Protection of A549 cells against the toxic effects of sulphur mustard by hexamethylenetetramine

The A549 cell line was used as a model of the deep lung to study the toxicity and mechanism of action of sulphur mustard (HD), using the neutral red (NR) dye retention and gentian violet (GV) assays as indices of cell viability. It was found that exposure to concentrations in excess of 40 μM HD resulted in a rapid onset of toxicity. Exposure to 1000 μM HD reduced viability in A549 cell cultures to 61% after 2 h (control cultures=100%), whereas exposure to 40 μM HD did not result in deleterious effects until 26 h at which point viability fell to only 84% (NR assay). Agarose gel electrophoresis of cell cultures exposed to 40 and 1000 μM HD and harvested at 4.5, 19 and 43 h after exposure to HD, indicated that cell death was due to necrosis, despite the observation that at the higher concentration of HD cells displayed many of the features common to cells undergoing apoptotic death. The ability of hexamethylenetetramine (HMT) to protect A549 cells against the effects of an LC50 challenge dose of HD was assessed using the GV and NR assays. It was found that HMT (15 mM) could protect cells against the effects of HD though HMT had to be present at the time of HD challenge. Cultures treated with HD only were 49% viable at 48 h after HD challenge, compared to 101% for protected cultures (NR assay) and 58% and 91% for unprotected and protected cultures respectively using the GV assay. Morphological observations of GV and NR stained cultures confirmed these findings. HMT concen trations of 2.5 to 25 mM were used. Maximal protection against the toxic effects of HD (LC50) was found at 10 to 25 mM HMT. Over this concentration range, HMT did not exert any toxic effects on A549 cells. Pretreatment of A549 cultures with HMT followed by its removal prior to HD challenge had no protective effect. Similarly, treating cultures with HD followed by addition of HMT did not increase the viability of the cultures, even if the HMT was added immediately after HD exposure. HMT was found to protect against the toxic effects ofHD, though it must be present at the time ofHD challenge. A549 cells were found to be a valuable experimental model for studying the toxicology of HD and other lung damaging agents, and for screening other compounds for potential therapeutic efficacy as a prelude to studies with non- transformed cell culture systems and in vivo models.

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Monoisopropylglutathione ester protects A549 cells from the cytotoxic effects of sulphur mustard

Human & Experimental Toxicology ◽

10.1177/096032719701601102 ◽

1997 ◽

Vol 16 (11) ◽

pp. 636-644 ◽

Cited By ~ 13

Author(s):

Christopher D Lindsay ◽

Joy L Hambrook ◽

Alison F Lailey

Keyword(s):

Cell Viability ◽

Cell Cultures ◽

A549 Cell ◽

High Performance ◽

Gentian Violet ◽

A549 Cells ◽

Rapid Onset ◽

Neutral Red ◽

Biochemical Basis ◽

Sulphur Mustard

1 The A549 cell line was used to assess the toxicity of sulphur mustard (HD), using gentian violet (GV) and neutral red (NR) dyes as indicators of cell viability. It was found that exposure to concentrations in excess of 40 ?M HD resulted in a rapid onset of toxicity. 2 The ability of monoisopropylglutathione ester (MIPE) to protect A549 cells against the effects of a 100 ?M challenge dose ofHD was determined using the NR and GV assays. It was found that MIPE (8 mM) could protect cells against the effects ofHD though MIPE had to be present at the time of HD challenge. Cultures protected with MIPE were two times more viable than HD exposed cells 48 h after HD challenge when using the GV and NR assays to assess viability. Observations by phase contrast microscopy of NR and GV stained cultures confirmed these findings. Addition of MIPE after previously exposing the A549 cultures to HD (for up to 5 min) maintained cell viability at 72% compared to 37% for unprotected cultures, after which time viability fell significantly so that at 10 min there was no difference in viability between the MIPE treated and untreated cultures. 3 Pretreating A549 cultures with MIPE for 1 h followed by its removal prior to HD challenge did not maintain cell viability. Treatment of cultures with HD for 1 h followed by addition of MIPE did not maintain the viability of the cultures, thus the window within which it was possible for MIPE to rescue cell cultures from the effects of HD was of short duration. 4 High performance liquid chromatography was used to determine the biochemical basis of the actions of MIPE. It was found that whilst intracellular levels of cysteine were increased up to 40-fold following treatment of A549 cell cultures with MIPE, levels of reduced glutathione did not rise. The lack of protection seen in cultures pretreated with MIPE for 1 h prior to HD exposure suggests that raising intracellular cysteine levels was not an effective strategy for protecting cells from the effects of HD. The protection observed is probably due to extra cellular inactivation of HD by MIPE.

