Signaling In A549 Cells Research Articles

HtrA1 Downregulation Induces Cisplatin Resistance in Lung Adenocarcinoma by Promoting Cancer Stem Cell‐Like Properties

Cisplatin (CDDP) resistance usually develops during lung adenocarcinoma (LAC) therapy. However, the comprehensive mechanisms remain largely unclear. In this study, we first established a CDDP-resistant LAC cell line-A549/CDDP from its parental cell line-A549. The results showed that CDDP resistance in A549/CDDP cells correlates with acquirement of cancer stem cell-like properties (increased percentage of CD133-expressing subpopulation, sphere formation and levels of some pluripotency-associated markers). HtrA1 expression at both mRNA and protein levels was reduced in CDDP-resistant A549/CDDP cells compared with that in A549 cells. Ectopic expression of HtrA1 in A549/CDDP cells reversed cancer stem cell-like properties and CDDP resistance. In A549 cells, stable knockdown of HtrA1 expression promoted cancer stem cell-like properties and CDDP insensitivity, however, these effects were blocked by inhibition of PI3K/Akt pathway using LY294002. Furthermore, HtrA1 knockdown could significantly stimulate PI3K/Akt signaling in A549 cells. In vivo studies, HtrA1 knockdown promoted tumorigenesis and conferred CDDP resistance in xenograft A549 tumors, which were reversed by intraperitoneal injection of LY294002. In conclusion, these results indicate that HtrA1 downregulation confers CDDP resistance by inducing cancer stem cell-like properties via PI3K/Akt-dependent pathway in A549 cells. Therefore, HtrA1 may be a potential target for overcoming CDDP resistance in LAC.

Surfactant protein D suppresses lung cancer progression by downregulation of epidermal growth factor signaling

Surfactant protein D (SP-D) is a member of the collectin family that has an important role in maintaining pulmonary homeostasis. In this study, we demonstrated that SP-D inhibited the proliferation, migration and invasion of A549 human lung adenocarcinoma cells. We found that SP-D suppressed epidermal growth factor (EGF) signaling in A549 cells, H441 human lung adenocarcinoma cells and human EGF receptor (EGFR) stable expression CHO-K1 cells. A binding study using (125)I-EGF demonstrated that SP-D downregulated the binding of EGF to EGFR. A ligand blot indicated that SP-D bound to EGFR, and a lectin blot suggested that EGFR in A549 cells had both high-mannose type and complex type N-glycans. We purified the recombinant extracellular domain of EGFR (soluble EGFR=soluble EGFR (sEGFR)), and demonstrated that SP-D directly bound to sEGFR in a Ca(2+)-dependent manner. The binding of SP-D to sEGFR was suppressed by EDTA, mannose or N-glycopeptidase F treatment. Mass spectrometric analysis indicated that N-glycans in domain III of EGFR were of a high-mannose type. These data suggest that SP-D reduces EGF binding to EGFR through the interaction between the carbohydrate recognition domain of SP-D and N-glycans of EGFR, and downregulates EGF signaling. Our finding suggests the novel type of regulation system of EGF signaling involving lectin-to-carbohydrate interaction and downregulation of ligand binding.

A Wnt/β-catenin negative feedback loop represses TLR-triggered inflammatory responses in alveolar epithelial cells

