina; Kim, Arthur Y; Lewis-Ximenez, Lia; Lauer, Georg M; Alter, Galit

2018-01-01

Hepatitis B virus (HBV) affects up to 400 million people worldwide and accounts for approximately one million deaths per year from liver pathologies. Current treatment regimens are effective in suppressing viremia but usually have to be taken indefinitely, warranting research into new therapeutic approaches. Acute HBV infection in adults almost universally results in resolution of viremia, with the exception of immunocompromised persons, suggesting that the immune response can functionally cure or even eradicate HBV infection. Because immunophenotypic and functional studies have implicated a role for Natural Killer (NK) cells in HBV clearance during acute infection, we hypothesized that a distinct NK-cell profile exists in acute HBV infection that could provide information for the mechanism of HBV clearance. Using multivariate flow cytometry, we evaluated the expression of key activating and inhibitory receptors on NK cells, and their ability to respond to classic target cell lines. Multivariate analysis revealed selective perturbation of the CD56 dim NK-cell subset during acute infection, displaying low levels of NKp46+, NKp30+, CD160+ and CD161+ cells. Intriguingly, the CD56 dim NK-cell profile predicted time to HBV surface antigen (HBsAg) clearance from the blood, and distinct NK-cell profiles predicted early (NKp30, CD94, CD161) and late clearance (KIR3DL1, CD158a, perforin, NKp46). Finally, functional analysis demonstrated that early and late clearance tracked with elevated degranulation (CD107a) or IFNγ production, respectively, in response to ADCC-mediated activation. The cytolytic CD56 dim NK-cell subset is selectively activated in acute HBV infection and displays distinct phenotypic and functional profiles associated with efficient and early control of HBV, implicating antibody-mediated cytolytic NK-cell responses in the early control and functional cure of HBV infection.

Luminescent passive-oxidized silicon quantum dots as biological staining labels and their cytotoxicity effects at high concentration

International Nuclear Information System (INIS)

Fujioka, Kouki; Manabe, Noriyoshi; Hanada, Sanshiro; Hoshino, Akiyoshi; Yamamoto, Kenji; Hiruoka, Masaki; Sato, Keisuke; Hirakuri, Kenji; Miyasaka, Ryosuke; Tilley, Richard D; Manome, Yoshinobu

2008-01-01

Semiconductor quantum dots (QDs) hold some advantages over conventional organic fluorescent dyes. Due to these advantages, they are becoming increasingly popular in the field of bioimaging. However, recent work suggests that cadmium based QDs affect cellular activity. As a substitute for cadmium based QDs, we have developed photoluminescent stable silicon quantum dots (Si-QDs) with a passive-oxidation technique. Si-QDs (size: 6.5 ± 1.5 nm) emit green light, and they have been used as biological labels for living cell imaging. In order to determine the minimum concentration for cytotoxicity, we investigated the response of HeLa cells. We have shown that the toxicity of Si-QDs was not observed at 112 μg ml -1 and that Si-QDs were less toxic than CdSe-QDs at high concentration in mitochondrial assays and with lactate dehydrogenase (LDH) assays. Especially under UV exposure, Si-QDs were more than ten times safer than CdSe-QDs. We suggest that one mechanism for the cytotoxicity is that Si-QDs can generate oxygen radicals and these radicals are associated with membrane damages. This work has demonstrated the suitability of Si-QDs for bioimaging in lower concentration, and their cytotoxicity and one toxicity mechanism at high concentration

Biological effects of a de novo designed myxoma virus peptide analogue: evaluation of cytotoxicity on tumor cells.

Directory of Open Access Journals (Sweden)

Taghrid S Istivan

Full Text Available BACKGROUND: The Resonant Recognition Model (RRM is a physico-mathematical model that interprets protein sequence linear information using digital signal processing methods. In this study the RRM concept was employed for structure-function analysis of myxoma virus (MV proteins and the design of a short bioactive therapeutic peptide with MV-like antitumor/cytotoxic activity. METHODOLOGY/PRINCIPAL FINDINGS: The analogue RRM-MV was designed by RRM as a linear 18 aa 2.3 kDa peptide. The biological activity of this computationally designed peptide analogue against cancer and normal cell lines was investigated. The cellular cytotoxicity effects were confirmed by confocal immunofluorescence microscopy, by measuring the levels of cytoplasmic lactate dehydrogenase (LDH and by Prestoblue cell viability assay for up to 72 hours in peptide treated and non-treated cell cultures. Our results revealed that RRM-MV induced a significant dose and time-dependent cytotoxic effect on murine and human cancer cell lines. Yet, when normal murine cell lines were similarly treated with RRM-MV, no cytotoxic effects were observed. Furthermore, the non-bioactive RRM designed peptide RRM-C produced negligible cytotoxic effects on these cancer and normal cell lines when used at similar concentrations. The presence/absence of phosphorylated Akt activity in B16F0 mouse melanoma cells was assessed to indicate the possible apoptosis signalling pathway that could be affected by the peptide treatment. So far, Akt activity did not seem to be significantly affected by RRM-MV as is the case for the original viral protein. CONCLUSIONS/SIGNIFICANCE: Our findings indicate the successful application of the RRM concept to design a bioactive peptide analogue (RRM-MV with cytotoxic effects on tumor cells only. This 2.345 kDa peptide analogue to a 49 kDa viral protein may be suitable to be developed as a potential cancer therapeutic. These results also open a new direction to the rational

Cationic lipid-coated PEI/DNA polyplexes with improved efficiency and reduced cytotoxicity for gene delivery into mesenchymal stem cells

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Song HM

2012-08-01

Full Text Available Hongmei Song, Gang Wang, Bin He, Li Li, Caixia Li, Yusi Lai, Xianghui Xu, Zhongwei GuNational Engineering Research Center for Biomaterials, Sichuan University, Chengdu, Sichuan, People's Republic of ChinaBackground: Effective gene transfection without serum deprivation is a prerequisite for successful stem cell-based gene therapy. Polyethylenimine (PEI is an efficient nonviral gene vector, but its application has been hindered by serum sensitivity and severe cytotoxicity.Methods: To solve this problem, a new family of lipopolyplexes was developed by coating PEI/DNA polyplexes with three serum-resistant cationic lipids, namely, lysinylated, histidylated, and arginylated cholesterol. The physical properties, transfection efficiency, cellular uptake, subcellular distribution, and cytotoxicity of the lipopolyplexes was investigated.Results: The outer coat composed of lysinylated or histidylated cholesterol remarkably improved the transfection efficiency of the polyplex with a low PEI/DNA ratio of 2 in the presence of serum. The resulting lysinylated and histidylated cholesterol lipopolyplexes were even more efficient than the best performing polyplex with a high PEI/DNA ratio of 10. Results from cellular uptake and subcellular distribution studies suggest that their higher transfection efficiency may result from accelerated DNA nuclear localization. The superiority of the lipopolyplexes over the best performing polyplex was also confirmed by delivering the therapeutic gene, hVEGF165. Equally importantly, the lipid coating removed the necessity of introducing excess free PEI chains into the transfection solution for higher efficiency, generating lipopolyplexes with no signs of cytotoxicity.Conclusion: Noncovalent modification of polyplexes with lysinylated and histidylated cholesterol lipids can simultaneously improve efficiency and reduce the toxicity of gene delivery under serum conditions, showing great promise for genetic modification of bone

The Cytotoxicity of Elderberry Ribosome-Inactivating Proteins Is Not Solely Determined by Their Protein Translation Inhibition Activity.

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Chenjing Shang

Full Text Available Although the protein translation inhibition activity of ribosome inactivating proteins (RIPs is well documented, little is known about the contribution of the lectin chain to the biological activity of these proteins. In this study, we compared the in vitro and intracellular activity of several S. nigra (elderberry RIPs and non-RIP lectins. Our data demonstrate that RIPs from elderberry are much more toxic to HeLa cells than to primary fibroblasts. Differences in the cytotoxicity between the elderberry proteins correlated with differences in glycan specificity of their lectin domain, cellular uptake efficiency and intracellular destination. Despite the fact that the bulk of the RIPs accumulated in the lysosomes and partly in the Golgi apparatus, we could demonstrate effective inhibition of protein synthesis in cellula. As we also observed cytotoxicity for non-RIP lectins, it is clear that the lectin chain triggers additional pathways heralding cell death. Our data suggest that one of these pathways involves the induction of autophagy.

Genetic Characterization of Adenoid Cystic Carcinoma of the Minor Salivary Glands

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Channir, Hani Ibrahim; Hansen, Thomas van Overeem; Andreasen, Simon

2017-01-01

Adenoid cystic carcinoma (AdCC) is a malignant salivary gland tumor. To date, no cases of AdCC in first-degree relatives have been reported in the literature. We present a 50-year-old female (Case 1) and this patients' father (Case 2), both of whom were diagnosed with AdCC of the minor salivary...... 18 germline variants in common between Case 1 and Case 2. However, none of the variants were associated with AdCC or other head and neck cancers. To our knowledge, we present the first potential case of familial AdCC. The presented genetic data may contribute to further investigations...

Cytotoxic glucosphingolipid from Celtis Africana.

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Perveen, Shagufta; Al-Taweel, Areej Mohammad; Fawzy, Ghada Ahmed; El-Shafae, Azza Muhammed; Khan, Afsar; Proksch, Peter

2015-05-01

Literature survey proved the use of the powdered sun-dried bark and roots of Celtis africana for the treatment of cancer in South Africa. The aim of this study was to do further isolation work on the ethyl acetate fraction and to investigate the cytotoxic activities of the various fractions and isolated compound. Cytotoxicity of petroleum ether, chloroform, ethyl acetate, n-butanol fractions and compound 1 were tested on mouse lymphoma cell line L5178Y using the microculture tetrazolium assay. One new glucosphingolipid 1 was isolated from the aerial parts of C. africana. The structure of the new compound was determined by extensive analysis by one-dimensional and two-dimensional nuclear magnetic resonance spectroscopy and mass spectrometry. The ethyl acetate fraction and compound 1 showed strong cytotoxic activity with an EC50 value of 8.3 μg/mL and 7.8 μg/mL, respectively, compared with Kahalalide F positive control (6.3 μg/mL). This is the first report of the occurrence of a cytotoxic glucosphingolipid in family Ulmaceae.

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

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Xie Y

2016-07-01

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

Genotoxic and Cytotoxic Studies of Beta-Sitosterol and Pteropodine in Mouse

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R. Paniagua-Pérez

2005-01-01

Full Text Available Beta-sitosterol (BS and pteropodine (PT are constituents of various plants with pharmacological activities potentially useful to man. The chemicals themselves possess biomedical properties related to the modulation of the immune and the nervous systems, as well as to the inflammatory process. Therefore, safety evaluation of the compounds is necessary in regard to their probable beneficial use in human health. The present study evaluates their genotoxic and cytotoxic potential by determining the capacity of the compounds to induce sister chromatid exchanges (SCE, or to alter cellular proliferation kinetics (CPK and the mitotic index (MI in mouse bone marrow cells. Besides, it also determines their capacity to increase the rate of micronucleated polychromatic erythrocytes (MNPE in peripheral mouse blood, and the relationship polychromatic erythrocytes/normochromatic erythrocytes (PE/NE as an index of cytotoxicity. For the first assay, four doses of each compound were tested: 200, 400, 600, and 1000 mg/kg in case of BS, and 100, 200, 300, and 600 mg/kg for PT. The results in regard to both agents showed no SCE increase induced by any of the tested doses, as well as no alteration in the CPK, or in the MI. With respect to the second assay, the results obtained with the two agents were also negative for both the MNPE and the PE/NE index along the daily evaluation made for four days. In the present study, the highest tested dose corresponded to 80% of the LD50 obtained for BS and to 78% in the case of PT. The results obtained establish that the studied agents have neither genotoxic nor cytotoxic effect on the model used, and therefore they encourage studies on their pharmacological properties.

Cellular islet autoimmunity associates with clinical outcome of islet cell transplantation.

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Volkert A L Huurman

2008-06-01

Full Text Available Islet cell transplantation can cure type 1 diabetes (T1D, but only a minority of recipients remains insulin-independent in the following years. We tested the hypothesis that allograft rejection and recurrent autoimmunity contribute to this progressive loss of islet allograft function.Twenty-one T1D patients received cultured islet cell grafts prepared from multiple donors and transplanted under anti-thymocyte globulin (ATG induction and tacrolimus plus mycophenolate mofetil (MMF maintenance immunosuppression. Immunity against auto- and alloantigens was measured before and during one year after transplantation. Cellular auto- and alloreactivity was assessed by lymphocyte stimulation tests against autoantigens and cytotoxic T lymphocyte precursor assays, respectively. Humoral reactivity was measured by auto- and alloantibodies. Clinical outcome parameters--including time until insulin independence, insulin independence at one year, and C-peptide levels over one year--remained blinded until their correlation with immunological parameters. All patients showed significant improvement of metabolic control and 13 out of 21 became insulin-independent. Multivariate analyses showed that presence of cellular autoimmunity before and after transplantation is associated with delayed insulin-independence (p = 0.001 and p = 0.01, respectively and lower circulating C-peptide levels during the first year after transplantation (p = 0.002 and p = 0.02, respectively. Seven out of eight patients without pre-existent T-cell autoreactivity became insulin-independent, versus none of the four patients reactive to both islet autoantigens GAD and IA-2 before transplantation. Autoantibody levels and cellular alloreactivity had no significant association with outcome.In this cohort study, cellular islet-specific autoimmunity associates with clinical outcome of islet cell transplantation under ATG-tacrolimus-MMF immunosuppression. Tailored immunotherapy targeting cellular

Cytotoxic and phenotypic effects of uranium and lead on osteoblastic cells are highly dependent on metal speciation

International Nuclear Information System (INIS)

Milgram, S.; Carriere, M.; Thiebault, C.; Malaval, L.; Gouget, B.

2008-01-01

Bone is one of the main retention organs for uranium (U) and lead (Pb). The clinical effects of U or Pb poisoning are well known: acute and chronic intoxications impair bone formation. However, only few studies dealt with the cellular and molecular mechanisms of their toxicity. The purpose of this study was to investigate acute cytotoxicity of U and Pb and their phenotypic effects on rat and human osteoblasts, the cells responsible for bone formation. The most likely species of the toxicants in contact with cells after blood contamination were selected for cell exposure. Results showed that the cytotoxic effect of U and Pb is highly dependent on their speciation. Thus, Pb was cytotoxic when left free in the exposure medium or when complexed with carbonate, cystein or citrate, but not when complexed with albumin or phosphate, under an insoluble form. U was cytotoxic whatever its speciation, but differences in sensitivity were observed as a function of speciation. Population growth recovery could be obtained after exposure to low doses of U or Pb, except for some U-carbonate complexes which had irreversible effects whatever the dose. The activation of two markers of bone formation and mineralization, osteocalcin and bone sialoprotein (BSP), was observed after exposure to non-toxic doses or non-toxic species of U or Pb while their inhibition was observed after toxic exposure to both metals. This work provides new elements to better understand the complex mechanisms of U and Pb toxicity to osteoblasts. Our results also illustrate the importance of a strictly controlled speciation of the metals in toxicological studies

Cellular redox homeostasis in endothelial cells treated with nonmodified and Fenton-modified nanodiamond powders.

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Solarska-Ściuk, K; Gajewska, A; Skolimowski, J; Gajek, A; Bartosz, G

2014-01-01

Diamond nanoparticles find numerous applications in pharmacy, medicine, cosmetics, and biotechnology. However, possible adverse cellular effects of diamond nanoparticle cells have been reported, which may limit their use. The aim of this study was to compare the effect of nonmodified diamond nanoparticles (D) and diamond nanoparticles modified by the Fenton reaction (D+OH) on human umbilical cord endothelial cells (HUVEC-ST). We found that both D and D+OH show time- and concentration-dependent cytotoxicity, inducing apoptosis and necrosis of HUVEC-ST. Interaction with D and D+OH also induced changes in the production of reactive oxygen and nitrogen species and changes in the level of glutathione and activities of antioxidant enzymes in the cells. These data demonstrate that diamond nanoparticles may induce oxidative stress in human endothelial cells, which contributes to their cytotoxic effects seen at higher concentrations of D and D+OH. © 2014 International Union of Biochemistry and Molecular Biology, Inc.

Quantification of cellular viability for the MTT method

International Nuclear Information System (INIS)

Altanes, M.

2001-01-01

In the last years, the scientists have been given to the task of finding new biomaterials whose biocompatibility with the human body allow them to substitute parts of the organism, as the bones and the junctures and also that they are little rejected. In the following work it was evaluated and quantified by the method of the MTT, a colorimetric, quick, simple and economic technical, the possible citohepatic effect of a watery extract of a biomaterials of polymeric origin (acrilamide and metacrilic acid) obtained for technical of gamma irradiation, consistent in culture medium 199 and veal serum, in cells Vero. In order to compare the answer of the analysis material, they were used the controls negative (polyethylene of high molecular weight), and positive (Sulphate of Streptomycin), just as they indicate it the established norms, being achieved the waited result of each one of them. In the observation made to the optic microscope, after 24 and 48 hours of contact between the cells and the extract of the analysis material, they were not perceived indicative of damage or cellular lysis, or morphologic changes in the structure of cells that it indicated us the possible cytotoxic effect of this biomaterials, therefore the qualitative analysis was not enough for the determination of the cytotoxic effect of the analysis sample

[Cytologic diagnosis of adenoid cystic carcinoma of salivary glands and distinction from basal cell adenoma].

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Bai, Y P; Zhang, Y; Tian, C; Xing, L; Liu, H G

2018-04-08

Objective: To describe the cytologic features of adenoid cystic carcinoma (ADCC) of salivary glands, and to identify distinguishing cytologic features of ADCC and basal cell adenoma (BCA). Methods: A retrospective review of cytology smears of 30 cases of ADCC and 12 cases of BCA of salivary glands were performed. All cases were collected from Beijing Tongren Hospital, Capital Medical University from January 2010 to January 2017. Except for 2 aspirate smears of ADCC, all were touch imprint smears. All cases had further histological confirmation. Results: Neoplastic ductal cells of ADCC were arranged in three-dimensional clusters, sheets and singles. Hyaline globules were found in most cases (20/30, 66.7%). The nuclei were round to oval, showing varying degrees of nuclear atypia. These included (1) the nuclei were hyperchromatic, demonstrating coarse or slightly coarse, irregularly distributed chromatin; (2) the nuclei were slightly large and vary in size; (3) appearance of the nuclei had a different degree of irregularity (often mild). Nucleoli were common seen (21/30, 70.0%), and were prominent in some cases. Mitosis and necrosis were rare. Cytologically, BCA showed cell arrangements and nuclear features overlapped with those of ADCC. The cytologic difference between these two tumors included: (1) the tumor cells presented rarely in singles; (2) hyaline globules were very uncommon (1/12) in BCA; (3) nuclei of BCA were hypochromatic or slightly hyperchromatic, homogeneous and uniform in appearance and size, overall without nuclear atypia and they were smaller and slender then those of ADCC and (4) individual cells of BCA showed relatively abundant cytoplasm. Conclusions: The cytologic features of ADCC and BCA both overlap and different from each other. Most cases can be diagnosed by cytologic examination. The presence of hyaline globules is an important diagnostic clue of ADCC, although not pathognomonic. Nuclear atypia of neoplastic ductal cells is an essential

Cellular defense against singlet oxygen-induced oxidative damage by cytosolic NADP+-dependent isocitrate dehydrogenase.

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Kim, Sun Yee; Park, Jeen-Woo

2003-03-01

Singlet oxygen (1O2) is a highly reactive form of molecular oxygen that may harm living systems by oxidizing critical cellular macromolecules. Recently, we have shown that NADP+-dependent isocitrate dehydrogenase is involved in the supply of NADPH needed for GSH production against cellular oxidative damage. In this study, we investigated the role of cytosolic form of NADP+-dependent isocitrate dehydrogenase (IDPc) against singlet oxygen-induced cytotoxicity by comparing the relative degree of cellular responses in three different NIH3T3 cells with stable transfection with the cDNA for mouse IDPc in sense and antisense orientations, where IDPc activities were 2.3-fold higher and 39% lower, respectively, than that in the parental cells carrying the vector alone. Upon exposure to singlet oxygen generated from photoactivated dye, the cells with low levels of IDPc became more sensitive to cell killing. Lipid peroxidation, protein oxidation, oxidative DNA damage and intracellular peroxide generation were higher in the cell-line expressing the lower level of IDPc. However, the cells with the highly over-expressed IDPc exhibited enhanced resistance against singlet oxygen, compared to the control cells. The data indicate that IDPc plays an important role in cellular defense against singlet oxygen-induced oxidative injury.

Portulaca oleracea Seed Oil Exerts Cytotoxic Effects on Human Liver Cancer (HepG2) and Human Lung Cancer (A-549) Cell Lines.

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Al-Sheddi, Ebtesam Saad; Farshori, Nida Nayyar; Al-Oqail, Mai Mohammad; Musarrat, Javed; Al-Khedhairy, Abdulaziz Ali; Siddiqui, Maqsood Ahmed

2015-01-01

Portulaca oleracea (Family: Portulacaceae), is well known for its anti-inflammatory, antioxidative, anti- bacterial, and anti-tumor activities. However, cytotoxic effects of seed oil of Portulaca oleracea against human liver cancer (HepG2) and human lung cancer (A-549) cell lines have not been studied previously. Therefore, the present study was designed to investigate the cytotoxic effects of Portulaca oleracea seed oil on HepG2 and A-549 cell lines. Both cell lines were exposed to various concentrations of Portulaca oleracea seed oil for 24h. After the exposure, percentage cell viability was studied by (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT), neutral red uptake (NRU) assays, and cellular morphology by phase contrast inverted microscopy. The results showed a concentration-dependent significant reduction in the percentage cell viability and an alteration in the cellular morphology of HepG2 and A-549 cells. The percentage cell viability was recorded as 73%, 63%, and 54% by MTT assay and 76%, 61%, and 50% by NRU assay at 250, 500, and 1000 μg/ml, respectively in HepG2 cells. Percentage cell viability was recorded as 82%, 72%, and 64% by MTT assay and 83%, 68%, and 56% by NRU assay at 250, 500, and 1000 μg/ml, respectively in A-549 cells. The 100 μg/ml and lower concentrations were found to be non cytotoxic to A-549 cells, whereas decrease of 14% and 12% were recorded by MTT and NRU assay, respectively in HepG2 cells. Both HepG2 and A-549 cell lines exposed to 250, 500, and 1000 μg/ ml of Portulaca oleracea seed oil lost their normal morphology, cell adhesion capacity, become rounded, and appeared smaller in size. The data from this study showed that exposure to seed oil of Portulaca oleracea resulted in significant cytotoxicity and inhibition of growth of the human liver cancer (HepG2) and human lung cancer (A-549) cell lines.

Induced pluripotent stem cell-derived limbal epithelial cells (LiPSC) as a cellular alternative for in vitro ocular toxicity testing.

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Aberdam, Edith; Petit, Isabelle; Sangari, Linda; Aberdam, Daniel

2017-01-01

Induced pluripotent stem cells hold great potential to produce unlimited amount of differentiated cells as cellular source for regenerative medicine but also for in vitro drug screening and cytotoxicity tests. Ocular toxicity testing is mandatory to evaluate the risks of drugs and cosmetic products before their application to human patients by preventing eye irritation or insult. Since the global ban to use animals, many human-derived alternatives have been proposed, from ex-vivo enucleated postmortem cornea, primary corneal cell culture and immortalized corneal epithelial cell lines. All of them share limitations for their routine use. Using an improved protocol, we derived limbal epithelial cells from human induced pluripotent stem cells, named LiPSC, that are able to be passaged and differentiate further into corneal epithelial cells. Comparative RT-qPCR, immunofluorescence staining, flow cytometry analysis and zymography assays demonstrate that LiPSC are morphologically and molecularly similar to the adult stem cells. Moreover, contrary to HCE, LiPSC and primary limbal cells display similarly sensitive to cytotoxicity treatment among passages. Our data strongly suggest that LiPSC could become a powerful alternative cellular model for cosmetic and drug tests.

Induced pluripotent stem cell-derived limbal epithelial cells (LiPSC as a cellular alternative for in vitro ocular toxicity testing.

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Edith Aberdam

Full Text Available Induced pluripotent stem cells hold great potential to produce unlimited amount of differentiated cells as cellular source for regenerative medicine but also for in vitro drug screening and cytotoxicity tests. Ocular toxicity testing is mandatory to evaluate the risks of drugs and cosmetic products before their application to human patients by preventing eye irritation or insult. Since the global ban to use animals, many human-derived alternatives have been proposed, from ex-vivo enucleated postmortem cornea, primary corneal cell culture and immortalized corneal epithelial cell lines. All of them share limitations for their routine use. Using an improved protocol, we derived limbal epithelial cells from human induced pluripotent stem cells, named LiPSC, that are able to be passaged and differentiate further into corneal epithelial cells. Comparative RT-qPCR, immunofluorescence staining, flow cytometry analysis and zymography assays demonstrate that LiPSC are morphologically and molecularly similar to the adult stem cells. Moreover, contrary to HCE, LiPSC and primary limbal cells display similarly sensitive to cytotoxicity treatment among passages. Our data strongly suggest that LiPSC could become a powerful alternative cellular model for cosmetic and drug tests.

Cytotoxicity of Different Excipients on RPMI 2650 Human Nasal Epithelial Cells

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Tamás Horváth

2016-05-01

Full Text Available The nasal route receives a great deal of attention as a non-invasive method for the systemic administration of drugs. For nasal delivery, specific formulations containing excipients are used. Because of the sensitive respiratory mucosa, not only the active ingredients, but also additives need to be tested in appropriate models for toxicity. The aim of the study was to measure the cytotoxicity of six pharmaceutical excipients, which could help to reach larger residence time, better permeability, and increased solubility dissolution rate. The following excipients were investigated on RPMI 2650 human nasal septum tumor epithelial cells: β-d-mannitol, sodium hyaluronate, α and β-cyclodextrin, polyvinyl alcohol and methylcellulose. 3-(4,5-dimethyltiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT dye conversion assay and real-time impedance analysis were used to investigate cytotoxicity. No excipient showed toxicity at 0.3% (w/v concentration or below while 1% concentration a significantly reduced metabolic activity was measured by MTT assay for methylcellulose and cyclodextrins. Using impedance measurements, only β-cyclodextrin (1% was toxic to cells. Mannitol at 1% concentration had a barrier opening effect on epithelial cells, but caused no cellular damage. Based on the results, all additives at 0.3%, sodium hyaluronate and polyvinyl alcohol at 1% concentrations can be safely used for nasal formulations.

Cytotoxic Effects of Bangladeshi Medicinal Plant Extracts

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Shaikh J. Uddin

2011-01-01

Full Text Available To investigate the cytotoxic effect of some Bangladeshi medicinal plant extracts, 16 Bangladeshi medicinal plants were successively extracted with n-hexane, dichloromethane, methanol and water. The methanolic and aqueous extracts were screened for cytotoxic activity against healthy mouse fibroblasts (NIH3T3 and three human cancer-cell lines (gastric: AGS; colon: HT-29; and breast: MDA-MB-435S using the MTT assay. Two methanolic extracts (Hygrophila auriculata and Hibiscus tiliaceous and one aqueous extract (Limnophila indica showed no toxicity against healthy mouse fibroblasts, but selective cytotoxicity against breast cancer cells (IC50 1.1–1.6 mg mL−1. Seven methanolic extracts from L. indica, Clerodendron inerme, Cynometra ramiflora, Xylocarpus moluccensis, Argemone mexicana, Ammannia baccifera and Acrostichum aureum and four aqueous extracts from Hygrophila auriculata, Bruguiera gymnorrhiza, X. moluccensis and Aegiceras corniculatum showed low toxicity (IC50 > 2.5 mg mL−1 against mouse fibroblasts but selective cytotoxicity (IC50 0.2–2.3 mg mL−1 against different cancer cell lines. The methanolic extract of Blumea lacera showed the highest cytotoxicity (IC50 0.01–0.08 mg mL−1 against all tested cell lines among all extracts tested in this study. For some of the plants their traditional use as anticancer treatments correlates with the cytotoxic results, whereas for others so far unknown cytotoxic activities were identified.

PD-L1 inhibition with avelumab for metastatic Merkel cell carcinoma.

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Gaiser, Maria Rita; Bongiorno, Michelle; Brownell, Isaac

2018-04-01

Merkel cell carcinoma (MCC) is a rare and aggressive neuroendocrine skin cancer that lacks durable responses to traditional chemotherapy. Areas covered: After MCC was shown to be an immunogenic tumor, small trials revealed high objective response rates to PD-1/PD-L1 checkpoint inhibitors. The JAVELIN Merkel 200 (NCT02155647) trial tested the use of avelumab, a human IgG1 monoclonal antibody against PD-L1, in metastatic MCC. Avelumab recently became the first approved drug for metastatic MCC. Expert commentary: By conducting broad phase I studies assessing the safety of avelumab and a small phase II study demonstrating efficacy in this rare orphan tumor type, avelumab gained accelerated approval for the treatment of metastatic MCC. Additional studies are needed to determine how the antibody-dependent cellular cytotoxicity (ADCC) competent Fc region of avelumab contributes to disease control. Remaining questions: Longer follow-up will determine the durability of checkpoint blockade in controlling metastatic MCC. Additional studies will assess the utility and safety of adjuvant checkpoint blockade in patients with excised MCC. How to increase response rates by combining PD-1/PD-L1 blockade with other treatment approaches needs to be explored. In addition, treatment options for MCC patients who fail or do not respond to avelumab need to be identified.

Origin of anti-tumor activity of the cysteine-containing GO peptides and further optimization of their cytotoxic properties

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Tyuryaeva, Irina I.; Lyublinskaya, Olga G.; Podkorytov, Ivan S.; Skrynnikov, Nikolai R.

2017-01-01

Antitumor GO peptides have been designed as dimerization inhibitors of prominent oncoprotein mucin 1. In this study we demonstrate that activity of GO peptides is independent of the level of cellular expression of mucin 1. Furthermore, these peptides prove to be broadly cytotoxic, causing cell death also in normal cells such as dermal fibroblasts and endometrial mesenchymal stem cells. To explore molecular mechanism of their cytotoxicity, we have designed and tested a number of new peptide sequences containing the key CxC or CxxC motifs. Of note, these sequences bear no similarity to mucin 1 except that they also contain a pair of proximal cysteines. Several of the new peptides turned out to be significantly more potent than their GO prototypes. The results suggest that cytotoxicity of these peptides stems from their (moderate) activity as disulfide oxidoreductases. It is expected that such peptides, which we have termed DO peptides, are involved in disulfide-dithiol exchange reaction, resulting in formation of adventitious disulfide bridges in cell proteins. In turn, this leads to a partial loss of protein function and rapid onset of apoptosis. We anticipate that coupling DO sequences with tumor-homing transduction domains can create a potentially valuable new class of tumoricidal peptides.

Extending in silico mechanism-of-action analysis by annotating targets with pathways: application to cellular cytotoxicity readouts.

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Liggi, Sonia; Drakakis, Georgios; Koutsoukas, Alexios; Cortes-Ciriano, Isidro; Martínez-Alonso, Patricia; Malliavin, Thérèse E; Velazquez-Campoy, Adrian; Brewerton, Suzanne C; Bodkin, Michael J; Evans, David A; Glen, Robert C; Carrodeguas, José Alberto; Bender, Andreas

2014-01-01

An in silico mechanism-of-action analysis protocol was developed, comprising molecule bioactivity profiling, annotation of predicted targets with pathways and calculation of enrichment factors to highlight targets and pathways more likely to be implicated in the studied phenotype. The method was applied to a cytotoxicity phenotypic endpoint, with enriched targets/pathways found to be statistically significant when compared with 100 random datasets. Application on a smaller apoptotic set (10 molecules) did not allowed to obtain statistically relevant results, suggesting that the protocol requires modification such as analysis of the most frequently predicted targets/annotated pathways. Pathway annotations improved the mechanism-of-action information gained by target prediction alone, allowing a better interpretation of the predictions and providing better mapping of targets onto pathways.

