p53 suppresses the inflammatory response following respiratory syncytial virus infection by inhibiting TLR2.

-Respiratory syncytial virus (RSV) is a pivotal virus leading to acute lower respiratory tract infections in children under 5 years old. This study aimed to explore the correlation between p53 and Toll-like receptors (TLRs) post RSV infection. p53 levels exhibited a substantial decrease in nasopharyngeal aspirates (NPAs) from infants with RSV infection compared to control group. Manipulating p53 expression had no significant impact on RSV replication or interferon signaling pathway. Suppression of p53 expression led to heightened inflammation following RSV infection in A549 cells or airways of BALB/c mice. while stabilizing p53 expression using Nutlin-3a mitigated the inflammatory response in A549 cells. Additionally, Inhibiting p53 expression significantly increased Toll-like receptor 2 (TLR2) expression in RSV-infected epithelial cells and BALB/c mice. Furthermore, the TLR2 inhibitor, C29, effectively reduced inflammation mediated by p53 in A549 cells. Collectively, our results indicate that p53 modulates the inflammatory response after RSV infection through TLR2.

Coronavirus M Protein Trafficking in Epithelial Cells Utilizes a Myosin Vb Splice Variant and Rab10.

The membrane (M) glycoprotein of coronaviruses (CoVs) serves as the nidus for virion assembly. Using a yeast two-hybrid screen, we identified the interaction of the cytosolic tail of Murine Hepatitis Virus (MHV-CoV) M protein with Myosin Vb (MYO5B), specifically with the alternative splice variant of cellular MYO5B including exon D (MYO5B+D), which mediates interaction with Rab10. When co-expressed in human lung epithelial A549 and canine kidney epithelial MDCK cells, MYO5B+D co-localized with the MHV-CoV M protein, as well as with the M proteins from Porcine Epidemic Diarrhea Virus (PEDV-CoV), Middle East Respiratory Syndrome (MERS-CoV) and Severe Acute Respiratory Syndrome 2 (SARS-CoV-2). Co-expressed M proteins and MYO5B+D co-localized with endogenous Rab10 and Rab11a. We identified point mutations in MHV-CoV M that blocked the interaction with MYO5B+D in yeast 2-hybrid assays. One of these point mutations (E121K) was previously shown to block MHV-CoV virion assembly and its interaction with MYO5B+D. The E to K mutation at homologous positions in PEDV-CoV, MERS-CoV and SARS-CoV-2 M proteins also blocked colocalization with MYO5B+D. The knockdown of Rab10 blocked the co-localization of M proteins with MYO5B+D and was rescued by re-expression of CFP-Rab10. Our results suggest that CoV M proteins traffic through Rab10-containing systems, in association with MYO5B+D.

Effect of WISP1 on paraquat-induced EMT.

BACKGROUND: Paraquat (PQ) can induce pulmonary fibrosis (PF) by modulating epithelial-mesenchymal transition (EMT) of alveolar epithelial cells, but the molecular mechanism is unknown. In this paper, the role of Wnt-inducible signaling protein-1 (WISP1) in PQ-induced EMT was inspected. METHODS: The morphology, apoptosis, and mortality of A549 cells were observed through a microscope. The mRNA and protein levels of WISP1, E-cadherin, and Vimentin were confirmed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot. RESULTS: With the increase of PQ concentration, the morphology of A549 cells was apparently changed, cell apoptosis and mortality were enhanced. Besides, the E-cadherin abundance was reduced (p < 0.01), however, WISP1 and Vimentin contents were boosted after PQ treatment (p < 0.01). With the increase of PQ treatment time, the epithelial index of cells first increased and then decreased. The expression of WISP1 gene increased significantly with the increase of PQ treatment time (p < 0.01). Silence of WISP1 abolished the effect of PQ treatment on E-cadherin and Vimentin levels (p < 0.01). Downregulation of WISP1 curbed morphology change and PQ-induced EMT in A549 cells. CONCLUSION: Knockdown of WISP1 inhibited PQ-induced EMT in A549 cells. This conclusion might provide a new therapeutic target for PQ poisoning treatment.

NAD(P)H Quinone Oxidoreductase-1 Expression Promotes Self-Renewal and Therapeutic Resistance in Non-Small Cell Lung Cancer.

