Human Gyrovirus Apoptin as a Potential Selective Anticancer Agent: An In Vitro Study

Amir Akbari; Rita Arabsolghar; Abbas Behzad Behbahani; Gholamreza Rafiei Dehbidi; Farahnaz Zare; Mahdieh Hadi

doi:10.15171/ps.2019.7

Human Gyrovirus Apoptin as a Potential Selective Anticancer Agent: An In Vitro Study

Pharmaceutical Sciences ◽

10.15171/ps.2019.7 ◽

2019 ◽

Vol 25 (1) ◽

pp. 44-49

Author(s):

Amir Akbari ◽

Rita Arabsolghar ◽

Abbas Behzad Behbahani ◽

Gholamreza Rafiei Dehbidi ◽

Farahnaz Zare ◽

...

Keyword(s):

Cell Line ◽

Cell Lines ◽

A549 Cells ◽

Chicken Anemia Virus ◽

Target Cells ◽

Hek 293 ◽

Control Plasmid ◽

Anemia Virus ◽

Apoptotic Effects ◽

Constructed Plasmids

Background: Selective therapy has always been the main challenge in cancer treatments. Recently, it has been shown that Human Gyrovirus-derived protein apoptin (HGV-Apoptin) has selective cytotoxic effects on cancer cells similar to its homologue, Chicken Anemia Virus-derived Apoptin (CAV-Apoptin). However, apoptotic effects of Human Gyrovirus apoptin have been only evaluated on a few cancerous cell lines and need to be further investigated. In this study, we have evaluated the apoptotic effects of HGV-Apoptin and CAV-Apoptin expression on lung cancer (A549) and normal (HEK-293) cell lines, in order to provide more information about the specificity of these proteins on cancerous cells. Methods: Target cells were transfected by the calcium-phosphate precipitation method with constructed plasmids expressing HGV-Apoptin and CAV-Apoptin proteins as well as the control plasmid. Transfection efficiency was followed and imaged by fluorescence microscopy. Quantification of apoptosis was performed by flow cytometry. Measurements were compared by paired Student t-test. Results: Cells were successfully transfected with control and constructed plasmids. Flowcytometry analysis showed that A549 cells transfected with HGV-Apoptin and CAV-Apoptin expressing plasmids, undergone the apoptosis compared to A549 cells transfected with control plasmid (P<0.001). None of the plasmids could induce apoptosis in HEK-293 cells. Conclusion: Human Gyrovirus-derived apoptin (HGV-Apoptin) similar to its homologue, chicken anemia virus derived Apoptin (CAV-Apoptin) can induce apoptosis in Non-small-cell lung carcinoma cell line A549, but not in normal human embryonic kidney cell line HEK-293, which can be introduced as a promising novel specific antitumor agent.

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Apoptotic Effects of N-(2-hydroxyphenyl)-2-propylpentanamide on U87-MG and U-2 OS Cells and Antiangiogenic Properties

Anti-Cancer Agents in Medicinal Chemistry ◽

10.2174/1871520620666200728125356 ◽

2020 ◽

Vol 20 ◽

Author(s):

Paola Castillo-Juárez ◽

Sebastián C. Sanchez ◽

Alma D. Chávez-Blanco ◽

Humberto Mendoza-Figueroa ◽

José Correa-Basurto

Keyword(s):