Delivery of paclitaxel across cellular barriers using a dendrimer-based nanocarrier.

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Teow, Huey Minn; Zhou, Zhengyuan; Najlah, Mohammad; Yusof, Siti R; Abbott, N Joan; D'Emanuele, Antony

2013-01-30

The aim of this study was to investigate the ability of a third-generation (G3) polyamidoamine (PAMAM) dendrimer-based carrier to enhance the permeability of paclitaxel (pac) and to overcome cellular barriers. G3 dendrimers were surface modified with lauryl chains (L) and conjugated with paclitaxel (pac) via a glutaric anhydride (glu) linker, followed by labeling with FITC. Biological evaluation of the dendrimer and conjugates was conducted using the human colon adenocarcinoma cell line (Caco-2) and primary cultured porcine brain endothelial cells (PBECs). LDH assay was used to evaluate the cytotoxicity of the dendrimer and conjugates. Cytotoxicity studies showed that the conjugation of lauryl chains and paclitaxel on G3 dendrimer significantly (pdendrimer-drug conjugates demonstrated an increase in the apparent permeability coefficient (P(app)) in both apical to basolateral A→B and basolateral to apical B→A directions across both cell monolayers compared to unmodified G3 and free drug. The B→A P(app) of paclitaxel was significantly (ptransporter system in both cell models. L6-G3-glu-pac conjugate had approximately 12-fold greater permeability across both cell monolayers than that of paclitaxel alone. Copyright © 2012 Elsevier B.V. All rights reserved.

Irradiation and various cytotoxic drugs enhance tyrosine phosphorylation and β1-integrin clustering in human A549 lung cancer cells in a substratum-dependent manner in vitro

International Nuclear Information System (INIS)

Cordes, N.; Beinke, C.; Beuningen, D. van; Plasswilm, L.

2004-01-01

Background and purpose: interactions of cells with a substratum, especially extracellular matrix proteins, initiate clustering of integrin receptors in the cell membrane. This process represents the initial step for the activation of signaling pathways regulating survival, proliferation, differentiation, adhesion, and migration, and could, furthermore, be important for cellular resistance-mediating mechanisms against radiation or cytotoxic drugs. The lack of data elucidating the impact of irradiation or cytotoxic drugs on this important phenomenon led to this study on human A549 lung cancer cells in vitro. Material and methods: the human lung carcinoma cell line A549 grown on polystyrene or fibronectin (FN) was irradiated with 0-8 Gy or treated with cisplatin (0.1-50 μM), paclitaxel (0.1-50 nM), or mitomycin (0.1-50 μM). Colony formation assays, immunofluorescence staining in combination with activation of integrin clustering using anti-β 1 -integrin antibodies (K20), and Western blotting for tyrosine phosphorylation under treatment of cells with the IC 50 for irradiation (2 Gy; IC 50 = 2.2 Gy), cisplatin (2 μM), paclitaxel (5 nM), or mitomycin (7 μM) were performed. Results: attachment of cells to FN resulted in a significantly reduced radio- and chemosensitivity compared to polystyrene. The clustering of β 1 -integrins examined by immunofluorescence staining was only stimulated by irradiation, cisplatin, paclitaxel, or mitomycin in case of cell attachment to FN. By contrast, tyrosine phosphorylation, as one of the major events following β 1 -integrin clustering, showed a 3.7-fold, FN-related enhancement, and treatment of cells with the IC 50 of radiation, cisplatin, paclitaxel, or mitomycin showed a substratum-dependent induction. Conclusion: for the first time, a strong influence of irradiation and a variety of cytotoxic drugs on the clustering of β 1 -integrins could be shown. This event is a prerequisite for tyrosine phosphorylation and, thus, the

Poly(d,l)-lactide-co-glycolide (PLGA) microspheres as immunoadjuvant for Brugia malayi antigens.

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Saini, Vinay; Verma, Shiv Kumar; Murthy, P Kalpana; Kohli, Dharmveer

2013-08-28

Recently we identified in Brugia malayi adult worm extract (BmA) a pro-inflammatory 54-68kDa SDS-PAGE resolved fraction F6 that protects the host from the parasite via Th1/Th2 type responses. We are currently investigating F6 as a potential source of vaccine candidate(s) and the present study is aimed at investigating the suitability of poly(d,l)-lactide-co-glycolide microspheres (PLGA-Ms) as immunoadjuvant for the antigen administration in a single dose. PLGA-Ms were prepared aseptically by a modified double emulsion (w/o/w) solvent evaporation technique and their size, shape, antigen adsorption efficiency, in-process stability, and antigen release were characterized. Swiss mice were immunized by a single subcutaneous administration of BmA and F6 adsorbed on PLGA-Ms (lactide:glycolide ratios 50:50 and 75:25) and the immune responses were compared with administration of 1 or 2 doses of plain BmA and F6. Specific IgG, IgG1, IgG2a, IgG2b, IgE levels in serum, cellular-proliferative response and release of IFN-γ, TNF-α and nitric oxide from the cells of immunized host in response to the antigens/LPS/Con A challenge and antibody-dependant cellular cytotoxicity (ADCC) to parasite life stages were determined. The average size of PLGA-Ms 50:50 was smaller than the size of PLGA-Ms 75:25 and the % antigen adsorption efficiency of PLGA-Ms 50:50 was greater than PLGA-Ms 75:25. Single shot injection of PLGA-Ms 50:50/75:25-BmA/F6 produced better and stronger IgG, IgG1/IgG2a and cell-mediated immune responses than even two injections of plain BmA or F6. Further, PLGA-Ms 50:50-F6 produced stronger responses than PLGA-Ms 50:50-BmA. Anti-PLGA-Ms 50:50-F6 antibodies elicited higher ADCC response to infective larval and microfilarial stages of the parasite than anti-PLGA-Ms 75:25-F6 antibodies. The findings demonstrate that PLGA-Ms 50:50 is an excellent adjuvant for use with F6 in a single administration. This is the first ever report on PLGA as immunoadjuvant for filarial antigens

Phase I dose escalation pharmacokinetic assessment of intravenous humanized anti-MUC1 antibody AS1402 in patients with advanced breast cancer.

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Pegram, Mark D; Borges, Virginia F; Ibrahim, Nuhad; Fuloria, Jyotsna; Shapiro, Charles; Perez, Susan; Wang, Karen; Schaedli Stark, Franziska; Courtenay Luck, Nigel

2009-01-01

MUC1 is a cell-surface glycoprotein that establishes a molecular barrier at the epithelial surface and engages in morphogenetic signal transduction. Alterations in MUC1 glycosylation accompany the development of cancer and influence cellular growth, differentiation, transformation, adhesion, invasion, and immune surveillance. A 20-amino-acid tandem repeat that forms the core protein of MUC1 is overexpressed and aberrantly glycosylated in the majority of epithelial tumors. AS1402 (formerly R1550) is a humanized IgG1k monoclonal antibody that binds to PDTR sequences within this tandem repeat that are not exposed in normal cells. AS1402 is a potent inducer of antibody-dependent cellular cytotoxicity (ADCC), specifically against MUC1-expressing tumor cells. The objective of this study was to determine the safety, tolerability, and pharmacokinetic (PK) characteristics of AS1402 monotherapy in patients with locally advanced or metastatic MUC1-positive breast cancer that had progressed after anthracyclines- and taxane-based therapy. Patients received AS1402 over a 1- to 3-hour intravenous (i.v.) infusion at doses between 1 and 16 mg/kg, with repeated dosing every 1 to 3 weeks (based on patient-individualized PK assessment) until disease progression. Serum AS1402 levels were measured at multiple times after i.v. administration. Human anti-human antibody (HAHA) responses were measured to determine the immunogenicity of AS1402. Noncompartmental pharmacokinetic parameters were determined and were used to assess dose dependency across the dose range studied. Twenty-six patients were treated. AS1402 was generally well tolerated. Two grade 3/4 drug-related adverse events were reported, both at the 3-mg/kg dose. Neither was observed in expanded or subsequent dosing cohorts. No anti-human antibodies were detected. Plasma concentrations of AS1402 appeared to be proportional to dose within the 1- to 16-mg/kg dose range assessed, with a mean terminal half-life of 115.4 +/- 37.1 hours

Cytotoxic constituents of ethyl acetate fraction from Dianthus superbus.

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Ding, Chengli; Zhang, Wu; Li, Jie; Lei, Jiachuan; Yu, Jianqing

2013-01-01

The ethyl acetate fraction (EE-DS) from Dianthus superbus was found to possess the cytotoxic activity against cancer cells in previous study. To investigate cytotoxic constituents, the bioassay-guided isolation of compounds from EE-DS was performed. Two dianthramides (1 and 2), three flavonoids (3-5), two coumarins (6 and 7) and three other compounds (8-10) were obtained. Structures of isolated compounds were identified by spectroscopic analysis. Cytotoxicity of the compounds against HepG2 cells was evaluated. Compound 1 showed the strongest cytotoxicity, compounds 10, 4, 3 and 5 had moderate cytotoxicity.

Structure-activity relationship analysis of cytotoxic cyanoguanidines: selection of CHS 828 as candidate drug

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Gullbo Joachim

2009-06-01

Full Text Available Abstract Background N-(6-(4-chlorophenoxyhexyl-N'-cyano-N''-4-pyridyl guanidine (CHS 828 is the first candidate drug from a novel group of anti-tumour agents – the pyridyl cyanoguanidines, shown to be potent compounds interfering with cellular metabolism (inhibition of nicotinamide phosphoribosyl transferase and NF-κB signalling. Substituted cyanoguanidines are also found in anti-hypertensive agents such as the potassium channel opener pinacidil (N-cyano-N'-(4-pyridyl-N''-(1,2,2-trimethylpropylguanidine and histamine-II receptor antagonists (e.g. cimetidine, N-cyano-N'-methyl-N''-[2-[[(5-methylimidazol-4-yl]methyl]thio]ethylguanidine. In animal studies, CHS 828 has shown very promising activity, and phase I and II studies resulted in further development of a with a water soluble prodrug. Findings To study the structural requirements for cyanoguanidine cytotoxicity a set of 19 analogues were synthesized. The cytotoxic effects were then studied in ten cell lines selected for different origins and mechanisms of resistance, using the fluorometric microculture cytotoxicity assay (FMCA. The compounds showed varying cytotoxic activity even though the dose-response curves for some analogues were very shallow. Pinacidil and cimetidine were found to be non-toxic in all ten cell lines. Starting with cyanoguanidine as the crucial core it was shown that 4-pyridyl substitution was more efficient than was 3-pyridyl substitution. The 4-pyridyl cyanoguanidine moiety should be linked by an alkyl chain, optimally a hexyl, heptyl or octyl chain, to a bulky end group. The exact composition of this end group did not seem to be of crucial importance; when the end group was a mono-substituted phenyl ring it was shown that the preferred position was 4-substitution, followed by 3- and, finally, 2-substitution as the least active. Whether the substituent was a chloro, nitro or methoxy substituent seemed to be of minor importance. Finally, the activity patterns in the

Cell-to-cell communication and cellular environment alter the somatostatin status of delta cells

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Kelly, Catriona, E-mail: catriona.kelly@qub.ac.uk [SAAD Centre for Pharmacy and Diabetes, School of Biomedical Sciences, University of Ulster, Coleraine (United Kingdom); Flatt, Peter R.; McClenaghan, Neville H. [SAAD Centre for Pharmacy and Diabetes, School of Biomedical Sciences, University of Ulster, Coleraine (United Kingdom)

2010-08-20

Research highlights: {yields} TGP52 cells display enhanced functionality in pseudoislet form. {yields} Somatostatin content was reduced, but secretion increased in high glucose conditions. {yields} Cellular interactions and environment alter the somatostatin status of TGP52 cells. -- Abstract: Introduction: Somatostatin, released from pancreatic delta cells, is a potent paracrine inhibitor of insulin and glucagon secretion. Islet cellular interactions and glucose homeostasis are essential to maintain normal patterns of insulin secretion. However, the importance of cell-to-cell communication and cellular environment in the regulation of somatostatin release remains unclear. Methods: This study employed the somatostatin-secreting TGP52 cell line maintained in DMEM:F12 (17.5 mM glucose) or DMEM (25 mM glucose) culture media. The effect of pseudoislet formation and culture medium on somatostatin content and release in response to a variety of stimuli was measured by somatostatin EIA. In addition, the effect of pseudoislet formation on cellular viability (MTT and LDH assays) and proliferation (BrdU ELISA) was determined. Results: TGP52 cells readily formed pseudoislets and showed enhanced functionality in three-dimensional form with increased E-cadherin expression irrespective of the culture environment used. However, culture in DMEM decreased cellular somatostatin content (P < 0.01) and increased somatostatin secretion in response to a variety of stimuli including arginine, calcium and PMA (P < 0.001) when compared with cells grown in DMEM:F12. Configuration of TGP52 cells as pseudoislets reduced the proliferative rate and increased cellular cytotoxicity irrespective of culture medium used. Conclusions: Somatostatin secretion is greatly facilitated by cell-to-cell interactions and E-cadherin expression. Cellular environment and extracellular glucose also significantly influence the function of delta cells.

Cell-to-cell communication and cellular environment alter the somatostatin status of delta cells

International Nuclear Information System (INIS)

Kelly, Catriona; Flatt, Peter R.; McClenaghan, Neville H.

2010-01-01

Research highlights: → TGP52 cells display enhanced functionality in pseudoislet form. → Somatostatin content was reduced, but secretion increased in high glucose conditions. → Cellular interactions and environment alter the somatostatin status of TGP52 cells. -- Abstract: Introduction: Somatostatin, released from pancreatic delta cells, is a potent paracrine inhibitor of insulin and glucagon secretion. Islet cellular interactions and glucose homeostasis are essential to maintain normal patterns of insulin secretion. However, the importance of cell-to-cell communication and cellular environment in the regulation of somatostatin release remains unclear. Methods: This study employed the somatostatin-secreting TGP52 cell line maintained in DMEM:F12 (17.5 mM glucose) or DMEM (25 mM glucose) culture media. The effect of pseudoislet formation and culture medium on somatostatin content and release in response to a variety of stimuli was measured by somatostatin EIA. In addition, the effect of pseudoislet formation on cellular viability (MTT and LDH assays) and proliferation (BrdU ELISA) was determined. Results: TGP52 cells readily formed pseudoislets and showed enhanced functionality in three-dimensional form with increased E-cadherin expression irrespective of the culture environment used. However, culture in DMEM decreased cellular somatostatin content (P < 0.01) and increased somatostatin secretion in response to a variety of stimuli including arginine, calcium and PMA (P < 0.001) when compared with cells grown in DMEM:F12. Configuration of TGP52 cells as pseudoislets reduced the proliferative rate and increased cellular cytotoxicity irrespective of culture medium used. Conclusions: Somatostatin secretion is greatly facilitated by cell-to-cell interactions and E-cadherin expression. Cellular environment and extracellular glucose also significantly influence the function of delta cells.

Quantitative structure-cytotoxicity relationship of phenylpropanoid amides.

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Shimada, Chiyako; Uesawa, Yoshihiro; Ishihara, Mariko; Kagaya, Hajime; Kanamoto, Taisei; Terakubo, Shigemi; Nakashima, Hideki; Takao, Koichi; Saito, Takayuki; Sugita, Yoshiaki; Sakagami, Hiroshi

2014-07-01

A total of 12 phenylpropanoid amides were subjected to quantitative structure-activity relationship (QSAR) analysis, based on their cytotoxicity, tumor selectivity and anti-HIV activity, in order to investigate on their biological activities. Cytotoxicity against four human oral squamous cell carcinoma (OSCC) cell lines and three human oral normal cells was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Tumor selectivity was evaluated by the ratio of the mean CC50 (50% cytotoxic concentration) against normal oral cells to that against OSCC cell lines. Anti-HIV activity was evaluated by the ratio of CC50 to EC50 (50% cytoprotective concentration from HIV infection). Physicochemical, structural, and quantum-chemical parameters were calculated based on the conformations optimized by the LowModeMD method followed by density functional theory (DFT) method. Twelve phenylpropanoid amides showed moderate cytotoxicity against both normal and OSCC cell lines. N-Caffeoyl derivatives coupled with vanillylamine and tyramine exhibited relatively higher tumor selectivity. Cytotoxicity against normal cells was correlated with descriptors related to electrostatic interaction such as polar surface area and chemical hardness, whereas cytotoxicity against tumor cells correlated with free energy, surface area and ellipticity. The tumor-selective cytotoxicity correlated with molecular size (surface area) and electrostatic interaction (the maximum electrostatic potential). The molecular size, shape and ability for electrostatic interaction are useful parameters for estimating the tumor selectivity of phenylpropanoid amides. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

New Benzimidazole-1,2,4-Triazole Hybrid Compounds: Synthesis, Anticandidal Activity and Cytotoxicity Evaluation

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Hülya Karaca Gençer

2017-03-01

Full Text Available Owing to the growing need for antifungal agents, we synthesized a new series 2-((5-(4-(5-substituted-1H-benzimidazol-2-ylphenyl-4-substituted-4H-1,2,4-triazol-3-ylthio-1-(substitutedphenylethan-1-one derivatives, which were tested against Candida species. The synthesized compounds were characterized and elucidated by FT-IR, 1H-NMR, 13C-NMR and HR-MS spectroscopies. The synthesized compounds were screened in vitro anticandidal activity against Candida species by broth microdiluation methods. In vitro cytotoxic effects of the final compounds were determined by MTT assay. Microbiological studies revealed that compounds 5m, 5o, 5r, 5t, 5y, 5ab, and 5ad possess a good antifungal profile. Compounds 5w was the most active derivative and showed comparable antifungal activity to those of reference drugs ketoconazole and fluconazole. Cytotoxicity evaluation of compounds 5m, 5o, 5r, 5w, 5y, 5ab and 5ad showed that compounds 5w and 5ad were the least cytotoxic agents. Effects of these two compounds against ergosterol biosynthesis were observed by LC-MS-MS method, which is based on quantification of ergosterol level in C. albicans. Compounds 5w and 5d inhibited ergosterol biosynthesis concentration dependently. A fluorescence microscopy study was performed to visualize effect of compound 5w against C. albicans at cellular level. It was determined that compound 5w has a membrane damaging effect, which may be related with inhibition of biosynthesis of ergosterol.

Characterisation of anifrolumab, a fully human anti-interferon receptor antagonist antibody for the treatment of systemic lupus erythematosus

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Rajan, Bhargavi; Zerrouki, Kamelia; Karnell, Jodi L; Sagar, Divya; Vainshtein, Inna; Farmer, Erika; Rosenthal, Kimberly; Morehouse, Chris; de los Reyes, Melissa; Schifferli, Kevin; Liang, Meina; Sanjuan, Miguel A; Sims, Gary P; Kolbeck, Roland

2018-01-01

Objective We investigated the mechanistic and pharmacological properties of anifrolumab, a fully human, effector-null, anti-type I interferon (IFN) alpha receptor 1 (IFNAR1) monoclonal antibody in development for SLE. Methods IFNAR1 surface expression and internalisation on human monocytes before and after exposure to anifrolumab were assessed using confocal microscopy and flow cytometry. The effects of anifrolumab on type I IFN pathway activation were assessed using signal transducer and activator of transcription 1 (STAT1) phosphorylation, IFN-stimulated response element–luciferase reporter cell assays and type I IFN gene signature induction. The ability of anifrolumab to inhibit plasmacytoid dendritic cell (pDC) function and plasma cell differentiation was assessed by flow cytometry and ELISA. Effector-null properties of anifrolumab were assessed in antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) assays with B cells. Results Anifrolumab reduced cell surface IFNAR1 by eliciting IFNAR1 internalisation. Anifrolumab blocked type I IFN-dependent STAT1 phosphorylation and IFN-dependent signalling induced by recombinant and pDC-derived type I IFNs and serum of patients with SLE. Anifrolumab suppressed type I IFN production by blocking the type I IFN autoamplification loop and inhibited proinflammatory cytokine induction and the upregulation of costimulatory molecules on stimulated pDCs. Blockade of IFNAR1 suppressed plasma cell differentiation in pDC/B cell co-cultures. Anifrolumab did not exhibit CDC or ADCC activity. Conclusions Anifrolumab potently inhibits type I IFN-dependent signalling, including the type I IFN autoamplification loop, and is a promising therapeutic for patients with SLE and other diseases that exhibit chronic dysfunctional type I IFN signalling. PMID:29644082

The human CD38 monoclonal antibody daratumumab shows anti-tumor activity and hampers leukemia-microenvironment interactions in chronic lymphocytic leukemia

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Matas-Céspedes, Alba; Vidal-Crespo, Anna; Rodriguez, Vanina; Villamor, Neus; Delgado, Julio; Giné, Eva; Roca-Ho, Heleia; Menéndez, Pablo; Campo, Elías; López-Guillermo, Armando; Colomer, Dolors; Roué, Gaël; Wiestner, Adrian; Parren, Paul W.H.I.; Doshi, Parul; van Bueren, Jeroen Lammerts; Pérez-Galán, Patricia

2016-01-01

Purpose To establish a proof-of-concept for the efficacy of the anti-CD38 antibody daratumumab in the poor prognosis CD38+ CLL subtype. Experimental design The mechanism of action of daratumumab was assessed in CLL primary cells and cell lines using peripheral blood mononuclear cells to analyze antibody-dependent cell cytotoxicity (ADCC), murine and human macrophages to study antibody-dependent cell phagocytosis (ADCP) or human serum to analyze complement-dependent cytotoxicity (CDC). The effect of daratumumab on CLL cell migration and adhesion to extracellular matrix was characterized. Daratumumab activity was validated in two in vivo models. Results Daratumumab demonstrated efficient lysis of patient-derived CLL cells and cell lines by ADCC in vitro and ADCP both in vitro and in vivo, while exhibited negligible CDC in these cells. To demonstrate the therapeutic effect of daratumumab in CLL, we generated a disseminated CLL mouse model with the CD38+ MEC2 cell line and CLL patient derived xenografts (CLL-PDX). Daratumumab significantly prolonged overall survival of MEC2 mice, completely eliminated cells from the infiltrated organs and significantly reduced disease burden in the spleen of CLL-PDX. The effect of daratumumab on patient-derived CLL cell dissemination was demonstrated in vitro by its effect on CXCL12-induced migration and in vivo by interfering with CLL cell homing to spleen in NSG mice. Daratumumab also reduced adhesion of CLL cells to VCAM-1, accompanied by down-regulation of the matrix metalloproteinase MMP9. Conclusions These unique and substantial effects of daratumumab on CLL viability and dissemination support the investigation of its use in a clinical setting of CLL. PMID:27637890

Preclinical activity of the type II CD20 antibody GA101 (obinutuzumab) compared with rituximab and ofatumumab in vitro and in xenograft models.

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Herter, Sylvia; Herting, Frank; Mundigl, Olaf; Waldhauer, Inja; Weinzierl, Tina; Fauti, Tanja; Muth, Gunter; Ziegler-Landesberger, Doris; Van Puijenbroek, Erwin; Lang, Sabine; Duong, Minh Ngoc; Reslan, Lina; Gerdes, Christian A; Friess, Thomas; Baer, Ute; Burtscher, Helmut; Weidner, Michael; Dumontet, Charles; Umana, Pablo; Niederfellner, Gerhard; Bacac, Marina; Klein, Christian

2013-10-01

We report the first preclinical in vitro and in vivo comparison of GA101 (obinutuzumab), a novel glycoengineered type II CD20 monoclonal antibody, with rituximab and ofatumumab, the two currently approved type I CD20 antibodies. The three antibodies were compared in assays measuring direct cell death (AnnexinV/PI staining and time-lapse microscopy), complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and internalization. The models used for the comparison of their activity in vivo were SU-DHL4 and RL xenografts. GA101 was found to be superior to rituximab and ofatumumab in the induction of direct cell death (independent of mechanical manipulation required for cell aggregate disruption formed by antibody treatment), whereas it was 10 to 1,000 times less potent in mediating CDC. GA101 showed superior activity to rituximab and ofatumumab in ADCC and whole-blood B-cell depletion assays, and was comparable with these two in ADCP. GA101 also showed slower internalization rate upon binding to CD20 than rituximab and ofatumumab. In vivo, GA101 induced a strong antitumor effect, including complete tumor remission in the SU-DHL4 model and overall superior efficacy compared with both rituximab and ofatumumab. When rituximab-pretreated animals were used, second-line treatment with GA101 was still able to control tumor progression, whereas tumors escaped rituximab treatment. Taken together, the preclinical data show that the glyoengineered type II CD20 antibody GA101 is differentiated from the two approved type I CD20 antibodies rituximab and ofatumumab by its overall preclinical activity, further supporting its clinical investigation. ©2013 AACR.

Cellular intrinsic mechanism affecting the outcome of AML treated with Ara-C in a syngeneic mouse model.

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Wenjun Zhao

Full Text Available The mechanisms underlying acute myeloid leukemia (AML treatment failure are not clear. Here, we established a mouse model of AML by syngeneic transplantation of BXH-2 derived myeloid leukemic cells and developed an efficacious Ara-C-based regimen for treatment of these mice. We proved that leukemic cell load was correlated with survival. We also demonstrated that the susceptibility of leukemia cells to Ara-C could significantly affect the survival. To examine the molecular alterations in cells with different sensitivity, genome-wide expression of the leukemic cells was profiled, revealing that overall 366 and 212 genes became upregulated or downregulated, respectively, in the resistant cells. Many of these genes are involved in the regulation of cell cycle, cellular proliferation, and apoptosis. Some of them were further validated by quantitative PCR. Interestingly, the Ara-C resistant cells retained the sensitivity to ABT-737, an inhibitor of anti-apoptosis proteins, and treatment with ABT-737 prolonged the life span of mice engrafted with resistant cells. These results suggest that leukemic load and intrinsic cellular resistance can affect the outcome of AML treated with Ara-C. Incorporation of apoptosis inhibitors, such as ABT-737, into traditional cytotoxic regimens merits consideration for the treatment of AML in a subset of patients with resistance to Ara-C. This work provided direct in vivo evidence that leukemic load and intrinsic cellular resistance can affect the outcome of AML treated with Ara-C, suggesting that incorporation of apoptosis inhibitors into traditional cytotoxic regimens merits consideration for the treatment of AML in a subset of patients with resistance to Ara-C.

Cellular Uptake and Delivery of Myeloperoxidase to Lysosomes Promote Lipofuscin Degradation and Lysosomal Stress in Retinal Cells*

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Yogalingam, Gouri; Lee, Amanda R.; Mackenzie, Donald S.; Maures, Travis J.; Rafalko, Agnes; Prill, Heather; Berguig, Geoffrey Y.; Hague, Chuck; Christianson, Terri; Bell, Sean M.; LeBowitz, Jonathan H.

2017-01-01

Neutrophil myeloperoxidase (MPO) catalyzes the H2O2-dependent oxidation of chloride anion to generate hypochlorous acid, a potent antimicrobial agent. Besides its well defined role in innate immunity, aberrant degranulation of neutrophils in several inflammatory diseases leads to redistribution of MPO to the extracellular space, where it can mediate tissue damage by promoting the oxidation of several additional substrates. Here, we demonstrate that mannose 6-phosphate receptor-mediated cellular uptake and delivery of MPO to lysosomes of retinal pigmented epithelial (RPE) cells acts to clear this harmful enzyme from the extracellular space, with lysosomal-delivered MPO exhibiting a half-life of 10 h. Lysosomal-targeted MPO exerts both cell-protective and cytotoxic functions. From a therapeutic standpoint, MPO catalyzes the in vitro degradation of N-retinylidene-N-retinylethanolamine, a toxic form of retinal lipofuscin that accumulates in RPE lysosomes and drives the pathogenesis of Stargardt macular degeneration. Furthermore, chronic cellular uptake and accumulation of MPO in lysosomes coincides with N-retinylidene-N-retinylethanolamine elimination in a cell-based model of macular degeneration. However, lysosomal-delivered MPO also disrupts lysosomal acidification in RPE cells, which coincides with nuclear translocation of the lysosomal stress-sensing transcription factor EB and, eventually, cell death. Based on these findings we predict that under periods of acute exposure, cellular uptake and lysosomal degradation of MPO mediates elimination of this harmful enzyme, whereas chronic exposure results in progressive accumulation of MPO in lysosomes. Lysosomal-accumulated MPO can be both cell-protective, by promoting the degradation of toxic retinal lipofuscin deposits, and cytotoxic, by triggering lysosomal stress and cell death. PMID:28115520

Cellular Uptake and Delivery of Myeloperoxidase to Lysosomes Promote Lipofuscin Degradation and Lysosomal Stress in Retinal Cells.

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Yogalingam, Gouri; Lee, Amanda R; Mackenzie, Donald S; Maures, Travis J; Rafalko, Agnes; Prill, Heather; Berguig, Geoffrey Y; Hague, Chuck; Christianson, Terri; Bell, Sean M; LeBowitz, Jonathan H

2017-03-10

Neutrophil myeloperoxidase (MPO) catalyzes the H 2 O 2 -dependent oxidation of chloride anion to generate hypochlorous acid, a potent antimicrobial agent. Besides its well defined role in innate immunity, aberrant degranulation of neutrophils in several inflammatory diseases leads to redistribution of MPO to the extracellular space, where it can mediate tissue damage by promoting the oxidation of several additional substrates. Here, we demonstrate that mannose 6-phosphate receptor-mediated cellular uptake and delivery of MPO to lysosomes of retinal pigmented epithelial (RPE) cells acts to clear this harmful enzyme from the extracellular space, with lysosomal-delivered MPO exhibiting a half-life of 10 h. Lysosomal-targeted MPO exerts both cell-protective and cytotoxic functions. From a therapeutic standpoint, MPO catalyzes the in vitro degradation of N -retinylidene- N -retinylethanolamine, a toxic form of retinal lipofuscin that accumulates in RPE lysosomes and drives the pathogenesis of Stargardt macular degeneration. Furthermore, chronic cellular uptake and accumulation of MPO in lysosomes coincides with N -retinylidene- N -retinylethanolamine elimination in a cell-based model of macular degeneration. However, lysosomal-delivered MPO also disrupts lysosomal acidification in RPE cells, which coincides with nuclear translocation of the lysosomal stress-sensing transcription factor EB and, eventually, cell death. Based on these findings we predict that under periods of acute exposure, cellular uptake and lysosomal degradation of MPO mediates elimination of this harmful enzyme, whereas chronic exposure results in progressive accumulation of MPO in lysosomes. Lysosomal-accumulated MPO can be both cell-protective, by promoting the degradation of toxic retinal lipofuscin deposits, and cytotoxic, by triggering lysosomal stress and cell death. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Dysregulated human Tyrosyl-DNA phosphodiesterase I acts as cellular toxin

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Cuya, Selma M.; Comeaux, Evan Q.; Wanzeck, Keith; Yoon, Karina J.; van Waardenburg, Robert C.A.M.