Identifying cellular drivers responsible for enhancing cancer cell resistance to therapeutics provides critical information for designing more effective drugs. Populations of slowly growing, self-renewing, chemo-resistant cells purportedly contribute to the development of therapeutic resistance in many solid tumors. In the current study, we implemented a tumor spheroid model to determine whether NAD(P)H quinone oxidoreductase-1 (NQO1) was requisite for self-renewal and promotion of the drug-resistant phenotype in non-small cell lung cancer (NSCLC). We found that stable depletion of NQO1 in A549 and H358 human NSCLC tumor models inhibits self-renewal capabilities, as demonstrated by a reduced ability to form primary, secondary, and tertiary spheroids. In contrast, the rescue of NQO1 expression restored the tumor cells' ability to form spheroids. Additionally, we discovered that NQO1 depletion renders cisplatin-refractory tumor spheroids highly susceptible to drug treatment. Together, these results suggest that NQO1 loss reduces the self-renewing capabilities of NSCLC cells and enhances their susceptibility to clinically relevant therapeutics. These findings describe a novel role for NQO1 and suggest that combining NQO1-inhibitors with conventional chemotherapeutics may enhance anti-tumor effects.

WITHDRAWN: Effect of shRNA­mediated knockdown EBF1 gene expression on the proliferation of lung cancer cell line A549 in vitro and in vivo.

The paper entitled "Effect of shRNA­mediated knockdown EBF1 gene expression on the proliferation of lung cancer cell line A549 in vitro and in vivo" by Lin Wang et al, which was published online on 16 March 2023, has been withdrawn at the authors' request.

Simultaneous Administration of Bevacizumab with Bee-Pollen Extract-Loaded Hybrid Protein Hydrogel NPs Is a Promising Targeted Strategy against Cancer Cells.

Bevacizumab (Bev) a humanized monoclonal antibody that fights vascular endothelial growth factor A (VEGF-A). It was the first specifically considered angiogenesis inhibitor and it has now become the normative first-line therapy for advanced non-small-cell lung cancer (NSCLC). In the current study, polyphenolic compounds were isolated from bee pollen (PCIBP) and encapsulated (EPCIBP) inside moieties of hybrid peptide-protein hydrogel nanoparticles in which bovine serum albumin (BSA) was combined with protamine-free sulfate and targeted with folic acid (FA). The apoptotic effects of PCIBP and its encapsulation (EPCIBP) were further investigated using A549 and MCF-7 cell lines, providing significant upregulation of Bax and caspase 3 genes and downregulation of Bcl2, HRAS, and MAPK as well. This effect was synergistically improved in combination with Bev. Our findings may contribute to the use of EPCIBP simultaneously with chemotherapy to strengthen the effectiveness and minimize the required dose.

Selenium Yeast and Fish Oil Combination Diminishes Cancer Stem Cell Traits and Reverses Cisplatin Resistance in A549 Sphere Cells.

Cisplatin is a prevalent chemotherapeutic agent used for non-small cell lung cancer (NSCLC) that is difficult to treat by targeted therapy, but the emergence of resistance severely limits its efficacy. Thus, an effective strategy to combat cisplatin resistance is required. This study demonstrated that, at clinically achievable concentrations, the combination of selenium yeast (Se-Y) and fish oil (FO) could synergistically induce the apoptosis of cancer stem cell (CSC)-like A549 NSCLC sphere cells, accompanied by a reversal of their resistance to cisplatin. Compared to parental A549 cells, sphere cells have higher cisplatin resistance and possess elevated CSC markers (CD133 and ABCG2), epithelial-mesenchymal transition markers (anexelekto (AXL), vimentin, and N-cadherin), and cytoprotective endoplasmic reticulum (ER) stress marker (glucose-regulated protein 78) and increased oncogenic drivers, such as yes-associated protein, transcriptional coactivator with PDZ-binding motif, ß-catenin, and cyclooxygenase-2. In contrast, the proapoptotic ER stress marker CCAAT/enhancer-binding protein homologous protein and AMP-activated protein kinase (AMPK) activity were reduced in sphere cells. The Se-Y and FO combination synergistically counteracted the above molecular features of A549 sphere cells and diminished their elevated CSC-like side population. AMPK inhibition by compound C restored the side population proportion diminished by this nutrient combination. The results suggest that the Se-Y and FO combination can potentially improve the outcome of cisplatin-treated NSCLC with phenotypes such as A549 cells.

