Acid In A549 Cells Research Articles

Diffractaic acid exhibits thioredoxin reductase 1 inhibition in lung cancer A549 cells.

Lung cancer is the leading cause of cancer-related deaths all over the world. Therefore, it has gained importance in the development of new chemotherapeutic strategies to identify anticancer agents with low side effects, reliable, high anticancer potential, and specific to lung cancer cells. Thioredoxin reductase 1 (TrxR1) is an important therapeutic target for lung cancer treatment because of its overexpression in tumor cells. Here, we aimed to examine the anticancer effect of diffractaic acid, a lichen secondary metabolite, in A549 cells by comparing it with the commercial chemotherapeutic drug carboplatin and also to investigate whether the anticancer effect of diffractaic acid occurs via TrxR1-targeting. The IC50 value of diffractaic acid on A549 cells was determined as 46.37 μg/mL at 48 h, and diffractaic acid had stronger cytotoxicity than carboplatin in A549 cells. qPCR results revealed that diffractaic acid promoted the intrinsic apoptotic pathway through the upregulation of the BAX/BCL2 ratio and P53 gene in A549 cells, which is consistent with the flow cytometry results. Furthermore, migration analysis results indicated that diffractaic acid impressively suppressed the migration of A549 cells. While the enzymatic activity of TrxR1 was inhibited by diffractaic acid in A549 cells, no changes were seen in the quantitative expression levels of gene and protein. These findings provide fundamental data on the anticancer effect of diffractaic acid on A549 cells targeting TrxR1 activity, suggesting that it could be considered a chemotherapeutic agent for lung cancer therapy.

Chlorogenic acid inhibits the proliferation of human lung cancer A549 cell lines by targeting annexin A2 in vitro and in vivo

Chlorogenic acid, an important active component of coffee with anti-tumor activities, has been found for a hundred years. However, the lack of understanding about its target proteins greatly limits the exploration of its anti-tumor molecular mechanisms and clinical applications. Here, in vitro and animal experiments showed that chlorogenic acid had a significant inhibitory effect on the proliferation of A549 cells. The ability of chlorogenic acid to naturally emit fluorescence was exploited to screen its target proteins while avoiding false positives brought about by chemical modifications when using fluorescent tags. Consequently, we identified and verified annexin A2 as a covalent binding target of chlorogenic acid in A549 cells. We also discovered that chlorogenic acid inhibits the binding of annexin A2 to p50 subunit thereby inhibiting the expression of downstream anti-apoptotic genes cIAP1 and cIAP2 of the NF-κB signaling pathway in A549 cells in vitro and in vivo. Moreover, we found that chlorogenic acid hindered the binding of annexin A2 to actin possibly causing inhibition of tumor cell cycle and migration. Thus, this work demonstrates that chlorogenic acid binds annexin A2, causing a decrease in the expression of NF-κB downstream anti-apoptotic genes, and inhibiting the proliferation of A549 cells in vivo and in vitro.

Phenolic compounds stage an interplay between the ubiquitin–proteasome system and ubiquitin signal autophagic degradation for the ubiquitin-based cancer chemoprevention

Inhibiting proteasome functional insufficiency plays an essential role in cancer prevention. Alternative strategies to target ubiquitin enzymes and ubiquitin-signal autophagic degradation can be the potential treatments. Phenolic compounds demonstrated anti-cancer effects. Phenolic compounds not only effectively inhibit E1, deubiquitinating enzyme, and 26S activity but also induce apoptosis in A549 cells. The IC50 of 26S activity and LD50 of A549 cells were nearly equivalent. Therefore, apoptosis may occur via the inhibition of the 26S proteasome activity. Inhibition of the proteasome by tannic acid in A549 cells resulted in reduction of S5a and 20S, accumulation of ubiquitin, and degradation of PARP, followed by induction of apoptosis. USP15 may play an essential role in the stability and activity of caspase-3. The levels of S5a, USP15 and USP47 were regulated by phenolic compounds, and the levels of procaspase-3 and polyADP-ribose polymerase were also decreased in A549 cells treated with phenolic compounds. Interestingly, tannic acid decreased Ub-conjugates while M + G + E increased the accumulation of Ub-conjugates in A549 reporter cells, whereas ubiquitin, E1, and USP47 levels were low in M + G + E-treated A549 reporter cells at 24 hours. Taken together, phenolic compounds may play an important role in mediating apoptosis by regulating the ubiquitin–proteasome pathway and autophagy system.

In vitro enhancement effect of humic acid on the progression of lung cancer cells

Humic acid, a group of polymer, resulting from the decomposition of organic matter has been implicated as a possible etiological factor for Black foot disease and cancer. However, the role of humic acid in carcinogenesis is not well clarified. In this study, we evaluated the enhancement effect of humic acid on the progression of A549 human lung cancer cells. Our findings showed that humic acid increased the migration, adhesion and invasion of A549 cells significantly after treatment at the concentration of 50 and 100 μg/ml for 24 and 48 h as compared with the untreated group. Results of zymography assay indicated that humic acid enhanced the activity of matrix metalloproteinase 9 (MMP-9). By western blotting analysis, humic acid increased the expression of phosphorylated focal adhesion kinase (FAK) and integrin α2 that may mediate cell motility and adhesion. Since the activation of signal pathways such as phosphoinositide 3-kinase/Akt (PI3K/Akt) and mitogen-activated protein kinases (MAPKs) may play a role in the cell invasion, we detected the expression of phosphorylated Akt and phosphorylated MAPKs including extracellular signal-regulated kinase (ERK), Jun N-terminus kinase (JNK), and p38. The result indicated that all kinases stated above were activated by humic acid in A549 cells. However, by adding an inhibitor respectively in the invasion assay, only Akt, ERK, and JNK pathways were found to be involved in the enhancing the invasion of A549 cells by humic acid. In addition, results of electrophoretic mobility shift assay (EMSA) demonstrated that humic acid increased nuclear extract binding to the DNA probe of activator protein 1 (AP-1) and nuclear factor κB (NFκB) respectively, implying that humic acid enhanced the progression of A549 lung cancer cells through activating multiple signaling pathways including ERK, JNK, and PI3K/Akt and increasing the transcription activation of AP-1 and NFκB.