2016-01-01

Tyrosyl-DNA phosphodiesterase I (TDP1) hydrolyzes the drug-stabilized 3’phospho-tyrosyl bond formed between DNA topoisomerase I (TOPO1) and DNA. TDP1-mediated hydrolysis uses a nucleophilic histidine (Hisnuc) and a general acid/base histidine (Hisgab). A Tdp1Hisgab to Arg mutant identified in patients with the autosomal recessive neurodegenerative disease SCAN1 causes stabilization of the TDP1-DNA intermediate. Based on our previously reported Hisgab-substitutions inducing yeast toxicity (Gajewski et al. J. Mol. Biol. 415, 741-758, 2012), we propose that converting TDP1 into a cellular poison by stabilizing the covalent enzyme-DNA intermediate is a novel therapeutic strategy for cancer treatment. Here, we analyzed the toxic effects of two TDP1 catalytic mutants in HEK293 cells. Expression of human Tdp1HisnucAla and Tdp1HisgabAsn mutants results in stabilization of the covalent TDP1-DNA intermediate and induces cytotoxicity. Moreover, these mutants display reduced in vitro catalytic activity compared to wild type. Co-treatment of Tdp1mutant with topotecan shows more than additive cytotoxicity. Overall, these results support the hypothesis that stabilization of the TDP1-DNA covalent intermediate is a potential anti-cancer therapeutic strategy. PMID:27893431

Cellular defense against UVB-induced phototoxicity by cytosolic NADP+-dependent isocitrate dehydrogenase

International Nuclear Information System (INIS)

Jo, Seung-Hee; Lee, So-Hyun; Suk Chun, Hang; Min Lee, Su; Koh, Ho-Jin; Lee, Sung-Eun; Chun, Jang-Soo; Park, Jeen-Woo; Huh, Tae-Lin

2002-01-01

Ultraviolet (UV) radiation is known as a major cause of skin photoaging and photocarcinogenesis. Many harmful effects of UV radiation are associated with the generation of reactive oxygen species. Recently, we have shown that NADP + -dependent isocitrate dehydrogenase is involved in the supply of NADPH needed for GSH production against cellular oxidative damage. In this study we investigated the role of cytosolic form of NADP + -dependent isocitrate dehydrogenase (IDPc) against UV radiation-induced cytotoxicity by comparing the relative degree of cellular responses in three different NIH3T3 cells with stable transfection with the cDNA for mouse IDPc in sense and antisense orientations, where IDPc activities were 2.3-fold higher and 39% lower, respectively, than that in the parental cells carrying the vector alone. Upon exposure to UVB (312 nm), the cells with low levels of IDPc became more sensitive to cell killing. Lipid peroxidation, protein oxidation, oxidative DNA damage, and intracellular peroxide generation were higher in the cell-line expressing the lower level of IDPc. However, the cells with the highly overexpressed IDPc exhibited enhanced resistance against UV radiation, compared to the control cells. The data indicate that IDPc plays an important role in cellular defense against UV radiation-induced oxidative injury

Cellular defense against UVB-induced phototoxicity by cytosolic NADP(+)-dependent isocitrate dehydrogenase.

Science.gov (United States)

Jo, Seung-Hee; Lee, So-Hyun; Chun, Hang Suk; Lee, Su Min; Koh, Ho-Jin; Lee, Sung-Eun; Chun, Jang-Soo; Park, Jeen-Woo; Huh, Tae-Lin

2002-03-29

Ultraviolet (UV) radiation is known as a major cause of skin photoaging and photocarcinogenesis. Many harmful effects of UV radiation are associated with the generation of reactive oxygen species. Recently, we have shown that NADP(+)-dependent isocitrate dehydrogenase is involved in the supply of NADPH needed for GSH production against cellular oxidative damage. In this study we investigated the role of cytosolic form of NADP(+)-dependent isocitrate dehydrogenase (IDPc) against UV radiation-induced cytotoxicity by comparing the relative degree of cellular responses in three different NIH3T3 cells with stable transfection with the cDNA for mouse IDPc in sense and antisense orientations, where IDPc activities were 2.3-fold higher and 39% lower, respectively, than that in the parental cells carrying the vector alone. Upon exposure to UVB (312 nm), the cells with low levels of IDPc became more sensitive to cell killing. Lipid peroxidation, protein oxidation, oxidative DNA damage, and intracellular peroxide generation were higher in the cell-line expressing the lower level of IDPc. However, the cells with the highly overexpressed IDPc exhibited enhanced resistance against UV radiation, compared to the control cells. The data indicate that IDPc plays an important role in cellular defense against UV radiation-induced oxidative injury. (c)2002 Elsevier Science (USA).

Aldose reductase regulates acrolein-induced cytotoxicity in human small airway epithelial cells.

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Yadav, Umesh C S; Ramana, K V; Srivastava, Satish K

2013-12-01

Aldose reductase (AR), a glucose-metabolizing enzyme, reduces lipid aldehydes and their glutathione conjugates with more than 1000-fold efficiency (Km aldehydes 5-30 µM) relative to glucose. Acrolein, a major endogenous lipid peroxidation product as well as a component of environmental pollutants and cigarette smoke, is known to be involved in various pathologies including atherosclerosis, airway inflammation, COPD, and age-related disorders, but the mechanism of acrolein-induced cytotoxicity is not clearly understood. We have investigated the role of AR in acrolein-induced cytotoxicity in primary human small airway epithelial cells (SAECs). Exposure of SAECs to varying concentrations of acrolein caused cell death in a concentration- and time-dependent manner. AR inhibition by fidarestat prevented the low-dose (5-10 µM) but not the high-dose (>10 µM) acrolein-induced SAEC death. AR inhibition protected SAECs from low-dose (5 µM) acrolein-induced cellular reactive oxygen species (ROS). Inhibition of acrolein-induced apoptosis by fidarestat was confirmed by decreased condensation of nuclear chromatin, DNA fragmentation, comet tail moment, and annexin V fluorescence. Further, fidarestat inhibited acrolein-induced translocation of the proapoptotic proteins Bax and Bad from the cytosol to the mitochondria and that of Bcl2 and BclXL from the mitochondria to the cytosol. Acrolein-induced cytochrome c release from mitochondria was also prevented by AR inhibition. The mitogen-activated protein kinases (MAPKs), such as extracellular signal-regulated kinases 1 and 2, stress-activated protein kinase/c-Jun NH2-terminal kinase, and p38MAPK, and c-Jun were transiently activated in airway epithelial cells by acrolein in a concentration- and time-dependent fashion, which was significantly prevented by AR inhibition. These results suggest that AR inhibitors could prevent acrolein-induced cytotoxicity in the lung epithelial cells. Copyright © 2013 Elsevier Inc. All rights

Aldose reductase regulates acrolein-induced cytotoxicity in human small airway epithelial cells

Science.gov (United States)

Yadav, Umesh CS; Ramana, KV; Srivastava, SK

2013-01-01

Aldose reductase (AR), a glucose metabolizing enzyme, reduces lipid aldehydes and their glutathione conjugates with more than 1000-fold efficiency (Km aldehydes 5-30μM) than glucose. Acrolein, a major endogenous lipid peroxidation product as well as component of environmental pollutant and cigarette smoke, is known to be involved in various pathologies including atherosclerosis, airway inflammation, COPD, and age-related disorders but the mechanism of acrolein-induced cytotoxicity is not clearly understood. We have investigated the role of AR in acrolein-induced cytotoxicity in primary human small airway epithelial cells SAECs. Exposure of SAECs to varying concentrations of acrolein caused cell-death in a concentration- and time-dependent manner. AR inhibition by fidarestat prevented the low (5 to 10 μM) but not high (>10 μM) concentrations of acrolein-induced SAECs cell death. AR inhibition protected SAECs from low dose (5 μM) acrolein-induced cellular reactive oxygen species (ROS). Inhibition of acrolein-induced apoptosis by fidarestat was confirmed by decreased condensation of nuclear chromatin, DNA fragmentation, comet tail-moment, and annexin-V fluorescence. Further, fidarestat inhibited acrolein-induced translocation of pro-apoptotic proteins Bax and Bad from cytosol to the mitochondria, and that of Bcl2 and BclXL from mitochondria to cytosol. Acrolein-induced cytochrome c release from mitochondria was also prevented by AR inhibition. The mitogen-activated protein kinases (MAPK) such as extracellular signal-regulated kinases 1 and 2 (ERK1/2), stress-activated protein kinases/c-jun NH2-terminal kinases (SAPK/JNK) and p38MAPK, and c-jun were transiently activated in airway epithelial cells by acrolein in a concentration and time-dependent fashion, which were significantly prevented by AR inhibition. These results suggest that AR inhibitors could prevent acrolein-induced cytotoxicity in the lung epithelial cells. PMID:23770200

Difference in effect of single immunosuppressive agents (cyclophosphamide, CCNU, 5-FU) on peripheral blood immune cell parameters and central nervous system immunoglobulin synthesis rate in patients with multiple sclerosis.

Science.gov (United States)

Shih, W W; Baumhefner, R W; Tourtellotte, W W; Haskell, C M; Korn, E L; Fahey, J L

1983-01-01

Cyclophosphamide (CY), 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) and 5-fluorouracil (5-FU) were given in single course schedules to chronic progressive multiple sclerosis (MS) patients clinically stable for 6 months. The following peripheral immune cellular parameters were measured before, during and after each drug administration: white blood count (WBC), polymorphonuclear count (PMN), lymphocyte count, percentage of T cells, T cell response to phytohaemagglutinin (PHA), percentage of B cells, percentage of cells bearing receptors for the Fc portion of immunoglobulin (% FcR cells), killer (K) cell activity defined by antibody-dependent cellular cytotoxicity (ADCC), and natural killer (NK) cell activity. Central nervous system (CNS) immunoglobulin G (IgG) synthesis was also measured. The patients were followed carefully by both quantitative and qualitative methods for any change in their neurologic condition. Selective reduction in NK activity was observed with CY and 5-FU while no significant alteration was seen in %FcR cells and K activity. CY differed from 5-FU in reducing lymphocyte count and B cell percentage while 5-FU decreased the percentage of T cells. CCNU, but not the other drugs, reduced T cell proliferative response to PHA. In addition, CCNU, which is known to penetrate well into the nervous system, caused a modest reduction in CNS IgG synthesis, while 5-FU had an uncertain effect. Clinically the patients were unchanged or continued to progress in their disability. The results suggest an independence of the CNS immune from the systemic immune system in MS in response to many immunosuppressive drugs. PMID:6603303

Natural cytolytic activity in mice with natural or induced cellular defects. I. Differential ability of in vitro interleukin-2 addition to augment natural cytolytic function

International Nuclear Information System (INIS)

Ades, E.W.; Hinson, A.; Butler, L.D.

1986-01-01

The ability of in vitro addition of recombinant interleukin 2 (rIL-2) to differentially enhance natural cytotoxicity was assessed using cells from mice with natural and induced cellular defects. In vivo treatment with most immunosuppressive or cytoreductive agents, anti-asialo-GM1 antibody, or gamma irradiation dramatically reduced in vitro cytotoxicity against natural killer (NK) sensitive targets by direct reduction in either percentage specific lysis or lytic units per spleen. In most cases, in vitro addition of rIL-2 (at concentrations causing augmented NK function in cells from naive Balb/C mice) enhanced cytotoxic activity of cells from treatment groups to a normal value but not within the rIL-2-enhanced range of nontreated animals. Additionally, cytotoxic activity of cells from animals treated with certain drugs or gamma irradiation could be augmented by rIL-2 when measured by percentage lysis but not lytic units per spleen. In vivo treatment with cyclosporin A did not affect natural cytotoxic activity and addition of rIL-2 augmented the NK activity in a similar fashion to the profile of naive cells. In experiments using cells from beige (C57Bl/6-bg) mice which have a natural defect in NK activity against YAC-1 targets, addition of rIL-2 (at concentrations causing augmented natural cytotoxic function in cells from C57Bl/6 mice) could not effectively enhance in vitro natural cytotoxic function

Natural cytolytic activity in mice with natural or induced cellular defects. I. Differential ability of in vitro interleukin-2 addition to augment natural cytolytic function

Energy Technology Data Exchange (ETDEWEB)

Ades, E.W.; Hinson, A.; Butler, L.D.

1986-08-01

The ability of in vitro addition of recombinant interleukin 2 (rIL-2) to differentially enhance natural cytotoxicity was assessed using cells from mice with natural and induced cellular defects. In vivo treatment with most immunosuppressive or cytoreductive agents, anti-asialo-GM1 antibody, or gamma irradiation dramatically reduced in vitro cytotoxicity against natural killer (NK) sensitive targets by direct reduction in either percentage specific lysis or lytic units per spleen. In most cases, in vitro addition of rIL-2 (at concentrations causing augmented NK function in cells from naive Balb/C mice) enhanced cytotoxic activity of cells from treatment groups to a normal value but not within the rIL-2-enhanced range of nontreated animals. Additionally, cytotoxic activity of cells from animals treated with certain drugs or gamma irradiation could be augmented by rIL-2 when measured by percentage lysis but not lytic units per spleen. In vivo treatment with cyclosporin A did not affect natural cytotoxic activity and addition of rIL-2 augmented the NK activity in a similar fashion to the profile of naive cells. In experiments using cells from beige (C57Bl/6-bg) mice which have a natural defect in NK activity against YAC-1 targets, addition of rIL-2 (at concentrations causing augmented natural cytotoxic function in cells from C57Bl/6 mice) could not effectively enhance in vitro natural cytotoxic function.

PLGA-soya lecithin based micelles for enhanced delivery of methotrexate: Cellular uptake, cytotoxic and pharmacokinetic evidences.

Science.gov (United States)

Singh, Anupama; Thotakura, Nagarani; Kumar, Rajendra; Singh, Bhupinder; Sharma, Gajanand; Katare, Om Prakash; Raza, Kaisar

2017-02-01

Biocompatible and biodegradable polymers like PLGA have revolutionized the drug delivery approaches. However, poor drug loading and substantially high lipophilicity, pave a path for further tailing of this promising agent. In this regard, PLGA was feathered with biocompatible phospholipid and polymeric micelles were developed for delivery of Methotrexate (MTX) to cancer cells. The nanocarriers (114.6nm±5.5nm) enhanced the cytotoxicity of MTX by 2.13 folds on MDA-MB-231 cells. Confocal laser scanning microscopy confirmed the increased intracellular delivery. The carrier decreased the protein binding potential and enhanced the bioavailable fraction of MTX. Pharmacokinetic studies vouched substantial enhancement in AUC and bioresidence time, promising an ideal carrier to effectively deliver the drug to the site of action. The developed nanocarriers offer potential to deliver the drug in the interiors of cancer cells in an effective manner for improved therapeutic action. Copyright © 2016 Elsevier B.V. All rights reserved.

Degenerate wave and capacitive coupling increase human MSC invasion and proliferation while reducing cytotoxicity in an in vitro wound healing model.

Directory of Open Access Journals (Sweden)

Michelle Griffin

Full Text Available Non-unions pose complications in fracture management that can be treated using electrical stimulation (ES. Bone marrow mesenchymal stem cells (BMMSCs are essential in fracture healing; however, the effect of different clinical ES waveforms on BMMSCs cellular activities remains unknown. We compared the effects of direct current (DC, capacitive coupling (CC, pulsed electromagnetic field (PEMF and degenerate wave (DW on cellular activities including cytotoxicity, proliferation, cell-kinetics and apoptosis by stimulating human-BMMSCs 3 hours a day, up to 5 days. In addition, migration and invasion were assessed using fluorescence microscopy and by quantifying gene and protein expression. We found that DW had the greatest proliferative and least apoptotic and cytotoxic effects compared to other waveforms. DC, DW and CC stimulations resulted in a higher number of cells in S phase and G(2/M phase as shown by cell cycle analysis. CC and DW caused more cells to invade collagen and showed increased MMP-2 and MT1-MMP expression. DC increased cellular migration in a scratch-wound assay and all ES waveforms enhanced expression of migratory genes with DC having the greatest effect. All ES treated cells showed similar progenitor potential as determined by MSC differentiation assay. All above findings were shown to be statistically significant (p<0.05. We conclude that ES can influence BMMSCs activities, especially DW and CC, which show greater invasion and higher cell proliferation compared to other types of ES. Application of DW or CC to the fracture site may help in the recruitment of BMMSCs to the wound that may enhance rate of bone healing at the fracture site.

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

International Nuclear Information System (INIS)

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

2004-01-01

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

Overexpression of the Anthocyanidin Synthase Gene in Strawberry Enhances Antioxidant Capacity and Cytotoxic Effects on Human Hepatic Cancer Cells.

Science.gov (United States)

Giampieri, Francesca; Gasparrini, Massimiliano; Forbes-Hernandez, Tamara Y; Mazzoni, Luca; Capocasa, Franco; Sabbadini, Silvia; Alvarez-Suarez, Josè M; Afrin, Sadia; Rosati, Carlo; Pandolfini, Tiziana; Molesini, Barbara; Sánchez-Sevilla, José F; Amaya, Iraida; Mezzetti, Bruno; Battino, Maurizio

2018-01-24

Food fortification through the increase and/or modulation of bioactive compounds has become a major goal for preventing several diseases, including cancer. Here, strawberry lines of cv. Calypso transformed with a construct containing an anthocyanidin synthase (ANS) gene were produced to study the effects on anthocyanin biosynthesis, metabolism, and transcriptome. Three strawberry ANS transgenic lines (ANS L5, ANS L15, and ANS L18) were analyzed for phytochemical composition and total antioxidant capacity (TAC), and their fruit extracts were assessed for cytotoxic effects on hepatocellular carcinoma. ANS L18 fruits had the highest levels of total phenolics and flavonoids, while those of ANS L15 had the highest anthocyanin concentration; TAC positively correlated with total polyphenol content. Fruit transcriptome was also specifically affected in the polyphenol biosynthesis and in other related metabolic pathways. Fruit extracts of all lines exerted cytotoxic effects in a dose/time-dependent manner, increasing cellular apoptosis and free radical levels and impairing mitochondrial functionality.

Improved cytotoxicity of paclitaxel loaded in nanosized lipid carriers by intracellular delivery

Energy Technology Data Exchange (ETDEWEB)

Miao, Jing, E-mail: joemj1005@163.com, E-mail: miaojing@zju.edu.cn [Zhejiang University, Department of Pharmacy, the First Affiliated Hospital, College of Medicine (China); Du, Yongzhong; Yuan, Hong [Zhejiang University, College of Pharmaceutical Sciences (China); Zhang, Xingguo; Li, Qian; Rao, Yuefeng [Zhejiang University, Department of Pharmacy, the First Affiliated Hospital, College of Medicine (China); Zhao, Mengdan [Zhejiang University, Women’s Hospital, College of Medicine (China); Hu, Fuqiang, E-mail: hufq@zju.edu.cn [Zhejiang University, College of Pharmaceutical Sciences (China)

2015-01-15

Nanosized lipid carriers (NLC) can improve the limited drug-loading (DL) capacity and drug expulsion during storage, and adjust the drug release profile of solid lipid nanoparticles (SLN). In this study, Paclitaxel (PTX)-loaded NLC were prepared by solvent diffusion method using monostearin as solid lipid and oleic acid (OA) as liquid lipid matrix. The blank NLC with different OA content (the size range was from 89.5 ± 7.4 to 160.2 ± 34.6 nm) showed smaller size than the blank SLN (the size was 272.7 ± 43.6 nm), while the PTX-loaded NLC (the size range was from 481.3 ± 29.8 to 561.7 ± 38.3 nm) showed little bigger size, higher DL capacity, and faster drug in vitro release rate comparing with SLN (the size was 437.3 ± 68.2 nm). The 50 % cellular growth inhibitions (IC{sub 50}) of PTX-loaded NLC with 0, 5, 10, and 20 wt % OA were 0.92 ± 0.06, 0.69 ± 0.04, 0.25 ± 0.02, and 0.12 ± 0.02 µg mL{sup −1}, respectively, while the IC{sub 50} of Taxol{sup TM} was 1.72 ± 0.09 µg mL{sup −1}. For analyzing cellular drug effect, cellular uptakes of fluorescent NLC and intracellular drug concentration were investigated. As the incorporation of OA into solid lipid matrix could accelerate both the cellular uptake and the PTX delivery, loaded by NLC, the cytotoxicity of PTX could be enhanced, and further enhanced by increasing OA content in NLC.

Improved cytotoxicity of paclitaxel loaded in nanosized lipid carriers by intracellular delivery

International Nuclear Information System (INIS)

Miao, Jing; Du, Yongzhong; Yuan, Hong; Zhang, Xingguo; Li, Qian; Rao, Yuefeng; Zhao, Mengdan; Hu, Fuqiang

2015-01-01

Nanosized lipid carriers (NLC) can improve the limited drug-loading (DL) capacity and drug expulsion during storage, and adjust the drug release profile of solid lipid nanoparticles (SLN). In this study, Paclitaxel (PTX)-loaded NLC were prepared by solvent diffusion method using monostearin as solid lipid and oleic acid (OA) as liquid lipid matrix. The blank NLC with different OA content (the size range was from 89.5 ± 7.4 to 160.2 ± 34.6 nm) showed smaller size than the blank SLN (the size was 272.7 ± 43.6 nm), while the PTX-loaded NLC (the size range was from 481.3 ± 29.8 to 561.7 ± 38.3 nm) showed little bigger size, higher DL capacity, and faster drug in vitro release rate comparing with SLN (the size was 437.3 ± 68.2 nm). The 50 % cellular growth inhibitions (IC 50 ) of PTX-loaded NLC with 0, 5, 10, and 20 wt % OA were 0.92 ± 0.06, 0.69 ± 0.04, 0.25 ± 0.02, and 0.12 ± 0.02 µg mL −1 , respectively, while the IC 50 of Taxol TM was 1.72 ± 0.09 µg mL −1 . For analyzing cellular drug effect, cellular uptakes of fluorescent NLC and intracellular drug concentration were investigated. As the incorporation of OA into solid lipid matrix could accelerate both the cellular uptake and the PTX delivery, loaded by NLC, the cytotoxicity of PTX could be enhanced, and further enhanced by increasing OA content in NLC

The antioxidant properties, cytotoxicity and monoamine oxidase ...

African Journals Online (AJOL)

ajl yemi

2011-11-28

Nov 28, 2011 ... and the nitroblue tetrazolium (NBT) assay. The cytotoxicity ... The antioxidant activity and cytotoxic effect of the extracts increased with increase ... supplements are concoctions of plants and/or plant .... In vitro antioxidant assay.

Putrescine-dependent re-localization of TvCP39, a cysteine proteinase involved in Trichomonas vaginalis cytotoxicity.

Directory of Open Access Journals (Sweden)

Bertha Isabel Carvajal-Gamez

Full Text Available Polyamines are involved in the regulation of some Trichomonas vaginalis virulence factors such as the transcript, proteolytic activity, and cytotoxicity of TvCP65, a cysteine proteinase (CP involved in the trichomonal cytotoxicity. In this work, we reported the putrescine effect on TvCP39, other CP that also participate in the trichomonal cytotoxicity. Parasites treated with 1,4-diamino-2-butanone (DAB (an inhibitor of putrescine biosynthesis, diminished the amount and proteolytic activity of TvCP39 as compared with untreated parasites. Inhibition of putrescine biosynthesis also reduced ∼ 80% the tvcp39 mRNA levels according to RT-PCR and qRT-PCR assays. Additionally, actinomycin D-treatment showed that the tvcp39 mRNA half-life decreased in the absence of putrescine. However, this reduction was restored by exogenous putrescine addition, suggesting that putrescine is necessary for tvcp39 mRNA stability. TvCP39 was localized in the cytoplasm but, in DAB treated parasites transferred into exogenous putrescine culture media, TvCP39 was re-localized to the nucleus and nuclear periphery of trichomonads. Interestingly, the amount and proteolytic activity of TvCP39 was recovered as well as the tvcp39 mRNA levels were restored when putrescine exogenous was added to the DAB-treated parasites. In conclusion, our data show that putrescine regulate the TvCP39 expression, protein amount, proteolytic activity, and cellular localization.

Cellular Uptake and Photo-Cytotoxicity of a Gadolinium(III-DOTA-Naphthalimide Complex “Clicked” to a Lipidated Tat Peptide

Directory of Open Access Journals (Sweden)

William I. O’Malley

2016-02-01

Full Text Available A new bifunctional macrocyclic chelator featuring a conjugatable alkynyl-naphthalimide fluorophore pendant group has been prepared and its Gd(III complex coupled to a cell-penetrating lipidated azido-Tat peptide derivative using Cu(I-catalysed “click” chemistry. The resulting fluorescent conjugate is able to enter CAL-33 tongue squamous carcinoma cells, as revealed by confocal microscopy, producing a very modest anti-proliferative effect (IC50 = 93 µM. Due to the photo-reactivity of the naphthalimide moiety, however, the conjugate’s cytotoxicity is significantly enhanced (IC50 = 16 µM upon brief low-power UV-A irradiation.

Preventing hepatocyte oxidative stress cytotoxicity with Mangifera indica L. extract (Vimang).

Science.gov (United States)

Remirez, Diadelis; Tafazoli, Shahrzad; Delgado, Rene; Harandi, Asghar A; O'Brien, Peter J

2005-01-01

Vimang is an aqueous extract of Mangifera indica used in Cuba to improve the quality of life in patients suffering from inflammatory diseases. In the present study we evaluated the effects of Vimang at preventing reactive oxygen species (ROS) formation and lipid peroxidation in intact isolated rat hepatocytes. Vimang at 20, 50 and 100 microg/ml inhibited hepatocyte ROS formation induced by glucose-glucose oxidase. Hepatocyte cytotoxicity and lipid peroxidation induced by cumene hydroperoxide was also inhibited by Vimang in a dose and time dependent manner at the same concentration. Vimang also inhibited superoxide radical formation by xanthine oxidase and hypoxanthine. The superoxide radical scavenging and antioxidant activity of the Vimang extract was likely related to its gallates, catechins and mangiferin content. To our knowledge, this is the first report of cytoprotective antioxidant effects of Vimang in cellular oxidative stress models.

Photolabile ruthenium complexes to cage and release a highly cytotoxic anticancer agent.

Science.gov (United States)

Wei, Jianhua; Renfrew, Anna K

2018-02-01

CHS-828 (N-(6-(4-chlorophenoxy)hexyl)-N'-cyano-N″-4-pyridyl guanidine) is an anticancer agent with low bioavailability and high systemic toxicity. Here we present an approach to improve the therapeutic profile of the drug using photolabile ruthenium complexes to generate light-activated prodrugs of CHS-828. Both prodrug complexes are stable in the dark but release CHS-828 when irradiated with visible light. The complexes are water-soluble and accumulate in tumour cells in very high concentrations, predominantly in the mitochondria. Both prodrug complexes are significantly less cyototoxic than free CHS-828 in the dark but their toxicity increases up to 10-fold in combination with visible light. The cellular responses to light treatment are consistent with release of the cytotoxic CHS-828 ligand. Copyright © 2017 Elsevier Inc. All rights reserved.

Cytotoxic effects of TBBPA and its interactions with signalling pathways in mammalian cells

Energy Technology Data Exchange (ETDEWEB)

Strack, S.; Sander, M.; Detzel, T.; Krug, H.F. [Forschungszentrum Kalsruhe (Germany). Inst. fuer Toxikologie und Genetik; Kuch, B. [Stuttgart Univ. (Germany). Inst. fuer Siedlungswasserbau und Wasserguetewirtschaft

2004-09-15

Toxic effects of TBBPA published so far have been recently reviewed by Birnbaum and Staskal The LC{sub 50} indicating the acute toxicity in vivo due to a single oral dose in mice and rats were higher than 4 to 5 g/kg, however, systematically long-term in vivo studies are missing. Weak estrogenic effects have been described by Meerts et al., demonstrating for TBBPA less pronounced activity than for other brominated bisphenols. The same group described competitive interactions in vitro with human transthyretin (TTR). In binding affinity assays they could demonstrate that TBBPA binds to TTR ten times more effectively than T{sub 4}. However, the available toxicological data are still extremely limited. For a comprehensive risk assessment valid data are insufficient. The aim of this study was to evaluate possible cytotoxic effects, and to gain insights into the underlying molecular mechanisms respectively the corresponding cellular signalling processes. This approach would allow to identify sensitive end-points of cellular toxicological responses. For these molecular toxicological investigations established cell lines should be used, in order to have a suitable model for appropriate toxicological studies.

Reducing the Cytotoxicity of Lipid Nanoparticles Associated with a Fusogenic Cationic Lipid in a Natural Killer Cell Line by Introducing a Polycation-Based siRNA Core.

Science.gov (United States)

Nakamura, Takashi; Yamada, Koharu; Fujiwara, Yuki; Sato, Yusuke; Harashima, Hideyoshi

2018-06-04

Introducing siRNA into human immune cells by an artificial delivery system continues to be a challenging issue. We previously developed a multifunctional envelope-type nanodevice (MEND) containing the YSK12-C4, a fusogenic cationic lipid, (YSK12-MEND) and succeeded in the efficient delivery of siRNA into human immune cell lines. Significant cytotoxicity, however, was observed at siRNA doses needed for gene silencing in NK-92 cells. NK-92 cells, a unique natural killer (NK) cell line, would be applicable for use in clinical NK therapy. Thus, reducing the cytotoxicity of the YSK12-MEND in NK-92 cells would strengthen the efficacy of NK-92 cell-based therapy. The amount of the YSK12-C4 in the MEND needed to be reduced to reduce the cytotoxicity, because the cytotoxicity was directly associated with the YSK12-C4. In the present study, we decreased the total amount of lipid, including the YSK12-C4, by introducing a core formed by electrostatic interactions of siRNA with a polycation (protamine) (siRNA core), which led to a decrease in cytotoxicity in NK-92 cells. We prepared a YSK12-MEND containing an siRNA core (YSK12-MEND/core) at charge ratios (CR: YSK12-C4/siRNA) of 10, 5, 3, and 2.5 and compared the YSK12-MEND/core with that for a YSK12-MEND (CR16.9). Cell viability was increased by more than 2 times at a CR5 or less. On the other hand, the YSK12-MEND/core (CR5) maintained the same gene silencing efficiency (60%) as the YSK12-MEND. Interestingly, the cellular uptake efficiency and hemolytic activity of the YSK12-MEND/core (CR5) was reduced compared to that for the YSK12-MEND. In calculating the silencing activity per cellular uptake efficiency and hemolytic activity, the value for the YSK12-MEND/core (CR5) was more than 2 times as high as that of the YSK12-MEND. The fact indicates that after endosomal escape, the process can be enhanced by using a YSK12-MEND/core (CR5). Thus, introducing an siRNA core into lipid nanoparticles can be a potent strategy for decreasing

Cellular compatibility of highly degradable bioactive ceramics for coating of metal implants.

Science.gov (United States)

Radetzki, F; Wohlrab, D; Zeh, A; Delank, K S; Mendel, T; Berger, G; Syrowatka, F; Mayr, O; Bernstein, A

2011-01-01

Resorbable ceramics can promote the bony integration of implants. Their rate of degradation should ideally be synchronized with bone regeneration. This study examined the effect of rapidly resorbable calcium phosphate ceramics 602020, GB14, 305020 on adherence, proliferation and morphology of human bone-derived cells (HBDC) in comparison to β-TCP. The in vitro cytotoxicity was determined by the microculture tetrazolium (MTT) assay. HBDC were grown on the materials for 3, 7, 11, 15 and 19 days and counted. Cell morphology, cell attachment, cell spreading and the cytoskeletal organization of HBDC cultivated on the substrates were investigated using laser scanning microscopy and environmental scanning electron microscopy. All substrates supported sufficient cellular growth for 19 days and showed no cytotoxicity. On each material an identical cell colonisation of well communicating, polygonal, vital cells with strong focal contacts was verified. HBDC showed numerous well defined stress fibres which give proof of well spread and strongly anchored cells. Porous surfaces encouraged the attachment and spreading of HBDC. Further investigations regarding long term biomaterial/cell interactions in vitro and in vivo are required to confirm the utility of the new biomaterials.