Increase in Anticancer Drug-Induced Toxicity by Fisetin in Lung Adenocarcinoma A549 Spheroid Cells Mediated by the Reduction of Claudin-2 Expression.

Claudin-2 (CLDN2), a component of tight junction, is involved in the reduction of anticancer drug-induced toxicity in spheroids of A549 cells derived from human lung adenocarcinoma. Fisetin, a dietary flavonoid, inhibits cancer cell growth, but its effect on chemosensitivity in spheroids is unknown. Here, we found that fisetin (20 µM) decreases the protein level of CLDN2 to 22.3%. Therefore, the expression mechanisms were investigated by real-time polymerase chain reaction and Western blotting. Spheroids were formed in round-bottom plates, and anticancer drug-induced toxicity was measured by ATP content. Fisetin decreased the phosphorylated-Akt level, and CLDN2 expression was decreased by a phosphatidylinositol 3-kinase (PI3K) inhibitor, suggesting the inhibition of PI3K/Akt signal is involved in the reduction of CLDN2 expression. Hypoxia level, one of the hallmarks of tumor microenvironment, was reduced by fisetin. Although fisetin did not change hypoxia inducible factor-1α level, it decreased the protein level of nuclear factor erythroid 2-related factor 2, a stress response factor, by 25.4% in the spheroids. The toxicity of doxorubicin (20 µM) was enhanced by fisetin from 62.8% to 40.9%, which was rescued by CLDN2 overexpression (51.7%). These results suggest that fisetin can enhance anticancer drug toxicity in A549 spheroids mediated by the reduction of CLDN2 expression.

Effect of the elastin-derived peptides (VGVAPG and VVGPGA) on breast (MCF-7) and lung (A549) cancer cell lines in vitro.

Tissues are subjected to dynamic communication between cells and the extracellular matrix (ECM), resulting in ECM remodeling. One of the ECM components is elastin, which releases elastin-derived peptides (EDPs) during the aging process. Therefore, the aim of the present study was to evaluate the impact of the VGVAPG hexapeptide and elastin-like peptide VVGPGA (control) on certain metabolism parameters in human breast adenocarcinoma (MCF-7) and human lung carcinoma (A549) cell lines. The results did not show a significant effect of the peptides on metabolic activity and caspase-3 activity. However, more specific analysis revealed that VGVAPG and VVGPGA were able to increase KI67 protein expression in both tested cell lines after 24-h treatment. Moreover, the same correlation was observed at the KI67 gene level. VGVAPG also increased the P53, ATM and SHH gene expression in the A549 cells up to 19.08%, 20.74%, and 28.77%, respectively. Interestingly, the VGVAPG peptide exerted an effect on the expression of antioxidant enzymes SOD2 and CAT in the A549 and MCF-7 cells, especially after the 24-h treatment. Lastly, both peptides influenced the CAV1 and CLTC1 expression. Our results show that the tested EDPs have an effect on both A549 and MCF-7 cells at the cellular level. This may be correlated with the multidrug-resistance (MDR) phenotype in these cancer cells, which is an emerging problem in the current anticancer treatment. However, more research is needed in this field.

Cytotoxic evaluation and LC-MS/MS analysis of aerial parts of Eryngium kotschyi Boiss. grown in Turkey

Abstract Increasing biological activity and phytochemical investigations on Eryngium species showed its potential as pharmaceutical approach. Eryngium kotschyi Boiss. is one of the species of Eryngium genus and is endemic to Turkey. It is known that this plant is traditionally used in the South-western part of Turkey for the treatment of various diseases. This study focuses on cytotoxic activities of methanol extract and ethyl acetate, n-butanol and water sub-extracts from E. kotschyi in A549, COLO 205 and MDA-MB-231 cell lines by Sulforhodamin B assay and qualitative and quantitative determination of phytochemical constituents in active extract by LC-MS/MS. From the result of the study, it was seen that E. kotschyi ethyl acetate (EKE) sub-extract showed the strongest cytotoxic effect with the low IC50 values (50.00; 31.96 and 22.26 µg/mL in A549; COLO 205 and MDA-MB-231 cells at 48 h, respectively). Preliminary examination of the mass spectrums revealed the presence of 15 phytochemical compounds in active sub-extract and 7 of them was quantified. According to quantitative analyses the main compounds of EKE sub-extract were rosmarinic acid (485.603 µg/mgextract), chlorogenic acid (62.355 µg/mgextract) and caffeic acid (59.266 µg/mgextract). Moreover, this preliminary study on inhibitory activity of EKE sub-extract suggests further toxicologic investigations and detailed investigation on cytotoxic effect of various combinations of determined compounds