Increasing evidence has demonstrated that the epithelial cells in the lung play crucial roles in regulating certain inflammatory responses by modulating Wnt signaling during microbial infection. However, the anti-microbial functions of Wnt signaling in alveolar epithelial cells remain elusive. In this report, we show that Wnt/β-catenin signaling is repressed in A549 alveolar epithelial cells during a Toll-like receptor ligand stimulation with Mycobacterium bovis Bacillus Calmette-Guerin (BCG) or lipopolysaccharide (LPS). In addition to activating TLR signaling, a stimulation of BCG or LPS led to the up-regulation of a Wnt receptor Frizzled-1, cytosolic GSK3β and Axin, and the down-regulation of nuclear β-catenin, lymphoid enhancer factor 1 and transcription factor 4. While an enhancement of β-catenin activity suppressed the TLR signal response, and substantially led to alleviate the TLR ligand-induced pro-inflammatory responses. Importantly, gain and loss of function studies by overexpressing or silencing of TLR signaling adaptor, myeloid differentiation primary response gene 88 (MyD88) further demonstrated an inverse relationship between TLR signaling and canonical Wnt signaling in A549 cells. These data imply that Wnt/β-catenin signaling acts as a negative feedback loop to suppress inflammation in alveolar epithelial cells, and averts cell injury from excessive inflammatory reactions. This study thus reveals a novel immunoregulatory mechanism in alveolar epithelial cells in response to bacterial infection.

Silver nanoparticles induced changes in the expression of NF-κB related genes are cell type specific and related to the basal activity of NF-κB

Silver nanoparticles (AgNPs) are widely used in industry and medicine but the recent evidence for their cytotoxicity rise a concern about the safety of their use. We have previously shown that human A549 cells are resistant to AgNPs cytotoxicity, as compared with similarly treated HepG2 cells. In order to check for the role of the NF-κB signaling pathway in response of A549 and HepG2 cell lines to the treatment with 20nm and 200nm AgNps, we analyzed the expression of 84 key genes related to the functionality of the NF-κB signaling pathway. We observed considerable alternations in gene expression in HepG2 cells treated with 20nm AgNPs, and minor changes when exposed to 200nm AgNPs. Surprisingly, no changes in gene expression were observed in A549 cells treated with both size AgNPs. Using the NF-κB luciferase reporter system, we further tested the basal activity and inducibility of the NF-κB pathway in both cell lines and found that the inducibility of NF-κB signaling in A549 cells is approximately 5 times lower than this of HepG2 cells, but the basal activity is approximately 3.5 times higher. In accordance, the NF-κB activation after AgNPs treatment was observed in HepG2 but not in A549. Altogether indicate that NF-kB mediated cellular response to AgNPs is cell type specific and related to the basal activity of NF-κB.

Protective Effect of Hypercapnic Acidosis in Ischemia-Reperfusion Lung Injury Is Attributable to Upregulation of Heme Oxygenase-1

Hypercapnic acidosis (HCA) has protective effects in animal models of acute lung injury, but the mechanism underlying the effect of HCA is unclear. Heme oxygenase-1 (HO-1) is an antioxidant enzyme that protects tissue from inflammation injury. We investigated whether HO-1 contributes to the protective effects of HCA in ischemia-reperfusion (IR)-induced lung injury. Typical acute lung injury in rats was successfully induced by 40 min of ischemia and 90 min of reperfusion in an isolated perfused lung model. The rat lungs were randomly assigned to the control group, IR group or IR + HCA group with or without zinc protoporphyrin IX (ZnPP), an HO-1 activity inhibitor. At the end of the experiment, bronchoalveolar lavage fluid (BALF) and lung tissues were collected to evaluate the degree of lung injury. In in vitro experiments, HO-1 siRNA transfected A549 cells were exposed to a normoxic or hypoxia-reoxygenation (H/R) environment in the presence or absence of HCA. IR caused significant increases in the pulmonary arterial pressure, lung weight to body weight and wet/dry ratios, lung weight gain, capillary filtration coefficient, lung injury scores, neutrophil infiltration, and concentrations of protein and TNF-α in the BALF. IR also induced degradation of inhibitor of nuclear factor (NF)-κB-α, increased IκB kinase (IKK)-β phosphorylation and nuclear translocation of NF-κB, and up-regulated HO-1 expression and activity. Furthermore, IR decreased Bcl-2 protein expression and increased the number of active caspase-3 stained cells. HCA treatment enhanced HO-1 expression and activity, and accordingly reduced IKK-NF-κB signaling, inhibited apoptosis, and significantly attenuated IR-induced changes. Treatment with ZnPP partially blocked the protective effect of HCA. In addition, HO-1 siRNA significantly reversed HCA-mediated inhibition of NF-κB signaling in A549 cells subjected to H/R. In conclusion, the protective effect of HCA in IR lung injury in rats was mediated in part by the anti-inflammatory and anti-apoptotic action of HO-1.