A cytotoxic serine proteinase isolated from mouse submandibular gland.

Science.gov (United States)

Shimamura, T; Nagumo, N; Ikigai, H; Murakami, K; Okubo, S; Toda, M; Ohnishi, R; Tomita, M

1989-08-01

We have isolated a novel cytotoxic factor from the submandibular glands of male BALB/c mice by Sephadex G-50 gel filtration chromatography and reverse-phase HPLC. The cytotoxic factor is a serine proteinase, which belongs to the mouse glandular kallikrein (mGK) family, with an Mr of approximately 27,000. The purified serine proteinase showed cytotoxic activity against mouse thymocytes in a dose-dependent manner, and a serine proteinase inhibitor, diisopropyl fluorophosphate, blocked its cytotoxic activity.

Aqueous Synthesis of PEGylated Quantum Dots with Increased Colloidal Stability and Reduced Cytotoxicity.

Science.gov (United States)

Ulusoy, Mehriban; Jonczyk, Rebecca; Walter, Johanna-Gabriela; Springer, Sergej; Lavrentieva, Antonina; Stahl, Frank; Green, Mark; Scheper, Thomas

2016-02-17

Ligands used on the surface of colloidal nanoparticles (NPs) have a significant impact on physiochemical properties of NPs and their interaction in biological environments. In this study, we report a one-pot aqueous synthesis of 3-mercaptopropionic acid (MPA)-functionalized CdTe/CdS/ZnS quantum dots (Qdots) in the presence of thiol-terminated methoxy polyethylene glycol (mPEG) molecules as a surface coordinating ligand. The resulting mPEG-Qdots were characterized by using ζ potential, FTIR, thermogravimetric (TG) analysis, and microscale thermophoresis (MST) studies. We investigated the effect of mPEG molecules and their grafting density on the Qdots photophysical properties, colloidal stability, protein binding affinity, and in vitro cellular toxicity. Moreover, cellular binding features of the resulting Qdots were examined by using three-dimensional (3D) tumor-like spheroids, and the results were discussed in detail. Promisingly, mPEG ligands were found to increase colloidal stability of Qdots, reduce adsorption of proteins to the Qdot surface, and mitigate Qdot-induced side effects to a great extent. Flow cytometry and confocal microscopy studies revealed that PEGylated Qdots exhibited distinctive cellular interactions with respect to their mPEG grafting density. As a result, mPEG molecules demonstrated a minimal effect on the ZnS shell deposition and the Qdot fluorescence efficiency at a low mPEG density, whereas they showed pronounced effect on Qdot colloidal stability, protein binding affinity, cytotoxicity, and nonspecific binding at a higher mPEG grafting amount.

Ceramide binding to anandamide increases its half-life and potentiates its cytotoxicity in human neuroblastoma cells.

Science.gov (United States)

Di Scala, Coralie; Mazzarino, Morgane; Yahi, Nouara; Varini, Karine; Garmy, Nicolas; Fantini, Jacques; Chahinian, Henri

2017-06-01

Anandamide (AEA) is a ubiquitous lipid that exerts neurotransmitter functions but also controls important biological functions such as proliferation, survival, or programmed cell death. The latter effects are also regulated by ceramide, a lipid enzymatically generated from sphingomyelin hydrolysis by sphingomyelinase. Ceramide has been shown to increase the cellular toxicity of AEA, but the mechanisms controlling this potentiating effect remained unclear. Here we have used a panel of in silico, physicochemical, biochemical and cellular approaches to study the crosstalk between AEA and ceramide apoptotic pathways. Molecular dynamics simulations indicated that AEA and ceramide could form a stable complex in phosphatidylcholine membranes. Consistent with these data, we showed that AEA can specifically insert into ceramide monolayers whereas it did not penetrate into sphingomyelin membranes. Then we have studied the effects of ceramide on AEA-induced toxicity of human neuroblastoma cells. In these experiments, the cells have been either naturally enriched in ceramide by neutral sphingomyelinase pre-incubation or treated with C2-ceramide, a biologically active ceramide analog. Both treatments significantly increased the cytotoxicity of AEA as assessed by the MTS mitochondrial toxicity assay. This effect was correlated with the concomitant accumulation of natural ceramide (or its synthetic analog) and AEA in the cells. A kinetic study of AEA hydrolysis showed that ceramide inhibited the fatty acid amino hydrolase (FAAH) activity in cell extracts. Taken together, these data suggested that ceramide binds to AEA, increases its half-life and potentiates its cytotoxicity. Overall, these mechanisms account for a functional cross-talk between AEA and ceramide apoptotic pathways. Copyright © 2017 Elsevier B.V. All rights reserved.

Analyses of the peripheral immunome following multiple administrations of avelumab, a human IgG1 anti-PD-L1 monoclonal antibody.

Science.gov (United States)

Donahue, Renee N; Lepone, Lauren M; Grenga, Italia; Jochems, Caroline; Fantini, Massimo; Madan, Ravi A; Heery, Christopher R; Gulley, James L; Schlom, Jeffrey

2017-01-01

Multiple anti-PD-L1/PD-1 checkpoint monoclonal antibodies (MAb) have shown clear evidence of clinical benefit. All except one have been designed or engineered to omit the possibility to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) as a second potential mode of anti-tumor activity; the reason for this is the concern of lysis of PD-L1 positive immune cells. Avelumab is a fully human IgG1 MAb which has been shown in prior in vitro studies to mediate ADCC versus a range of human tumor cells, and clinical studies have demonstrated anti-tumor activity versus a range of human cancers. This study was designed to investigate the effect on immune cell subsets in the peripheral blood of cancer patients prior to and following multiple administrations of avelumab. One hundred twenty-three distinct immune cell subsets in the peripheral blood of cancer patients ( n  = 28) in a phase I trial were analyzed by flow cytometry prior to and following one, three, and nine cycles of avelumab. Changes in soluble (s) CD27 and sCD40L in plasma were also evaluated. In vitro studies were also performed to determine if avelumab would mediate ADCC of PBMC. No statistically significant changes in any of the 123 immune cell subsets analyzed were observed at any dose level, or number of doses, of avelumab. Increases in the ratio of sCD27:sCD40L were observed, suggesting potential immune activation. Controlled in vitro studies also showed lysis of tumor cells by avelumab versus no lysis of PBMC from five donors. These studies demonstrate the lack of any significant effect on multiple immune cell subsets, even those expressing PD-L1, following multiple cycles of avelumab. These results complement prior studies showing anti-tumor effects of avelumab and comparable levels of adverse events with avelumab versus other anti-PD-1/PD-L1 MAbs. These studies provide the rationale to further exploit the potential ADCC mechanism of action of avelumab as well as other human IgG1 checkpoint

Sorafenib enhances proteasome inhibitor-mediated cytotoxicity via inhibition of unfolded protein response and keratin phosphorylation

International Nuclear Information System (INIS)

Honma, Yuichi; Harada, Masaru

2013-01-01

Hepatocellular carcinoma (HCC) is highly resistant to conventional systemic therapies and prognosis for advanced HCC patients remains poor. Recent studies of the molecular mechanisms responsible for tumor initiation and progression have identified several potential molecular targets in HCC. Sorafenib is a multi-kinase inhibitor shown to have survival benefits in advanced HCC. It acts by inhibiting the serine/threonine kinases and the receptor type tyrosine kinases. In preclinical experiments sorafenib had anti-proliferative activity in hepatoma cells and it reduced tumor angiogenesis and increased apoptosis. Here, we demonstrate for the first time that the cytotoxic mechanisms of sorafenib include its inhibitory effects on protein ubiquitination, unfolded protein response (UPR) and keratin phosphorylation in response to endoplasmic reticulum (ER) stress. Moreover, we show that combined treatment with sorafenib and proteasome inhibitors (PIs) synergistically induced a marked increase in cell death in hepatoma- and hepatocyte-derived cells. These observations may open the way to potentially interesting treatment combinations that may augment the effect of sorafenib, possibly including drugs that promote ER stress. Because sorafenib blocked the cellular defense mechanisms against hepatotoxic injury not only in hepatoma cells but also in hepatocyte-derived cells, we must be careful to avoid severe liver injury. -- Graphical abstract: Display Omitted -- Highlights: •We examined the cytotoxic mechanisms of sorafenib in hepatoma cells. •Sorafenib induces cell death via apoptotic and necrotic fashion. •Sorafenib inhibits protein ubiquitination and unfolded protein response. •Autophagy induced by sorafenib may affect its cytotoxicity. •Sorafenib inhibits keratin phosphorylation and cytoplasmic inclusion formation

Evaluation of the cytotoxicity of dihydroxytryptamines and 5-hydroxytryptamine antagonists as cytotoxic agents in dimethylhydrazine-induced adenocarcinomata.

Science.gov (United States)

Tutton, P J; Barkla, D H

1978-01-01

The cytotoxicity of 5,6-dihydroxytryptamine (5,6-DHT), 5,7-dihydroxytryptamine (5,7-DHT), bromolysergic acid diethylamide (BOL), methysergide, and cyproheptadine, and also of 5,6-DHT together with either BOL, methysergide, or cyproheptadine in dimethylhydrazine-induced (DMH) carcinomata of rat colon was evaluated by estimating the percentage of necrotic cells in histological sections of tissues taken 15 h after injection of each of the drugs. In addition, the influence of methysergide and cyproheptadine on the tumour cell mitotic rate was estimated by means of a stathmokinetic technique. Both 5,6-DHT and 5,7-DHT were cytotoxic at each dose tested and for each of these agents the percentage of necrotic cells was directly correlated with the dose of drug used. BOL was not found to be cytotoxic to the colonic carcinomata, whereas both methysergide and cyproheptadine did cause detectable tumour cell necrosis. Methysergide was also found to accelerate tumour cell proliferation, whereas cyproheptadine did not. BOL competitively inhibited the cytotoxicity of 5,6-DHT and neither methysergide nor cyproheptadine potentiated the effect of 5,6 DHT.

Cellular uptake, nuclear localization and cytotoxicity of 125I-labelled DNA minor groove binding ligands in K562, human erythroleukaemia cells

International Nuclear Information System (INIS)

Karagiannis, T.C.; Lobachevsky, P.N.; Martin, R.F.

2000-01-01

Full text: Iodine-125 decays by orbital electron capture and internal conversion resulting in the emission of numerous Auger electrons which produce a highly localised radiochemical damage in the immediate vicinity of the site of decay. Given the requirement to deliver 125 I to the nuclear DNA, a minor groove binding bibenzimidazole, 125 I-iodoHoechst 33258 was investigated. It has been noted that this analogue may be prone to de-iodination in vitro and in vivo, given the presence of an orthoiodophenol moiety which is analogous to that in thyroxins. Therefore, an 125 I -iodoHoechst analogue without the hydroxyl group was also studied. The 125 I -iodoHoechst 33258 analogue was prepared by direct iodination of Hoechst 33258 and 125 I iodoHoechst was prepared by demetallation of a trimethylstannyl precursor. DNA binding studies indicated that both iodo-analogues bind to calf thymus DNA, K D = 89 ± 30nM, n = 0.018 bp - 1 for iodoHoechst 33258 and K D = 121 ± 31nM, n = 0.024 bp -1 for iodoHoechst. Similarly, nuclear localization following incubation with 5μM of either ligand at 37 deg C was observed in K562 cells by fluorescence microscopy. Flow cytometry was used to investigate the kinetics of drug uptake and efflux in K562 cells. The results indicated that when 10 6 cells were incubated with 5μM ligand at 37 deg C, the uptake reached a plateau at approximately 43 minutes for iodoHoechst 33258 and approximately 52 minutes for iodoHoechst. Ligand efflux results indicated two-phase kinetics. The initial phase which involves 50-60% of drug was characterised by a half-life time (t 1/2 ) of 55.4 minutes for efflux of iodoHoechst 33258 and a t 1/2 of 10.3 minutes for efflux of iodoHoechst, at 37 deg C. Furthermore, the results suggested that the DNA binding sites in a 10 6 cell/ml suspension were saturated by incubation with 3μM iodoHoechst 33258 and 5μM iodoHoechst. In the initial cytotoxicity experiments using 125 I-iodoHoechst 33258, K562 cells were incubated for 1

Cystathionine metabolic enzymes play a role in the inflammation resolution of human keratinocytes in response to sub-cytotoxic formaldehyde exposure

Energy Technology Data Exchange (ETDEWEB)

Lee, Eunyoung [College of Pharmacy, Seoul National University, Seoul 08826 (Korea, Republic of); Natural Products Research Institute, Seoul National University, Seoul 08826 (Korea, Republic of); Kim, Hyoung-June [Basic Research and Innovation Division, AmorePacific Corporation R& D Center, Yongin, Gyeounggi-do 17074 (Korea, Republic of); Lee, Moonyoung [College of Pharmacy, Seoul National University, Seoul 08826 (Korea, Republic of); Jin, Sun Hee; Hong, Soo Hyun; Ahn, Seyeon; Kim, Sae On [College of Pharmacy, Seoul National University, Seoul 08826 (Korea, Republic of); Natural Products Research Institute, Seoul National University, Seoul 08826 (Korea, Republic of); Shin, Dong Wook [Basic Research and Innovation Division, AmorePacific Corporation R& D Center, Yongin, Gyeounggi-do 17074 (Korea, Republic of); Lee, Seung-Taek [Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 03722 (Korea, Republic of); Noh, Minsoo, E-mail: minsoonoh@snu.ac.kr [College of Pharmacy, Seoul National University, Seoul 08826 (Korea, Republic of); Natural Products Research Institute, Seoul National University, Seoul 08826 (Korea, Republic of)

2016-11-01

Low-level formaldehyde exposure is inevitable in industrialized countries. Although daily-life formaldehyde exposure level is practically impossible to induce cell death, most of mechanistic studies related to formaldehyde toxicity have been performed in cytotoxic concentrations enough to trigger cell death mechanism. Currently, toxicological mechanisms underlying the sub-cytotoxic exposure to formaldehyde are not clearly elucidated in skin cells. In this study, the genome-scale transcriptional analysis in normal human keratinocytes (NHKs) was performed to investigate cutaneous biological pathways associated with daily life formaldehyde exposure. We selected the 175 upregulated differentially expressed genes (DEGs) and 116 downregulated DEGs in NHKs treated with 200 μM formaldehyde. In the Gene Ontology (GO) enrichment analysis of the 175 upregulated DEGs, the endoplasmic reticulum (ER) unfolded protein response (UPR) was identified as the most significant GO biological process in the formaldeyde-treated NHKs. Interestingly, the sub-cytotoxic formaldehyde affected NHKs to upregulate two enzymes important in the cellular transsulfuration pathway, cystathionine γ-lyase (CTH) and cystathionine-β-synthase (CBS). In the temporal expression analysis, the upregulation of the pro-inflammatory DEGs such as MMP1 and PTGS2 was detected earlier than that of CTH, CBS and other ER UPR genes. The metabolites of CTH and CBS, L-cystathionine and L-cysteine, attenuated the formaldehyde-induced upregulation of pro-inflammatory DEGs, MMP1, PTGS2, and CXCL8, suggesting that CTH and CBS play a role in the negative feedback regulation of formaldehyde-induced pro-inflammatory responses in NHKs. In this regard, the sub-cytotoxic formaldehyde-induced CBS and CTH may regulate inflammation fate decision to resolution by suppressing the early pro-inflammatory response. - Highlights: • Sub-cytotoxic formaldehyde upregulates ER UPR-associated genes in NHKs. • Formaldehyde-induced ER UPR

Anesthetic propofol overdose causes endothelial cytotoxicity in vitro and endothelial barrier dysfunction in vivo

International Nuclear Information System (INIS)

Lin, Ming-Chung; Chen, Chia-Ling; Yang, Tsan-Tzu; Choi, Pui-Ching; Hsing, Chung-Hsi; Lin, Chiou-Feng

2012-01-01

An overdose and a prolonged treatment of propofol may cause cellular cytotoxicity in multiple organs and tissues such as brain, heart, kidney, skeletal muscle, and immune cells; however, the underlying mechanism remains undocumented, particularly in vascular endothelial cells. Our previous studies showed that the activation of glycogen synthase kinase (GSK)-3 is pro-apoptotic in phagocytes during overdose of propofol treatment. Regarding the intravascular administration of propofol, we therefore hypothesized that propofol overdose also induces endothelial cytotoxicity via GSK-3. Propofol overdose (100 μg/ml) inhibited growth in human arterial and microvascular endothelial cells. After treatment, most of the endothelial cells experienced caspase-independent necrosis-like cell death. The activation of cathepsin D following lysosomal membrane permeabilization (LMP) determined necrosis-like cell death. Furthermore, propofol overdose also induced caspase-dependent apoptosis, at least in part. Caspase-3 was activated and acted downstream of mitochondrial transmembrane potential (MTP) loss; however, lysosomal cathepsins were not required for endothelial cell apoptosis. Notably, activation of GSK-3 was essential for propofol overdose-induced mitochondrial damage and apoptosis, but not necrosis-like cell death. Intraperitoneal administration of a propofol overdose in BALB/c mice caused an increase in peritoneal vascular permeability. These results demonstrate the cytotoxic effects of propofol overdose, including cathepsin D-regulated necrosis-like cell death and GSK-3-regulated mitochondrial apoptosis, on endothelial cells in vitro and the endothelial barrier dysfunction by propofol in vivo. Highlights: ► Propofol overdose causes apoptosis and necrosis in endothelial cells. ► Propofol overdose triggers lysosomal dysfunction independent of autophagy. ► Glycogen synthase kinase-3 facilitates propofol overdose-induced apoptosis. ► Propofol overdose causes an increase

Anesthetic propofol overdose causes endothelial cytotoxicity in vitro and endothelial barrier dysfunction in vivo

Energy Technology Data Exchange (ETDEWEB)

Lin, Ming-Chung [Institute of Clinical Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan (China); Department of Anesthesiology, Chi Mei Medical Center, Liouying, Tainan, Taiwan (China); Chen, Chia-Ling [Center of Infectious Disease and Signaling Research, College of Medicine, National Cheng Kung University, Tainan, Taiwan (China); Yang, Tsan-Tzu; Choi, Pui-Ching [Institute of Clinical Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan (China); Hsing, Chung-Hsi [Department of Anesthesiology, Chi Mei Medical Center, Tainan, Taiwan (China); Department of Anesthesiology, College of Medicine, Taipei Medical University, Taipei, Taiwan (China); Lin, Chiou-Feng, E-mail: cflin@mail.ncku.edu.tw [Institute of Clinical Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan (China); Center of Infectious Disease and Signaling Research, College of Medicine, National Cheng Kung University, Tainan, Taiwan (China); Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University, Tainan, Taiwan (China); Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan, Taiwan (China)

2012-12-01

An overdose and a prolonged treatment of propofol may cause cellular cytotoxicity in multiple organs and tissues such as brain, heart, kidney, skeletal muscle, and immune cells; however, the underlying mechanism remains undocumented, particularly in vascular endothelial cells. Our previous studies showed that the activation of glycogen synthase kinase (GSK)-3 is pro-apoptotic in phagocytes during overdose of propofol treatment. Regarding the intravascular administration of propofol, we therefore hypothesized that propofol overdose also induces endothelial cytotoxicity via GSK-3. Propofol overdose (100 μg/ml) inhibited growth in human arterial and microvascular endothelial cells. After treatment, most of the endothelial cells experienced caspase-independent necrosis-like cell death. The activation of cathepsin D following lysosomal membrane permeabilization (LMP) determined necrosis-like cell death. Furthermore, propofol overdose also induced caspase-dependent apoptosis, at least in part. Caspase-3 was activated and acted downstream of mitochondrial transmembrane potential (MTP) loss; however, lysosomal cathepsins were not required for endothelial cell apoptosis. Notably, activation of GSK-3 was essential for propofol overdose-induced mitochondrial damage and apoptosis, but not necrosis-like cell death. Intraperitoneal administration of a propofol overdose in BALB/c mice caused an increase in peritoneal vascular permeability. These results demonstrate the cytotoxic effects of propofol overdose, including cathepsin D-regulated necrosis-like cell death and GSK-3-regulated mitochondrial apoptosis, on endothelial cells in vitro and the endothelial barrier dysfunction by propofol in vivo. Highlights: ► Propofol overdose causes apoptosis and necrosis in endothelial cells. ► Propofol overdose triggers lysosomal dysfunction independent of autophagy. ► Glycogen synthase kinase-3 facilitates propofol overdose-induced apoptosis. ► Propofol overdose causes an increase

Synthetic Lignan Secoisolariciresinol Diglucoside (LGM2605 Reduces Asbestos-Induced Cytotoxicity in an Nrf2-Dependent and -Independent Manner

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Ralph A. Pietrofesa

2018-03-01

Full Text Available Asbestos exposure triggers inflammatory processes associated with oxidative stress and tissue damage linked to malignancy. LGM2605 is the synthetic lignan secoisolariciresinol diglucoside (SDG with free radical scavenging, antioxidant, and anti-inflammatory properties in diverse inflammatory cell and mouse models, including exposure to asbestos fibers. Nuclear factor-E2 related factor 2 (Nrf2 activation and boosting of endogenous tissue defenses were associated with the protective action of LGM2605 from asbestos-induced cellular damage. To elucidate the role of Nrf2 induction by LGM2605 in protection from asbestos-induced cellular damage, we evaluated LGM2605 in asbestos-exposed macrophages from wild-type (WT and Nrf2 disrupted (Nrf2−/− mice. Cells were pretreated with LGM2605 (50 µM and 100 µM and exposed to asbestos fibers (20 µg/cm2 and evaluated 8 h and 24 h later for inflammasome activation, secreted cytokine levels (interleukin-1β (IL-1β, interleukin-18 (IL-18, interleukin-6 (IL-6, and tumor necrosis factor alpha (TNFα, cytotoxicity and cell death, nitrosative stress, and Nrf2-regulated enzyme levels. Asbestos exposure induced robust oxidative and nitrosative stress, cell death and cytotoxicity, which were equally mitigated by LGM2605. Inflammasome activation was significantly attenuated in Nrf2−/− macrophages compared to WT, and the protective action of LGM2605 was seen only in WT cells. In conclusion, in a cell model of asbestos-induced toxicity, LGM2605 acts via protective mechanisms that may not involve Nrf2 activation.

Synthetic Lignan Secoisolariciresinol Diglucoside (LGM2605) Reduces Asbestos-Induced Cytotoxicity in an Nrf2-Dependent and -Independent Manner

Science.gov (United States)

Pietrofesa, Ralph A.; Chatterjee, Shampa; Park, Kyewon; Arguiri, Evguenia; Albelda, Steven M.; Christofidou-Solomidou, Melpo

2018-01-01

Asbestos exposure triggers inflammatory processes associated with oxidative stress and tissue damage linked to malignancy. LGM2605 is the synthetic lignan secoisolariciresinol diglucoside (SDG) with free radical scavenging, antioxidant, and anti-inflammatory properties in diverse inflammatory cell and mouse models, including exposure to asbestos fibers. Nuclear factor-E2 related factor 2 (Nrf2) activation and boosting of endogenous tissue defenses were associated with the protective action of LGM2605 from asbestos-induced cellular damage. To elucidate the role of Nrf2 induction by LGM2605 in protection from asbestos-induced cellular damage, we evaluated LGM2605 in asbestos-exposed macrophages from wild-type (WT) and Nrf2 disrupted (Nrf2−/−) mice. Cells were pretreated with LGM2605 (50 µM and 100 µM) and exposed to asbestos fibers (20 µg/cm2) and evaluated 8 h and 24 h later for inflammasome activation, secreted cytokine levels (interleukin-1β (IL-1β), interleukin-18 (IL-18), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNFα)), cytotoxicity and cell death, nitrosative stress, and Nrf2-regulated enzyme levels. Asbestos exposure induced robust oxidative and nitrosative stress, cell death and cytotoxicity, which were equally mitigated by LGM2605. Inflammasome activation was significantly attenuated in Nrf2−/− macrophages compared to WT, and the protective action of LGM2605 was seen only in WT cells. In conclusion, in a cell model of asbestos-induced toxicity, LGM2605 acts via protective mechanisms that may not involve Nrf2 activation. PMID:29498660

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

Science.gov (United States)

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.

Interleukin-2 activation of cytotoxic cells in postmastectomy seroma.

Science.gov (United States)

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

1996-02-15

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

Photoluminescent Gold Nanoclusters in Cancer Cells: Cellular Uptake, Toxicity, and Generation of Reactive Oxygen Species.

Science.gov (United States)

Matulionyte, Marija; Dapkute, Dominyka; Budenaite, Laima; Jarockyte, Greta; Rotomskis, Ricardas

2017-02-10

In recent years, photoluminescent gold nanoclusters have attracted considerable interest in both fundamental biomedical research and practical applications. Due to their ultrasmall size, unique molecule-like optical properties, and facile synthesis gold nanoclusters have been considered very promising photoluminescent agents for biosensing, bioimaging, and targeted therapy. Yet, interaction of such ultra-small nanoclusters with cells and other biological objects remains poorly understood. Therefore, the assessment of the biocompatibility and potential toxicity of gold nanoclusters is of major importance before their clinical application. In this study, the cellular uptake, cytotoxicity, and intracellular generation of reactive oxygen species (ROS) of bovine serum albumin-encapsulated (BSA-Au NCs) and 2-(N-morpholino) ethanesulfonic acid (MES)capped photoluminescent gold nanoclusters (Au-MES NCs) were investigated. The results showed that BSA-Au NCs accumulate in cells in a similar manner as BSA alone, indicating an endocytotic uptake mechanism while ultrasmall Au-MES NCs were distributed homogeneously throughout the whole cell volume including cell nucleus. The cytotoxicity of BSA-Au NCs was negligible, demonstrating good biocompatibility of such BSA-protected Au NCs. In contrast, possibly due to ultrasmall size and thin coating layer, Au-MES NCs exhibited exposure time-dependent high cytotoxicity and higher reactivity which led to highly increased generation of reactive oxygen species. The results demonstrate the importance of the coating layer to biocompatibility and toxicity of ultrasmall photoluminescent gold nanoclusters.

Photoluminescent Gold Nanoclusters in Cancer Cells: Cellular Uptake, Toxicity, and Generation of Reactive Oxygen Species

Directory of Open Access Journals (Sweden)

Marija Matulionyte

2017-02-01

Full Text Available In recent years, photoluminescent gold nanoclusters have attracted considerable interest in both fundamental biomedical research and practical applications. Due to their ultrasmall size, unique molecule-like optical properties, and facile synthesis gold nanoclusters have been considered very promising photoluminescent agents for biosensing, bioimaging, and targeted therapy. Yet, interaction of such ultra-small nanoclusters with cells and other biological objects remains poorly understood. Therefore, the assessment of the biocompatibility and potential toxicity of gold nanoclusters is of major importance before their clinical application. In this study, the cellular uptake, cytotoxicity, and intracellular generation of reactive oxygen species (ROS of bovine serum albumin-encapsulated (BSA-Au NCs and 2-(N-morpholino ethanesulfonic acid (MEScapped photoluminescent gold nanoclusters (Au-MES NCs were investigated. The results showed that BSA-Au NCs accumulate in cells in a similar manner as BSA alone, indicating an endocytotic uptake mechanism while ultrasmall Au-MES NCs were distributed homogeneously throughout the whole cell volume including cell nucleus. The cytotoxicity of BSA-Au NCs was negligible, demonstrating good biocompatibility of such BSA-protected Au NCs. In contrast, possibly due to ultrasmall size and thin coating layer, Au-MES NCs exhibited exposure time-dependent high cytotoxicity and higher reactivity which led to highly increased generation of reactive oxygen species. The results demonstrate the importance of the coating layer to biocompatibility and toxicity of ultrasmall photoluminescent gold nanoclusters.

Cytotoxicity evaluation of extracts and fractions of five marine sponges from the Persian Gulf and HPLC fingerprint analysis of cytotoxic extracts

Institute of Scientific and Technical Information of China (English)

Davood; Mahdian; Milad; Iranshahy; Abolfazl; Shakeri; Azar; Hoseini; Hoda; Yavari; Melika; Nazemi; Mehrdad; Iranshahi

2015-01-01

Objective: To screen the cytotoxic effects of some marine sponges extracts on HeLa and PC12 cells.Methods: Five marine sponges including Ircinia echinata(I. echinata), Dysidea avara,Axinella sinoxea, Haliclona tubifera and Haliclona violacea were collected from the Persian Gulf(Hengam Island). The cytotoxic effect of these sponges was evaluated by using MTT assay. The metabolic high performance liquid chromatography fingerprint of I. echinata was also carried out at two wavelengths(254 and 280 nm).Results: Among the sponges tested in this study, the extracts of I. echinata and Dysidea avara possessed the cytotoxic effect on HeLa and PC12 cells. The obtained fractions from high performance liquid chromatography were evaluated for their cytotoxic properties against the cell lines. The isolated fractions did not show significant cytotoxic properties.Conclusions: I. echinata could be considered as a potential extract for chemotherapy.Further investigation is needed to determine the accuracy of mechanism.

Cytotoxicity evaluation of extracts and fractions of ifve marine sponges from the Persian Gulf and HPLC ifngerprint analysis of cytotoxic extracts

Institute of Scientific and Technical Information of China (English)

Davood Mahdian; Milad Iranshahy; Abolfazl Shakeri; Azar Hoseini; Hoda Yavari; Melika Nazemi; Mehrdad Iranshahi

2015-01-01

Objective:To screen the cytotoxic effects of some marine sponges extracts on HeLa and PC12 cells. Methods: Five marine sponges including Ircinia echinata (I. echinata), Dysidea avara, Axinella sinoxea, Haliclona tubifera and Haliclona violacea were collected from the Persian Gulf (Hengam Island). The cytotoxic effect of these sponges was evaluated by using MTT assay. The metabolic high performance liquid chromatography fingerprint of I. echinata was also carried out at two wavelengths (254 and 280 nm). Results:Among the sponges tested in this study, the extracts of I. echinata and Dysidea avara possessed the cytotoxic effect on HeLa and PC12 cells. The obtained fractions from high performance liquid chromatography were evaluated for their cytotoxic properties against the cell lines. The isolated fractions did not show significant cytotoxic properties. Conclusions:I. echinata could be considered as a potential extract for chemotherapy. Further investigation is needed to determine the accuracy of mechanism.

Characterization of three human cell line models for high-throughput neuronal cytotoxicity screening.