Maf1 suppression of ATF5-dependent mitochondrial unfolded protein response contributes to rapamycin-induced radio-sensitivity in lung cancer cell line A549.

mTOR is well known to promote tumor growth but its roles in enhancing chemotherapy and radiotherapy have not been well studied. mTOR inhibition by rapamycin can sensitize cancer cells to radiotherapy. Here we show that Maf1 is required for rapamycin to increase radio-sensitivity in A549 lung cancer cells. In response to ionizing radiation (IR), Maf1 is inhibited by Akt-dependent re-phosphorylation, which activates mitochondrial unfolded protein response (UPRmt) through ATF5. Rapamycin suppresses IR-induced Maf1 re-phosphorylation and UPRmt activation in A549 cells, resulting in increased sensitivity to IR-mediated cytotoxicity. Consistently, Maf1 knockdown activates ATF5-transcription of mtHSP70 and HSP60, enhances mitochondrial membrane potential, reduces intracellular ROS levels and dampens rapamycin's effect on increasing IR-mediated cytotoxicity. In addition, Maf1 overexpression suppresses ethidium bromide-induced UPRmt and enhances IR-mediated cytotoxicity. Supporting our cell-based studies, elevated expression of UPRmt makers (mtHSP70 and HSP60) are associated with poor prognosis in patients with lung adenocarcinoma (LAUD). Together, our study reveals a novel role of Maf1-UPRmt axis in mediating rapamycin's enhancing effect on IR sensitivity in A549 lung cancer cells.

iTRAQ­based proteomic analysis of the interaction of A549 human lung epithelial cells with Aspergillus fumigatus conidia.

Severe invasive aspergillosis infection occurs when human immune function is impaired. The interaction between Aspergillus fumigatus (A. fumigatus) conidia and type II lung epithelial cells serves an important role in disease progression. The present study compared the proteomes of A549 human lung epithelial cells with and without A. fumigatus infection. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and protein interaction analyses were performed, and differential protein expression was verified by western blotting and reverse transcription­quantitative PCR (RT­qPCR). In addition, the RNA interference method, an internalization assay and ELISA were performed. Isobaric tags for relative and absolute quantification analysis detected a total of 1,582 proteins, from which 111 proteins with differential expression were obtained (fold change >1.5 or <0.75). Among them, 18 proteins were upregulated and 93 proteins were downregulated in A549 cells challenged with A. fumigatus. GO and KEGG analyses revealed that the altered proteins were mainly involved in biological functions, such as cell metabolism, synthesis, the cellular stress response, metabolic pathways and pyruvate metabolism. N­myc downstream­regulated gene 1 (NDRG1) expression was upregulated 1.88­fold, while CD44 expression was downregulated 0.47­fold following A. fumigatus infection. The expression levels of specific proteins were verified by western blotting and RT­qPCR. The internalization efficiency was affected by NDRG1 gene silencing. The secretion of IL­6 and IL­8 was affected when CD44 was inhibited. These results indicated that A. fumigatus affects lung epithelial cell metabolism and biological synthetic functions. A number of novel molecules, including NDRG1 and CD44, were found to be related to A. fumigatus infection.

High frequency acoustic cell stimulation promotes exosome generation regulated by a calcium-dependent mechanism.

Exosomes are promising disease diagnostic markers and drug delivery vehicles, although their use in practice is limited by insufficient homogeneous quantities that can be produced. We reveal that exposing cells to high frequency acoustic irradiation stimulates their generation without detriment to cell viability by exploiting their innate membrane repair mechanism, wherein the enhanced recruitment of calcium ions from the extracellular milieu into the cells triggers an ESCRT pathway known to orchestrate exosomal production. Given the high post-irradiation cell viabilities (≈95%), we are able to recycle the cells through iterative irradiation and post-excitation incubation steps, which facilitate high throughput production of a homogeneous population of exosomes-a particular challenge for translating exosome therapy into clinical practice. In particular, we show that approximately eight- to ten-fold enrichment in the number of exosomes produced can be achieved with just 7 cycles over 280 mins, equivalent to a yield of around 1.7-2.1-fold/h.