Cell Line ◽

Cell Lines ◽

Cancer Cell ◽

Histone Deacetylases ◽

Biochemical Mechanism ◽

Antiproliferative Effects ◽

Human Osteosarcoma ◽

Antiproliferative Agent ◽

Induced Apoptosis ◽

Apoptotic Effects

Background and Objective: Histone deacetylases (HDACs) are important therapeutic targets for many types of human cancers. A derivative of valproic acid, N-(2-hydroxyphenyl)-2-propylpentanamide (HO-AAVPA), has antiproliferative properties on some cancer cell lines and inhibits the HDAC1 isoform. Materials and Methods: In this work, HO-AAVPA was tested as an antiproliferative agent in U87-MG (human glioblastoma) and U-2 OS cells (human osteosarcoma), which are types of cancer that are difficult to treat, and its antiangiogenic properties were explored. Results: HO-AAVPA had antiproliferative effects at 48 h with an IC50 = 0.655 mM in U87-MG cells and an IC50 = 0.453 mM in U-2 OS cells. Additionally, in the colony formation assay, HO-AAVPA decreased the number of colonies by approximately 99% in both cell lines and induced apoptosis by 31.3% in the U-2 OS cell line and by 78.2% in the U87-MG cell line. Additionally, HO-AAVPA reduced the number of vessels in chorioallantoid membranes (CAMs) by approximately 67.74% and IL-6 levels in both cell lines suggesting that the biochemical mechanism on cancer cell of HO-AAVPA is different compared to VPA. Conclusion: HO-AAVPA has antiproliferative effects on glioblastoma and osteosarcoma and antiangiogenic properties.

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The effectiveness of antiviral agents with broad-spectrum activity against chikungunya virus varies between host cell lines

Antiviral Chemistry and Chemotherapy ◽

10.1177/2040206618807580 ◽

2018 ◽

Vol 26 ◽

pp. 204020661880758 ◽

Cited By ~ 14

Author(s):

Evelyn J Franco ◽

Jaime L Rodriquez ◽

Justin J Pomeroy ◽

Kaley C Hanrahan ◽

Ashley N Brown

Keyword(s):

Antiviral Activity ◽

Host Cell ◽

Cell Line ◽

Cell Lines ◽

Broad Spectrum ◽

Chikungunya Virus ◽

Plaque Assay ◽

A549 Cells ◽

Western Hemisphere ◽

Viral Burden

Chikungunya virus (CHIKV) is a mosquito-borne virus that has recently emerged in the Western Hemisphere. Approved antiviral therapies or vaccines for the treatment or prevention of CHIKV infections are not available. This study aims to evaluate the antiviral activity of commercially available broad-spectrum antivirals against CHIKV. Due to host cell-specific variability in uptake and intracellular processing of drug, we evaluated the antiviral effects of each agent in three cell lines. Antiviral activities of ribavirin (RBV), interferon-alfa (IFN-α) and favipiravir (FAV) were assessed in CHIKV-infected Vero, HUH-7, and A549 cells. CHIKV-infected cells were treated with increasing concentrations of each agent for three days and viral burden was quantified by plaque assay on Vero cells. Cytotoxic effects of RBV, FAV and IFN-α were also evaluated. Antiviral activity differed depending on the cell line used for evaluation. RBV had the greatest antiviral effect in HUH-7 cells (EC50 = 2.575 µg/mL); IFN-α was most effective in A549 cells (EC50 = 4.235 IU/mL); and FAV in HUH-7 cells (EC50 = 20.00 μg/mL). The results of our study show FAV and IFN-α are the most promising candidates, as their use led to substantial reductions in viral burden at clinically achievable concentrations in two human-derived cell lines. FAV is an especially attractive candidate for further investigation due to its oral bioavailability. These findings also highlight the importance of cell line selection for preclinical drug trials.

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Epithelium-Specific Ets-Like Transcription Factor 1, ESE-1, Regulates ICAM-1 Expression in Cultured Lung Epithelial Cell Lines

Mediators of Inflammation ◽

10.1155/2015/547928 ◽

2015 ◽

Vol 2015 ◽

pp. 1-8 ◽

Cited By ~ 3

Author(s):

Zhiqi Yu ◽

Jun Xu ◽

Jinbao Liu ◽

Jing Wu ◽

Chan Mi Lee ◽

...

Keyword(s):