Science.gov (United States)

Tong, Zhi-Bin; Hogberg, Helena; Kuo, David; Sakamuru, Srilatha; Xia, Menghang; Smirnova, Lena; Hartung, Thomas; Gerhold, David

2017-02-01

More than 75 000 man-made chemicals contaminate the environment; many of these have not been tested for toxicities. These chemicals demand quantitative high-throughput screening assays to assess them for causative roles in neurotoxicities, including Parkinson's disease and other neurodegenerative disorders. To facilitate high throughput screening for cytotoxicity to neurons, three human neuronal cellular models were compared: SH-SY5Y neuroblastoma cells, LUHMES conditionally-immortalized dopaminergic neurons, and Neural Stem Cells (NSC) derived from human fetal brain. These three cell lines were evaluated for rapidity and degree of differentiation, and sensitivity to 32 known or candidate neurotoxicants. First, expression of neural differentiation genes was assayed during a 7-day differentiation period. Of the three cell lines, LUHMES showed the highest gene expression of neuronal markers after differentiation. Both in the undifferentiated state and after 7 days of neuronal differentiation, LUHMES cells exhibited greater cytotoxic sensitivity to most of 32 suspected or known neurotoxicants than SH-SY5Y or NSCs. LUHMES cells were also unique in being more susceptible to several compounds in the differentiating state than in the undifferentiated state; including known neurotoxicants colchicine, methyl-mercury (II), and vincristine. Gene expression results suggest that differentiating LUHMES cells may be susceptible to apoptosis because they express low levels of anti-apoptotic genes BCL2 and BIRC5/survivin, whereas SH-SY5Y cells may be resistant to apoptosis because they express high levels of BCL2, BIRC5/survivin, and BIRC3 genes. Thus, LUHMES cells exhibited favorable characteristics for neuro-cytotoxicity screening: rapid differentiation into neurons that exhibit high level expression neuronal marker genes, and marked sensitivity of LUHMES cells to known neurotoxicants. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Pentachlorophenol-Induced Cytotoxic, Mitogenic, and Endocrine-Disrupting Activities in Channel Catfish, Ictalurus punctatus

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Paul B. Tchounwou

2004-09-01

Full Text Available Pentachlorophenol (PCP is an organochlorine compound that has been widely used as a biocide in several industrial, agricultural, and domestic applications. Although it has been shown to induce systemic toxicity and carcinogenesis in several experimental studies, the literature is scarce regarding its toxic mechanisms of action at the cellular and molecular levels. Recent investigations in our laboratory have shown that PCP induces cytotoxicity and transcriptionally activates stress genes in human liver carcinoma (HepG2 cells [1]. In this research, we hypothesize that environmental exposure to PCP may trigger cytotoxic, mitogenic, and endocrine-disrupting activities in aquatic organisms including fish. To test this hypothesis, we carried out in vitro cultures of male channel catfish hepatocytes, and performed the fluorescein diacetate assay (FDA to assess for cell viability, and the Western Blot analysis to assess for vitellogenin expression following exposure to PCP. Data obtained from FDA experiments indicated a strong dose-response relationship with respect to PCP cytotoxicity. Upon 48 hrs of exposure, the chemical dose required to cause 50% reduction in cell viability (LD50 was computed to be 1,987.0 + 9.6 μg PCP/mL. The NOAEL and LOAEL were 62.5 + 10.3 μg PCP/mL and 125.0+15.2 μg PCP/mL, respectively. At lower levels of exposure, PCP was found to be mitogenic, showing a strong dose- and time-dependent response with regard to cell proliferation. Western Blot analysis demonstrated the potential of PCP to cause endocrine-disrupting activity, as evidenced by the up regulation of the 125-kDa vitellogenin protein the hepatocytes of male channel catfish.

Cytotoxicity of alkylating agents towards sensitive and resistant strains of Escherichia coli in relation to extent and mode of alkylation of cellular macromolecules and repair of alkylation lesions in deoxyribonucleic acids.

Science.gov (United States)

Lawley, P D; Brookes, P

1968-09-01

1. A quantitative study was made of the relationship between survival of colony-forming ability in Escherichia coli strains B/r and B(s-1) and the extents of alkylation of cellular DNA, RNA and protein after treatment with mono- or di-functional sulphur mustards, methyl methanesulphonate or iodoacetamide. 2. The mustards and methyl methanesulphonate react with nucleic acids in the cells, in the same way as found previously from chemical studies in vitro, and with proteins. Iodoacetamide reacts only with protein, principally with the thiol groups of cysteine residues. 3. The extents of alkylation of cellular constituents required to prevent cell division vary widely according to the strain of bacteria and the nature of the alkylating agent. 4. The extents of alkylation of the sensitive and resistant strains at a given dose of alkylating agent do not differ significantly. 5. Removal of alkyl groups from DNA of cells of the resistant strains B/r and 15T(-) after alkylation with difunctional sulphur mustard was demonstrated; the product di(guanin-7-ylethyl) sulphide, characteristic of di- as opposed to mono-functional alkylation, was selectively removed; the time-scale of this effect suggests an enzymic rather than a chemical mechanism. 6. The sensitive strain B(s-1) removed alkyl groups from DNA in this way only at very low extents of alkylation. When sensitized to mustard action by treatment with iodoacetamide, acriflavine or caffeine, the extent of alkylation of cellular DNA corresponding to a mean lethal dose was decreased to approximately 3 molecules of di(guanin-7-ylethyl) sulphide in the genome of this strain. 7. Relatively large numbers of monofunctional alkylations per genome can be withstood by this sensitive strain. Iodoacetamide had the weakest cytotoxic action of the agents investigated; methyl methanesulphonate was significantly weaker in effect than the monofunctional sulphur mustard, which was in turn weaker than the difunctional sulphur mustard. 8

A novel llama antibody targeting Fn14 exhibits anti-metastatic activity in vivo.

Science.gov (United States)

Trebing, Johannes; Lang, Isabell; Chopra, Martin; Salzmann, Steffen; Moshir, Mahan; Silence, Karen; Riedel, Simone S; Siegmund, Daniela; Beilhack, Andreas; Otto, Christoph; Wajant, Harald

2014-01-01

Expression of fibroblast growth factor (FGF)-inducible 14 (Fn14), a member of the tumor necrosis factor receptor superfamily, is typically low in healthy adult organisms, but strong Fn14 expression is induced in tissue injury and tissue remodeling. High Fn14 expression is also observed in solid tumors, which is why this receptor is under consideration as a therapeutic target in oncology. Here, we describe various novel mouse-human cross-reactive llama-derived recombinant Fn14-specific antibodies (5B6, 18D1, 4G5) harboring the human IgG1 Fc domain. In contrast to recombinant variants of the established Fn14-specific antibodies PDL192 and P4A8, all three llama-derived antibodies efficiently bound to the W42A and R56P mutants of human Fn14. 18D1 and 4G5, but not 5B6, efficiently blocked TNF-like weak inducer of apoptosis(TWEA K) binding at low concentrations (0.2–2 μg/ml). Oligomerization and Fcγ receptor (FcγR) binding converted all antibodies into strong Fn14 agonists. Variants of 18D1 with enhanced and reduced antibody-dependent cell-mediated cytotoxicity (ADCC) activity were further analyzed in vivo with respect to their effect on metastasis. In a xenogeneic model using human colon carcinoma cancer cells, both antibody variants were effective in reducing metastasis to the liver. In contrast, only the 18D1 variant with enhanced ADCC activity, but not its ADCC-defective counterpart, suppressed lung metastasis in the RE NCA model. In sum, this suggests that Fn14 targeting might primarily act by triggering of antibody effector functions, but also by blockade of TWEA K-Fn14 interaction in some cases

TNFα-Induced Mucin 4 Expression Elicits Trastuzumab Resistance in HER2-Positive Breast Cancer.

Science.gov (United States)

Mercogliano, María F; De Martino, Mara; Venturutti, Leandro; Rivas, Martín A; Proietti, Cecilia J; Inurrigarro, Gloria; Frahm, Isabel; Allemand, Daniel H; Deza, Ernesto Gil; Ares, Sandra; Gercovich, Felipe G; Guzmán, Pablo; Roa, Juan C; Elizalde, Patricia V; Schillaci, Roxana

2017-02-01

Although trastuzumab administration improved the outcome of HER2-positive breast cancer patients, resistance events hamper its clinical benefits. We demonstrated that TNFα stimulation in vitro induces trastuzumab resistance in HER2-positive breast cancer cell lines. Here, we explored the mechanism of TNFα-induced trastuzumab resistance and the therapeutic strategies to overcome it. Trastuzumab-sensitive breast cancer cells, genetically engineered to stably overexpress TNFα, and de novo trastuzumab-resistant tumors, were used to evaluate trastuzumab response and TNFα-blocking antibodies effectiveness respectively. Immunohistochemistry and antibody-dependent cell cytotoxicity (ADCC), together with siRNA strategy, were used to explore TNFα influence on the expression and function of its downstream target, mucin 4 (MUC4). The clinical relevance of MUC4 expression was studied in a cohort of 78 HER2-positive breast cancer patients treated with adjuvant trastuzumab. TNFα overexpression turned trastuzumab-sensitive cells and tumors into resistant ones. Histopathologic findings revealed mucin foci in TNFα-producing tumors. TNFα induced upregulation of MUC4 that reduced trastuzumab binding to its epitope and impaired ADCC. Silencing MUC4 enhanced trastuzumab binding, increased ADCC, and overcame trastuzumab and trastuzumab-emtansine antiproliferative effects in TNFα-overexpressing cells. Accordingly, administration of TNFα-blocking antibodies downregulated MUC4 and sensitized de novo trastuzumab-resistant breast cancer cells and tumors to trastuzumab. In HER2-positive breast cancer samples, MUC4 expression was found to be an independent predictor of poor disease-free survival (P = 0.008). We identified TNFα-induced MUC4 expression as a novel trastuzumab resistance mechanism. We propose MUC4 expression as a predictive biomarker of trastuzumab efficacy and a guide to combination therapy of TNFα-blocking antibodies with trastuzumab. Clin Cancer Res; 23(3); 636-48. �

Characteristics of medication errors with parenteral cytotoxic drugs

OpenAIRE

Fyhr, A; Akselsson, R

2012-01-01

Errors involving cytotoxic drugs have the potential of being fatal and should therefore be prevented. The objective of this article is to identify the characteristics of medication errors involving parenteral cytotoxic drugs in Sweden. A total of 60 cases reported to the national error reporting systems from 1996 to 2008 were reviewed. Classification was made to identify cytotoxic drugs involved, type of error, where the error occurred, error detection mechanism, and consequences for the pati...

Cytotoxic activity of four Mexican medicinal plants.

Science.gov (United States)

Vega-Avila, Elisa; Espejo-Serna, Adolfo; Alarcón-Aguilar, Francisco; Velasco-Lezama, Rodolfo

2009-01-01

Ibervillea sonorae Greene, Cucurbita ficifolia Bouché, Tagetes lucida Cav and Justicia spicigera Scheltdd are Mexican native plants used in the treatment of different illnesses. The ethanolic extract of J. spicigera and T. lucida as well as aqueous extracts from I. sonorae, C. ficifolia, T. lucida and J. spicigera were investigated using sulforhodamine B assay. These extracts were assessed using two cell line: T47D (Human Breast cancer) and HeLa (Human cervix cancer). Colchicine was used as the positive control. Data are presented as the dose that inhibited 50% control growth (ED50). All of the assessed extracts were cytotoxic (ED50 < 20 microg/ml) against T47D cell line, meanwhile only the aqueous extract from T. lucida and the ethanolic extract from J. spicigera were cytotoxic to HeLa cell line. Ethanolic extract from J. spicigera presented the best cytotoxic effect. The cytotoxic activity of J. spicigera correlated with one of the popular uses, the treatment of cancer.

Effect of radiotherapy on lymphocyte cytotoxicity in vitro

Energy Technology Data Exchange (ETDEWEB)

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

1975-11-01

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

Cytotoxicity and Hsp 70 induction in Hep G2 cells in response to zearalenone and cytoprotection by sub-lethal heat shock

International Nuclear Information System (INIS)

Hassen, Wafa; Golli, Emna El; Baudrimont, Isabelle; Mobio, A. Theophile; Ladjimi, M. Moncef; Creppy, E. Edmond; Bacha, Hassen

2005-01-01

Zearalenone (ZEN) is a mycotoxin with several adverse effects in laboratory and domestic animals. The mechanism of ZEN toxicity that involves mainly binding to oestrogen receptors and inhibition of macromolecules synthesis is not fully understood. Using human hepatocytes Hep G2 cells as a model, the aim of this work was (i) to investigate the ability of ZEN to induce heat shock proteins Hsp 70 and (ii) to find out the mechanisms of ZEN cytotoxicity by examining cell proliferation and protein synthesis. Our study demonstrated that ZEN induces Hsp 70 expression in a time and dose-dependant manner; this induction occurs at non-cytotoxic concentrations, it could be therefore considered as a biomarker of toxicity. A cytoprotective effect of Hsp 70 was elicited when Hep G2 cells were exposed to Sub-Lethal heat shock prior to ZEN treatment and evidenced by a reduced ZEN cytolethality. This cytoprotection suggests that Hsp 70 may constitute an important cellular defence mechanism. Finally, our data show that ZEN is cytotoxic in Hep G2 cells by inhibiting cell proliferation and total protein synthesis and pointed out oxidative damage as possible pathway involved in ZEN toxicity; however, other investigations are needed to further confirm Zen induced oxidative stress

Cytotoxicity of cardiotonic steroids in sensitive and multidrug-resistant leukemia cells and the link with Na(+)/K(+)-ATPase.

Science.gov (United States)

Zeino, Maen; Brenk, Ruth; Gruber, Lisa; Zehl, Martin; Urban, Ernst; Kopp, Brigitte; Efferth, Thomas

2015-06-01

Cardiotonic steroids have long been in clinical use for treatment of heart failure and are now emerging as promising agents in various diseases, especially cancer. Their main target is Na(+)/K(+)-ATPase, a membrane protein involved in cellular ion homeostasis. Na(+)/K(+)-ATPase has been implicated in cancer biology by affecting several cellular events and signaling pathways in both sensitive and drug-resistant cancer cells. Hence, we investigated the cytotoxic activities of 66 cardiotonic steroids and cardiotonic steroid derivatives in sensitive CCRF-CEM and multidrug-resistant CEM/ADR5000 leukemia cells. Data were then subjected to quantitative structure-activity relationship analysis (QSAR) and molecular docking into Na(+)/K(+)-ATPase, which both indicated a possible differential expression of the pump in the mentioned cell lines. This finding was confirmed by western blotting, intracellular potassium labeling and next generation sequencing which showed that Na(+)/K(+)-ATPase was less expressed in multidrug-resistant than in sensitive cells. Copyright © 2015 Elsevier Ltd. All rights reserved.

Phytochemical and Cytotoxic Investigations of Alpinia mutica Rhizomes

Directory of Open Access Journals (Sweden)

Kae Shin Sim

2011-01-01

Full Text Available The methanol and fractionated extracts (hexane, ethyl acetate and water of Alpinia mutica (Zingiberaceae rhizomes were investigated for their cytotoxic effect against six human carcinoma cell lines, namely KB, MCF7, A549, Caski, HCT116, HT29 and non-human fibroblast cell line (MRC 5 using an in vitro cytotoxicity assay. The ethyl acetate extract possessed high inhibitory effect against KB, MCF7 and Caski cells (IC50 values of 9.4, 19.7 and 19.8 µg/mL, respectively. Flavokawin B (1, 5,6-dehydrokawain (2, pinostrobin chalcone (3 and alpinetin (4, isolated from the active ethyl acetate extract were also evaluated for their cytotoxic activity. Of these, pinostrobin chalcone (3 and alpinetin (4 were isolated from this plant for the first time. Pinostrobin chalcone (3 displayed very remarkable cytotoxic activity against the tested human cancer cells, such as KB, MCF7 and Caski cells (IC50 values of 6.2, 7.3 and 7.7 µg/mL, respectively. This is the first report of the cytotoxic activity of Alpinia mutica.

Identification of stable cytotoxic factors in the gas phase extract of cigarette smoke and pharmacological characterization of their cytotoxicity.

Science.gov (United States)

Noya, Yoichi; Seki, Koh-Ichi; Asano, Hiroshi; Mai, Yosuke; Horinouchi, Takahiro; Higashi, Tsunehito; Terada, Koji; Hatate, Chizuru; Hoshi, Akimasa; Nepal, Prabha; Horiguchi, Mika; Kuge, Yuji; Miwa, Soichi

2013-12-06

Smoking is a major risk factor for atherosclerotic vascular diseases, but the mechanism for its genesis is unknown. We have recently shown that the gas phase of cigarette smoke (nicotine- and tar-free cigarette smoke extract; CSE) likely to reach the systemic circulation contains stable substances which cause cytotoxicity like plasma membrane damage and cell death in cultured cells, and also that the plasma membrane damage is caused through sequential activation of protein kinase C (PKC) and NADPH oxidase (NOX) and the resulting generation of reactive oxygen species (PKC/NOX-dependent mechanism), whereas cell death is caused through PKC/NOX-dependent and -independent mechanisms. To identify these stable substances, the CSE was prepared by passing the main-stream smoke of 10 cigarettes through a Cambridge glass fiber filter, trapping of the smoke in a vessel cooled at -80°C, and subsequent dissolution in 10ml of water. The CSE was fractionated into nine fractions using reversed-phase HPLC, and each fraction was screened for cytotoxicity in cultured cells, using propidium iodide uptake assay for cell membrane damage and MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] reduction assay for cell viability. The cytotoxicity was positive in two of the nine fractions (Fr2 and Fr5). After extraction of the active fractions into dichloromethane, GC/MS analysis identified 2-cyclopenten-1-one (CPO) in Fr5 but none in Fr2. After derivatization of the active fractions with O-(2,3,4,5,6-pentafluorobenzyl) hydroxylamine hydrochloride, GC/MS analysis identified acrolein, acetone and propionaldehyde in Fr2, and methyl vinyl ketone (MVK) in Fr5. After 4-h incubation, authentic acrolein and MVK induced concentration-dependent cytotoxicity with EC50 values of 75.9±8.2 and 47.0±8.0μM (mean±SEM; n=3), respectively, whereas acetone, propionaldehyde and CPO were without effect. However, after 24-h incubation, CPO induced concentration

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

Directory of Open Access Journals (Sweden)

Qiujing Yu

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

Cytotoxicity of fluorographene

Czech Academy of Sciences Publication Activity Database

Teo, W. Z.; Sofer, Z.; Šembera, Filip; Janoušek, Zbyněk; Pumera, M.

2015-01-01

Roč. 5, č. 129 (2015), s. 107158-107165 ISSN 2046-2069 R&D Projects: GA ČR(CZ) GA15-09001S Institutional support: RVO:61388963 Keywords : fluorinated graphene * viability assays * cytotoxicity Subject RIV: CC - Organic Chemistry Impact factor: 3.289, year: 2015

N-glycan engineering of a plant-produced anti-CD20-hIL-2 immunocytokine significantly enhances its effector functions.

Science.gov (United States)

Marusic, Carla; Pioli, Claudio; Stelter, Szymon; Novelli, Flavia; Lonoce, Chiara; Morrocchi, Elena; Benvenuto, Eugenio; Salzano, Anna Maria; Scaloni, Andrea; Donini, Marcello

2018-03-01

Anti-CD20 recombinant antibodies are among the most promising therapeutics for the treatment of B-cell malignancies such as non-Hodgkin lymphomas. We recently demonstrated that an immunocytokine (2B8-Fc-hIL2), obtained by fusing an anti-CD20 scFv-Fc antibody derived from C2B8 mAb (rituximab) to the human interleukin 2 (hIL-2), can be efficiently produced in Nicotiana benthamiana plants. The purified immunocytokine (IC) bearing a typical plant protein N-glycosylation profile showed a CD20 binding activity comparable to that of rituximab and was efficient in eliciting antibody-dependent cell-mediated cytotoxicity (ADCC) of human PBMC against Daudi cells, indicating its fuctional integrity. In this work, the immunocytokine devoid of the typical xylose/fucose N-glycosylation plant signature (IC-ΔXF) and the corresponding scFv-Fc-ΔXF antibody not fused to the cytokine, were obtained in a glyco-engineered ΔXylT/FucT N. benthamiana line. Purification yields from agroinfiltrated plants amounted to 20-35 mg/kg of leaf fresh weight. When assayed for interaction with FcγRI and FcγRIIIa, IC-ΔXF exhibited significantly enhanced binding affinities if compared to the counterpart bearing the typical plant protein N-glycosylation profile (IC) and to rituximab. The glyco-engineered recombinant molecules also exhibited a strongly improved ADCC and complement-dependent cytotoxicity (CDC). Notably, our results demonstrate a reduced C1q binding of xylose/fucose carrying IC and scFv-Fc compared to versions that lack these sugar moieties. These results demonstrate that specific N-glycosylation alterations in recombinant products can dramatically affect the effector functions of the immunocytokine, resulting in an overall improvement of the biological functions and consequently of the therapeutic potential. © 2017 Wiley Periodicals, Inc.

Prostate-specific membrane antigen PET imaging and immunohistochemistry in adenoid cystic carcinoma-@]@a preliminary analysis

Energy Technology Data Exchange (ETDEWEB)

Klein Nulent, Thomas J.W.; Es, Robert J.J. van [Utrecht Cancer Center, University Medical Center Utrecht, Department of Head and Neck Surgical Oncology, P.O. Box 85500, Utrecht (Netherlands); University Medical Center Utrecht, Department of Oral and Maxillofacial Surgery, Utrecht (Netherlands); Krijger, Gerard C.; Keizer, Bart de [University Medical Center Utrecht, Department of Radiology and Nuclear Medicine, Utrecht (Netherlands); Bree, Remco de [Utrecht Cancer Center, University Medical Center Utrecht, Department of Head and Neck Surgical Oncology, P.O. Box 85500, Utrecht (Netherlands); Willems, Stefan M. [University Medical Center Utrecht, Department of Pathology, Utrecht (Netherlands)

2017-09-15

Adenoid cystic carcinoma (AdCC) of the head and neck is an uncommon malignant epithelial tumour of the secretory glands. Many patients develop slowly growing local recurrence and/or distant metastasis, for which treatment options are limited. A retrospective analysis of 9 AdCC patients was conducted to analyse the visualization of AdCC on PSMA PET/CT and to investigate the expression of PSMA on primary, recurrent and metastatic AdCC tumour tissue using immunohistochemistry. Local recurrence occurred in six patients and eight developed distant metastasis. All PET/CTs depicted PSMA-ligand uptake. Four PSMA PET/CTs showed suspected residual disease, eight scans depicted uptake in areas suspected of distant metastasis. Median Maximum Standardized Uptake Value (SUV{sub max}) in local recurrent and distant metastatic AdCC was 2.52 (IQR 2.41-5.95) and 4.01 (IQR 2.66-8.71), respectively. All primary tumours showed PSMA expression on immunohistochemistry (5-90% expression), as well as all available specimens of local recurrence and distant metastases. PSMA PET/CT is able to detect and visualize local recurrent and distant metastatic AdCC. PSMA-specific targeting is supported by PSMA expression on immunohistochemistry. (orig.)

The future of cytotoxic therapy: selective cytotoxicity based on biology is the key

International Nuclear Information System (INIS)

Bono, Johann S de; Tolcher, Anthony W; Rowinsky, Eric K

2003-01-01

Although mortality from breast cancer is decreasing, 15% or more of all patients ultimately develop incurable metastatic disease. It is hoped that new classes of target-based cytotoxic therapeutics will significantly improve the outcome for these patients. Many of these novel agents have displayed cytotoxic activity in preclinical and clinical evaluations, with little toxicity. Such preferential cytotoxicity against malignant tissues will remain tantamount to the Holy Grail in oncologic therapeutics because this portends improved patient tolerance and overall quality of life, and the capacity to deliver combination therapy. Combinations of such rationally designed target-based therapies are likely to be increasingly important in treating patients with breast carcinoma. The anticancer efficacy of these agents will, however, remain dependent on the involvement of the targets of these agents in the biology of the individual patient's disease. Results of DNA microarray analyses have raised high hopes that the analyses of RNA expression levels can successfully predict patient prognosis, and indicate that the ability to rapidly 'fingerprint' the oncogenic profile of a patient's tumor is now possible. It is hoped that these studies will support the identification of the molecules driving a tumor's growth, and the selection of the appropriate combination of targeted agents in the near future

Autonomously bioluminescent mammalian cells for continuous and real-time monitoring of cytotoxicity.

Science.gov (United States)

Xu, Tingting; Close, Dan M; Webb, James D; Ripp, Steven A; Sayler, Gary S

2013-10-28

Mammalian cell-based in vitro assays have been widely employed as alternatives to animal testing for toxicological studies but have been limited due to the high monetary and time costs of parallel sample preparation that are necessitated due to the destructive nature of firefly luciferase-based screening methods. This video describes the utilization of autonomously bioluminescent mammalian cells, which do not require the destructive addition of a luciferin substrate, as an inexpensive and facile method for monitoring the cytotoxic effects of a compound of interest. Mammalian cells stably expressing the full bacterial bioluminescence (luxCDABEfrp) gene cassette autonomously produce an optical signal that peaks at 490 nm without the addition of an expensive and possibly interfering luciferin substrate, excitation by an external energy source, or destruction of the sample that is traditionally performed during optical imaging procedures. This independence from external stimulation places the burden for maintaining the bioluminescent reaction solely on the cell, meaning that the resultant signal is only detected during active metabolism. This characteristic makes the lux-expressing cell line an excellent candidate for use as a biosentinel against cytotoxic effects because changes in bioluminescent production are indicative of adverse effects on cellular growth and metabolism. Similarly, the autonomous nature and lack of required sample destruction permits repeated imaging of the same sample in real-time throughout the period of toxicant exposure and can be performed across multiple samples using existing imaging equipment in an automated fashion.

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

Science.gov (United States)

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

2016-07-01

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

Phenolics, Antiradical Assay and Cytotoxicity of Processed Mango ...

African Journals Online (AJOL)

Phenolics, Antiradical Assay and Cytotoxicity of Processed Mango ( Mangifera indica ) and Bush Mango ( Irvingia gabonensis ) Kernels. ... Nigerian Food Journal ... Phenolic constituents (total phenols, flavonoids, tannins, and anthocyanins), comparative antiradical potency and cytotoxicity of processed mango (Mangifera ...

Cytotoxic and genotoxic affects of acid mine drainage on fish Channa punctata (Bloch).

Science.gov (United States)

Talukdar, B; Kalita, H K; Basumatary, S; Saikia, D J; Sarma, D

2017-10-01

The investigation deals with the effects of Acid Mine Drainage (AMD) of coal mine on fish Channa punctata (Bloch) by examining the incidence of haematological, morphological, histological changes and DNA fragmentation in tissues of C. punctata in laboratory condition. For this study fishes were exposed to 10% of AMD for a period of 30 days. The fusion of the primary and secondary gill lamellae, distortion, loss of alignment, deposition of worn out tissues and mucous on the surface of the lamella in the gills; degeneration of morphological architecture, loss of alignment of tubules, mucous deposition in the kidney; cellular damage, cellular necrosis, extraneous deposition on the surface, pore formation in the liver are some important changes detected by scanning electron microscopy. Fishes of AMD treated group showed gradual significant decrease in TEC, Hb and, increase in TLC and DLC as compared to that of the control. DNA fragmentation observed in kidney of fishes from treated group indicates an intricate pollutant present in the AMD. The high incidence of morphological and histological alterations, haematological changes along with DNA breakage in C. punctata is an evidence of the cytotoxic and genotoxic potential of AMD of coal mines. Copyright © 2017 Elsevier Inc. All rights reserved.

Cytotoxic effects of commonly used nanomaterials and microplastics on cerebral and epithelial human cells.

Science.gov (United States)

Schirinzi, Gabriella F; Pérez-Pomeda, Ignacio; Sanchís, Josep; Rossini, Cesare; Farré, Marinella; Barceló, Damià

2017-11-01

Plastic wastes are among the major inputs of detritus into aquatic ecosystems. Also, during recent years the increasing use of new materials such as nanomaterials (NMs) in industrial and household applications has contributed to the complexity of waste mixtures in aquatic systems. The current effects and the synergism and antagonisms of mixtures of microplastics (MPLs), NMs and organic compounds on the environment and in human health have, to date, not been well understood but instead they are a cause for general concern. The aim of this work is to contribute to a better understanding of the cytotoxicity of NMs and microplastics/nanoplastics (MPLs/NPLs), at cell level in terms of oxidative stress (evaluating Reactive Oxygen Species effect) and cell viability. Firstly, the individual cytotoxicity of metal nanoparticles (NPs) (AgNPs and AuNPs), of metal oxide NPs (ZrO 2 NPs, CeO 2 NPs, TiO 2 NPs, and Al 2 O 3 NPs), carbon nanomaterials (C 60 fullerene, graphene), and MPLs of polyethylene (PE) and polystyrene (PS) has been evaluated in vitro. Two different cellular lines T98G and HeLa, cerebral and epithelial human cells, respectively, were employed. The cells were exposed during 24-48h to different levels of contaminants, from 10ng/mL to 10µg/mL, under the same conditions. Secondly, the synergistic and antagonistic relationships between fullerenes and other organic contaminants, including an organophosphate insecticide (malathion), a surfactant (sodium dodecylbenzenesulfonate) and a plasticiser (diethyl phthalate) were assessed. The obtained results confirm that oxidative stress is one of the mechanisms of cytotoxicity at cell level, as has been observed for both cell lines and contributes to the current knowledge of the effects of NMs and MPLs-NPLs. Copyright © 2017 Elsevier Inc. All rights reserved.

Antitumor and cytotoxic properties of a humanized antibody specific for the GM3(Neu5Gc) ganglioside.

Science.gov (United States)

Dorvignit, Denise; García-Martínez, Liliana; Rossin, Aurélie; Sosa, Katya; Viera, Justo; Hernández, Tays; Mateo, Cristina; Hueber, Anne-Odile; Mesa, Circe; López-Requena, Alejandro

2015-12-01

Gangliosides are sialic acid-bearing glycosphingolipids expressed on all mammalian cell membranes, and participate in several cellular processes. During malignant transformation their expression changes, both at the quantitative and qualitative levels. Of particular interest is the overexpression by tumor cells of Neu5Gc-gangliosides, which are absent, or detected in trace amounts, in human normal cells. The GM3(Neu5Gc) ganglioside in particular has been detected in many human tumors, and it is considered one of the few tumor specific antigen. We previously demonstrated that a humanized antibody specific for this molecule, named 14F7hT, retained the binding and cytotoxic properties of the mouse antibody. In this work, we confirm that 14F7hT exerts a non-apoptotic cell death mechanism in vitro and shows its potent in vivo antitumor activity on a solid mouse myeloma model. Also, we demonstrate, in contrast to the murine counterpart, the capacity of this antibody to induce antibody-dependent cell-mediated cytotoxicity using human effector cells, which increases its potential for the treatment of GM3(Neu5Gc)-expressing human tumors. Copyright © 2015 Elsevier GmbH. All rights reserved.

Cytotoxicity of cadmium-free quantum dots and their use in cell bioimaging.

Science.gov (United States)

Soenen, Stefaan J; Manshian, Bella B; Aubert, Tangi; Himmelreich, Uwe; Demeester, Jo; De Smedt, Stefaan C; Hens, Zeger; Braeckmans, Kevin

2014-06-16

The use of quantum dots (QDots) as bright and photostable probes for long-term fluorescence imaging is gaining more interest. Thus far, (pre)clinical use of QDots remains limited, which is primarily caused by the potential toxicity of QDots. Most QDots consist of Cd2+ ions, which are known to cause high levels of toxicity. In order to overcome this problem, several strategies have been tested, such as the generation of cadmium-free QDots. In the present study, two types of cadmium-free QDots, composed of ZnSe/ZnS (QDotZnSe) and InP/ZnS (QDotInP), were studied with respect to their cytotoxicity and cellular uptake in a variety of cell types. A multiparametric cytotoxicity approach is used, where the QDots are studied with respect to cell viability, oxidative stress, cell morphology, stem cell differentiation, and neurite outgrowth. The data reveal slight differences in uptake levels for both types of QDots (maximal for QDotZnSe), but clear differences in cytotoxicity and cell functionality effects exist, with highest toxicity for QDotZnSe. Differences between cell types and between both types of QDots can be explained by the intrinsic sensitivity of certain cell types and chemical composition of the QDots. At concentrations at which no toxic effects can be observed, the functionality of the QDots for fluorescence cell visualization is evaluated, revealing that the higher brightness of QDotZnSe overcomes most of the toxicity issues compared to that of QDotInP. Comparing the results obtained with common Cd2+-containing QDots tested under identical conditions, the importance of particle functionality is demonstrated, revealing that cadmium-free QDots tested in this study are not significantly better than Cd2+-containing QDots for long-term cell imaging and that more work needs to be performed in optimizing the brightness and surface chemistry of cadmium-free QDots for them to replace currently used Cd2+-containing QDots.

