scholarly journals Paving the way towards understanding the inflammatory pathways triggered by giant viruses in mammalian cells: effect of mimivirus-cell interactions on IκBα degradation

2021 ◽  
Author(s):  
Juliana dos Santos Oliveira ◽  
Dahienne Ferreira Oliveira ◽  
Victor Alejandro Essus ◽  
Gabriel Henrique Pereira Nunes ◽  
Leandro Honorato ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
A549 Cells ◽  
Giant Virus ◽  
Inflammatory Pathways ◽  
Large Size ◽  
Adenocarcinoma Cells ◽  
Human Lung Adenocarcinoma Cells ◽  
Giant Viruses ◽  
Cellular Immune ◽  
Acanthamoeba Polyphaga Mimivirus

AbstractEven after two decades since the identification of the first giant virus, the Acanthamoeba polyphaga mimivirus (APMV), it still elude scientists. Their gigantic size and genome are unique in the whole virosphere, and many aspects of their biology are still unknown, including their possible hosts. They are cultivated in laboratories using Acanthamoeba cells as hosts, but little is known about the infectivity of these giant viruses in vertebrate cells. However, there is evidence of the possible involvement of APMV in pneumonia and activation of inflammatory pathways. Among the hundreds of prospected giant viruses members is Tupanvirus, isolated in Brazil. Its particles have a characteristically large size varying between 1.2 to 2 μm and are covered by fibrils. In the present work, we aim to study the consequences of the incubation of APMV and Tupanvirus with mammalian cells. These cells express Toll-like receptors (TLR) that are capable of recognizing lipopolysaccharides, favoring the internalization of the antigen and activation of the inflammatory system. We used a lineage of human lung adenocarcinoma cells (A549) to evaluate possible effects of TLR activation by the giant viruses and if we could detect the probable cause of the said giant-virus dependent pneumonia. Our results show that APMV and Tupanvirus (TPV) activate cellular receptors related to the Toll-like 4 type-induced inflammatory response and that the A549 cells are capable of internalizing the latter virus. Therefore, this study brings new insights into the possible interactions established between mimiviruses (here represented by APMV and Tupanvirus) and members of the innate cellular immune response.

scholarly journals Emodin Inhibits ATP-Induced Proliferation and Migration by Suppressing P2Y Receptors in Human Lung Adenocarcinoma Cells

2017 ◽  
Vol 44 (4) ◽  
pp. 1337-1351 ◽  
Author(s):  
Xia Wang ◽  
Long Li ◽  
Ruijuan Guan ◽  
Danian Zhu ◽  
Nana Song ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Lung Adenocarcinoma ◽  
Human Lung ◽  
A549 Cells ◽  
P2y Receptors ◽  
Mesenchymal Transition ◽  
Human Lung Adenocarcinoma ◽  
Adenocarcinoma Cells ◽  
Human Lung Adenocarcinoma Cells ◽  
Lung Adenocarcinoma Cells

Background/Aims: Extracellular ATP performs multiple important functions via activation of P2 receptors on the cell surface. P2Y receptors play critical roles in ATP evoked response in human lung adenocarcinoma cells (A549 cells). Emodin is an anthraquinone derivative originally isolated from Chinese rhubarb, possesses anticancer properties. In this study we examined the inhibiting effects of emodin on proliferation, migration and epithelial-mesenchymal transition (EMT) by suppressing P2Y receptors-dependent Ca2+ increase and nuclear factor-κB (NF-KB) signaling in A549 cells. Methods: A549 cells were pretreated with emodin before stimulation with ATP for the indicated time. Then, intracellular Ca2+ concentration ([Ca2+]i) was measured by Fluo-8/AM staining. Cell proliferation and cell cycle progression were tested by CCK8 assay and flow cytometry In addition, wound healing and western blot were performed to determine cell migration and related protein levels (Bcl-2, Bax, claudin-1, NF-κB). Results: Emodin blunted ATP/UTP-induced increase of [Ca2+]i and cell proliferation concentration-dependently Meanwhile, it decreased ATP-induced cells accumulation in the S phase. Furthermore, emodin altered protein abundance of Bcl-2, Bax and claudin-1 and attenuated EMT caused by ATP. Such ATP-induced cellular reactions were also inhibited by a nonselective P2Y receptors antagonist, suramin, in a similar way to emodin. Besides, emodin could inhibit activation of NF-κB, thus suppressed ATP-induced proliferation, migration and EMT. Conclusion: Our results demonstrated that emodin inhibits ATP-induced proliferation, migration, EMT by suppressing P2Y receptors-mediated [Ca2+]i increase and NF-κB signaling in A549 cells.