Robust induction of interferon and interferon-stimulated gene expression by influenza B/Yamagata lineage virus infection of A549 cells.

Influenza B virus (IBV) belongs to the Orthomyxoviridae family and generally causes sporadic epidemics but is occasionally deadly to individuals. The current research mainly focuses on clinical and pathological characteristics of IBV. However, to better prevent or treat the disease, one must determine the strategies developed by IBV to invade and disrupt cellular proteins and approach to replicate itself, to suppress antiviral innate immunity, and understand how the host responds to IBV infection. The B/Shanghai/PD114/2018 virus was able to infect alveolar epithelial cells (A549) cells, with good potential for replication. To identify host cellular responses against IBV infection, differentially expressed genes (DEGs) were obtained using RNA sequencing. The GO and KEGG pathway term enrichment analyses with the DEGs were performed, and we found that the DEGs were primary involved in metabolic processes and cellular function, which may be related to the host response, including the innate immune response against the virus. Our transcriptome analysis results demonstrated robust induction of interferon and interferon-stimulated gene expression by IBV in human cells during the early stages of infection, providing a foundation for further studies focused on antiviral drug development and interactions between the virus and host.

Photosensitive nanocarriers for specific delivery of cargo into cells.

Nanoencapsulation is a rapidly expanding technology to enclose cargo into inert material at the nanoscale size, which protects cargo from degradation, improves bioavailability and allows for controlled release. Encapsulation of drugs into functional nanocarriers enhances their specificity, targeting ability, efficiency, and effectiveness. Functionality may come from cell targeting biomolecules that direct nanocarriers to a specific cell or tissue. Delivery is usually mediated by diffusion and erosion mechanisms, but in some cases, this is not sufficient to reach the expected therapeutic effects. This work reports on the development of a new photoresponsive polymeric nanocarrier (PNc)-based nanobioconjugate (NBc) for specific photo-delivery of cargo into target cells. We readily synthesized the PNcs by modification of chitosan with ultraviolet (UV)-photosensitive azobenzene molecules, with Nile red and dofetilide as cargo models to prove the encapsulation/release concept. The PNcs were further functionalized with the cardiac targeting transmembrane peptide and efficiently internalized into cardiomyocytes, as a cell line model. Intracellular cargo-release was dramatically accelerated upon a very short UV-light irradiation time. Delivering cargo in a time-space controlled fashion by means of NBcs is a promising strategy to increase the intracellular cargo concentration, to decrease dose and cargo side effects, thereby improving the effectiveness of a therapeutic regime.

MiR-424 overexpression protects alveolar epithelial cells from LPS-induced apoptosis and inflammation by targeting FGF2 via the NF-κB pathway.

Acute respiratory distress syndrome (ARDS) is a multifactorial, inflammatory lung injury disease with high morbidity and mortality. However, the underlying pathogenic mechanism remains unknown. In this study, lipopolysaccharide (LPS)-stimulated alveolar epithelial cells were used to mimic the inflammatory pathogenesis of ARDS in vitro. We here investigated the role of miR-424 in LPS-stimulated alveolar epithelial cells and found it to be substantially downregulated. Overexpression of miR-424 inhibited apoptosis and inflammation in LPS-stimulated alveolar epithelial cells, and the miR-424 inhibitor exhibited the opposite effect. A bioinformatic analysis revealed a potential binding site of miR-424 in the 3'-UTR of fibroblast growth factor 2 (FGF2). A luciferase reporter assay suggested that miR-424 targeted FGF2 in alveolar epithelial cells. The level of FGF2 protein was inhibited by miR-424 mimic, whereas was significantly upregulated after miR-424 suppression in LPS-stimulated alveolar epithelial cells. MiR-424 also exhibited the protective role in LPS-induced apoptosis and inflammation by directly targeting FGF2 via the NF-κB pathway. In conclusion, our results demonstrate that miR-424 had a protective role in LPS-induced apoptosis and inflammation of alveolar epithelial cells by targeting FGF2 via regulating NF-κB pathway. This might contribute novel evidence to help identify a therapeutic target for treating ARDS.