Study of apoptotic mechanisms induced by all-trans retinoic acid and its 13-cis isomer on cellular lines of human hepato carcinoma Hep3B and HepG2

International Nuclear Information System (INIS)

Arce Vargas, Frederick

2006-01-01

Two cellular lines of liver cancer (Hep3B and HepG2) were incubated during different periods of time with some concentrations of two retinoic acid isomers (ATRA and 13-cis AR) and with 5-fu chemotherapeutic agents, cisplatin and paclitaxel. It was determined if these substances leaded cytotoxicity, apoptosis and if they modified the expression of different genes related to cellular death by apoptosis, in order to explain the hepatocellular carcinoma resistance to these drugs. HepG2 cells showed more resistance than Hep3B cells to 72 hours of treatment, as much ATRA as the 13-cis AR were toxic and produced apoptosis in two cellular lines. This type of cellular death seems to be mediated by a decrease in Bcl-xL concentration in Hep3B cells treated with both retinoids an increase in bax concentration in HepG2 cells treated with 13-cis AR. It were observed 3 and 8 proteolysis of procaspase in Hep3B cells, suggesting extrinsic via activation of the apoptosis, while cellular death in HepG2 cells seems to be independent of caspases. Cisplatin and paclitaxel leaded cytotoxicity to 48 hours of treatment, with significant differences between two cellular lines only in case of paclitaxel. Hep3B cells treated with cisplatin and HepG2 cells treated with paclytaxel suffered apoptosis. 5-FU produced toxicity only when it was used to high concentrations and the mechanism of cellular death induced by this agent seems to be primarily necrosis in Hep3B cells and apoptosis in HepG2. There was decrease in the Bcl-xL concentration in two cellular lines when it was treated with cisplatin and in HepG2 cells treated with 5-FU. Bax concentration there no was modified with no treatment. Activation of the 3 caspases seems to happen only in HepG2 cells with 5-FU and paclytaxel. These two agents, also, decreased the survivin concentration of HepG2 cells. Treatments of the three drugs produced an increase in the expression of this gen in Hep3B cells, which might explain partially the resistance

Anti-inflammatory and cytotoxic activities of five Veronica species.

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Harput, U Sebnem; Saracoglu, Iclal; Inoue, Makoto; Ogihara, Yukio

2002-04-01

Biological activities of five Veronica species (Scrophulariaceae), V. cymbalaria, V. hederifolia, V. pectinata var. glandulosa, V. persica and V. polita were studied for their anti-inflammatory and cytotoxic activities. Their methanol extracts showed both the inhibitory activity of nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated macrophages and cytotoxic activity against KB epidermoid carcinoma and B16 melanoma. When the methanol extracts were fractionated between water and chloroform, water fractions significantly inhibited NO production without any cytotoxicity, while chloroform fractions showed cytotoxicity dose-dependently. When the radical scavenging activity was determined using 2,2-diphenyl-1-picryl-hydrazyl (DPPH), water fractions of the five Veronica species scavenged free radicals effectively, suggesting that the inhibitory effect of this species on NO production was due to their radical scavenging activity. On the other hand, chloroform fractions of Veronica species except for V. cymbalaria showed similar cytotoxic activity against KB and B16 melanoma cells.

A fluorescence-based rapid screening assay for cytotoxic compounds

International Nuclear Information System (INIS)

Montoya, Jessica; Varela-Ramirez, Armando; Estrada, Abril; Martinez, Luis E.; Garza, Kristine; Aguilera, Renato J.

2004-01-01

A simple fluorescence-based assay was developed for the rapid screening of potential cytotoxic compounds generated by combinatorial chemistry. The assay is based on detection of nuclear green fluorescent protein (GFP) staining of a human cervical cancer cell line (HeLa) carrying an integrated histone H2B-GFP fusion gene. Addition of a cytotoxic compound to the HeLa-GFP cells results in the eventual degradation of DNA and loss of the GFP nuclear fluorescence. Using this assay, we screened 11 distinct quinone derivatives and found that several of these compounds were cytotoxic. These compounds are structurally related to plumbagin an apoptosis-inducing naphthoquinone isolated from Black Walnut. In order to determine the mechanism by which cell death was induced, we performed additional experiments with the most cytotoxic quinones. These compounds were found to induce morphological changes (blebbing and nuclear condensation) consistent with induction of apoptosis. Additional tests revealed that the cytotoxic compounds induce both necrotic and apoptotic modes of death

HIV antibodies for treatment of HIV infection.

Science.gov (United States)

Margolis, David M; Koup, Richard A; Ferrari, Guido

2017-01-01

The bar is high to improve on current combination antiretroviral therapy (ART), now highly effective, safe, and simple. However, antibodies that bind the HIV envelope are able to uniquely target the virus as it seeks to enter new target cells, or as it is expressed from previously infected cells. Furthermore, the use of antibodies against HIV as a therapeutic may offer advantages. Antibodies can have long half-lives, and are being considered as partners for long-acting antiretrovirals for use in therapy or prevention of HIV infection. Early studies in animal models and in clinical trials suggest that such antibodies can have antiviral activity but, as with small-molecule antiretrovirals, the issues of viral escape and resistance will have to be addressed. Most promising, however, are the unique properties of anti-HIV antibodies: the potential ability to opsonize viral particles, to direct antibody-dependent cellular cytotoxicity (ADCC) against actively infected cells, and ultimately the ability to direct the clearance of HIV-infected cells by effector cells of the immune system. These distinctive activities suggest that HIV antibodies and their derivatives may play an important role in the next frontier of HIV therapeutics, the effort to develop treatments that could lead to an HIV cure. Published 2017. This article is a U.S. Government work and is in the public domain in the USA.

Ex vivo assessment of genotoxicity and cytotoxicity in murine fibroblasts exposed to white MTA or white Portland cement with 15% bismuth oxide.

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Zeferino, E G; Bueno, C E S; Oyama, L M; Ribeiro, D A

2010-10-01

To evaluate whether white mineral trioxide aggregate (MTA) or white Portland cement with 15% bismuth oxide were able to induce genetic damage and cellular death ex vivo. Aliquots of 1 × 10(4) murine fibroblasts were incubated at 37 °C for 3 h with MTA (white) or white Portland cement with 15% bismuth oxide, at final concentrations ranging from 10 to 1000 μg mL(-1) individually. Data of three independent repeats from the comet assay and the trypan blue exclusion test were assessed by the one-way anova followed by Tukey's test. Mineral trioxide aggregate or Portland cement containing bismuth oxide did not produce genotoxic effects with respect to the single-cell gel (comet) assay data for all concentrations evaluated. Furthermore, no cytotoxicity was observed for MTA or Portland cement. White MTA or white Portland cement containing 15% bismuth oxide were not genotoxic and cytotoxic. © 2010 International Endodontic Journal.

Silver Nanoparticle-Mediated Cellular Responses in Various Cell Lines: An in Vitro Model

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Xi-Feng Zhang

2016-09-01

Full Text Available Silver nanoparticles (AgNPs have attracted increased interest and are currently used in various industries including medicine, cosmetics, textiles, electronics, and pharmaceuticals, owing to their unique physical and chemical properties, particularly as antimicrobial and anticancer agents. Recently, several studies have reported both beneficial and toxic effects of AgNPs on various prokaryotic and eukaryotic systems. To develop nanoparticles for mediated therapy, several laboratories have used a variety of cell lines under in vitro conditions to evaluate the properties, mode of action, differential responses, and mechanisms of action of AgNPs. In vitro models are simple, cost-effective, rapid, and can be used to easily assess efficacy and performance. The cytotoxicity, genotoxicity, and biocompatibility of AgNPs depend on many factors such as size, shape, surface charge, surface coating, solubility, concentration, surface functionalization, distribution of particles, mode of entry, mode of action, growth media, exposure time, and cell type. Cellular responses to AgNPs are different in each cell type and depend on the physical and chemical nature of AgNPs. This review evaluates significant contributions to the literature on biological applications of AgNPs. It begins with an introduction to AgNPs, with particular attention to their overall impact on cellular effects. The main objective of this review is to elucidate the reasons for different cell types exhibiting differential responses to nanoparticles even when they possess similar size, shape, and other parameters. Firstly, we discuss the cellular effects of AgNPs on a variety of cell lines; Secondly, we discuss the mechanisms of action of AgNPs in various cellular systems, and try to elucidate how AgNPs interact with different mammalian cell lines and produce significant effects; Finally, we discuss the cellular activation of various signaling molecules in response to AgNPs, and conclude with

Cytotoxic effect of betulinic acid and betulinic acid acetate isolated ...

African Journals Online (AJOL)

Cytotoxic effect of betulinic acid and betulinic acid acetate isolated from Melaleuca cajuput on human myeloid leukemia (HL-60) cell line. ... The cytotoxic effect of betulinic acid (BA), isolated from Melaleuca cajuput a Malaysian plant and its four synthetic derivatives were tested for their cytotoxicity in various cell line or ...

Cellular uptake of glucoheptoamidated poly(amidoamine) PAMAM G3 dendrimer with amide-conjugated biotin, a potential carrier of anticancer drugs.

Science.gov (United States)

Uram, Łukasz; Szuster, Magdalena; Filipowicz, Aleksandra; Zaręba, Magdalena; Wałajtys-Rode, Elżbieta; Wołowiec, Stanisław

2017-01-15

In search for soluble derivatives of PAMAM dendrimers as potential carriers for hydrophobic drugs, the conjugates of PAMAM G3 with biotin, further converted into glycodendrimer with d-glucoheptono-1,4-lactone, were prepared. Polyamidoamine dendrimer (PAMAM) of third generation, G3 was functionalized with four biotin equivalents covalently attached to terminal amine nitrogens via amide bond G3 4B . The remaining 28 amine groups were blocked by glucoheptoamide substituents (gh) to give G3 4B28gh or with one fluorescein equivalent (attached by reaction of G3 4B with fluorescein isothiocyanate, FITC) via thiourea bond as FITC followed by exhaustive glucoheptoamidation to get G3 4B27gh1F . As a control the G3 substituted totally with 32 glucoheptoamide residues, G3 gh and its fluorescein labeled analogue G3 31gh1F were synthesized. The glucoheptoamidation of PAMAM G0 dendrimer with glucoheptono-1,4-lactone was performed in order to fully characterize the 1 H NMR spectra of glucoheptoamidated PAMAM dendrimers and to control the derivatization of G3 with glucoheptono-1,4-lactone. Another two derivatives of G3, namely G3 4B28gh1F' and G3 32ghF' , with ester bonded fluorescein were also obtained. Biological properties of obtained dendrimer conjugates were estimated in vitro with human cell lines: normal fibroblast (BJ) and two cancer glioblastoma (U-118 MG) and squamous carcinoma (SCC-15), including cytotoxicity by reduction of XTT and neutral red (NR) assays. Cellular uptake of dendrimer conjugates was evaluated with confocal microscopy. Obtained results confirmed, that biotinylated bioconjugates have always lower cytotoxicity and 3-4 times higher cellular uptake than non-biotinylated dendrimer conjugates in all cell lines. Comparison of various cell lines revealed different dose-dependent cell responses and the lower cytotoxicity of examined dendrimer conjugates for normal fibroblasts and squamous carcinoma, as compared with much higher cytotoxic effects seen in

Effect of enhanced expression of connexin 43 on sunitinib-induced cytotoxicity in mesothelioma cells

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Miaki Uzu

2015-05-01

Full Text Available Connexin (Cx makes up a type of intercellular channel called gap junction (GJ. GJ plays a regulatory role in cellular physiology. The Cx expression level is often decreased in cancer cells compared to that in healthy ones, and the restoration of its expression has been shown to exert antiproliferative effects. This work aims to evaluate the effect of the restoration of connexin 43 (Cx43 (the most ubiquitous Cx subtype expression on sunitinib (SU-induced cytotoxicity in malignant mesothelioma (MM cells. Increased Cx43 expression in an MM cell line (H28 improved the ability of SU to inhibit receptor tyrosine kinase (RTK signaling. Moreover, higher Cx43 expression promoted SU-induced apoptosis. The cell viability test revealed that Cx43 enhanced the cytotoxic effect of SU in a GJ-independent manner. The effect of Cx43 on a proapoptotic factor, Bax, was then investigated. The interaction between Cx43 and Bax was confirmed by immunoprecipitation. Furthermore, higher Cx43 expression increased the production of a cleaved (active form of Bax during SU-induced apoptosis with no alteration in total Bax expression. These findings indicate that Cx43 most likely increases sensitivity to SU in H28 through direct interaction with Bax. In conclusion, we found that Cx43 overcame the chemoresistance of MM cells.

Cytotoxicity and cellular uptake of different sized gold nanoparticles in ovarian cancer cells

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Kumar, Dhiraj; Mutreja, Isha; Chitcholtan, Kenny; Sykes, Peter

2017-11-01

Nanomedicine has advanced the biomedical field with the availability of multifunctional nanoparticles (NPs) systems that can target a disease site enabling drug delivery and helping to monitor the disease. In this paper, we synthesised the gold nanoparticles (AuNPs) with an average size 18, 40, 60 and 80 nm, and studied the effect of nanoparticles size, concentration and incubation time on ovarian cancer cells namely, OVCAR5, OVCAR8, and SKOV3. The size measured by transmission electron microscopy images was slightly smaller than the hydrodynamic diameter; measured size by ImageJ as 14.55, 38.13, 56.88 and 78.56 nm. The cellular uptake was significantly controlled by the AuNPs size, concentration, and the cell type. The nanoparticles uptake increased with increasing concentration, and 18 and 80 nm AuNPs showed higher uptake ranging from 1.3 to 5.4 μg depending upon the concentration and cell type. The AuNPs were associated with a temporary reduction in metabolic activity, but metabolic activity remained more than 60% for all sample types; NPs significantly affected the cell proliferation activity in first 12 h. The increase in nanoparticle size and concentration induced the production of reactive oxygen species in 24 h.

Cytotoxic activity of plants from East Azarbaijan province, Iran

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M. Irani

2017-11-01

Full Text Available Background and objectives: Due to the high cancer mortality rates and side effects of different types of cancer treatments, discovering effective treatments without or with fewer side effects is the main purpose of many researchers all around the world. Plants play an important role in the discovery of new drugs. Iran owns rich and varied vegetation but the majority of these plants have not yet undergone chemical, pharmacological and toxicological studies. In the present study, some species from East Azarbaijan province of Iran were evaluated for cytotoxicity effects. Methods: Total methanol extract of 29 plants from 18 families were screened for their cytotoxic activities. The inhibition of cell growth for these extracts was evaluated against MCF-7, A-549, Hep-G2, HT-29 and MDBK cell lines. Their 50% inhibitions of growth (IC50 were determined by MTT assay. Moreover, cytotoxic evaluation of different fractions (ether de petrol, chloroform and methanol of the most potent species was performed. Results: Total extracts and fractions of Bryonia aspera, Centaurea salicifolia, Cuscuta chinensis, Ecbalium elaterium, Gypsophila ruscifolia, Ononis spinosa exhibited potent cytotoxic activity against one or more of the cell lines. Three of the mentioned total extracts presented cytotoxicity effects exclusively against HT-29 cells. Also three fractions (one ether de petrol and two chloroform fractions demonstrated selective cytotoxicity effects against MCF-7cells. Conclusion: It was concluded that these 6 potent species were proper candidates for identification and isolation of active ingredients with cytotoxic effects  and further studies about these species are recommended.

Antibacterial and Cytotoxic Activities of Acacia nilotica Lam ...

African Journals Online (AJOL)

Erah

that had maximum bactericidal activity against all the tested isolates, but showed < 30 % host cell cytotoxicity. Conclusion: The lysate of Acacia nilotica ... cytotoxic effects on human cells. EXPERIMENTAL. Plant material. Acacia nilotica Lam .... a detergent that permeabilizes eukaryotic cells and results in HBMEC damage.

Identification of SlpB, a Cytotoxic Protease from Serratia marcescens.

Science.gov (United States)

Shanks, Robert M Q; Stella, Nicholas A; Hunt, Kristin M; Brothers, Kimberly M; Zhang, Liang; Thibodeau, Patrick H

2015-07-01

The Gram-negative bacterium and opportunistic pathogen Serratia marcescens causes ocular infections in healthy individuals. Secreted protease activity was characterized from 44 ocular clinical isolates, and a higher frequency of protease-positive strains was observed among keratitis isolates than among conjunctivitis isolates. A positive correlation between protease activity and cytotoxicity to human corneal epithelial cells in vitro was determined. Deletion of prtS in clinical keratitis isolate K904 reduced, but did not eliminate, cytotoxicity and secreted protease production. This indicated that PrtS is necessary for full cytotoxicity to ocular cells and implied the existence of another secreted protease(s) and cytotoxic factors. Bioinformatic analysis of the S. marcescens Db11 genome revealed three additional open reading frames predicted to code for serralysin-like proteases noted here as slpB, slpC, and slpD. Induced expression of prtS and slpB, but not slpC and slpD, in strain PIC3611 rendered the strain cytotoxic to a lung carcinoma cell line; however, only prtS induction was sufficient for cytotoxicity to a corneal cell line. Strain K904 with deletion of both prtS and slpB genes was defective in secreted protease activity and cytotoxicity to human cell lines. PAGE analysis suggests that SlpB is produced at lower levels than PrtS. Purified SlpB demonstrated calcium-dependent and AprI-inhibited protease activity and cytotoxicity to airway and ocular cell lines in vitro. Lastly, genetic analysis indicated that the type I secretion system gene, lipD, is required for SlpB secretion. These genetic data introduce SlpB as a new cytotoxic protease from S. marcescens. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

The in vitro effect of gefitinib ('Iressa' alone and in combination with cytotoxic chemotherapy on human solid tumours

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Knight Louise A

2004-11-01

Full Text Available Abstract Background Activation of the epidermal growth factor receptor (EGFR triggers downstream signaling pathways that regulate many cellular processes involved in tumour survival and growth. Gefitinib ('Iressa' is an orally active tyrosine kinase inhibitor (TKI targeted to the ATP-binding domain of EGFR (HER1; erbB1. Methods In this study we have used a standardised ATP-based tumour chemosensitivity assay (ATP-TCA to measure the activity of gefitinib alone or in combination with different cytotoxic drugs (cisplatin, gemcitabine, oxaliplatin and treosulfan against a variety of solid tumours (n = 86, including breast, colorectal, oesophageal and ovarian cancer, carcinoma of unknown primary site, cutaneous and uveal melanoma, non-small cell lung cancer (NSCLC and sarcoma. The IC50 and IC90 were calculated for each single agent or combination. To allow comparison between samples the IndexSUM was calculated based on the percentage tumour growth inhibition (TGI at each test drug concentration (TDC. Gefitinib was tested at concentrations ranging from 0.0625–2 microM (TDC = 0.446 microg/ml. This study represents the first use of a TKI in the assay. Results There was heterogeneity in the degree of TGI observed when tumours were tested against single agent gefitinib. 7% (6/86 of tumours exhibited considerable inhibition, but most showed a more modest response resulting in a low TGI. The median IC50 value for single agent gefitinib in all tumours tested was 3.98 microM. Interestingly, gefitinib had both positive and negative effects when used in combination with different cytotoxics. In 59% (45/76 of tumours tested, the addition of gefitinib appeared to potentiate the effect of the cytotoxic agent or combination (of these, 11% (5/45 had a >50% decrease in their IndexSUM. In 38% of tumours (29/76, the TGI was decreased when the combination of gefitinib + cytotoxic was used in comparison to the cytotoxic alone. In the remaining 3% (2/76 there was no

Mitochondrial electron transport is the cellular target of the oncology drug elesclomol.

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Ronald K Blackman

Full Text Available Elesclomol is a first-in-class investigational drug currently undergoing clinical evaluation as a novel cancer therapeutic. The potent antitumor activity of the compound results from the elevation of reactive oxygen species (ROS and oxidative stress to levels incompatible with cellular survival. However, the molecular target(s and mechanism by which elesclomol generates ROS and subsequent cell death were previously undefined. The cellular cytotoxicity of elesclomol in the yeast S. cerevisiae appears to occur by a mechanism similar, if not identical, to that in cancer cells. Accordingly, here we used a powerful and validated technology only available in yeast that provides critical insights into the mechanism of action, targets and processes that are disrupted by drug treatment. Using this approach we show that elesclomol does not work through a specific cellular protein target. Instead, it targets a biologically coherent set of processes occurring in the mitochondrion. Specifically, the results indicate that elesclomol, driven by its redox chemistry, interacts with the electron transport chain (ETC to generate high levels of ROS within the organelle and consequently cell death. Additional experiments in melanoma cells involving drug treatments or cells lacking ETC function confirm that the drug works similarly in human cancer cells. This deeper understanding of elesclomol's mode of action has important implications for the therapeutic application of the drug, including providing a rationale for biomarker-based stratification of patients likely to respond in the clinical setting.

Cytotoxic triterpenoid saponins from Clematis tangutica.

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Zhao, Min; Da-Wa, Zhuo-Ma; Guo, Da-Le; Fang, Dong-Mei; Chen, Xiao-Zhen; Xu, Hong-Xi; Gu, Yu-Cheng; Xia, Bing; Chen, Lei; Ding, Li-Sheng; Zhou, Yan

2016-10-01

Eight previously undescribed oleanane-type triterpenoid saponins, clematangoticosides A-H, together with eight known saponins, were isolated from the whole plants of Clematis tangutica (Maxim.) Korsh. Their structures were elucidated by extensive spectroscopic analysis, in combination with chemical methods (acid hydrolysis and mild alkaline hydrolysis). Clematangoticosides D-G were found to be unusual 23, 28-bidesmosidic glycosides. The cytotoxic activities of all of the isolated saponins were evaluated against the four human cancer cell lines SGC-7901, HepG2, HL-60 and U251MG. Clematoside S, sapindoside B, kalopanax saponin A, and koelreuteria saponin A exhibited cytotoxicity against all of the test cancer cell lines with IC50 values in the range of 1.88-27.20 μM, while clematangoticoside D and F showed selective cytotoxicity against SGC-7901 with IC50 values of 24.22 and 21.35 μM, respectively. Copyright © 2016 Elsevier Ltd. All rights reserved.

Comparative cytotoxic response of nickel ferrite nanoparticles in human liver HepG2 and breast MFC-7 cancer cells.

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Ahamed, Maqusood; Akhtar, Mohd Javed; Alhadlaq, Hisham A; Khan, M A Majeed; Alrokayan, Salman A

2015-09-01

Nickel ferrite nanoparticles (NPs) have received much attention for their potential applications in biomedical fields such as magnetic resonance imaging, drug delivery and cancer hyperthermia. However, little is known about the toxicity of nickel ferrite NPs at the cellular and molecular levels. In this study, we investigated the cytotoxic responses of nickel ferrite NPs in two different types of human cells (i.e., liver HepG2 and breast MCF-7). Nickel ferrite NPs induced dose-dependent cytotoxicity in both types of cells, which was demonstrated by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazoliumbromide (MTT), neutral red uptake (NRU) and lactate dehydrogenase (LDH) assays. Nickel ferrite NPs were also found to induce oxidative stress, which was evident by the depletion of glutathione and the induction of reactive oxygen species (ROS) and lipid peroxidation. The mitochondrial membrane potential due to nickel ferrite NP exposure was also observed. The mRNA levels for the tumor suppressor gene p53 and the apoptotic genes bax, CASP3 and CASP9 were up-regulated, while the anti-apoptotic gene bcl-2 was down-regulated following nickel ferrite NP exposure. Furthermore, the activities of apoptotic enzymes (caspase-3 and caspase-9) were also higher in both types of cells treated with nickel ferrite NPs. Cytotoxicity induced by nickel ferrite was efficiently prevented by N-acetyl cysteine (ROS scavenger) treatment, which suggested that oxidative stress might be one of the possible mechanisms of nickel ferrite NP toxicity. We also observed that MCF-7 cells were slightly more susceptible to nickel ferrite NP exposure than HepG2 cells. This study warrants further investigation to explore the potential mechanisms of different cytotoxic responses of nickel ferrite NPs in different cell lines. Copyright © 2015 Elsevier Ltd. All rights reserved.

Determining the size and concentration dependence of gold nanoparticles in vitro cytotoxicity (IC50) test using WST-1 assay

International Nuclear Information System (INIS)

Rosli, Nur Shafawati binti; Rahman, Azhar Abdul; Aziz, Azlan Abdul; Shamsuddin, Shaharum

2015-01-01

Gold nanoparticles (AuNPs) received a great deal of attention for biomedical applications, especially in diagnostic imaging and therapeutics. Even though AuNPs have potential benefits in biomedical applications, the impact of AuNPs on human and environmental health still remains unclear. The use of AuNPs which is a high-atomic-number materials, provide advantages in terms of radiation dose enhancement. However, before this can become a clinical reality, cytotoxicity of the AuNPs has to be carefully evaluated. Cytotoxicity test is a rapid, standardized test that is very sensitive to determine whether the nanoparticles produced are harmful or benign on cellular components. In this work the size and concentration dependence of AuNPs cytotoxicity in breast cancer cell lines (MCF-7) are tested by using WST-1 assay. The sizes of AuNPs tested were 13 nm, 50 nm, and 70 nm. The cells were seeded in the 96-well plate and were treated with different concentrations of AuNPs by serial dilution for each size of AuNPs. The high concentration of AuNPs exhibit lower cell viability compared to low concentration of AuNPs. We quantified the toxicity of AuNPs in MCF-7 cell lines by determining the IC 50 values in WST-1 assays. The IC 50 values (inhibitory concentrations that effected 50% growth inhibition) of 50 nm AuNPs is lower than 13 nm and 70 nm AuNPs. Mean that, 50nm AuNPs are more toxic to the MCF-7 cells compared to smaller and larger sizes AuNPs. The presented results clearly indicate that the cytotoxicity of AuNPs depend not only on the concentration, but also the size of the nanoparticles

Laboratory determination of chemotherapeutic drug resistance in tumor cells from patients with leukemia, using a fluorometric microculture cytotoxicity assay (FMCA).

Science.gov (United States)

Larsson, R; Kristensen, J; Sandberg, C; Nygren, P

1992-01-21

An automated fluorometric microculture cytotoxicity assay (FMCA) based on the measurement of fluorescence generated from cellular hydrolysis of fluorescein diacetate (FDA) to fluorescein was employed for chemotherapeutic-drug-sensitivity testing of tumor-cell suspensions from patients with leukemia. Fluorescence was linearly related to cell number, and reproducible measurements of drug sensitivity could be performed using fresh or cryopreserved leukemia cells. A marked heterogeneity with respect to chemotherapeutic drug sensitivity was observed for a panel of cytotoxic drugs tested in 43 samples from 35 patients with treated or untreated acute and chronic leukemia. For samples obtained from patients with chronic lymphocytic and acute myelocytic leukemia, sensitivity profiles for standard drugs corresponded to known clinical activity and the assay detected primary and acquired drug resistance. Individual in vitro/in vivo correlations indicated high specificity with respect to the identification of drug resistance. The results suggest that the FMCA may be a simple and rapid method for in vivo-representative determinations of chemotherapeutic drug resistance in tumor cells obtained from patients with leukemia.

Involvement of ER stress and activation of apoptotic pathways in fisetin induced cytotoxicity in human melanoma.

Science.gov (United States)

Syed, Deeba N; Lall, Rahul K; Chamcheu, Jean Christopher; Haidar, Omar; Mukhtar, Hasan

2014-12-01

The prognosis of malignant melanoma remains poor in spite of recent advances in therapeutic strategies for the deadly disease. Fisetin, a dietary flavonoid is currently being investigated for its growth inhibitory properties in various cancer models. We previously showed that fisetin inhibited melanoma growth in vitro and in vivo. Here, we evaluated the molecular basis of fisetin induced cytotoxicity in metastatic human melanoma cells. Fisetin treatment induced endoplasmic reticulum (ER) stress in highly aggressive A375 and 451Lu human melanoma cells, as revealed by up-regulation of ER stress markers including IRE1α, XBP1s, ATF4 and GRP78. Time course analysis indicated that the ER stress was associated with activation of the extrinsic and intrinsic apoptotic pathways. Fisetin treated 2-D melanoma cultures displayed autophagic response concomitant with induction of apoptosis. Prolonged treatment (16days) with fisetin in a 3-D reconstituted melanoma model resulted in inhibition of melanoma progression with significant apoptosis, as evidenced by increased staining of cleaved Caspase-3 in the treated constructs. However, no difference in the expression of autophagic marker LC-3 was noted between treated and control groups. Fisetin treatment to 2-D melanoma cultures resulted in phosphorylation and activation of the multifunctional AMP-activated protein kinase (AMPK) involved in the regulation of diverse cellular processes, including autophagy and apoptosis. Silencing of AMPK failed to prevent cell death indicating that fisetin induced cytotoxicity is mediated through both AMPK-dependent and -independent mechanisms. Taken together, our studies confirm apoptosis as the primary mechanism through which fisetin inhibits melanoma cell growth and that activation of both extrinsic and intrinsic pathways contributes to fisetin induced cytotoxicity.

An in vitro strategy to evaluate the phototoxicity of solar UV at the molecular and cellular level: application to photoprotection assessment.

Science.gov (United States)

Marrot, L; Belaidi, J P; Chaubo, C; Meunier, J R; Perez, P; Agapakis-Causse, C

1998-09-01

Skin cancers are among the most common human cancers and have an increasing incidence. The ultraviolet radiation components of sunlight play a major role in skin tumor induction and development. Cellular DNA has been identified as a target for most of the biological effects of UV, and the induction of photodamage is considered as the initiating step of photocarcinogenesis. Thus, effective photoprotection of DNA against harmful overex-posure to solar UV is a critical issue. The efficiency of a sunscreen is usually tested with respect to its ability to prevent skin erythema, but conceivably, more data are required at the molecular and cellular level in order to ascertain protection against photocarcinogenic risk. In the present study, we define a strategy based on the use of various in vitro models and solar-simulated light to evaluate photodamage and photoprotection: -Supercoiled circular plasmid DNA for detection of structural alterations. -The yeast Saccharomyces cerevisiae to evaluate cytotoxicity and genotoxicity. -The single-cell gel electrophoresis or comet assay to determine DNA damage and DNA repair in human keratinocytes. -p53 expression as a hallmark for genotoxic stress. -Induction of pigmentation in human melanocytes. In conditions where light source, spectrum and control of radiation delivery were precisely defined, we have demonstrated that the wide spectrum UVA sunscreen Mexoryl SX protects from the cytotoxicity and genotoxicity of solar UV.

Cellular function of neuropathy target esterase in lysophosphatidylcholine action

International Nuclear Information System (INIS)

Vose, Sarah C.; Fujioka, Kazutoshi; Gulevich, Alex G.; Lin, Amy Y.; Holland, Nina T.; Casida, John E.