Differential Effect of Schisandrin B Stereoisomers on ATR-Mediated DNA Damage Checkpoint Signaling

We have previously reported that schisandrin B (SchB) is a specific inhibitor of ATR (ataxia telangiectasia and Rad-3-related) protein kinase. Since SchB consists of a mixture of its diastereomers gomisin N (GN) and γ-schisandrin (γ-Sch), the inhibitory action of SchB might result from a stereospecific interaction between one of the stereoisomers of SchB and ATR. Therefore, we investigated the effect of GN and γ-Sch on UV (UVC at 254 nm)-induced activation of DNA damage checkpoint signaling in A549 cells. UV-induced cell death (25 – 75 J/m2) was amplified by the presence of the diastereomers, especially GN. At the same time, GN, but not γ-Sch, inhibited the phosphorylation of checkpoint proteins such as p53, structural maintenance of chromosomes 1, and checkpoint kinase 1 in UV-irradiated cells. Moreover, GN inhibited the G2/M checkpoint during UV-induced DNA damage. The in vitro kinase activity of immunoaffinity-purified ATR was dose-dependently inhibited by GN (IC50: 7.28 μM) but not by γ-Sch. These results indicate that GN is the active component of SchB and suggest that GN inhibits the DNA damage checkpoint signaling by stereospecifically interacting with ATR.

Effects of Brazilian green propolis on double‐stranded RNA‐mediated induction of interferon‐inducible gene and inhibition of recruitment of polymorphonuclear cells

Propolis is a bee product with various biological properties, including an antiviral activity when taken orally. However, its mechanisms at the cellular and molecular level are not well understood. We investigated the effect of propolis on antiviral signaling in A549 cells transfected with double-stranded RNA (dsRNA), a model for viral infection. Pretreatment of the cells with propolis inhibited poly I:C (synthetic dsRNA)-induced interferon (IFN)-β expression. Propolis had no effect on the dsRNA-induced expression of RIG-I-like receptors (RLRs), which are known as intracellular viral RNA sensors. As to the effect on antiviral executor genes, propolis enhanced myxovirus resistance 1 (MX1) expression, whereas interferon-inducible gene 6-16 (G1P3) and 2'-5'-oligoadenylate synthetase (OAS) were unaffected. All of these genes belong to the IFN-inducible genes, suggesting that the effect of propolis on antiviral signaling is not necessarily mediated by the autocrine regulation by IFN-β. Propolis pretreatment inhibited dsRNA-induced interleukin-8 (IL8) and CCL5 expression, and consequently lowered polymorphonuclear leukocyte (PMN) chemotactic activity in the cell-conditioned medium. Taken together, these results suggest that propolis may suppress excess inflammatory responses without affecting the innate immunity during viral infection.

Abstract LB-430: Hedgehog signaling is required for the survival and tumorigenicity of cancer cells.