MicroRNA-31/184 is involved in transforming growth factor-ß-induced apoptosis in A549 human alveolar adenocarcinoma cells.

AIMS: TGF-ß-induced alveolar epithelial cells apoptosis were involved in idiopathic pulmonary fibrosis (IPF). This study aimed to explore potential targets and mechanisms of IPF. MAIN METHODS: mRNA and microRNA arrays were used to analyze differentially expressed genes and miRNAs. Several essential targets of TGF-ß-SMADs and TGF-ß-PI3K-AKT pathways were detected. KEY FINDINGS: miR-31 and miR-184 expression levels were positively correlated with smad6 and smad2/akt expression levels in IPF patients. TGF-ß could induce miR-31 and suppress miR-184 levels in A549 cells. miR-31 was confirmed to bind to the smad6-3'UTR and functionally suppress its expression. Down-regulated SMAD6 enhanced SMAD2/SMAD4 dimer formation and translocation due to its failure to prevent SMAD2 phosphorylation. In contrast, anti-fibrotic functions of miR-184 were abolished due to TGF-ß directly suppressing miR-184 levels in A549 cells. When A549 was stimulated by TGF-ß combined with or without miR-31 inhibitor/miR-184 mimic, it was showed that depleted miR-31 and/or increased miR-184 significantly ameliorated TGF-ß-induced viability of A549 cells, as well as inhibited the expression of profibrotic factors, MMP7 and RUNX2. SIGNIFICANCE: Inhibiting miR-31 and/or promoting miR-184 protect against TGF-ß-induced fibrogenesis by respectively repressing the TGF-ß-SMAD2 and TGF-ß-PI3K-AKT signaling pathways, implying that miR-31/184 are potential targets and suggesting a new management strategy for IPF.

A new ocotillol-type ginsenoside from stems and leaves of Panax quinquefolium L. and its anti-oxidative effect on hydrogen peroxide exposed A549 cells.

A new ocotillol-type ginsenoside, namely 12-one-pseudoginsenoside F11 (12-one-PF11), was isolated from stems and leaves of Panax quinquefolium, whose structure was elucidated 6-O-[α-L-rhamnopyranosyl-(1-2)-ß-D-glucopyranosyl]-dammar-12-one-20S,24R-epoxy-3ß,6α,25-triol. 12-one-PF11 significantly suppressed hydrogen peroxide induced oxidative stress in human lung carcinoma A549 cells. As compared with model group, 12-one-PF11 improved cell viability of A549 cells in a dose-dependent manner, and significantly decreased the generation of malondialdehyde (MDA) and increased production of superoxide dismutase (SOD) and glutathione (GSH) and protein expression levels of nuclear related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) in A549 cells.

Glycolytic suppression dramatically changes the intracellular metabolic profile of multiple cancer cell lines in a mitochondrial metabolism-dependent manner.

Most cancer cells rely on glycolysis to generate ATP, even when oxygen is available. However, merely inhibiting the glycolysis is insufficient for the eradication of cancer cells. One main reason for this is that cancer cells have the potential to adapt their metabolism to their environmental conditions. In this study, we investigated how cancer cells modify their intracellular metabolism when glycolysis is suppressed, using PANC-1 pancreatic cancer cells and two other solid tumor cell lines, A549 and HeLa. Our study revealed that glycolytically suppressed cells upregulated mitochondrial function and relied on oxidative phosphorylation (OXPHOS) to obtain the ATP necessary for their survival. Dynamic changes in intracellular metabolic profiles were also observed, reflected by the reduced levels of TCA cycle intermediates and elevated levels of most amino acids. Glutamine and glutamate were important for this metabolic reprogramming, as these were largely consumed by influx into the TCA cycle when the glycolytic pathway was suppressed. During the reprogramming process, activated autophagy was involved in modulating mitochondrial function. We conclude that upon glycolytic suppression in multiple types of tumor cells, intracellular energy metabolism is reprogrammed toward mitochondrial OXPHOS in an autophagy-dependent manner to ensure cellular survival.