2008-01-01

Neuropathy target esterase (NTE) plays critical roles in embryonic development and maintenance of peripheral axons. It is a secondary target of some organophosphorus toxicants including analogs of insecticides and chemical warfare agents. Although the mechanistic role of NTE in vivo is poorly defined, it is known to hydrolyze lysophosphatidylcholine (LPC) in vitro and may protect cell membranes from cytotoxic accumulation of LPC. To determine the cellular function of NTE, Neuro-2a and COS-7 cells were transfected with a full-length human NTE-containing plasmid yielding recombinant NTE (rNTE). We find the same inhibitor sensitivity and specificity profiles for rNTE assayed with LPC or phenyl valerate (a standard NTE substrate) and that this correlation extends to the LPC hydrolases of human brain, lymphocytes and erythrocytes. All of these LPC hydrolases are therefore very similar to each other in respect to a conserved inhibitor binding site conformation. NTE is expressed in brain and lymphocytes and contributes to LPC hydrolase activities in these tissues. The enzyme or enzymes responsible for erythrocyte LPC hydrolase activity remain to be identified. We also show that rNTE protects Neuro-2a and COS-7 cells from exogenous LPC cytotoxicity. Expression of rNTE in Neuro-2a cells alters their phospholipid balance (analyzed by liquid chromatography-mass spectrometry with single ion monitoring) by lowering LPC-16:0 and LPC-18:0 and elevating glycerophosphocholine without a change in phosphatidylcholine-16:0/18:1 or 16:0/18:2. NTE therefore serves an important function in LPC homeostasis and action

A chemical genetic screen for modulators of asymmetrical 2,2'-dimeric naphthoquinones cytotoxicity in yeast.

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Ashkan Emadi

Full Text Available BACKGROUND: Dimeric naphthoquinones (BiQ were originally synthesized as a new class of HIV integrase inhibitors but have shown integrase-independent cytotoxicity in acute lymphoblastic leukemia cell lines suggesting their use as potential anti-neoplastic agents. The mechanism of this cytotoxicity is unknown. In order to gain insight into the mode of action of binaphthoquinones we performed a systematic high-throughput screen in a yeast isogenic deletion mutant array for enhanced or suppressed growth in the presence of binaphthoquinones. METHODOLOGY/PRINCIPAL FINDINGS: Exposure of wild type yeast strains to various BiQs demonstrated inhibition of yeast growth with IC(50s in the microM range. Drug sensitivity and resistance screens were performed by exposing arrays of a haploid yeast deletion mutant library to BiQs at concentrations near their IC(50. Sensitivity screens identified yeast with deletions affecting mitochondrial function and cellular respiration as having increased sensitivity to BiQs. Corresponding to this, wild type yeast grown in the absence of a fermentable carbon source were particularly sensitive to BiQs, and treatment with BiQs was shown to disrupt the mitochondrial membrane potential and lead to the generation of reactive oxygen species (ROS. Furthermore, baseline ROS production in BiQ sensitive mutant strains was increased compared to wild type and could be further augmented by the presence of BiQ. Screens for resistance to BiQ action identified the mitochondrial external NAD(PH dehydrogenase, NDE1, as critical to BiQ toxicity and over-expression of this gene resulted in increased ROS production and increased sensitivity of wild type yeast to BiQ. CONCLUSIONS/SIGNIFICANCE: In yeast, binaphthoquinone cytotoxicity is likely mediated through NAD(PH:quonine oxidoreductases leading to ROS production and dysfunctional mitochondria. Further studies are required to validate this mechanism in mammalian cells.

Caveolin-1 enhances resveratrol-mediated cytotoxicity and transport in a hepatocellular carcinoma model

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Yang Hui-ling

2009-03-01

Full Text Available Abstract Background Resveratrol (RES, an estrogen analog, is considered as a potential cancer chemo-preventive agent. However, it remains unclear how RES is transported into cells. In this study, we observed that Caveolin-1(CAV1 expression can increase the cytotoxic and pro-apoptotic activity of RES in a dose- and time-dependent manner both in vitro and in vivo in a Hepatocellular Carcinoma animal model. Methods High performance liquid chromatography (HPLC demonstrated that RES intra-cellular concentration is increased about 2-fold in cells stably expressing CAV1 or CAVM1 (a scaffolding domain (81-101AA-defective CAV1 mutant compared to the untransduced human Hepatoblastoma cell line (HepG2 or after transduction with the green fluorescent protein (GFP control vector. The increased intra-cellular transport of RES was abolished in cells stably expressing CAVM2 (a cholesterol shuttle domain (143-156AA-defective CAV1 mutant or CAVRNAi. In order to further characterize CAV1-dependent RES transport, we synthesized RES-dansyl chloride derivatives as fluorescent probes to visualize the transport process, which demonstrated a distribution consistent with that of CAV1 in HepG2 cells. Results In addition, RES endocytosis was not mediated by estrogen receptor (ER α and β, as suggested by lack of competitive inhibition by estrogen or Tamoxifen. Pathway analysis showed that RES can up-regulate the expression of endogenous CAV1; this activates further the MAPK pathway and caspase-3 expression. Discussion This study provides novel insights about the role played by CAV1 in modulating cellular sensitivity to RES through enhancement of its internalization and trafficking.

Comparative cytotoxic and genotoxic potential of 13 drinking water disinfection by-products using a microplate-based cytotoxicity assay and a developed SOS/umu assay.

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Zhang, Shao-Hui; Miao, Dong-Yue; Tan, Li; Liu, Ai-Lin; Lu, Wen-Qing

2016-01-01

The implications of disinfection by-products (DBPs) present in drinking water are of public health concern because of their potential mutagenic, carcinogenic and other toxic effects on humans. In this study, we selected 13 main DBPs found in drinking water to quantitatively analyse their cytotoxicity and genotoxicity using a microplate-based cytotoxicity assay and a developed SOS/umu assay in Salmonella typhimurium TA1535/pSK1002. With the developed SOS/umu test, eight DBPs: 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-fura3-chloro-4-(dichloromethyl)-5-hydroxy-2-[5H]-furanone (MX), dibromoacetonitrile (DBN), iodoacetic acid (IA), bromochloroacetonitrile (BCN), bromoacetic acid (BA), trichloroacetonitrile (TCN), dibromoacetic acid (DBA) and dichloroacetic acid (DCA) were significantly genotoxic to S. typhimurium. Three DBPs: chloroacetic acid (CA), trichloroacetic acid (TCA) and dichloroacetonitrile (DCN) were weakly genotoxic, whereas the remaining DBPs: chloroacetonitrile (CN) and chloral hydrate (CH) were negative. The rank order in decreasing genotoxicity was as follows: MX > DBN > IA > BCN > BA > TCN > DBA > DCA > CA, TCA, DCN > CN, CH. MX was approximately 370 000 times more genotoxic than DCA. In the microplate-based cytotoxicity assay, cytotoxic potencies of the 13 DBPs were compared and ranked in decreasing order as follows: MX > IA > DBN > BCN > BA > TCN > DCN > CA > DCA > DBA > CN > TCA > CH. MX was approximately 19 200 times more cytotoxic than CH. A statistically significant correlation was found between cytotoxicity and genotoxicity of the 13 DBPs in S. typhimurium. Results suggest that microplate-based cytotoxicity assay and the developed SOS/umu assay are feasible tools for analysing the cytotoxicity and genotoxicity of DBPs, particularly for comparing their toxic intensities quantitatively. © The Author 2015. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e

Measuring T cell-mediated cytotoxicity using fluorogenic caspase substrates.

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

2003-10-01

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

Cytotoxicity potentials of eleven Bangladeshi medicinal plants.

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Khatun, Amina; Rahman, Mahmudur; Haque, Tania; Rahman, Md Mahfizur; Akter, Mahfuja; Akter, Subarna; Jhumur, Afrin

2014-01-01

Various forms of cancer are rising all over the world, requiring newer therapy. The quest of anticancer drugs both from natural and synthetic sources is the demand of time. In this study, fourteen extracts of different parts of eleven Bangladeshi medicinal plants which have been traditionally used for the treatment of different types of carcinoma, tumor, leprosy, and diseases associated with cancer were evaluated for their cytotoxicity for the first time. Extraction was conceded using methanol. Phytochemical groups like reducing sugars, tannins, saponins, steroids, gums, flavonoids, and alkaloids were tested using standard chromogenic reagents. Plants were evaluated for cytotoxicity by brine shrimp lethality bioassay using Artemia salina comparing with standard anticancer drug vincristine sulphate. All the extracts showed potent to moderate cytotoxicity ranging from LC50 2 to 115 µg/mL. The highest toxicity was shown by Hygrophila spinosa seeds (LC50 = 2.93 µg/mL) and the lowest by Litsea glutinosa leaves (LC50 = 114.71 µg/mL) in comparison with standard vincristine sulphate (LC50 = 2.04 µg/mL). Among the plants, the plants traditionally used in different cancer and microbial treatments showed highest cytotoxicity. The results support their ethnomedicinal uses and require advanced investigation to elucidate responsible compounds as well as their mode of action.

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

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

2014-06-01

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

Evaluation of the Cytotoxicity of Structurally Correlated p-Menthane Derivatives

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Luciana Nalone Andrade

2015-07-01

Full Text Available Compounds isolated from essential oils play an important role in the prevention and treatment of cancer. Monoterpenes are natural products, and the principal constituents of many essential oils. The aim of this study was to investigate the cytotoxic potential of p-menthane derivatives. Additionally, analogues of perillyl alcohol, a monoterpene with known anticancer activity, were evaluated to identify the molecular characteristics which contribute to their cytotoxicity, which was tested against OVCAR-8, HCT-116, and SF-295 human tumor cell lines, using the MTT assay. The results of this study showed that (−-perillaldehyde 8,9-epoxide exhibited the highest percentage inhibition of cell proliferation (GI = 96.32%–99.89%. Perillyl alcohol exhibited high cytotoxic activity (90.92%–95.82%, while (+-limonene 1,2-epoxide (GI = 58.48%–93.10%, (−-perillaldehyde (GI = 59.28%–83.03%, and (−-8-hydroxycarvotanacetone (GI = 61.59%–94.01% showed intermediate activity. All of the compounds tested were less cytotoxic than perillyl alcohol, except (−-perillaldehyde 8,9-epoxide (IC50 = 1.75–1.03 µL/mg. In general, replacement of C-C double bonds by epoxide groups in addition to the aldehyde group increases cytotoxicity. Furthermore, stereochemistry seems to play an important role in cytotoxicity. We have demonstrated the cytotoxic influence of chemical substituents on the p-menthane structure, and analogues of perillyl alcohol.

Determining the size and concentration dependence of gold nanoparticles in vitro cytotoxicity (IC{sub 50}) test using WST-1 assay

Energy Technology Data Exchange (ETDEWEB)

Rosli, Nur Shafawati binti; Rahman, Azhar Abdul [School of Physics, Universiti Sains Malaysia, 11800, Pulau Pinang (Malaysia); Aziz, Azlan Abdul [School of Physics, Universiti Sains Malaysia, 11800, Pulau Pinang (Malaysia); Nano-Biotechnology Research and Innovation (NanoBRI), Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, 11800, Pulau Pinang (Malaysia); Shamsuddin, Shaharum [Nano-Biotechnology Research and Innovation (NanoBRI), Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, 11800, Pulau Pinang (Malaysia); School of Health Sciences, Health Campus, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan (Malaysia)

2015-04-24

Gold nanoparticles (AuNPs) received a great deal of attention for biomedical applications, especially in diagnostic imaging and therapeutics. Even though AuNPs have potential benefits in biomedical applications, the impact of AuNPs on human and environmental health still remains unclear. The use of AuNPs which is a high-atomic-number materials, provide advantages in terms of radiation dose enhancement. However, before this can become a clinical reality, cytotoxicity of the AuNPs has to be carefully evaluated. Cytotoxicity test is a rapid, standardized test that is very sensitive to determine whether the nanoparticles produced are harmful or benign on cellular components. In this work the size and concentration dependence of AuNPs cytotoxicity in breast cancer cell lines (MCF-7) are tested by using WST-1 assay. The sizes of AuNPs tested were 13 nm, 50 nm, and 70 nm. The cells were seeded in the 96-well plate and were treated with different concentrations of AuNPs by serial dilution for each size of AuNPs. The high concentration of AuNPs exhibit lower cell viability compared to low concentration of AuNPs. We quantified the toxicity of AuNPs in MCF-7 cell lines by determining the IC{sub 50} values in WST-1 assays. The IC{sub 50} values (inhibitory concentrations that effected 50% growth inhibition) of 50 nm AuNPs is lower than 13 nm and 70 nm AuNPs. Mean that, 50nm AuNPs are more toxic to the MCF-7 cells compared to smaller and larger sizes AuNPs. The presented results clearly indicate that the cytotoxicity of AuNPs depend not only on the concentration, but also the size of the nanoparticles.

Preparation, characterization and cytotoxic evaluation of bovine serum albumin nanoparticles encapsulating 5-methylmellein: A secondary metabolite isolated from Xylaria psidii.

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Arora, Divya; Kumar, Amit; Gupta, Prasoon; Chashoo, Gousia; Jaglan, Sundeep

2017-12-01

In this study, 5-methylmellein (5-MM) loaded bovine serum albumin nanoparticles (BSA NPs) were developed using desolvation technique. The developed nanoparticles were characterized for their mean particle size, polydispersity, zeta potential, loading efficiency, X-ray diffractometry (XRD), differential scanning calorimetry (DSC) and release profile. The developed nanoparticles were spherical in shape under transmission electron microscopy (TEM) and atomic force microscopy (AFM). The developed 5-MM loaded BSA NPs demonstrated a mean particle size with a diameter of 154.95 ± 4.44 nm. The results from XRD and DSC studies demonstrated that the crystal state of the 5-MM was converted to an amorphous state in polymeric matrix. The encapsulation and loading efficiency was found to be 73.26 ± 4.48% and 7.09 ± 0.43%. The in vitro cytotoxicity in human prostate cancer cell line (PC-3), human colon cancer cells (HCT-116) and human breast adenocarcinoma cell line (MCF-7) cells demonstrated enhanced cytotoxicity of 5-MM BSA NPs as compared to native 5-MM after 72-h treatment. The enhancement in cytotoxicity of 5-MM BSA NPs was also supported by increase in cellular apoptosis, mitochondrial membrane potential loss and generation of high reactive oxygen species (ROS). In conclusion, these findings collectively indicated that BSA nanoparticles may serve as promising drug delivery system for improving the efficacy of 5-methylmellein. Copyright © 2017 Elsevier Ltd. All rights reserved.

Study of Stevia rebaudiana Bertoni antioxidant activities and cellular properties.

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Bender, Cecilia; Graziano, Sara; Zimmermann, Benno F

2015-01-01

The aim of our study was to determine the antioxidant activities, cytotoxicity and proliferative properties in Stevia rebaudiana leaves and stems. Leaves extracts exhibited a higher antioxidant activity than stems extract, through oxygen radical absorbance capacity (ORAC) and cellular antioxidant activity (CAA) assays. Stevioside and rebaudioside A, the main sweetening metabolites in stevia leaves, exhibited a low ORAC value in comparison with plant extracts, while did not elicit any CAA. Stevia rebaudiana did not exhibit toxicity against HepG2 (hepatocellular carcinoma) human cells. No proliferative nor catalase modulations were observed in cells treated with such extracts. Our findings support the promising role of stevia that, apart from its sweetness, can act as a source of antioxidants, even at the intracellular level. This activity makes S. rebaudiana crude extract an interesting resource of natural sweetness with antioxidant properties which may find numerous applications in foods and nutritional supplements industries.

Functionalized graphene oxide/Fe3O4 hybrids for cellular magnetic resonance imaging and fluorescence labeling.

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Zhou, Chaohui; Wu, Hui; Wang, Mingliang; Huang, Chusen; Yang, Dapeng; Jia, Nengqin

2017-09-01

In this work, we developed a T 2 -weighted contrast agent based on graphene oxide (GO)/Fe 3 O 4 hybrids for efficient cellular magnetic resonance imaging (MRI). The GO/Fe 3 O 4 hybrids were obtained by combining with co-precipitation method and pyrolysis method. The structural, surface and magnetic characteristics of the hybrids were systematically characterized by transmission electron microscopy (TEM), vibrating sample magnetometer (VSM), AFM, Raman, FT-IR and XRD. The GO/Fe 3 O 4 hybrids were functionalized by modifying with anionic and cationic polyelectrolyte through layer-by-layer assembling. The fluorescence probe fluorescein isothiocyanate (FITC) was further loaded on the surface of functionalized GO/Fe 3 O 4 hybrids to trace the location of GO/Fe 3 O 4 hybrids in cells. Functionalized GO/Fe 3 O 4 hybrids possess good hydrophilicity, less cytotoxicity, high MRI enhancement with the relaxivity (r 2 ) of 493mM -1 s -1 as well as cellular MRI contrast effect. These obtained results indicated that the functionalized GO/Fe 3 O 4 hybrids could have great potential to be utilized as cellular MRI contrast agents for tumor early diagnosis and monitoring. Copyright © 2017 Elsevier B.V. All rights reserved.

Cytotoxic activity of Agave lechuguilla Torr | Casillas | African ...

African Journals Online (AJOL)

The cytotoxic activity of extract and isolated saponin from leaves of Agave lechuguilla was investigated. Ethanol extract from leaves of A. lechuguilla exhibited cytotoxic activity against HeLa cells in vitro (50% inhibitory concentration (IC50) = 89 μg/ml). Bioassay-guided fractionation of this extract had led to the isolation of 5-β ...

Real-time monitoring of cisplatin cytotoxicity on three-dimensional spheroid tumor cells

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Baek NH

2016-07-01

Full Text Available NamHuk Baek,1,* Ok Won Seo,1,* Jaehwa Lee,1 John Hulme,2 Seong Soo A An2 1Department of Research and Development, NanoEntek Inc., Seoul, 2Department of BioNano Technology, Gachon University, Gyeonggi-do, Korea *These authors contributed equally to this work Abstract: Three-dimensional (3D cell cultivation is a powerful technique for monitoring and understanding diverse cellular mechanisms in developmental cancer and neuronal biology, tissue engineering, and drug development. 3D systems could relate better to in vivo models than two-dimensional (2D cultures. Several factors, such as cell type, survival rate, proliferation rate, and gene and protein expression patterns, determine whether a particular cell line can be adapted to a 3D system. The 3D system may overcome some of the limitations of 2D cultures in terms of cell–cell communication and cell networks, which are essential for understanding differentiation, structural organization, shape, and extended connections with other cells or organs. Here, the effect of the anticancer drug cisplatin, also known as cis-diamminedichloroplatinum (II or CDDP, on adenosine triphosphate (ATP generation was investigated using 3D spheroid-forming cells and real-time monitoring for 7 days. First, 12 cell lines were screened for their ability to form 3D spheroids: prostate (DU145, testis (F9, embryonic fibroblast (NIH-3T3, muscle (C2C12, embryonic kidney (293T, neuroblastoma (SH-SY5Y, adenocarcinomic alveolar basal epithelial cell (A549, cervical cancer (HeLa, HeLa contaminant (HEp2, pituitary epithelial-like cell (GH3, embryonic cell (PA317, and osteosarcoma (U-2OS cells. Of these, eight cell lines were selected: NIH-3T3, C2C12, 293T, SH-SY5Y, A549, HeLa, PA317, and U-2OS; and five underwent real-time monitoring of CDDP cytotoxicity: HeLa, A549, 293T, SH-SY5Y, and U-2OS. ATP generation was blocked 1 day after addition of 50 µM CDDP, but cytotoxicity in HeLa, A549, SH-SY5Y, and U-2OS cells could be

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

International Nuclear Information System (INIS)

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

2005-01-01

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

In vitro study on porous silver scaffolds prepared by electroplating method using cellular carbon skeleton as the substrate

International Nuclear Information System (INIS)

Guo, M.; Wang, X.; Zhou, H.M.; Li, L.; Nie, F.L.; Cheng, Y.; Zheng, Y.F.

2012-01-01

Porous silver scaffolds, with the porosity ranging from 68% to 81% and the apparent density ranging from 0.4 to 1 g⋅cm −3 were prepared by electroplating method using cellular carbon skeleton as the substrate. The microstructure, mechanical property, cytotoxicity and antibacterial activity of the prepared porous silver scaffold were studied. The present porous silver scaffolds had a highly three-dimensional trabecular porous structure with the porosity and the apparent density close to that of the cancellous bone. Furthermore, the mechanical property such as elastic modulus and yield strength of the porous silver scaffolds were lower than that of commercial available porous Ti and porous Ti alloys but much closer to that of the cancellous bone and porous Ta. In addition, study of in vitro behavior showed that the porous silver scaffold possessed significant antibacterial capability of inhibition of bacterial proliferation and adherence against Staphylococcus aureus and Staphylococcus epidermidis, and little cytotoxicity to Mg-63 cell line and NIH-3T3 cell line. Consequently, the porous silver scaffolds prepared by electrodeposition possess a promising application for bone implants. - Highlights: ► Porous Ag scaffolds were produced by electroplating Ag on cellular carbon skeleton. ► Porous Ag scaffolds have the porosity 68–81% and the apparent density 0.4–1 g⋅cm −3 . ► The mechanical property of porous Ag is close to cancellous bone and porous Ta. ► Porous Ag inhibits the proliferation and adherence of S. aureus and S. epidermidis.

Light-triggered liposomal cargo delivery platform incorporating photosensitizers and gold nanoparticles for enhanced singlet oxygen generation and increased cytotoxicity

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Kautzka, Zofia; Clement, Sandhya; Goldys, Ewa M.; Deng, Wei

2018-02-01

We developed light-triggered liposomes incorporating gold nanoparticles and Rose Bengal (RB), a well-known photosensitizer used for photodynamic therapy. Singlet oxygen generated by these liposomes with 532 nm light illumination was characterized by adjusting the molar ratio of lipids and gold nanoparticles while keeping the amount of RB constant. Gold nanoparticles were found to enhance the singlet oxygen generation rate, with a maximum enhancement factor of 1.75 obtained for the molar ratio of HSPC: PE-NH2: gold of 57:5:17 compared with liposomes loaded with RB alone. The experimental results could be explained by the local electric field enhancement caused by gold nanoparticles. We further assessed cellular cytotoxicity of these liposomes by encapsulating an antitumor drug, doxorubicin (Dox); such Dox loaded liposomes were applied to human colorectal cancer cells, HCT116, and exposed to light. Gold-loaded liposomes containing RB and Dox where Dox release was triggered by light were found to exhibit higher cytotoxicity, compared to the liposomes loaded with RB and Dox alone. Our results indicate that gold-loaded liposomes incorporating photosensitizers may have improved therapeutic efficacy in photodynamic therapy and chemotherapy.

Norartocarpetin from a folk medicine Artocarpus communis plays a melanogenesis inhibitor without cytotoxicity in B16F10 cell and skin irritation in mice.

Science.gov (United States)

Ko, Horng-Huey; Tsai, Yi-Ting; Yen, Ming-Hong; Lin, Chun-Ching; Liang, Chan-Jung; Yang, Tsung-Han; Lee, Chiang-Wen; Yen, Feng-Lin

2013-12-10

Many natural products used in preventive medicine have also been developed as cosmeceutical ingredients in skin care products, such as Scutellaria baicalensis and Gardenia jasminoides. Norartocarpetin is one of the antioxidant and antityrosinase activity compound in Artocarpus communis; however, the cytotoxicity, skin irritation and antimelanogenesis mechanisms of norartocarpetin have not been investigated yet. In the present study, cell viability in vitro and skin irritation in vivo are used to determine the safety of norartocarpetin. The melanogenesis inhibition of norartocarpetin was determined by cellular melanin content and tyrosinase in B16F10 melanoma cell. Moreover, we examined the related-melanogenesis protein by western blot analysis for elucidating the antimelanogenesis mechanism of norartocarpin. The result of the present study demonstrated that norartocarpetin not only present non-cytotoxic in B16F10 and human fibroblast cells but also non-skin irritation in mice. Moreover, our result also first found that norartocarpetin downregulated phospho-cAMP response element-binding (phospho-CREB) and microphthalmia-associated transcription factor (MITF) expression, which in turn decreased both synthesis of tyrosinases (TRP-1 and TRP-2) and cellular melanin content. This process is dependent on norartocarpetin phosphorylation by mitogen-activated protein kinases such as phospho-JNK and phospho-p38, and it results in decreased melanogenesis. The present study suggests that norartocarpetin could be used as a whitening agent in medicine and/or cosmetic industry and need further clinical study.

In vitro determination of cytotoxic drug response in ovarian carcinoma using the fluorometric microculture cytotoxicity assay (FMCA).

Science.gov (United States)

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

1997-09-17

The fluorometric microculture cytotoxicity assay (FMCA), a short-term in vitro assay based on the concept of total tumor cell kill, was used for testing the cytotoxic drug sensitivity of tumor cells from patients with ovarian carcinoma. A total of 125 fresh specimens was obtained, 98 (78%) of which were analyzed successfully. Data from 45 patients were available for clinical correlations. The FMCA appeared to yield clinically relevant cytotoxic drug sensitivity data for ovarian carcinoma as indicated by a comparison with tumor samples obtained from patients with non-Hodgkin's lymphoma or kidney carcinoma. Considering the most active single agent in vitro actually given in vivo, and using the median drug activity among all ovarian carcinoma samples as a cut-off, the sensitivity of the assay and its specificity were 75 and 52%, respectively. Cross-resistance in vitro was frequently observed between standard drugs but not between standard drugs and Taxol. Ten percent of the specimens showed an extreme resistance for at least 4 of 6 of the drugs investigated.

Biocompatible transferrin-conjugated sodium hexametaphosphate-stabilized gold nanoparticles: synthesis, characterization, cytotoxicity and cellular uptake

International Nuclear Information System (INIS)

Parab, Harshala J; Huang, Jing-Hong; Liu, Ru-Shi; Lai, Tsung-Ching; Jan, Yi-Hua; Wang, Jui-Ling; Hsiao, Michael; Chen, Chung-Hsuan; Hwu, Yeu-Kuang; Tsai, Din Ping; Chuang, Shih-Yi; Pang, Jong-Hwei S

2011-01-01

The feasibility of using gold nanoparticles (AuNPs) for biomedical applications has led to considerable interest in the development of novel synthetic protocols and surface modification strategies for AuNPs to produce biocompatible molecular probes. This investigation is, to our knowledge, the first to elucidate the synthesis and characterization of sodium hexametaphosphate (HMP)-stabilized gold nanoparticles (Au-HMP) in an aqueous medium. The role of HMP, a food additive, as a polymeric stabilizing and protecting agent for AuNPs is elucidated. The surface modification of Au-HMP nanoparticles was carried out using polyethylene glycol and transferrin to produce molecular probes for possible clinical applications. In vitro cell viability studies performed using as-synthesized Au-HMP nanoparticles and their surface-modified counterparts reveal the biocompatibility of the nanoparticles. The transferrin-conjugated nanoparticles have significantly higher cellular uptake in J5 cells (liver cancer cells) than control cells (oral mucosa fibroblast cells), as determined by inductively coupled plasma mass spectrometry. This study demonstrates the possibility of using an inexpensive and non-toxic food additive, HMP, as a stabilizer in the large-scale generation of biocompatible and monodispersed AuNPs, which may have future diagnostic and therapeutic applications.

Biocompatible transferrin-conjugated sodium hexametaphosphate-stabilized gold nanoparticles: synthesis, characterization, cytotoxicity and cellular uptake

Energy Technology Data Exchange (ETDEWEB)

Parab, Harshala J; Huang, Jing-Hong; Liu, Ru-Shi [Department of Chemistry, National Taiwan University, Taipei 106, Taiwan (China); Lai, Tsung-Ching; Jan, Yi-Hua; Wang, Jui-Ling; Hsiao, Michael; Chen, Chung-Hsuan [Genomics Research Center, Academia Sinica, Taipei 115, Taiwan (China); Hwu, Yeu-Kuang [Institute of Physics, Academia Sinica, Taipei 115, Taiwan (China); Tsai, Din Ping [Department of Physics, National Taiwan University, Taipei 106, Taiwan (China); Chuang, Shih-Yi; Pang, Jong-Hwei S, E-mail: rsliu@ntu.edu.tw, E-mail: mhsiao@gate.sinica.edu.tw [Graduate Institute of Clinical Medical Sciences, Chang Gung University, Tao-Yuan, Taiwan (China)

2011-09-30

The feasibility of using gold nanoparticles (AuNPs) for biomedical applications has led to considerable interest in the development of novel synthetic protocols and surface modification strategies for AuNPs to produce biocompatible molecular probes. This investigation is, to our knowledge, the first to elucidate the synthesis and characterization of sodium hexametaphosphate (HMP)-stabilized gold nanoparticles (Au-HMP) in an aqueous medium. The role of HMP, a food additive, as a polymeric stabilizing and protecting agent for AuNPs is elucidated. The surface modification of Au-HMP nanoparticles was carried out using polyethylene glycol and transferrin to produce molecular probes for possible clinical applications. In vitro cell viability studies performed using as-synthesized Au-HMP nanoparticles and their surface-modified counterparts reveal the biocompatibility of the nanoparticles. The transferrin-conjugated nanoparticles have significantly higher cellular uptake in J5 cells (liver cancer cells) than control cells (oral mucosa fibroblast cells), as determined by inductively coupled plasma mass spectrometry. This study demonstrates the possibility of using an inexpensive and non-toxic food additive, HMP, as a stabilizer in the large-scale generation of biocompatible and monodispersed AuNPs, which may have future diagnostic and therapeutic applications.

Analogues of luteinizing hormone-releasing hormone containing cytotoxic groups.

Science.gov (United States)

Janáky, T; Juhász, A; Bajusz, S; Csernus, V; Srkalovic, G; Bokser, L; Milovanovic, S; Redding, T W; Rékási, Z; Nagy, A

1992-02-01

In an attempt to produce better cytotoxic analogues, chemotherapeutic antineoplastic radicals including an alkylating nitrogen mustard derivative of D-phenylalanine (D-melphalan), reactive cyclopropane, anthraquinone derivatives [2-(hydroxymethyl)anthraquinone and the anticancer antibiotic doxorubicin], and an antimetabolite (methotrexate) were coupled to suitably modified agonists and antagonists of luteinizing hormone-releasing hormone (LH-RH). Analogues with D-lysine6 and D-ornithine6 or N epsilon-(2,3-diaminopropionyl)-D-lysine and N delta-(2,3-diaminopropionyl)-D-ornithine were used as carriers for one or two cytotoxic moieties. The enhanced biological activities produced by the incorporation of D amino acids into position 6 of the agonistic analogues were further increased by the attachment of hydrophobic cytotoxic groups, resulting in compounds with 10-50 times higher activity than LH-RH. Most of the monosubstituted agonistic analogues showed high affinities for the membrane receptors of human breast cancer cells, while the receptor binding affinities of peptides containing two cytotoxic side chains were lower. Antagonistic carriers [Ac-D-Nal(2)1,D-Phe(4Cl)2,D-Trp3,Arg5,D-Lys6,D-Ala10] LH-RH [where Nal(2) is 3-(2-naphthyl)alanine], [Ac-D-Nal(2)1,D-Phe(4Cl)2,D-Trp3,Arg5,N epsilon-(2,3-diaminopropionyl)-D-Lys6,D-Ala10]LH-RH, and their D-Pal(3)3 homologs [Pal(3) is 3-(3-pyridyl)alanine] as well as [Ac-D-Nal(2)1,D-Phe(4Cl)2,D-Pal(3)3,Tyr5,N epsilon-(2,3-diamino-propionyl)-D-Lys6,D-Ala10]LH-RH were linked to cytotoxic compounds. The hybrid molecules inhibited ovulation in rats at doses of 10 micrograms and suppressed LH release in vitro. The receptor binding of cytotoxic analogues was decreased compared to the precursor peptides, although analogues with 2-(hydroxymethyl)anthraquinone hemiglutarate had high affinities. All of the cytotoxic analogues tested inhibited [3H]thymidine incorporation into DNA in cultures of human breast and prostate cancer cell lines

Novel molecular, cytotoxical, and immunological study on promising and selective anticancer activity of Mung bean sprouts

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Hafidh Rand R

2012-11-01

Full Text Available Abstract Background The anticancer and immunomodulatory activity of mung bean sprouts (MBS and the underlying mechanisms against human cervical and hepatocarcinoma cancer cells were explored. Methods MBS cytotoxicity and MBS-induced anticancer cytokines, TNF-α and IFN-β from cancer cells, and immunological cytokines, IL-4, IFN-γ, and IL-10 from peripheral mononuclear cells (PMNC were assessed by MTS and ELISA assays. Apoptotic cells were investigated by flow cytometry. The expression level of apoptotic genes (Bax, BCL-2, Capsases 7–9 and cell cycle regulatory genes (cyclin D, E, and A and tumor suppressor proteins (p27, p21, and p53 was assessed by real-time qPCR in the cancer cells treated with extract IC50. Results The cytotoxicity on normal human cells was significantly different from HeLa and HepG2 cells, 163.97 ± 5.73, 13.3 ± 0.89, and 14.04 ± 1.5 mg/ml, respectively. The selectivity index (SI was 12.44 ± 0.83 for HeLa and 11.94 ± 1.2 for HepG2 cells. Increased levels of TNF-α and IFN-β were observed in the treated HeLa and HepG2 culture supernatants when compared with untreated cells. MBS extract was shown to be an immunopolarizing agent by inducing IFNγ and inhibiting IL-4 production by PBMC; this leads to triggering of CMI and cellular cytotoxicity. The extract induced apoptosis, in a dose and time dependent manner, in treated HeLa and HepG2, but not in untreated, cells (P Conclusion MBS extract was shown to be a potent anticancer agent granting new prospects of anticancer therapy using natural products.

induced acute cytotoxicity in human cervical epithelial carcinoma cells

African Journals Online (AJOL)

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

Effect of surface modification of silica nanoparticles on toxicity and cellular uptake by human peripheral blood lymphocytes in vitro.