Abstract Background: Using small molecule smoothened (SMO) antagonists, Hedgehog (HH) signaling had been implicated to play an important role in a variety of cancers (e.g. Brain, Lung, Pancreas, digestive tract etc.). Recently, based on studies in a selected set of pancreatic and colorectal cancer cells it has been suggested: a) earlier observation of HH signaling dependence of various cancers by others and us is due to non-specific effect of SMO antagonists; b) HH producing cancer cells do not have a functional HH pathway; c) HH signaling is not required for the growth or survival of cancer cells rather it affects the growth of tumor by activating HH pathway in stroma cells. Given the reported prevalence of HH signaling in cancers and the suggested implication of this latter work on devising a successful cancer therapy, we decided to investigate the role of HH signaling in cancer in a manner independent of SMO antagonist. Lung cancer, being one of the deadliest cancers with the reported higher incidence of HH signaling, was used as a test system to evaluate the basic assumptions of this recently proposed model of HH role in cancer. Objective: Define the role of HH signaling in cancer cells. Methodology: The human non-small cell lung carcinoma (NSCLC) cell lines (HOP62, A549, U1752, H23, H157, H522) were used as a model system to study the relevance of HH signaling in cancer cells. The HH signaling was modulated by shRNA mediated knockdown of various HH signaling components (e.g. SMO, GLI1, SHH). The lentiviruses expressing shRNA were made and used to deliver the shRNA in NSCLC cells. HH responsiveness (quantitative real time RT-PCR and western blotting), cell proliferation (ATP quantitation), survival (Annexin-V labeling), anchorage independent growth (soft agar assay) and tumorigenesis (xenografts in Nu/Nu nude mice) were evaluated following shRNA mediated attenuation of HH signaling. Currently, HH pathway activation in cancer and stroma cells of primary lung cancer samples is being evaluated by immunohistochemistry (IHC) and RNA in situ hybridization (ISH). Preliminary Results and Conclusions: Exogenous expression of SHH in HOP62 and A549 induced the HH target gene GLI1 and PTCH1 expression while shRNA mediated knockdown of HH pathway components (e.g. SMO, SHH) inhibited the HH target gene expression. Further more, knockdown of HH pathway components, attenuated the proliferation, increased the apoptosis while decreased the anchorage independent growth of tested lung cancer cell lines. GLI1 shRNA mediated attenuation of HH signaling in A549 cells, greatly reduced their tumorigenicity in nude mice. We anticipate seeing activated HH signaling in cancer and stroma cells of primary lung tumor samples by IHC and ISH. Together, these results would indicate that cancer cells do elaborate a functional HH signaling pathway and require HH signaling for growth, survival and tumorigenicity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-430. doi:10.1158/1538-7445.AM2011-LB-430

Cell Proliferation and Epidermal Growth Factor Signaling in Non-small Cell Lung Adenocarcinoma Cell Lines Are Dependent on Rin1

Rin1 is a Rab5 guanine nucleotide exchange factor that plays an important role in Ras-activated endocytosis and growth factor receptor trafficking in fibroblasts. In this study, we show that Rin1 is expressed at high levels in a large number of non-small cell lung adenocarcinoma cell lines, including Hop62, H650, HCC4006, HCC827, EKVX, HCC2935, and A549. Rin1 depletion from A549 cells resulted in a decrease in cell proliferation that was correlated to a decrease in epidermal growth factor receptor (EGFR) signaling. Expression of wild type Rin1 but not the Rab5 guanine nucleotide exchange factor-deficient Rin1 (Rin1Delta) complemented the Rin1 depletion effects, and overexpression of Rin1Delta had a dominant negative effect on cell proliferation. Rin1 depletion stabilized the cell surface levels of EGFR, suggesting that internalization was necessary for robust signaling in A549 cells. In support of this conclusion, introduction of either dominant negative Rab5 or dominant negative dynamin decreased A549 proliferation and EGFR signaling. These data demonstrate that proper internalization and endocytic trafficking are critical for EGFR-mediated signaling in A549 cells and suggest that up-regulation of Rin1 in A549 cell lines may contribute to their proliferative nature.