Effect of simvastatin on the uptake and metabolic conversion of palmitic, dihomo- γ-linoleic and α-linolenic acids in A549 cells

It is well known that simvastatin affects cholesterol synthesis. Furthermore it inhibits growth and proliferation and perturbs fatty acid metabolism in some cell lines. We have studied the effects of simvastatin on the uptake and metabolism of exogenous fatty acid in the human lung adenocarcinoma A549 cells. Simvastatin inhibited the proliferation of A549, and caused an increment in phospholipid/cholesterol ratio due to an increment in phospholipid content without affecting cholesterol content. All the fatty acids were uptaken and metabolized in both control and treated cells. The conversion of palmitic, linoleic and dihomo- γ-linoleic acids to their metabolites and products/precursor ratios for the desaturation and elongation reactions showed that simvastatin enhanced the Δ5 desaturation step and altered some elongating steps. The machinery for unsaturated fatty acid synthesis in A549 is quite sensitive to simvastatin and its effects could have important implication taking into account that highly unsaturated fatty acids are involved in the regulation of diverse cellular functions by themselves or through their metabolites.

Lipocortin 1 and the control of cPLA 2 activity in A549 cells : Glucocorticoids block EGF stimulation of cPLA 2 phosphorylation

Epidermal growth factor (EGF) rapidly stimulates the release of arachidonic acid in A549 cells by a mechanism that is sensitive to pertussis toxin [1]. We show that EGF treatment of A549 cells stimulates phosphorylation of cytosolic phospholipase A 2 (cPLA 2) through a mechanism that is similarly inhibited by pertussis toxin. The level of cPLA 2 expression is, apparently, not changed during this period. Pretreatment of cells with dexamethasone (10–100 nM) for 3 hr prevents this activation of cPLA 2 by EGF, without changing the level of cPLA 2 expression. The effect of dexamethasone is reversed in the presence of the neutralizing antilipocortin Mab 1A but not by the nonneutralizing antilipocortin 1 control Mab 1B. This strongly suggests that lipocortin 1 mediates the effect of dexamethasone by inhibiting activation of cPLA 2. This concept is supported by the fact that a peptide Lc13-25 (10–200 μg/mL), derived from the N-terminus of lipocortin 1, also inhibits activation of cPLA 2 by EGF in these cells.

Differential role of extra- and intracellular calcium in bradykinin and interleukin 1α stimulation of arachidonic acid release from A549 cells

The release of arachidonic acid in A549 cells was stimulated in a time- and dose-dependent manner by the Ca 2+ ionophore ionomycin ( t 1 2 = 4 min ), thapsigargin ( t 1 2 = 8 min , bradykinin ( t 1 2 = 12 min , EC 50 = 3 nM), and interleukin 1α ( t 1 2 = 28 min , EC 50 = 0.3 ng/ ml). Bradykinin (10 nM) and interleukin 1α (1 ng/ml) stimulation was blocked by the bradykinin B 2 receptor antagonist, D-Arg,[Hyp 3, Thi 5,8, D-Phe 7]bradykinin and interleukin 1 receptor antagonist ( IC 50 = 30 mM and 20 ng/ ml, respectively), suggesting receptor mediation. Diacylglycerol release was < 10% of total arachidonic acid release in all cases, suggesting activation of phospholipase A 2 activity was greater than phosopholipase C activation by these agents. The effects of ionomycin (3 μM) and thapsigargin (0.3 μM) were abolished in Ca 2+-free buffer with and without 0.5 mM EGTA. Bradykinin (10 nM) stimulation was reduced by 50% in Ca 2+-free buffer whereas interleukin 1α (1 ng/ml) stimulation remained unaffected. However, the presence of EGTA completely abolished bradykinin stimulation and partially blocked the effect of interleukin 1α (43% inhibition). In the presence of extracellular Ca 2+, ionomycin (3 mM), thapsigargin (0.3 mM), bradykinin (10 nM), and interleukin 1α (1 ng/ml) stimulation of arachidonic acid release was blocked by the Ca 2+ influx blocker LaCl 3 (29, 44, 35, and 41% inhibition, respectively). Nifedipine also blocked ionomycin and thapsigargin stimulation but only partially blocked bradykinin and interleukin 1α stimulation. These results suggest that following B 2 receptor activation, cytosolic phospholipase A 2 is stimulated by a rise in intracellular Ca 2+ levels which are sensitive to the action of EGTA, whereas interleukin 1α stimulation of cytosolic phospholipase A 2 is mediated by a rise in intracellular Ca 2+ from both EGTA-sensitive and resistant pools. Furthermore the results of ionomycin and thapsigargin indicate that extracellular Ca 2+ is important for activation of cytosolic phospholipase A 2 in A549 cells.