Transcription Factor ◽

Airway Inflammation ◽

Cell Lines ◽

Leukemia Cell ◽

A549 Cells ◽

Leukemia Cell Line ◽

Intercellular Adhesion Molecule ◽

Cytokine Signaling ◽

Target Cells ◽

Transcription Factor 1

Cystic fibrosis (CF) patients suffer from chronic airway inflammation with excessive neutrophil infiltration. Migration of neutrophils to the lung requires chemokine and cytokine signaling as well as cell adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1), which plays an important role in mediating adhesive interactions between effector and target cells in the immune system. In this study, we investigated the relationship betweenICAM-1and epithelium-specific ETS-like transcription factor 1 (ESE-1) and found thatICAM-1expression is upregulated in cell lines of CF (IB3-1) as well as non-CF (BEAS-2B and A549) epithelial origin in response to inflammatory cytokine stimulation. SinceESE-1is highly expressed in A549 cells without stimulation, we examined the effect ofESE-1knockdown onICAM-1expression in these cells. We found thatICAM-1expression was downregulated whenESE-1was knocked down in A549 cells. We also tested the effect ofESE-1knockdown on cell-cell interactions and demonstrate that the knocking downESE-1in A549 cells reduce their interactions with HL-60 cells (human promyelocytic leukemia cell line). These results suggest thatESE-1may play a role in regulating airway inflammation by regulatingICAM-1expression.

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Expression of 9-O- and 7,9-O-Acetyl Modified Sialic Acid in Cells and Their Effects on Influenza Viruses

mBio ◽

10.1128/mbio.02490-19 ◽

2019 ◽

Vol 10 (6) ◽

Cited By ~ 8

Author(s):

Karen N. Barnard ◽

Brian R. Wasik ◽

Justin R. LaClair ◽

David W. Buchholz ◽

Wendy S. Weichert ◽

...

Keyword(s):

Cell Lines ◽

Influenza A ◽

A549 Cells ◽

Sialic Acids ◽

Influenza Viruses ◽

Obvious Effect ◽

Hek 293 ◽

Chemically Modified ◽

Modified Forms ◽

In Cells

ABSTRACT Sialic acids (Sia) are widely displayed on the surfaces of cells and tissues. Sia come in a variety of chemically modified forms, including those with acetyl modifications at the C-7, C-8, and C-9 positions. Here, we analyzed the distribution and amounts of these acetyl modifications in different human and canine cells. Since Sia or their variant forms are receptors for influenza A, B, C, and D viruses, we examined the effects of these modifications on virus infections. We confirmed that 9-O-acetyl and 7,9-O-acetyl modified Sia are widely but variably expressed across cell lines from both humans and canines. Although they were expressed on the cell surfaces of canine MDCK cell lines, they were located primarily within the Golgi compartment of human HEK-293 and A549 cells. The O-acetyl modified Sia were expressed at low levels of 1 to 2% of total Sia in these cell lines. We knocked out and overexpressed the sialate O-acetyltransferase gene (CasD1) and knocked out the sialate O-acetylesterase gene (SIAE) using CRISPR/Cas9 editing. Knocking out CasD1 removed 7,9-O- and 9-O-acetyl Sia expression, confirming previous reports. However, overexpression of CasD1 and knockout of SIAE gave only modest increases in 9-O-acetyl levels in cells and no change in 7,9-O-acetyl levels, indicating that there are complex regulations of these modifications. These modifications were essential for influenza C and D infection but had no obvious effect on influenza A and B infection. IMPORTANCE Sialic acids are key glycans that are involved in many different normal cellular functions, as well as being receptors for many pathogens. However, Sia come in diverse chemically modified forms. Here, we examined and manipulated the expression of 7,9-O- and 9-O-acetyl modified Sia on cells commonly used in influenza virus and other research by engineering the enzymes that produce or remove the acetyl groups.

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Megakaryocytic cell line-specific hyperploidy by cytotoxic necrotizing factor bacterial toxins

Blood ◽

10.1182/blood.v88.9.3465.bloodjournal8893465 ◽

1996 ◽

Vol 88 (9) ◽

pp. 3465-3473 ◽

Cited By ~ 4

Author(s):

KM Hudson ◽

NC Denko ◽

E Schwab ◽

E Oswald ◽

A Weiss ◽

...

