Reactive Oxygen Species In A549 Cells Research Articles

NRF2 protects lung epithelial cells from wood smoke particle toxicity

Wildfire smoke is a potential source of oxidative stress in lung epithelial tissue. The response to oxidative stress is controlled by the transcription factor NRF2, which is the central regulator of antioxidant gene expression. If wood smoke particle (WSP) exposure induces reactive oxygen species (ROS) in epithelial cells, then NRF2 may protect against pathological conditions resulting from increased oxidative stress through changes in gene expression. We used two lung epithelial cell lines to test this hypothesis in vitro: A549, which harbor a mutation resulting in constitutive activation of NRF2, and BEAS-2B, which show limited NRF2 activity under basal conditions, but high inducibility during oxidative stress. In BEAS-2B cells, WSP exposure leads to increased cellular ROS, activation of NRF2, and upregulation of the NRF2 target genes NQO1, GCLM, and SRXN1. WSP exposure also increased ROS in A549 cells, although NRF2 activation and antioxidant gene upregulation were less robust as both were basally high in this cell line. Overall, the degree of ROS induction by WSP across cell lines is dependent upon NRF2 activity, and a similar pattern was observed for WSP cytotoxicity. WSP also sensitized both cell lines to the ferroptosis inducer erastin in a manner that is correlated with NRF2 activity. Knockout of NRF2 in A549 resulted in higher WSP-induced ROS generation, cytotoxicity, and erastin sensitivity. Taken together, these results suggest that NRF2 serves as a protective factor against wood smoke induced ROS and oxidative stress in lung epithelial cells.

Luteolin Alleviates Oxidative Stress in Chronic Obstructive Pulmonary Disease Induced by Cigarette Smoke via Modulation of the TRPV1 and CYP2A13/NRF2 Signaling Pathways.

The current study aims to investigate the therapeutic potential of luteolin (Lut), a naturally occurring flavonoid found in various medicinal plants, for treating chronic obstructive pulmonary disease (COPD) through both in vitro and in vivo studies. The results demonstrated that Lut increased body weight, reduced lung tissue swelling and lung damage indices, mitigated systemic oxidative stress levels, and decreased alveolar fusion in cigarette smoke (CS)- and lipopolysaccharide (LPS)-induced COPD mice. Additionally, Lut was observed to downregulate the expression of the TRPV1 and CYP2A13 proteins while upregulating SIRT6 and NRF2 protein expression in CS + LPS-induced COPD mice and cigarette smoke extract (CSE)-treated A549 cells. The concentrations of total reactive oxygen species (ROS) and mitochondrial ROS in A549 cells induced by CSE significantly increased. Moreover, CSE caused a notable elevation of intracellular Ca2+ levels in A549 cells. Importantly, Lut exhibited inhibitory effects on the inward flow of Ca2+ and attenuated the overproduction of mitochondrial and intracellular ROS in A549 cells treated with CSE. In conclusion, Lut demonstrated a protective role in alleviating oxidative stress and inflammation in CS + LPS-induced COPD mice and CSE-treated A549 cells by regulating TRPV1/SIRT6 and CYP2A13/NRF2 signaling pathways.

CD44-mediated tumor homing of hyaluronic acid nanogels for hypoxia-activated photodynamic therapy against tumor