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Diisopropylglutathione ester protects A549 cells from the cytotoxic effects of sulphur mustard

Human & Experimental Toxicology ◽

10.1177/096032719801701104 ◽

1998 ◽

Vol 17 (11) ◽

pp. 606-612 ◽

Cited By ~ 6

Author(s):

Christopher D Lindsay ◽

Joy L Hambrook

Keyword(s):

Phase Contrast ◽

Gentian Violet ◽

A549 Cells ◽

Neutral Red ◽

A549 Cell Line ◽

Cytotoxic Effects ◽

Sulphur Mustard ◽

Culture Viability ◽

Contrast Microscopy ◽

Optimal Effect

The A549 cell line was used to assess the ability of diisopropylglutathione (DIPE) to protect against a 100 mM challenge dose of sulphur mustard (HD) using gentian violet (GV), thiazolyl blue (MTT) and neutral red (NR) assays as indicators of cell culture viability. As part of a continuing study of the efficacy of protective nucleophiles as candidate treatments for HD poisoning, several different combinations of protectant and HD were used to determine the optimal means of protecting A549 cells from the effects of HD. It was found that DIPE (4 mM) could protect cells against the effects of HD though for optimal effect, DIPE had to be present at the time of HD challenge. Cultures protected with DIPE were up to 2.9- fold more viable than HD exposed cells 48 h after HD challenge when using the GV, MTT and NR assays to assess viability. Observations by phase contrast microscopy of GV stained cultures confirmed these findings. Pretreating A549 cultures with DIPE for 1 h followed by its removal prior to HD challenge did maintain cell viability, though at a relatively low level (only up to 1.4- fold more viable than HD only exposed cells). DIPE was also able to protect HD exposed A549 cultures when added to cell cultures at intervals of up to 12 to 15 min after the initial HD exposure, though viability tended to decrease over this period, so that at 1 h, addition of DIPE did not maintain the viability of the cultures. This is the first such report of the anti-HD protectant properties of DIPE in A549 cells. It is concluded that the protection observed against HD is probably largely due to extracellular inactivation of HD by DIPE.

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Group B Coxsackievirus Diabetogenic Phenotype Correlates with Replication Efficiency

Journal of Virology ◽

10.1128/jvi.02361-05 ◽

2006 ◽

Vol 80 (11) ◽

pp. 5637-5643 ◽

Cited By ~ 42

Author(s):

Toru Kanno ◽

Kisoon Kim ◽

Ken Kono ◽

Kristen M. Drescher ◽

Nora M. Chapman ◽

...

Keyword(s):

Beta Cell ◽

Cell Cultures ◽

Virulent Strain ◽

Nod Mice ◽

Cell Types ◽

Rapid Onset ◽

Infectious Dose ◽

Group B ◽

Islet Cell Types

ABSTRACT Group B coxsackieviruses can initiate rapid onset type 1 diabetes (T1D) in old nonobese diabetic (NOD) mice. Inoculating high doses of poorly pathogenic CVB3/GA per mouse initiated rapid onset T1D. Viral protein was detectable in islets shortly after inoculation in association with beta cells as well as other primary islet cell types. The virulent strain CVB3/28 replicated to higher titers more rapidly than CVB3/GA in the pancreas and in established beta cell cultures. Exchange of 5′-nontranslated regions between the two CVB3 strains demonstrated a variable impact on replication in beta cell cultures and suppression of in vivo replication for both strains. While any CVB strain may be able to induce T1D in prediabetic NOD mice, T1D onset is linked both to the viral replication rate and infectious dose.

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Quantitative Chemical Proteomics Reveals Resveratrol Inhibition of A549 Cell Migration Through Binding Multiple Targets to Regulate Cytoskeletal Remodeling and Suppress EMT

Frontiers in Pharmacology ◽

10.3389/fphar.2021.636213 ◽

2021 ◽

Vol 12 ◽

Author(s):

Xiao Chen ◽

Yutong Wang ◽

Jing Tian ◽

Yurou Shao ◽

Bo Zhu ◽

...