Science.gov (United States)

Lankoff, Anna; Arabski, Michal; Wegierek-Ciuk, Aneta; Kruszewski, Marcin; Lisowska, Halina; Banasik-Nowak, Anna; Rozga-Wijas, Krystyna; Wojewodzka, Maria; Slomkowski, Stanislaw

2013-05-01

Silica nanoparticles have an interesting potential in drug delivery, gene therapy and molecular imaging due to the possibility of tailoring their surface reactivity that can be obtained by surface modification. Despite these potential benefits, there is concern that exposure of humans to certain types of silica nanomaterials may lead to significant adverse health effects. The motivation of this study was to determine the kinetics of cellular binding/uptake of the vinyl- and the aminopropyl/vinyl-modified silica nanoparticles into peripheral blood lymphocytes in vitro, to explore their genotoxic and cytotoxic properties and to compare the biological properties of modified silica nanoparticles with those of the unmodified ones. Size of nanoparticles determined by SEM varied from 10 to 50 nm. The average hydrodynamic diameter and zeta potential also varied from 176.7 nm (+18.16 mV) [aminopropyl/vinyl-modified] and 235.4 nm (-9.49 mV) [vinyl-modified] to 266.3 (-13.32 mV) [unmodified]. Surface-modified silica particles were internalized by lymphocytes with varying efficiency and expressed no cytotoxic nor genotoxic effects, as determined by various methods (cell viability, apoptosis/necrosis, oxidative DNA damage, chromosome aberrations). However, they affected the proliferation of the lymphocytes as indicated by a decrease in mitotic index value and cell cycle progression. In contrast, unmodified silica nanoparticles exhibited cytotoxic and genotoxic properties at high doses as well as interfered with cell cycle.

A cell-microelectronic sensing technique for profiling cytotoxicity of chemicals

International Nuclear Information System (INIS)

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

2008-01-01

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

A cell-microelectronic sensing technique for profiling cytotoxicity of chemicals

Energy Technology Data Exchange (ETDEWEB)

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

2008-05-12

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

Cytotoxicity Potentials of Eleven Bangladeshi Medicinal Plants

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Amina Khatun

2014-01-01

Full Text Available Various forms of cancer are rising all over the world, requiring newer therapy. The quest of anticancer drugs both from natural and synthetic sources is the demand of time. In this study, fourteen extracts of different parts of eleven Bangladeshi medicinal plants which have been traditionally used for the treatment of different types of carcinoma, tumor, leprosy, and diseases associated with cancer were evaluated for their cytotoxicity for the first time. Extraction was conceded using methanol. Phytochemical groups like reducing sugars, tannins, saponins, steroids, gums, flavonoids, and alkaloids were tested using standard chromogenic reagents. Plants were evaluated for cytotoxicity by brine shrimp lethality bioassay using Artemia salina comparing with standard anticancer drug vincristine sulphate. All the extracts showed potent to moderate cytotoxicity ranging from LC50 2 to 115 µg/mL. The highest toxicity was shown by Hygrophila spinosa seeds (LC50=2.93 µg/mL and the lowest by Litsea glutinosa leaves (LC50=114.71 µg/mL in comparison with standard vincristine sulphate (LC50=2.04 µg/mL. Among the plants, the plants traditionally used in different cancer and microbial treatments showed highest cytotoxicity. The results support their ethnomedicinal uses and require advanced investigation to elucidate responsible compounds as well as their mode of action.

HUMORAL AND CELLULAR IMMUNITY PARAMETERS IN CHILDREN BEFORE AND AFTER ADENOTONSILLECTOMY

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M. H. Baradaranfar

2007-08-01

Full Text Available Adenoids and tonsils are active lymphoid organs and play an important role ‎against invading antigens of upper aerodigestive tract in children. ‎The present study analyzes the changes in cellular and humoral immunity of children six ‎months after adenotonsillectomy. The study population consisted of 30 children whit chronic adenotonsillar hypertrophy and 30 age-matched healthy children. ‎In all children serum level of IgM and IgG, percentage of T lymphocytes (CD3, T ‎helper cells (CD4, T cytotoxic ‎cells (CD8 and B lymphocytes (CD20 were measured before surgery. These parameters were ‎remeasured in patients 6 months after adenotonsillectomy. ‎Before the operation, a reduction in percentage of T lymphocytes (CD3,TCD4,TC8 ‎and B CD20 was seen compared to control group. This reduction was only significant in T ‎lymphocytes (CD3.The serum IgM and IgG levels were not different in two groups. Six months after ‎operation, the percentage of lymphocytes T CD3, T CD8 and BCD20 was increased and ‎reached the control group. The IgM level was also significantly decreased in patients after ‎operation. ‎Our results indicate that cellular and humoral immunity decreases in children ‎with chronic adenotonsiller hypertrophy preoperatively and increases to healthy children ‎level, six months postoperatively. It means that chronic adenotosillar hypertrophy affect ‎some parameters of cellular and humoral immunity and adenotonsillectomy by removing ‎chronic stimulations and reverses these changes without any negative effect on immune ‎function of patients.

Comparison of UVA induced cytotoxicity by iodoHoechst isomers

International Nuclear Information System (INIS)

Karagiannis, T.C.; Lobachevsky, P.N.; Martin, R.F.

2003-01-01

Full text: Isomers of the DNA minor groove binding ligand, iodoHoechst, have been shown to sensitise DNA to cleavage by ultraviolet type A (UVA). The DNA damage has been attributed to formation of a carbon-centred radical upon UVA induced dehalogenation of the drugs. Comparison of the efficacy of the ligands in inducing DNA single strand breaks in plasmid DNA has indicated that the ortho isomer is more efficient than the para- and meta-isomers, mainly due to a greater cross-section for dehalogenation, and to some extent from increased efficiency of DNA damage per dehalogenation event. In the present study, the efficiency of dehalogenation and cytotoxicity of the three iodoHoechst isomers has been compared in human erythroleukemic, K562 cells. The uptake of the iodoHoechst compounds in K562 nuclei has been measured, and the photoefficiency of the cellular associated dehalogenation by UVA has been established for the three isomers. The results indicate that the sensitivity to UVA mediated dehalogenation is much higher for the ortho analogue compared to the para and meta-analogues. Values of the UVA D37 doses for the ortho, para and meta isomers are 49 ± 2, 327 ± 29 and 251 ± 32 J/m 2 , respectively. Clonogenic survival assays have been used to compare the efficiency of sensitisation of cells to UVA irradiation by the analogues. The ortho analogue exhibits higher efficiency compared to the meta and para analogues. The numbers of dehalogenation events required for cell kill have been calculated from the clonogenic survival at various levels of drug uptake, and the results for the ortho, para and meta isomers are 1.2x10 4 , 3.9x10 4 and 11.6x10 4 , respectively. These results indicate that the ortho analogue is the most efficient isomer in sensitising cell kill by UVA irradiation due to both the high quantum yield for dehalogenation and the higher cytotoxic efficiency of dehalogenation events

Cytotoxic effects of psychotropic benzofuran derivatives, N-methyl-5-(2-aminopropyl)benzofuran and its N-demethylated derivative, on isolated rat hepatocytes.

Science.gov (United States)

Nakagawa, Yoshio; Suzuki, Toshinari; Tada, Yukie; Inomata, Akiko

2017-03-01

The novel psychoactive compounds derived from amphetamine have been illegally abused as recreational drugs, some of which are known to be hepatotoxic in humans and experimental animals. The cytotoxic effects and mechanisms of 5-(2-aminopropyl)benzofuran (5-APB) and N-methyl-5-(2-aminopropyl)benzofuran (5-MAPB), both of which are benzofuran analogues of amphetamine, and 3,4-methylenedioxy-N-methamphetamine (MDMA) were studied in freshly isolated rat hepatocytes. 5-MAPB caused not only concentration-dependent (0-4.0 mm) and time-dependent (0-3 h) cell death accompanied by the depletion of cellular ATP and reduced glutathione and protein thiol levels, but also accumulation of oxidized glutathione. Of the other analogues examined at a concentration of 4 mm, 5-MAPB/5-APB-induced cytotoxicity with the production of reactive oxygen species and loss of mitochondrial membrane potential was greater than that induced by MDMA. In isolated rat liver mitochondria, the benzofurans resulted in a greater increase in the rate of state 4 oxygen consumption than did MDMA, with a decrease in the rate of state 3 oxygen consumption. Furthermore, the benzofurans caused more of a rapid mitochondrial swelling dependent on the mitochondrial permeability transition than MDMA. 5-MAPB at a weakly toxic level (1 mm) was metabolized slowly: levels of 5-MAPB and 5-APB were approximately 0.9 mm and 50 μm, respectively, after 3 h incubation. Taken collectively, these results indicate that mitochondria are target organelles for the benzofuran analogues and MDMA, which elicit cytotoxicity through mitochondrial failure, and the onset of cytotoxicity may depend on the initial and/or residual concentrations of 5-MAPB rather than on those of its metabolite 5-APB. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Microchip screening platform for single cell assessment of NK cell cytotoxicity

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Karolin eGuldevall

2016-04-01

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

Microchip Screening Platform for Single Cell Assessment of NK Cell Cytotoxicity

Science.gov (United States)

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

2016-01-01

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

Lineage-related cytotoxicity and clonogenic profile of 1,4-benzoquinone-exposed hematopoietic stem and progenitor cells

Energy Technology Data Exchange (ETDEWEB)

Chow, Paik Wah [Biomedical Science Programme, School of Diagnostic & Applied Health Sciences, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Abdul Muda Aziz, 50300 Kuala Lumpur, Wilayah Persekutuan (Malaysia); Toxicology Laboratory, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur (Malaysia); Abdul Hamid, Zariyantey, E-mail: zyantey@ukm.edu.my [Biomedical Science Programme, School of Diagnostic & Applied Health Sciences, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Abdul Muda Aziz, 50300 Kuala Lumpur, Wilayah Persekutuan (Malaysia); Toxicology Laboratory, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur (Malaysia); Chan, Kok Meng [Environmental Health and Industrial Safety Programme, School of Diagnostic & Applied Health Sciences, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Abdul Muda Aziz, 50300 Kuala Lumpur, Wilayah Persekutuan (Malaysia); Toxicology Laboratory, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur (Malaysia); Inayat-Hussain, Salmaan Hussain [Environmental Health and Industrial Safety Programme, School of Diagnostic & Applied Health Sciences, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Abdul Muda Aziz, 50300 Kuala Lumpur, Wilayah Persekutuan (Malaysia); Rajab, Nor Fadilah [Biomedical Science Programme, School of Diagnostic & Applied Health Sciences, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Abdul Muda Aziz, 50300 Kuala Lumpur, Wilayah Persekutuan (Malaysia); Toxicology Laboratory, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur (Malaysia)

2015-04-01

Hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) are sensitive targets for benzene-induced hematotoxicity and leukemogenesis. The impact of benzene exposure on the complex microenvironment of HSCs and HPCs remains elusive. This study aims to investigate the mechanism linking benzene exposure to targeting HSCs and HPCs using phenotypic and clonogenic analyses. Mouse bone marrow (BM) cells were exposed ex vivo to the benzene metabolite, 1,4-benzoquinone (1,4-BQ), for 24 h. Expression of cellular surface antigens for HSC (Sca-1), myeloid (Gr-1, CD11b), and lymphoid (CD45, CD3e) populations were confirmed by flow cytometry. The clonogenicity of cells was studied using the colony-forming unit (CFU) assay for multilineage (CFU-GM and CFU-GEMM) and single-lineage (CFU-E, BFU-E, CFU-G, and CFU-M) progenitors. 1,4-BQ demonstrated concentration-dependent cytotoxicity in mouse BM cells. The percentage of apoptotic cells increased (p < 0.05) following 1,4-BQ exposure. Exposure to 1,4-BQ showed no significant effect on CD3e{sup +} cells but reduced the total counts of Sca-1{sup +}, CD11b{sup +}, Gr-1{sup +}, and CD45{sup +} cells at 7 and 12 μM (p < 0.05). Furthermore, the CFU assay showed reduced (p < 0.05) clonogenicity in 1,4-BQ-treated cells. 1,4-BQ induced CFU-dependent cytotoxicity by significantly inhibiting colony growth for CFU-E, BFU-E, CFU-G, and CFU-M starting at a low concentration of exposure (5 μM); whereas for the CFU-GM and CFU-GEMM, the inhibition of colony growth was remarkable only at 7 and 12 μM of 1,4-BQ, respectively. Taken together, 1,4-BQ caused lineage-related cytotoxicity in mouse HPCs, demonstrating greater toxicity in single-lineage progenitors than in those of multi-lineage. - Highlights: • We examine 1,4-BQ toxicity targeting mouse hematopoietic cell lineages. • 1,4-BQ induces concentration-dependent cytotoxicity in bone marrow (BM) cells. • 1,4-BQ shows lineage-related toxicity on hematopoietic stem and

Lineage-related cytotoxicity and clonogenic profile of 1,4-benzoquinone-exposed hematopoietic stem and progenitor cells

International Nuclear Information System (INIS)

Chow, Paik Wah; Abdul Hamid, Zariyantey; Chan, Kok Meng; Inayat-Hussain, Salmaan Hussain; Rajab, Nor Fadilah

2015-01-01

Hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) are sensitive targets for benzene-induced hematotoxicity and leukemogenesis. The impact of benzene exposure on the complex microenvironment of HSCs and HPCs remains elusive. This study aims to investigate the mechanism linking benzene exposure to targeting HSCs and HPCs using phenotypic and clonogenic analyses. Mouse bone marrow (BM) cells were exposed ex vivo to the benzene metabolite, 1,4-benzoquinone (1,4-BQ), for 24 h. Expression of cellular surface antigens for HSC (Sca-1), myeloid (Gr-1, CD11b), and lymphoid (CD45, CD3e) populations were confirmed by flow cytometry. The clonogenicity of cells was studied using the colony-forming unit (CFU) assay for multilineage (CFU-GM and CFU-GEMM) and single-lineage (CFU-E, BFU-E, CFU-G, and CFU-M) progenitors. 1,4-BQ demonstrated concentration-dependent cytotoxicity in mouse BM cells. The percentage of apoptotic cells increased (p < 0.05) following 1,4-BQ exposure. Exposure to 1,4-BQ showed no significant effect on CD3e + cells but reduced the total counts of Sca-1 + , CD11b + , Gr-1 + , and CD45 + cells at 7 and 12 μM (p < 0.05). Furthermore, the CFU assay showed reduced (p < 0.05) clonogenicity in 1,4-BQ-treated cells. 1,4-BQ induced CFU-dependent cytotoxicity by significantly inhibiting colony growth for CFU-E, BFU-E, CFU-G, and CFU-M starting at a low concentration of exposure (5 μM); whereas for the CFU-GM and CFU-GEMM, the inhibition of colony growth was remarkable only at 7 and 12 μM of 1,4-BQ, respectively. Taken together, 1,4-BQ caused lineage-related cytotoxicity in mouse HPCs, demonstrating greater toxicity in single-lineage progenitors than in those of multi-lineage. - Highlights: • We examine 1,4-BQ toxicity targeting mouse hematopoietic cell lineages. • 1,4-BQ induces concentration-dependent cytotoxicity in bone marrow (BM) cells. • 1,4-BQ shows lineage-related toxicity on hematopoietic stem and progenitors. • 1,4-BQ

Permeation of cytotoxic formulations through swatches from selected medical gloves.

Science.gov (United States)

Klein, Michael; Lambov, Nikolai; Samev, Nikola; Carstens, Gerhard

2003-05-15

The permeability of selected medical glove materials to various cytotoxic agents is described. Fifteen cytotoxic agents were prepared at the highest concentrations normally encountered by hospital personnel. Four single-layer and two double-layer glove systems made of two materials--latex and neoprene--were exposed to the drugs for 30, 60, 90, 120, 150, and 180 minutes. Circular sections of the glove material were cut from the cuff and evaluated without any pretreatment. Permeability tests were conducted in an apparatus consisting of a donor chamber containing the cytotoxic solution and a collection chamber filled with water (the acceptor medium). The two sections were separated by the glove material. Permeating portions, collected in water as the acceptor medium, were analyzed by either ultraviolet-visible light spectrophotometry or high-performance liquid chromatography (HPLC). Permeation rates were calculated on the basis of the concentration of the cytotoxic agent in the acceptor medium. Spectrophotometric measurements were taken every 30 minutes, and HPLC analysis was performed at the end of the three-hour period. Average permeation rates for 14 drugs were low (materials. All glove materials tested were impermeable to most of the cytotoxic agents over a period of three hours. Carmustine was the only agent that substantially permeated single-layer latex glove materials. Permeation of most tested cytotoxic formulations was low through swatches of material from various medical gloves.

Cytotoxic activity of some medicinal plants from hamedan district of iran.

Science.gov (United States)

Behzad, Sahar; Pirani, Atefeh; Mosaddegh, Mahmoud

2014-01-01

Medicinal plants have been investigated for possible anti-cancer effects. The aim of the present study was to examine the cytotoxic activity of several medicinal plants on different tumor cell lines. 11 selected plant species which have been used in folkloric prescriptions were collected from different sites of Hamedan district of Iran. The methanolic extracts of the plants were prepared and their cytotoxic effects on four human cancer cell lines (A549, human lung adenocarcinoma; MCF7, human breast adenocarcinoma; HepG2, hepatocellular carcinoma and HT-29, human colon carcinoma) and one normal cell line (MDBK, bovine kidney) were examined using the MTT assay. Three of these were exhibited antiproliferative activity against one or more of the cell lines. The extract from Primula auriculata demonstrated the highest cytotoxicity with IC50 of 25.79, 35.79 and 43.34 μg.mL-1 against MCF7, HepG2 and HT- 29 cells, respectively. For some of the plants, their traditional use was correlated with the cytotoxic results, whereas for others the results may support the non-cytotoxicity of species used traditionally as natural remedies. The cytotoxic species could be considered as potential of anticancer compounds.

Acid Sphingomyelinase Promotes Cellular Internalization of Clostridium perfringens Iota-Toxin.

Science.gov (United States)

Nagahama, Masahiro; Takehara, Masaya; Miyamoto, Kazuaki; Ishidoh, Kazumi; Kobayashi, Keiko

2018-05-20

Clostridium perfringens iota-toxin is a binary actin-ADP-ribosylating toxin composed of the enzymatic component Ia and receptor binding component Ib. Ib binds to a cell surface receptor, forms Ib oligomer in lipid rafts, and associates with Ia. The Ia-Ib complex then internalizes by endocytosis. Here, we showed that acid sphingomyelinase (ASMase) facilitates the cellular uptake of iota-toxin. Inhibitions of ASMase and lysosomal exocytosis by respective blockers depressed cell rounding induced by iota-toxin. The cytotoxicity of the toxin increased in the presence of Ca 2+ in extracellular fluids. Ib entered target cells in the presence but not the absence of Ca 2+ . Ib induced the extracellular release of ASMase in the presence of Ca 2+ . ASMase siRNA prevented the cell rounding induced by iota-toxin. Furthermore, treatment of the cells with Ib resulted in the production of ceramide in cytoplasmic vesicles. These observations showed that ASMase promotes the internalization of iota-toxin into target cells.

HPLC-DAD PROFILE OF PHENOLIC COMPOUNDS, CYTOTOXICITY, ANTIOXIDANT AND ANTI-INFLAMMATORY ACTIVITIES OF THE AMAZON FRUIT Caryocar villosum

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Klenicy K. L. Yamaguchi

Full Text Available Piquiá (Caryocar villosum - Caryocaraceae is a native fruit from the Amazon region rich in bioactive substances. Fruit pulp extracts were analyzed by HPLC, together with extracts obtained from fruit pulp industry residual parts, byproducts such as husks (shells and seeds. Extracts were prepared with two ethanolic solvent systems. Phenolic substances ellagic and gallic acids were detected with standards and quantified by HPLC. Cytotoxic, antioxidant and anti-inflammatory activities in vitro were also evaluated. Shell extracts showed free radicals scavenger capacity in ABTS (IC50: 3.93 ± 0.12 µg mL-1 and DPPH models (IC50: 7.81 ± 0.34 µg mL-1, low cytotoxicity in human fibroblasts, but high at tumor strains, and also a high anti-inflammatory potential observed by the inhibition of nitric oxide production. At low concentrations (20 µg mL-1, excellent antioxidant activities were verified in cellular assays, with percentages of 70.69 ± 2.77%, 79.89 ± 6.50% and 79.48 ± 8.6% for shell, pulp and seed extracts, respectively. With this set of results, C. villosum fruit extracts become a high potential raw material to be used in pharmaceutical and cosmetic applications.

JS-K, a Nitric Oxide Prodrug, Has Enhanced Cytotoxicity in Colon Cancer Cells with Knockdown of Thioredoxin Reductase 1

Science.gov (United States)

Edes, Kornelia; Cassidy, Pamela; Shami, Paul J.; Moos, Philip J.

2010-01-01

Background The selenoenzyme thioredoxin reductase 1 has a complex role relating to cell growth. It is induced as a component of the cellular response to potentially mutagenic oxidants, but also appears to provide growth advantages to transformed cells by inhibiting apoptosis. In addition, selenocysteine-deficient or alkylated forms of thioredoxin reductase 1 have also demonstrated oxidative, pro-apoptotic activity. Therefore, a greater understanding of the role of thioredoxin reductase in redox initiated apoptotic processes is warranted. Methodology The role of thioredoxin reductase 1 in RKO cells was evaluated by attenuating endogenous thioredoxin reductase 1 expression with siRNA and then either inducing a selenium-deficient thioredoxin reductase or treatment with distinct redox challenges including, hydrogen peroxide, an oxidized lipid, 4-hydroxy-2-nonenol, and a nitric oxide donating prodrug. Thioredoxin redox status, cellular viability, and effector caspase activity were measured. Conclusions/Significance In cells with attenuated endogenous thioredoxin reductase 1, a stably integrated selenocysteine-deficient form of the enzyme was induced but did not alter either the thioredoxin redox status or the cellular growth kinetics. The oxidized lipid and the nitric oxide donor demonstrated enhanced cytotoxicity when thioredoxin reductase 1 was knocked-down; however, the effect was more pronounced with the nitric oxide prodrug. These results are consistent with the hypothesis that attenuation of the thioredoxin-system can promote apoptosis in a nitric oxide-dependent manner. PMID:20098717

Cytotoxicity and in vivo efficacy of a novel antimicrobial peptoid against Staphylococcus pseudintermedius

DEFF Research Database (Denmark)

Damborg, Peter Panduro; Skindersø, Mette E; Hansen, Paul Robert

the in vivo efficacy of a novel peptoid (D2) previously shown to be effective against S. pseudintermedius in vitro. Methods: D2 was subjected to an array of cytotoxicity assays targeting proliferation, cellular stress, apoptosis and cell cycle in Jurkat cells. D2 was then formulated into a gel...... or fusidic acid ointment (day 1-3). Mice were euthanized on day 4. Affected skin was homogenized and CFU/ml was quantified by plate dilution on selective media. Results: D2 did not mediate apoptosis and showed limited effect on Jurkat cell viability and cell cycle at a concentration similar to that used...... time in this murine skin infection model and appeared to colonize well. The lacking effect of D2 in vivo could be due to either a too low concentration or an inhibitory effect of the gel formulation used. The next step will be to find the optimal concentration working in mice and assess toxicity...

The cyto- and genotoxicity of organotin compounds is dependent on the cellular uptake capability

International Nuclear Information System (INIS)

Dopp, E.; Hartmann, L.M.; Recklinghausen, U. von; Florea, A.M.; Rabieh, S.; Shokouhi, B.; Hirner, A.V.; Obe, G.; Rettenmeier, A.W.

2007-01-01

Organotin compounds have been widely used as stabilizers and anti-fouling agents with the result that they are ubiquitously distributed in the environment. Organotins accumulate in the food chain and potential effects on human health are disquieting. It is not known as yet whether cell surface adsorption or accumulation within the cell, or indeed both is a prerequisite for the toxicity of organotin compounds. In this study, the alkylated tin derivatives monomethyltin trichloride (MMT), dimethyltin dichloride (DMT), trimethyltin chloride (TMT) and tetramethyltin (TetraMT) were investigated for cyto- and genotoxic effects in CHO-9 cells in relation to the cellular uptake. To identify genotoxic effects, induction of micronuclei (MN), chromosome aberrations (CA) and sister chromatid exchanges (SCE) were analyzed and the nuclear division index (NDI) was calculated. The cellular uptake was assessed using ICP-MS analysis. The toxicity of the tin compounds was also evaluated after forced uptake by electroporation. Our results show that uptake of the organotin compounds was generally low but dose-dependent. Only weak genotoxic effects were observed after exposure of cells to DMT and TMT. MMT and TetraMT were negative in the test systems. After forced uptake by electroporation MMT, DMT and TMT induced significant DNA damage at non-cytotoxic concentrations. The results presented here indicate a considerable toxicological potential of some organotin species but demonstrate clearly that the toxicity is modulated by the cellular uptake capability

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

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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.

Cerebrospinal fluid cytotoxicity does not affect survival in amyotrophic lateral sclerosis.

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Galán, L; Matías-Guiu, J; Matias-Guiu, J A; Yáñez, M; Pytel, V; Guerrero-Sola, A; Vela-Souto, A; Arranz-Tagarro, J A; Gómez-Pinedo, U; García, A G

2017-09-01

Cerebrospinal fluid (CSF) from some patients with amyotrophic lateral sclerosis (ALS) has been demonstrated to significantly reduce the neuronal viability of primary cell cultures of motor neurons. We aimed to study the potential clinical consequences associated with the cytotoxicity of CSF in a cohort of patients with ALS. We collected CSF from thirty-one patients with ALS. We analysed cytotoxicity by incubating it into the primary cultures of motor cortex neurons. Neural viability was quantified after 24 hours using the colorimetric MTT reduction assay. All patients were followed up from the moment of diagnosis to death, and a complete evaluation during disease progression and survival was performed, including gastrostomy and respiratory assistance. Twenty-one patients (67.7%) presented a cytotoxic CSF. There were no significant differences between patients with and without cytotoxicity regarding mean time from symptom onset to the diagnosis, from the diagnosis to death, from the diagnosis to respiratory assistance with BIPAP, from diagnosis to gastrostomy and from the onset of symptoms to death. In Cox regression analysis, bulbar onset, but not cytotoxicity, gender or age at onset, was associated with a lower risk of survival. Cerebrospinal fluid cytotoxicity was not associated with differential survival rates. This suggests that the presence of cytotoxicity in CSF, measured through neuronal viability in primary cultures of motor cortex neurons, could reflect different mechanisms of the disease, but it does not predict disease outcome. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Effect of varying incubation periods on cytotoxicity and virucidal ...

African Journals Online (AJOL)

Backgrounds: Justicia gendarussa Burm.f. has an anti-HIV activity. This study was conducted to evaluate the effects of incubation periods on the cytotoxicity and virucidal activities of the J. gendarussa leaves extract on MOLT-4 cells. Materials and Methods: The cytotoxicity assay was evaluated by using the WST-1 test with ...

Cytotoxic diterpenoids from Jatropha curcas cv. nigroviensrugosus CY Yang Roots.

Science.gov (United States)

Liu, JieQing; Yang, YuanFeng; Xia, JianJun; Li, XuYang; Li, ZhongRong; Zhou, Lin; Qiu, MingHua

2015-09-01

An investigation of phytochemicals from the roots of Jatropha curcas cv. nigroviensrugosus resulted in the isolation of twenty diterpenoids, including lathyranlactone, an unusual diterpenoid lactone possessing a 5/13/3 tricyclic skeleton, jatrocurcasenones A-E and jatrophodiones B-E, as well as 10 known analogues. All isolates were evaluated for cytotoxicity against the HL-60, SMMC-772, A-549, MCF-7 and SW480 human tumor cell lines using the MTS viability assay. Four of the known analogues showed cytotoxic activity in these cell lines, with IC50 values ranging from 2.0 to 23.0 μM. Moreover, the assessment of their cytotoxic structure-activity relationships showed the epoxy ring between C-5 and C-6 and the hydroxyl group at C-2 were the key functionalities for cytotoxicity. Copyright © 2015 Elsevier Ltd. All rights reserved.

Three-dimensional culture conditions lead to decreased radiation induced cytotoxicity in human mammary epithelial cells

International Nuclear Information System (INIS)

Sowa, Marianne B.; Chrisler, William B.; Zens, Kyra D.; Ashjian, Emily J.; Opresko, Lee K.

2010-01-01

For both targeted and non-targeted exposures, the cellular responses to ionizing radiation have predominantly been measured in two-dimensional monolayer cultures. Although convenient for biochemical analysis, the true interactions in vivo depend upon complex interactions between cells themselves and the surrounding extracellular matrix. This study directly compares the influence of culture conditions on radiation induced cytotoxicity following exposure to low-LET ionizing radiation. Using a three-dimensional (3D) human mammary epithelial tissue model, we have found a protective effect of 3D cell culture on cell survival after irradiation. The initial state of the cells (i.e., 2D versus 3D culture) at the time of irradiation does not alter survival, nor does the presence of extracellular matrix during and after exposure to dose, but long term culture in 3D which offers significant reduction in cytotoxicity at a given dose (e.g. ∼4-fold increased survival at 5 Gy). The cell cycle delay induced following exposure to 2 and 5 Gy was almost identical between 2D and 3D culture conditions and cannot account for the observed differences in radiation responses. However the amount of apoptosis following radiation exposure is significantly decreased in 3D culture relative to the 2D monolayer after the same dose. A likely mechanism of the cytoprotective effect afforded by 3D culture conditions is the down regulation of radiation induced apoptosis in 3D structures.