In this study, unique hypoxia-activated hyaluronic acid nanogels (HANGs) were reported for CD44-targeted delivery of photosensitizers (chlorin e6, Ce6) for diagnostic imaging and photodynamic therapy (PDT) of cancers. Through the use of a hypoxia-responsive cross-linker (AZO-CDI), the HANGs were prepared by chemically cross-linking primary amine groups-functionalized hyaluronic acid (HA). Under normoxic condition, fluorescence of Ce6 conjugated on the HANGs was highly quenched, and level of reactive oxygen species (ROS) generated from the HANGs was rather low after laser irradiation. However, under hypoxic condition, the HANGs underwent rapid disassociation, and fluorescence of Ce6 conjugated on the HANGs was recovered, triggering high-level singlet oxygen generation after laser irradiation. Due to the presence of HA, the HANGs showed much higher cellular uptake by CD44-positive cancer cells (A549 cells) than that by CD44-negative cancer cells (HepG2 cells). In addition, the HANGs could generate higher level of ROS in A549 cells because of improved cancer cell uptake. This excellent tumor-targeting and singlet oxygen-generating ability of the HANGs was favorable to hypoxia-activated PDT of CD44-positive cancers with significant inhibition of tumor growth within the whole treatment period. Taken together, the HANGs are safe and effective tools in treating CD44-positive cancers.

Thimerosal, a competitive thioredoxin reductase 1 (TrxR1) inhibitor discovered via high-throughput screening

Thioredoxin reductase 1 (TrxR1) is considered as an important anti-cancer drug target, inhibition of which can induce reactive oxygen species (ROS)-mediated apoptosis of human cancer cells. Here, we developed and optimized a high-throughput screening (HTS) assay based on enzyme kinetics for the discovery of TrxR1 inhibitors. By utilizing this assay, we performed a HTS for 2500 compounds from an in-house library against TrxR1. We found that a vaccine preservative, thimerosal, strongly inhibited TrxR1 in a competitive and reversible manner with an IC50 of 24.08 ± 0.86 nM. In addition, we determined that thiomersal has an inhibitory effect on the proliferation of A549 lung cancer cell line, with a GI50 of 6.81 ± 0.09 μM, slightly more potent than auranofin (GI50 = 11.85 ± 0.56 μM). Furthermore, we showed by flow cytometer that thimerosal effectively increased the content of ROS in A549 cells. Therefore, our work provided a high-throughput screening assay to quickly and effectively discover TrxR1 inhibitors, identifying thiomersal as a novel TrxR1 inhibitor and chemical probe.

Fructose Induces Pulmonary Fibrotic Phenotype Through Promoting Epithelial-Mesenchymal Transition Mediated by ROS-Activated Latent TGF-β1.

Fructose is a commonly used food additive and has many adverse effects on human health, but it is unclear whether fructose impacts pulmonary fibrosis. TGF-β1, a potent fibrotic inducer, is produced as latent complexes by various cells, including alveolar epithelial cells, macrophages, and fibroblasts, and must be activated by many factors such as reactive oxygen species (ROS). This study explored the impact of fructose on pulmonary fibrotic phenotype and epithelial-mesenchymal transition (EMT) using lung epithelial cells (A549 or BEAS-2B) and the underlying mechanisms. Fructose promoted the cell viability of lung epithelial cells, while N-Acetyl-l-cysteine (NAC) inhibited such. Co-treatment of fructose and latent TGF-β1 could induce the fibrosis phenotype and the epithelial-mesenchymal transition (EMT)-related protein expression, increasing lung epithelial cell migration and invasion. Mechanism analysis shows that fructose dose-dependently promoted the production of total and mitochondrial ROS in A549 cells, while NAC eliminated this promotion. Notably, post-administration with NAC or SB431542 (a potent TGF-β type I receptor inhibitor) inhibited fibrosis phenotype and EMT process of lung epithelial cells co-treated with fructose and latent TGF-β1. Finally, the fibrosis phenotype and EMT-related protein expression of lung epithelial cells were mediated by the ROS-activated latent TGF-β1/Smad3 signal. This study revealed that high fructose promoted the fibrotic phenotype of human lung epithelial cells by up-regulating oxidative stress, which enabled the latent form of TGF-β1 into activated TGF-β1, which provides help and reference for the diet adjustment of healthy people and patients with fibrosis.

Anticancer mechanism of breviscapine in non-small cell lung cancer A549 cells acts via ROS-mediated upregulation of IGFBP4.