Keyword(s):

Cell Migration ◽

A549 Cell ◽

A549 Cells ◽

Multiple Targets ◽

Chemical Proteomics ◽

Protein Targets ◽

Cytoskeletal Remodeling ◽

Quantitative Chemical

Resveratrol (RSV), a health-promoting natural product, has been shown to affect various cellular processes in tumor cells. However, the specific protein targets of RSV and the mechanism of action (MOA) of its anticancer effect remain elusive. In this study, the pharmacological activity of RSV was first evaluated in A549 cells, and the results showed that RSV significantly inhibited A549 cell migration but did not affect cell viability. To elucidate the underlying mechanism, a quantitative chemical proteomics approach was employed to identify the protein targets of RSV. A total of 38 target proteins were identified, and proteomic analysis showed that the targets were mainly involved in cytoskeletal remodeling and EMT, which were verified by subsequent in vitro and in vivo assays. In conclusion, RSV inhibits A549 cell migration by binding to multiple targets to regulate cytoskeletal remodeling and suppress EMT.

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RNAseq reveals extensive metabolic disruptions in the sensitive SF-295 cell line treated with schweinfurthins

Scientific Reports ◽

10.1038/s41598-021-04117-7 ◽

2022 ◽

Vol 12 (1) ◽

Author(s):

J. S. Weissenrieder ◽

J. D. Weissenkampen ◽

J. L. Reed ◽

M. V. Green ◽

C. Zheng ◽

...

Keyword(s):

Cell Line ◽

Cell Lines ◽

A549 Cell ◽

Protein Metabolism ◽

A549 Cells ◽

Hedgehog Pathway ◽

Late Time ◽

A549 Cell Line ◽

Significant Interaction Effect

AbstractThe schweinfurthin family of natural compounds exhibit a unique and potent differential cytotoxicity against a number of cancer cell lines and may reduce tumor growth in vivo. In some cell lines, such as SF-295 glioma cells, schweinfurthins elicit cytotoxicity at nanomolar concentrations. However, other cell lines, like A549 lung cancer cells, are resistant to schweinfurthin treatment up to micromolar concentrations. At this time, the precise mechanism of action and target for these compounds is unknown. Here, we employ RNA sequencing of cells treated with 50 nM schweinfurthin analog TTI-3066 for 6 and 24 h to elucidate potential mechanisms and pathways which may contribute to schweinfurthin sensitivity and resistance. The data was analyzed via an interaction model to observe differential behaviors between sensitive SF-295 and resistant A549 cell lines. We show that metabolic and stress-response pathways were differentially regulated in the sensitive SF-295 cell line as compared with the resistant A549 cell line. In contrast, A549 cell had significant alterations in response genes involved in translation and protein metabolism. Overall, there was a significant interaction effect for translational proteins, RNA metabolism, protein metabolism, and metabolic genes. Members of the Hedgehog pathway were differentially regulated in the resistant A549 cell line at both early and late time points, suggesting a potential mechanism of resistance. Indeed, when cotreated with the Smoothened inhibitor cyclopamine, A549 cells became more sensitive to schweinfurthin treatment. This study therefore identifies a key interplay with the Hedgehog pathway that modulates sensitivity to the schweinfurthin class of compounds.

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In vivo and in vitro effects of hyperplasia suppressor gene on the proliferation and apoptosis of lung adenocarcinoma A549 cells

Bioscience Reports ◽

10.1042/bsr20180391 ◽

2018 ◽

Vol 38 (5) ◽

Author(s):

Zhen-Qing Sun ◽

Gang Chen ◽

Qiang Guo ◽

He-Fei Li ◽

Zhou Wang

Keyword(s):