Role of DACH1 on proliferation, invasion, and apoptosis in human lung adenocarcinoma cells

2021 ◽  
Vol 21 ◽  
Author(s):  
Junjie Yu ◽  
Ping Jiang ◽  
Ke Zhao ◽  
Zhiguo Chen ◽  
Tao Zuo ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Lung Adenocarcinoma ◽  
Tumor Cells ◽  
Human Lung ◽  
A549 Cells ◽  
Cancer Tissue ◽  
Lung Cancer Tissue ◽  
Adenocarcinoma Cells ◽  
Human Lung Adenocarcinoma Cells ◽  
Lung Adenocarcinoma Cells

Objective: To investigate DACH1 protein expression in lung cancer tissue and matched paracancerous tissue, and explore its effect on proliferation, invasion, and apoptosis in human lung adenocarcinoma cells (HLACs). Methods: Tumor tissue and matched paracancerous tissue was collected from 46 patients with pathologically diagnosed lung cancer. RT-PCR was perfomed to detect DACH1 mRNA expression and immunohistochemistry to measured DACH1 protein expression. To determine the effect of DACH1 on lung cancer behavior, small interfering RNA (siRNA) was used to silence DACH1 expression in A549 cells. The impact on the proliferation of tumor cells was then observed by MTT assay, changes in the invasion of tumor cells were identified using transwell chamber assay, and the effects on apoptosis in the cell line were detected using flow cytometry. Results: The expression of DACH1 mRNA and DACH1 protein were significantly decreased in lung cancer tissue versus matched paracancerous control tissue. Silencing of DACH1 expression in A549 cells significantly enhanced cell proliferation, significantly increased cell invasion and significantly reduced spontaneous apoptosis. Conclusion: DACH1 is downregulated in lung adenocarcinoma tissue. In vitro assessment shows that DACH1 functions as a tumor suppressor, suggesting its potential use as new target for lung cancer treatment.

scholarly journals Thapsigargin Induces Apoptosis by Impairing Cytoskeleton Dynamics in Human Lung Adenocarcinoma Cells

2014 ◽  
Vol 2014 ◽  
pp. 1-7 ◽  
Author(s):  
Fei Wang ◽  
Da-zhong Liu ◽  
Hao Xu ◽  
Yi Li ◽  
Wei Wang ◽  
...  
Keyword(s):  
Lung Adenocarcinoma ◽  
Human Lung ◽  
A549 Cells ◽  
Dependent Manner ◽  
Human Lung Adenocarcinoma ◽  
Adenocarcinoma Cells ◽  
Human Lung Adenocarcinoma Cells ◽  
Cytoskeletal Dynamics ◽  
Lung Adenocarcinoma Cells ◽  
Dose Dependent

The objective of this study was performed to investigate the effects of thapsigargin on apoptosis, actin cytoskeletal dynamics, and actin cytoskeletal proteins in human lung adenocarcinoma cell. Thapsigargin is a specific irreversible inhibitor of ER calcium-ATPase, which may promote ER stress by depletion of lumenal calcium stores and show potential to induce cell death. The effects of thapsigargin on the apoptosis in A549 cells were assayed by Hoechst staining. Moreover, the F-actin staining by Rhodamine-phalloidin and RhoA antibody for cytoskeleton organizations were applied to A549 cells. To confirm the impairment of cytoskeletal dynamics treated with thapsigargin, western blots were applied to analyze the protein levels of p-Cofilin-1 (Ser3), Cofilin-1, and pPaxillin (Tyr118), as well as RhoA and pS6 (S240/244). Results suggest that thapsigargin may induce cell death in A549 cells with a time- and dose-dependent manner. The F-actin fibers and RhoA signals are also reduced with a time- and dose-dependent manner by thapsigargin treatment. The phosphorylation forms of Cofilin-1 and paxillin are attenuated by 1 μM thapsigargin treatment for 24 h. These alternations may be caused by the inhibition of of mTORC1 activities (indicated by pS6 (Ser240/244)) and RhoA pathways after thapsigargin treatment. The present findings highlight important roles of calcium entry in cytoskeleton organization and apoptosis in human lung adenocarcinoma cells and will help to set a stage to the clinical treatment of cancer cell metastasis.