BackgroundThe overall 5-year survival rate of non-small cell lung cancer (NSCLC) is less than 15% because of multiple drug resistance to chemotherapy and the limitations of early diagnosis. Thus, safe and effective drugs to treat NSCLC are required. The present study aimed to investigate the effects of breviscapine (BVP) on NSCLC cell apoptosis and proliferation, and to study its possible mechanisms.MethodsUsing the NSCLC A549 cell line and BVP (0, 25, 50, and 100 µM), the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to detect A549 cell proliferation, and flow cytometry was used to assess cell apoptosis. Insulin-like growth factor binding protein 4 (IGFBP4) levels was assessed using enzyme-linked immunosorbent assays and western blotting. Flow cytometry of hydrogen peroxide and superoxide was used to assess intracellular reactive oxygen species (ROS) generation. Western blotting was used to assess the levels of BCL2-associated X, apoptosis regulator (BAX) and B-cell CLL/lymphoma 2 (BCL2). Quantitative real-time reverse transcription PCR (qRT-PCR) was used to assess IGFBP4 mRNA expression.ResultsBVP induced apoptosis, inhibited cell proliferation, and increased ROS in A549 cells. Western blotting and qRT-PCR showed that BVP increased IGFBP4 protein and mRNA expressions in A549 cells. Compared with BVP treatment alone, IGFBP4 expression decreased in A549 cells treated with BVP and the ROS scavenger N-acetylcysteine. IGFBP4 overexpression increased BVP-induced proliferation inhibition, while increasing BAX expression and decreasing BCL2 expression. Silencing IGFBP4 had the opposite effects.ConclusionsBVP could inhibit the growth of NSCLC A549 cells by promoting apoptosis via ROS-mediated upregulation of IGFBP4.

Chrysotile asbestos induces apoptosis via activation of the p53-regulated mitochondrial pathway mediated by ROS in A549 cells

Long-term exposure to chrysotile asbestos has been associated with an increased risk of pulmonary fibrosis, lung cancer, ovarian cancer and peritoneal mesothelioma. Although numerous pathophysiological mechanisms account for chrysotile asbestos-induced pulmonary toxicity, apoptosis has attracted considerable attention and has not yet been fully elucidated. Therefore, the goal of the current study was to determine whether chrysotile asbestos could cause oxidative stress and apoptosis in A549 cells and the potential mechanism of reactive oxygen species (ROS)-induced cell apoptosis. The experimental results showed that chrysotile asbestos significantly inhibited the proliferation of A549 cells. Additionally, chrysotile asbestos increased ROS in a dose-dependent manner. Excessive ROS injured the antioxidant defense system and caused lipid peroxidation, of which glutathione (GSH) and superoxide dismutase (SOD) were dramatically decreased while malondialdehyde (MDA) was significantly increased. Excessive production of ROS upregulated the expression of pro-apoptotic proteins p53 and Bax to depress the mitochondrial membrane potential (MMP) and promote cytochrome c (Cyt c) release into the cytosol, which triggered the downstream apoptotic protein cleave-caspase-3 to induce cell apoptosis. Furthermore, N-acetyl-L-cysteine (NAC) was applied to inhibit the production of ROS. NAC pretreatment hindered the decrease of MMP, reduced expression of p53, Bax and Cyt c, downregulated cleave-caspase-3 and reduced cellular apoptosis. In conclusion, chrysotile asbestos caused mitochondrial dysfunction and cell oxidative stress to induce apoptosis via the p53-regulated mitochondrial pathway. Our research results provided a fundamental basis for a toxicity discussion and rational utilization of chrysotile asbestos.