Cell Proliferation ◽

Lung Adenocarcinoma ◽

Gene Silencing ◽

Nude Mice ◽

A549 Cell ◽

A549 Cells ◽

Suppressor Gene ◽

Proliferation And Apoptosis ◽

Lung Adenocarcinoma A549

Lung adenocarcinoma is the most common subtype of non-small cell lung cancer (NSCLC). Hyperplasia suppressor gene (HSG) has been reported to inhibit cell proliferation, migration, and remodeling in cardiovascular diseases. However, there lacks systematic researches on the effect of HSG on the apoptosis and proliferation of lung adenocarcinoma A549 cells and data of in vivo experiments. The present study aims to investigate the effects of HSG gene silencing on proliferation and apoptosis of lung adenocarcinoma A549 cells. The human lung adenocarcinoma A549 cell was selected to construct adenovirus vector. Reverse transcription-quantitative PCR (RT-qPCR) and Western blot analysis were conducted to detect expressions of HSG and apoptosis related-proteins. Cell Counting Kit (CCK)-8 assay was performed to assess A549 cell proliferation and flow cytometry to analyze cell cycle and apoptosis rate. The BALB/C nude mice were collected to establish xenograft model. Silenced HSG showed decreased mRNA and protein expressions of HSG, and elevated A549 cell survival rates at the time point of 24, 48, and 72 h. The ratio of cells at G0/G1 phase and apoptosis rate decreased and the ratio of cells at S- and G2/M phases increased following the silencing of HSG. There were decreases of B cell lymphoma-2 (Bcl-2)-associated X protein (Bax), Caspase-3, and Caspase-8 expressions but increases in Bcl-2 induced by silenced HSG. As for the xenograft in nude mice, tumor volume increased, and apoptosis index (AI) decreased after HSG silencing. These results indicate that HSG gene silencing may promote the proliferation of A549 cells and inhibit the apoptosis. HSG may be a promising target for the treatment of lung adenocarcinoma.

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Edelfosine: An Antitumor Drug Prototype

Anti-Cancer Agents in Medicinal Chemistry ◽

10.2174/1871520618666180105165431 ◽

2018 ◽

Vol 18 (6) ◽

pp. 865-874 ◽

Cited By ~ 3

Author(s):

Sarah F. Teixeira ◽

Cecilia P. Rodrigues ◽

Cícero J.S. Costa ◽

Thais N. Pettinati ◽

Ricardo A. de Azevedo ◽

...

Keyword(s):

Lung Cancer ◽

Flow Cytometry ◽

Mitochondrial Membrane ◽

3D Culture ◽

A549 Cells ◽

Antitumor Drug ◽

New Drugs ◽

Cytotoxic Effects ◽

In Vivo Models

Background: Lung cancer is the most prevalent cancer and a high fatality disease. Despite of all available therapeutic approaches, drug resistance of chemotherapy agents for patients remain as an obstacle. New drugs integrating immunotherapeutic and conventional cytotoxic effects is a powerful strategy for the treatment of cancer to overcome this limitation. Antineoplastic phospholipids combine both of these activities by affecting lipid metabolism and signaling through lipid rafts. Therefore, they emerge as interesting scaffolds for designing new drugs. Objective: We aimed to evaluate antineoplastic phospholipids as scaffolds for designing new drugs for lung cancer treatment. Methods: The initial screening in A549 cells was performed by MTT assay. Others cytotoxic effects were evaluated in A549 cells by clonogenic assay, Matrigel 3D culture and flow cytometry analyses of cell cycle, apoptosis, mitochondrial membrane electronic potential and superoxide production. Immunological effects of ED were accessed on dendritic cells (DCs) and the expression of some markers were evaluated by flow cytometry. In vivo lung colonization analysis was performed after intravenously injection of A549 cells and daily treatment with ED. Results: Herein, ED showed to be the most efficient compound concerning cytotoxic, thereby, ED was selected for following tests. ED showed a cytotoxic profile in both monolayer and 3D culture and also in vivo models using A549 cells. This profile is due to G0/G1 phase cellular arrest and apoptosis drove by mitochondrial membrane depolarization and superoxide overproduction. Moreover, ED modulated DCs toward an activated pattern by the increased expression of CD83 and a remarkable decreased expression of PD-L1/CD274 on DCs membrane. Conclusions: Thus, ED is an interesting antitumor drug prototype due to not only its direct cellular cytotoxicity but also given its immunological features.

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In vitro and in vivo effects of the mTOR inhibitors everolimus (EVE) and temsirolimus (TEM).