Keyword(s):

Cell Line ◽

Cell Lines ◽

Phorbol Ester ◽

Dna Content ◽

Nuclear Dna ◽

Nuclear Dna Content ◽

Adherent Cell ◽

Target Cells ◽

Suspension Cells ◽

Cytotoxic Necrotizing Factor

Cytotoxic necrotizing factor (CNF) toxins, isolated from certain Escherichia coli strains known to cause intestinal and extra intestinal infections, induce reorganization of the actin cytoskeleton and generate hyperploidy in adherent cell lines. We have examined the effect of CNF toxin on one of the few cell types that naturally increase nuclear DNA content, megakaryocytes. Our studies show that only hematopoietic cells capable of differentiating along the megakaryocyte lineage responded to the CNF2 toxin by becoming polyploid and by reorganizing actin. The K562, HEL, and CHRF-288–11 cell lines can be induced with phorbol ester to differentiate along the megakaryocyte lineage, and these cells also respond to the toxin with increased DNA content and actin cytoskeletal rearrangements. Interestingly, treatment of the K562 and HEL cell lines with CNF2 does not result in an increase in production of the megakaryocytic marker glycoprotein IIIa, unlike phorbol ester treatment. Conversely, two T-cell leukemic cell lines, CEM and Molt4, and the promyelocytic HL-60 cell line, which do not differentiate along the megakaryocyte lineage in response to phorbol myristate acetate, do not respond to CNF2, by increased expression of gpIIIa, increased nuclear DNA content, or actin reorganization. A potential target of these toxins, RhoA, is expressed by both megakaryocytic and nonmegakaryocytic cell lines, as shown by reverse transcription-polymerase chain reaction and Western blot. Although it is clear that the CNF toxins can affect a wide variety of adherent nonhematopoietic cell lines, we propose that the response to CNF, in terms of reorganizing actin structure and increase in DNA content in hematologic suspension cells, correlates with the capability of these target cells to differentiate along the megakaryocytic lineage.

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Neuropilin-1 expression in adenocarcinoma and squamous cell carcinoma of the lung is differentially regulated by hypoxia

Journal of Clinical Oncology ◽

10.1200/jco.2006.24.18_suppl.17152 ◽

2006 ◽

Vol 24 (18_suppl) ◽

pp. 17152-17152

Author(s):

G. P. Pidgeon ◽

M. P. Barr ◽

M. C. Cathcart ◽

S. Gray ◽

K. J. O’Byrne

Keyword(s):

Lung Cancer ◽

Squamous Cell Carcinoma ◽

Cell Carcinoma ◽

Cell Line ◽

Cell Lines ◽

Squamous Cell ◽

A549 Cells ◽

Lung Tumours ◽

Western Analysis ◽

Neuropilin 1

17152 Background: Neuropilin-1 (NRP-1) is an isoform-specific receptor for VEGF165 and semaphorin3A, initially discovered on migrating neurons. NRP-1 expression has been reported on a number of tumour cell lines in the absence of the other VEGF receptors, where it mediates survival signals. In this study we examined the regulation of NRP-1 by hypoxia, its effect on survival in a panel of lung cancer cell lines and its potential as a biomarker in retrospective human lung tumours. Methods: A549, SK-MES1, H460 and H647 cells were grown in serum depleted media (0.5%) in normoxic or hypoxic (0.1% O2) conditions and screened for NRP-1 expression by western and immunocytochemistry analysis. Cell survival and apoptosis was determined using BrdU and Annexin-V/PI staining respectively following treatment with an antibody to the extracellular NRP-1 domain. A panel of 100 retrospective resected lung tumours and matched normal samples were stained for NRP-1 expression by immunhistochemistry. Results: A549, SKMES-1 and H647 cell lines all expressed NRP-1 and displayed reduced survival following treatment with NRP-1 antibody (1ug/ml) compared to controls (A549 46%, SKMES-1 61%, H647 53%). H460 did not express NRP-1 and no survival inhibition was seen in the cell line (104%). Reduced survival was accompanied by increased apoptosis in all NRP-1 positive cell lines. Hypoxia strongly increased NRP-1 expression in the A549 adenocarcinoma (AC) cell line, while NRP-1 was decreased in SKMES-1 squamous cell carcinoma (SCC) following hypoxia. Neutralisation of NRP-1 had a greater effect in A549 cells under hypoxia (37%), with a lesser effect in SKMES-1 cells (82%). Western analysis of matched frozen normal and lung cancer biopsies showed NRP-1 overexpression in AC and decreased expression in SCC relative to normal. High NRP-1 expression was confirmed in AC and large cell carcinoma by immunohistochemistry, relative to normal. However, SCCs had a lower level of NRP-1 staining, supporting the results by western analysis and following hypoxia in vitro. Conclusions: These results implicate NRP-1 as an important survival pathway in lung cancer. Hypoxia differentially regulated NRP-1 mediated survival implicating this pathway as a potential therapeutic strategy in AC. No significant financial relationships to disclose.