The Role of TSP-1-CD47 in ROS-mediated Pulmonary Fibrosis Induced by Paraquat

Objective: Taking human A549 cells as the research object, to construct the paraquat-induced pulmonary fibrosis model in vitro, and to explore the role of TSP-1 (Thrombospondin-1, TSP-1) and its receptor CD47 in PQ-induced pulmonary fibrosis. Methods: Human A549 cells were cultured in vitro, divided into normal control group, PQ group, Anti-TSP1 group (PQ with neutralizing anti-TSP1 antibody at a final concentration of 10 μg/ml) . A549 cells were stimulated with different concentrations (50, 100, 200, 400, 600, 800, 1 000 μmol/L) for different time (12, 24, 48 h) , and then CCK8 method was used to detect the cell viability to screen out the concentration and time of half cell viability. The subsequent test will be performed at this concentration point.The morphology of the cells was observed under inverted microscope. The expression levels of Fibronectin (FN) and type I collagen were determined by Enzyme Linked Immunosorbent Assay (ELISA) . Immunocytochemistry (ICC) and Immunofluorescence (IF) were used to observe the expression of TSP-1 and CD47 protein and the co-expression.The mRNA expression of TSP-1 and CD47 was detected by Real Time PCR (RT-PCR) . The protien expression of TSP-1 and CD47 was detected by Western Blot (WB) . The levels of Reactive Oxygen Species (ROS) were measured by flow cytometry. Results: Before neutralizing anti-TSP1 antibody intervention: (1) When the time of PQ was constant, the cell viability decreased with the increase of PQ concentration. (2) The cells in the control group were closely connected, cobble-like, arranged neatly; with the increase of PQ concentration, the cell gap of PQ group gradually increased, spindle shape or long spindle shape. (3) With the increase of PQ concentration, the relative expression of FN and I collagen in PQ group was gradually increased compared with the control group in a concentration-dependent manner, and 200 μmol/L is the most obvious. (4) Compared with the control group, the mRNA level and the protein expression of TSP-1 and CD47 in PQ group was significantly increased, and 200 μmol/L is the most obvious, and Immunofluorescence showed they were co-expression in cytoplasm. (5) Compared with the normal group, the level of ROS in A549 cells was significantly increased at 24 h after PQ stimulation. (6) Compared with PQ group, the cell viability of Anti-TSP1 group was significantly increased, and the morphology changed to normal cell morphology, and the mRNA level and the protein expression of TSP-1 and CD47 decreased, and the overexpression of ROS was inhibited, and the relative expression of FN and I collagen decreased. Conclusion: PQ stimulation induced morphological changes of A549 cells, increased expression of TSP-1, CD47, FN and type I collagen, and increased production of ROS.Neutralizing anti-TSP1 antibodies against TSP-1 can partially improve the above lesions. TSP-1-CD47 may be associated with oxidative stress-mediated PQ-induced pulmonary fibrosis.

Phenylalkyl isoselenocyanates vs phenylalkyl isothiocyanates: Thiol reactivity and its implications

Phenylalkyl isoselenocyanate (ISC) compounds were recently designed in our laboratory by incorporating the anticancer element selenium into a panel of phenylalkyl isothiocyanates (ITCs), known to have anticancer properties. A structural activity investigation was carried out to compare the ISC and ITC panels. Cell viability assay and Annexin V staining for apoptosis showed ISC compounds to be more potent in killing A549 lung adenocarcinoma cells. Both ITCs and ISCs were able to deplete reduced glutathione (GSH) in cells, ISCs more rapidly, but ITCs to a greater extent. ISC compounds had a higher rate of reaction to thiol (–SH) groups as determined by pseudo first order kinetics than the corresponding carbon chain length ITC. The equilibrium concentrations of the GSH and protein thiol conjugates did not differ significantly when comparing sulfur to selenium compounds of the same carbon chain length, and did follow the same trend of displaying decreasing reactivity with increasing carbon chain length for both ITCs and ISCs. Furthermore, only ITCs were able to induce cell cycle arrest, suggesting that protein targets inside the cell may differ for the S and Se panels. Finally, the panels were tested for their ability to redox cycle when reacted with GSH to form superoxide and other reactive oxygen species (ROS). ISC compounds showed a much greater ability to redox cycle than corresponding ITCs, and were able to induce higher levels of ROS in A549 cells. Also, the direct pro-apoptotic effects of ISCs and ITCs were inhibited by GSH and potentiated by depletion of intracellular GSH by buthionine sulfoximine. In conclusion, our studies suggest that the redox-cycling capabilities of ISCs and thus generation of higher levels of ROS may be contributing to the increased cytotoxicity of ISC compounds in A549 cells, compared to that of the corresponding ITCs.