Journal of Clinical Oncology ◽

10.1200/jco.2014.32.4_suppl.466 ◽

2014 ◽

Vol 32 (4_suppl) ◽

pp. 466-466

Author(s):

David Chen ◽

Michael Terence O'Reilly ◽

Paul M.J. McSheehy

Keyword(s):

Cell Proliferation ◽

Antitumor Activity ◽

A549 Cells ◽

Human Tumor ◽

Mtor Inhibitors ◽

In Vivo Models ◽

Hct 116 ◽

Xenograft Models

466 Background: Two mTOR inhibitors are approved for treating mRCC: EVE following the failure of VEGF-targeted therapy and TEM as first-line therapy in poor-risk patients. Both agents exert their clinical effect by binding to FKBP-12, which then interacts with mTOR to inhibit its kinase activity. We compared the activity of EVE and TEM in in vitro and in vivo models. Methods: EVE and TEM binding to mTOR was assessed using time-resolved fluorescence resonance energy transfer. Inhibition of cell proliferation in A549, NCI-H460, and MCF7 human tumor cell lines was assessed by methylene blue protein staining. Phosphorylation of the downstream mTOR target pS6 was assessed via immunohistochemistry in A549 cells. Antitumor activity of EVE (oral) and TEM (intraperitoneal) at doses between 0.1 and 2.5 mg/kg once daily was assessed in vivo in A549, KB-31, KB-8511, and HCT-116 human tumor xenograft models. Results: The binding efficiency of TEM for mTOR was reduced 10-fold compared with that of EVE (EC50, 56 nM vs 6 nM; p < 0.01). EVE demonstrated 6- to 7-fold greater inhibition of cell proliferation than TEM (IC50, 1.0 nM vs 6.5 nM in A549 [p < 0.001], 0.7 nM vs 4.7 nM in NCI-H460 [p < 0.01], and 19.4 nM vs 150 nM in MCF7 [p < 0.001]). Complete inhibition of pS6 phosphorylation in A549 cells at 24 hours was achieved with 6.7 nM EVE, but required 20 nM TEM. In all xenograft models, EVE and TEM showed a dose-response relationship over the range of 0.1-2.5 mg/kg/day. EVE was significantly more potent than TEM in the A549 model (EC50, 0.11 mg/kg vs 0.51 mg/kg; p = 0.002); no appreciable differences between EVE and TEM were observed in the KB-31, KB-8511, and HCT-116 xenograft models. However, correcting for drug exposure suggests increased potency of EVE over TEM. Conclusions: Compared with TEM, EVE had a higher affinity for the molecular target of FKBP-12. This was consistent with more potent antitumor activity in vitro and in vivo. Whether these data would translate into a better therapeutic index for EVE is unknown. However, the results suggest that these mTOR inhibitors may not be clinically interchangeable.

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Lipopolysaccharide Administration Alters Extracellular Vesicles in Cell Lines and Mice

Current Microbiology ◽

10.1007/s00284-021-02348-5 ◽

2021 ◽

Author(s):

Leandra B. Jones ◽

Sanjay Kumar ◽

Courtnee’ R. Bell ◽

Brennetta J. Crenshaw ◽

Mamie T. Coats ◽

...

Keyword(s):

Membrane Protein ◽

Bacterial Infection ◽

Cell Lines ◽

Extracellular Vesicles ◽

A549 Cells ◽

Diagnostic Tools ◽

In Vivo Models ◽

The Impact

AbstractExtracellular vesicles (EVs) play a fundamental role in cell and infection biology and have the potential to act as biomarkers for novel diagnostic tools. In this study, we explored the in vitro impact of bacterial lipopolysaccharide administration on cell lines that represents a target for bacterial infection in the host. Administration of lipopolysaccharide at varying concentrations to A549 and BV-2 cell lines caused only modest changes in cell death, but EV numbers were significantly changed. After treatment with the highest concentration of lipopolysaccharide, EVs derived from A549 cells packaged significantly less interleukin-6 and lysosomal-associated membrane protein 1. EVs derived from BV-2 cells packaged significantly less tumor necrosis factor after administration of lipopolysaccharide concentrations of 0.1 µg/mL and 1 µg/mL. We also examined the impact of lipopolysaccharide administration on exosome biogenesis and cargo composition in BALB/c mice. Serum-isolated EVs from lipopolysaccharide-treated mice showed significantly increased lysosomal-associated membrane protein 1 and toll-like receptor 4 levels compared with EVs from control mice. In summary, this study demonstrated that EV numbers and cargo were altered using these in vitro and in vivo models of bacterial infection.

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