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Acrylamide-derived cytotoxic, anti-proliferative, and apoptotic effects on A549 cells

Human & Experimental Toxicology ◽

10.1177/0960327117712386 ◽

2017 ◽

Vol 37 (5) ◽

pp. 468-474 ◽

Cited By ~ 11

Author(s):

S Kacar ◽

D Vejselova ◽

HM Kutlu ◽

V Sahinturk

Keyword(s):

Confocal Microscopy ◽

Cell Lines ◽

Mtt Assay ◽

A549 Cells ◽

Morphological Characteristics ◽

Annexin V ◽

Fluorescein Isothiocyanate ◽

Morphological Alterations ◽

Lung Adenocarcinoma Cell Line ◽

Apoptotic Effects

Background: Acrylamide is a very common compound even reaching up to our daily foods. It has been studied in a wealth of cell lines on which it proved to have various toxic effects. Among these cell lines, human lung adenocarcinoma cell line (A549) is one of that on which acrylamide’s toxicity has not been studied well yet. Aim: We intended to determine the half maximal inhibitory concentration (IC50) dose of acrylamide and to investigate its cytotoxic, anti-proliferative and apoptotic effects on A549 cells. Methods: We determined the IC50 dose by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Then, the mode of cell death was evaluated by flow cytometry using Annexin-V fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining. Next, we performed transmission electron microscopy (TEM) and confocal microscopy analyses for morphological alterations and apoptotic indices. Results: According to the MTT assay results, A549 cell viability decreases proportionally with increasing acrylamide concentrations and IC50 for A549 was 4.6 mM for 24 h. Annexin-V FITC/PI assay results indicated that acrylamide induces apoptosis in 64% of the A549 cells. TEM and confocal microscopy analyses showed nuclear condensations, fragmentations, cytoskeleton laceration, and membrane blebbing, which are morphological characteristics of apoptosis. Conclusion: Our research suggests that acrylamide causes cytotoxic, anti-proliferative, and apoptotic effects on A549 cells at 4.6 mM IC50 dose in 24 h.

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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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Multiple RSV strains infecting HEp-2 and A549 cells reveal cell line-dependent differences in resistance to RSV infection.

10.1101/2021.06.15.448622 ◽

2021 ◽

Author(s):

Anubama Rajan ◽

Felipe-Andres Piedra ◽

Letisha Aideyan ◽

Trevor McBride ◽

Matthew J Robertson ◽

...

Keyword(s):

Gene Expression ◽

Cell Line ◽

Cell Lines ◽

A549 Cells ◽

Viral Gene Expression ◽

Model Systems ◽

Viral Gene ◽

Transformed Cell ◽

Syncytial Virus ◽

Transformed Cell Lines

Respiratory syncytial virus (RSV) is a leading cause of pediatric acute respiratory infection worldwide. There are currently no approved vaccines or antivirals to combat RSV disease. A few transformed cell lines and two historic strains have been extensively used to study RSV. Here we report a thorough molecular and cell biological characterization of HEp-2 and A549 cells infected with four strains of RSV representing both major subgroups as well as historic and more contemporaneous genotypes -- [RSV/A/Tracy (GA1), RSV/A/Ontario (ON), RSV/B/18537 (GB1), RSV/B/Buenos Aires (BA)] -- via measurements of viral replication kinetics and viral gene expression, immunofluorescence-based imaging of gross cellular morphology and cell-associated RSV, and measurements of host response including transcriptional changes and levels of secreted cytokines and growth factors. Our findings strongly suggest 1) the existence of a conserved difference in gene expression between RSV subgroups A and B; 2) the A549 cell line is a more stringent and natural host of replicating RSV than the HEp-2 cell line; and 3) consistent with previous studies, determining the full effects of viral genetic variation in RSV pathogenesis requires model systems as tractable as transformed cell lines but better representative of the human host.

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