scholarly journals Initial Steps for the Development of a Phage-Mediated Gene Replacement Therapy Using CRISPR-Cas9 Technology

2020 ◽  
Vol 9 (5) ◽  
pp. 1498
Author(s):  
Jordi Yang Zhou ◽  
Keittisak Suwan ◽  
Amin Hajitou
Keyword(s):  
Lung Adenocarcinoma ◽  
Replacement Therapy ◽  
Human Lung ◽  
Negative Impact ◽  
Quantitative Polymerase Chain Reaction ◽  
Human Lung Adenocarcinoma ◽  
Large Size ◽  
Adenocarcinoma Cells ◽  
Human Lung Adenocarcinoma Cells ◽  
Lung Adenocarcinoma Cells

p53 gene (TP53) replacement therapy has shown promising results in cancer gene therapy. However, it has been hampered, mostly because of the gene delivery vector of choice. CRISPR-Cas9 technology (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) can knock out the mutated TP53 (mutTP53), but due to its large size, many viral vectors are not suitable or require implemented strategies that lower the therapeutic efficiency. Here, we introduced a bacteriophage or phage-based vector with the ability to target cancer cells and aimed to investigate the feasibility of using this vector to deliver CRISPR-Cas9 transgene in human lung adenocarcinoma cells. First, we produced a tumour-targeted bacteriophage carrying a CRISPR-Cas9 transgene cassette. Next, we investigated any negative impact on vector titers via quantitative polymerase chain reaction (qPCR) and colony-forming agar plate. Last, we combined Western blot analysis and immunofluorescence staining to prove cell transduction in vitro. We showed that the tumour-targeted bacteriophage can package a large-size vector genome, ~10 kb, containing the CRISPR-Cas9 sequence without any negative impact on the active or total number of bacteriophage particles. Then, we detected expression of the Cas9 in human lung adenocarcinoma cells in a targeted and efficient manner. Finally, we proved loss of p53 protein expression when a p53 gRNA was incorporated into the CRISPR-Cas9 phage DNA construct. These proof-of-concept findings support the use of engineered bacteriophage for TP53 replacement therapy in lung cancer.

scholarly journals Annexin A2 Mediates Mycoplasma pneumoniae Community-Acquired Respiratory Distress Syndrome Toxin Binding to Eukaryotic Cells

mBio ◽  
2014 ◽  
Vol 5 (4) ◽  
Author(s):  
Sudha R. Somarajan ◽  
Fadi Al-Asadi ◽  
Kumaraguruparan Ramasamy ◽  
Lavanya Pandranki ◽  
Joel B. Baseman ◽  
...  
Keyword(s):  
Respiratory Distress Syndrome ◽  
Respiratory Distress ◽  
Mammalian Cells ◽  
Distress Syndrome ◽  
Protein A ◽  
A549 Cells ◽  
Surfactant Protein ◽  
Annexin A2 ◽  
Toxin Binding ◽  
Cards Toxin