Redox regulation in radiation-induced cytochrome c release from mitochondria of human lung carcinoma A549 cells

Mitochondria in mammalian cells are well-known to play an important role in the intrinsic pathway of genotoxic-agent-induced apoptosis by releasing cytochrome c into cytosol and to be a major source of reactive oxygen species (ROS). The aim of this study was to examine whether mitochondrial ROS are involved in radiation-induced apoptotic signaling in A549 cells. Post-irradiation treatment with N-acetyl- l-cystein (NAC) inhibited cytochrome c release from mitochondria but did not affect expression levels of Bcl-2, Bcl-X L and Bax, suggesting that late production of ROS triggered cytochrome c release. Experiments using DCFDA (a classical ROS fluorescence probe) and MitoAR (a novel mitochondrial ROS probe) demonstrated that intracellular and mitochondrial ROS were enhanced 6 h after X irradiation. Furthermore, the O 2 - production ability of mitochondria isolated from A549 cells was evaluated by ESR spectroscopy combined with a spin-trapping reagent (CYPMPO). When isolated mitochondria were incubated with NADH, succinate and CYPMPO, an ESR spectrum due to CYPMPO–OOH was detected. This NADH/succinate-dependent O 2 - production from mitochondria of irradiated cells was significantly increased in comparison with that of unirradiated cells. These results indicate that ionizing radiation enhances O 2 - production from mitochondria to trigger cytochrome c release in A549 cells.

Soluble nickel inhibits HIF‐prolyl‐hydroxylases creating persistent hypoxic signaling in A549 cells

Soluble nickel compounds are carcinogenic to humans although the mechanism by which they cause cancer remains unclear. One major consequence of exposure to nickel is the stabilization of hypoxia inducible factor-1alpha (HIF-1alpha), a protein known to be overexpressed in a variety of cancers. In this study, we report a persistent stabilization of HIF-1alpha by nickel chloride up to 72 h after the removal of nickel from the culture media. In addition, we show that the HIF-prolyl hydroxylases (PHD's) are inhibited when cells are exposed to nickel and that they remain repressed for up to 72 h after nickel is removed. We then show that nickel can inhibit purified HIF-PHD's 2 in vitro, through direct interference with the enzyme. Through theoretical calculations, we also demonstrate that nickel may be able to replace the iron in the active site of this enzyme, providing a plausible mechanism for the persistent inhibition of HIF-PHD's by nickel. The data presented suggest that nickel can interfere with HIF-PHD directly and does not inhibit the enzyme by simply depleting cellular factors, such as iron or ascorbic acid. Understanding the mechanisms by which nickel can inhibit HIF-PHD's and stabilize HIF-1alpha may be important in the treatment of cancer and ischemic diseases.

2-Aminoethoxydiphenyl borate perturbs hormone-sensitive calcium stores and blocks store-operated calcium influx pathways independent of cytoskeletal disruption in human A549 lung cancer cells

Recent studies have identified novel actions for 2-aminoethoxydiphenyl borate (2-APB) in triggering calcium release and enhancing calcium influx induced by the depletion of intracellular calcium stores. In this study, we have examined the effects of 2-APB on the human lung adenocarcinoma A549 cell line, which we have previously shown displays a unique calcium influx response, when ER calcium stores are depleted by thapsigargin (TG) treatment. Here, we show that low concentrations of 2-APB failed to induce the rapid augmentation of TG-activated calcium influx previously reported for other cell types. We observed that store-operated calcium (SOC) channels in the A549 cell line exhibited short-term sensitivity to low doses of 2-APB, perhaps reflecting a delayed augmentation of SOC channel activity or the recruitment of 2-APB-insensitive SOC channels. In both intact and permeabilized cells, 2-APB effectively discharged a subset of A549 calcium pools corresponding to the hormone-sensitive intracellular calcium stores. The 2-APB-induced calcium release produced a long-lasting perturbation of the adenosine triphosphate (ATP)-releasable calcium pools, effectively uncoupling ATP-activated calcium release even, when stores are replenished with calcium. In contrast to previous reports, we found that disruption of either the actin or microtubule-based cytoskeleton failed to block the 2-APB-induced effects on calcium signaling in A549 cells. Our study describes novel cytoskeletal-independent effects of 2-APB on Ca 2+-signaling pathways, revealing differentially sensitive Ca 2+-influx pathways and long-term perturbation of hormone-sensitive Ca 2+ stores.