Lipopolysaccharide induces apoptotic insults to human alveolar epithelial A549 cells through reactive oxygen species-mediated activation of an intrinsic mitochondrion-dependent pathway

Alveolar type II epithelial cells can regulate immune responses to sepsis-induced acute lung injury. Lipopolysaccharide (LPS), an outer membrane component of Gram-negative bacteria, can cause septic shock. This study was designed to evaluate the cytotoxic effects of LPS on human alveolar epithelial A549 cells and its possible molecular mechanisms. Exposure of A549 cells to LPS decreased cell viability in concentration- and time-dependent manners. In parallel, LPS concentration- and time-dependently induced apoptosis of A549 cells. Meanwhile, LPS only at a high concentration of 10 μg/ml caused mildly necrotic insults to A549 cells. In terms of the mechanism, exposure of A549 cells to LPS increased the levels of cellular nitric oxide and reactive oxygen species (ROS). Pretreatment with N-acetylcysteine (NAC), an antioxidant, significantly lowered LPS-caused enhancement of intracellular ROS in A549 cells and simultaneously attenuated the apoptotic insults. Sequentially, treatment of A549 cells with LPS caused significant decreases in the mitochondrial membrane potential and biosynthesis of adenosine triphosphate. In succession, LPS triggered the release of cytochrome c from the mitochondria to the cytoplasm. Activities of caspase-9 and caspase-6 were subsequently augmented following LPS administration. Consequently, exposure of A549 cells induced DNA fragmentation in a time-dependent manner. Pretreatment of A549 cells with NAC significantly ameliorated LPS-caused alterations in caspase-9 activation and DNA damage. Therefore, this study shows that LPS specifically induces apoptotic insults to human alveolar epithelial cells through ROS-mediated activation of the intrinsic mitochondrion-cytochrome c-caspase protease mechanism.

Silica-Triggered Cellular Signaling Without Immediate Increase of ROS in A549 Cells

Silica has been known to be a factor in acute cell injury and chronic pulmonary fibrosis. To date, many studies have emphasized the reactive oxygen species (ROS) as a primary cause of this pathogenesis. Previously, we demonstrated that silica induced acute radical generation and intracellular Ca2+ increase in Rat2 cells. Moreover, increases in intracellular Ca2+ directly affect the rapid degradation of peroxiredoxin (Prx), a newly discovererd antioxidant enzyme family. In the present study, we investigated the mechanism by which silica transfers its cellular signal downstream of the cell by using human lung epithelial cells, which are known to contribute to the inflammatory response after exposure to silica in the lung. In A549 cells, silica induced the generation of ROS immediately, although immediate responses were observed in C6 cells or in Rat2 cells. Nevertheless, the immediate production of ROS in response to silica in A549 cells was not clearly detected, but the initial step for NF-κB signaling, such as rapid degradation of IκB-α, was sensitive to the addition of silica. Additionally, silica triggered the rapid degradation of cytoplasmic antioxidant enzymes such as PrxI and PrxII, indicating that this may accelerate silica-induced cellular damage. Moreover, silica was observed to increase intracellular Ca2+ concentration. This report suggests that silica transduces diverse pathways, such as the rapid increase of intracellular Ca2+ concentration, NF-κB signaling or modulation of redox potential by destruction of antioxidant enzymes, without the immediate generation of ROS in A549 cells.