ABSTRACT Mycoplasma pneumoniae synthesizes a novel human surfactant protein A (SP-A)-binding cytotoxin, designated community-acquired respiratory distress syndrome (CARDS) toxin, that exhibits ADP-ribosylating and vacuolating activities in mammalian cells and is directly linked to a range of acute and chronic airway diseases, including asthma. In our attempt to detect additional CARDS toxin-binding proteins, we subjected the membrane fraction of human A549 airway cells to affinity chromatography using recombinant CARDS toxin as bait. A 36-kDa A549 cell membrane protein bound to CARDS toxin and was identified by time of flight (TOF) mass spectroscopy as annexin A2 (AnxA2) and verified by immunoblotting with anti-AnxA2 monoclonal antibody. Dose-dependent binding of CARDS toxin to recombinant AnxA2 reinforced the specificity of the interaction, and further studies revealed that the carboxy terminus of CARDS toxin mediated binding to AnxA2. In addition, pretreatment of viable A549 cells with anti-AnxA2 monoclonal antibody or AnxA2 small interfering RNA (siRNA) reduced toxin binding and internalization. Immunofluorescence analysis of CARDS toxin-treated A549 cells demonstrated the colocalization of CARDS toxin with cell surface-associated AnxA2 upon initial binding and with intracellular AnxA2 following toxin internalization. HepG2 cells, which express low levels of AnxA2, were transfected with a plasmid expressing AnxA2 protein, resulting in enhanced binding of CARDS toxin and increased vacuolization. In addition, NCI-H441 cells, which express both AnxA2 and SP-A, upon AnxA2 siRNA transfection, showed decreased binding and subsequent vacuolization. These results indicate that CARDS toxin recognizes AnxA2 as a functional receptor, leading to CARDS toxin-induced changes in mammalian cells. IMPORTANCE Host cell susceptibility to bacterial toxins is usually determined by the presence and abundance of appropriate receptors, which provides a molecular basis for toxin target cell specificities. To perform its ADP-ribosylating and vacuolating activities, community-acquired respiratory distress syndrome (CARDS) toxin must bind to host cell surfaces via receptor-mediated events in order to be internalized and trafficked effectively. Earlier, we reported the binding of CARDS toxin to surfactant protein A (SP-A), and here we show how CARDS toxin uses an alternative receptor to execute its pathogenic properties. CARDS toxin binds selectively to annexin A2 (AnxA2), which exists both on the cell surface and intracellularly. Since AnxA2 regulates membrane dynamics at early stages of endocytosis and trafficking, it serves as a distinct receptor for CARDS toxin binding and internalization and enhances CARDS toxin-induced vacuolization in mammalian cells.

scholarly journals Apoptosis Induction of Agave lechuguilla Torrey Extract on Human Lung Adenocarcinoma Cells (SK-LU-1)

2018 ◽  
Vol 19 (12) ◽  
pp. 3765 ◽  
Author(s):  
Luis Anguiano-Sevilla ◽  
Eugenia Lugo-Cervantes ◽  
Cynthia Ordaz-Pichardo ◽  
Jorge Rosas-Trigueros ◽  
María Jaramillo-Flores
Keyword(s):  
Cell Death ◽  
Caspase 3 ◽  
Ethanol Extract ◽  
Fragmentation Pattern ◽  
Intrinsic Pathway ◽  
Adenocarcinoma Cells ◽  
Human Lung Adenocarcinoma Cells ◽  
Caspase 7 ◽  
Mcf 7 ◽  
Apoptotic Signal

In this study, an ethanol extract of Agave lechuguilla was evaluated against six carcinogenic cell lines (HCT-15, MCF-7, PC-3, U-251, SK-LU-1 and K-562) with an inhibition of 75.7 ± 2.3% against the SK-LU-1 line. Based on the previous result, the extract was hydrolyzed and fractionated, to which the IC50 was determined; the cell line was more sensitive to the fractionated extract with an IC50 6.96 ± 0.15 µg/mL. Characterization by mass spectrometry showed the presence of kaempferol, quercetin and a flavonoid dimer formed by afzelechin-4β-8-quercetin, according to the generated fragmentation pattern. The fractionated extract presented cell death by apoptosis with 39.8% at 24 h. Molecular docking was performed with the molecules found to try to describe cell death by apoptosis through death receptors such as FasCD95, TNF-R1, DR4/5 and blocking signaling on the EGFR and K-Ras MAPK/ERK pathway, as well as through the intrinsic pathway activating tBID, which promotes the amplification of the apoptotic signal due to the activation of caspase-3, and consequently caspase-7. In addition to the activation of the IIb complex associated with cell death due to necroptosis.

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