A549 Cells In Response Research Articles

Burkholderia pseudomallei survival in lung epithelial cells benefits from miRNA-mediated suppression of ATG10

Burkholderia pseudomallei is the causative agent of melioidosis, a disease with high mortality, which is prevalent in tropical regions of the world. A recent study shows that B. pseudomallei can survive inside mammalian cells because of its ability to actively evade cell autophagy. However, the underlying mechanisms remain unclear. In the present study, based on microarray screening, we found that ATG10 was downregulated following B. pseudomallei infection in A549 human lung epithelial cells. Forced expression of ATG10 accelerated the elimination of intracellular B. pseudomallei by enhancing the process of autophagy. Moreover, MIR4458, MIR4667-5p, and MIR4668-5p were found, by microarray screening, to be upregulated in response to B. pseudomallei infection. These 3 novel miRNAs, MIR4458, MIR4667-5p, and MIR4668-5p, targeted to the 3′-untranslated region of ATG10 in different time-course and spatial manners. Upregulation of these miRNAs reduced the level of ATG10 and inhibited autophagy, leading to increasing survival rate of intracellular B. pseudomallei. Furthermore, the increase of these miRNAs was correlated with the reduced promoter methylation status in A549 cells in response to B. pseudomallei infection. Our results reveal that 3 novel miRNAs regulate autophagy-mediated elimination of B. pseudomallei by targeting ATG10, and provide potential targets for clinical treatment.

Effects of histone deacetylase inhibitor trichostatin A combined with cisplatin on apoptosis of A549 cell line.

BackgroundHistone deacetylase (HDAC) inhibitors combined with other drugs for the treatment of malignant tumors are used more and more widely. In this study, we investigated the effect of trichostatin A (TSA), a HDAC inhibitor, in combination with cisplatin, a cytotoxic chemotherapy agent, on the apoptosis of lung cancer A549 cells.MethodsA549 cells were treated with TSA alone, cisplatin alone or the two drugs combined. Cell viability and apoptosis were evaluated using a light microscope, methyl thiazolyl tetrazolium (MTT) (3-[4, 5-dimethylthiazol-2-yl] −2, 5-diphenyltetrazolium bromide) assay and Hochst33258 staining. Moreover, Western blot analysis was employed to examine the alterations of apoptosis protein: cellular Fas-associated death domain-like interleukin-1 β converting enzyme inhibitory protein (cFLIP) and caspase-8 in A549 cells in response to the different exogenous stimuli.ResultsCompared with single-agent treatment, the co-treatment of A549 cells with TSA and cisplatin synergistically inhibited cell proliferation, induced apoptosis, and increased the inhibition rate. Treatment with TSA and cisplatin led to a significant decrease of cFLIP expression. Furthermore, the treatment of A549 cells with TSA and cisplatin resulted in a significant decrease of pro-caspase-8 and a significant increase of caspase-8.ConclusionsSynergistic anti-tumor effects are observed between cisplatin and TSA in lung cancer cells. The combination of TSA with cisplatin may be a more effective method in human lung cancer treatment.

Enhanced phenotypic alterations of alveolar type II cells in response to Aflatoxin G1 -induced lung inflammation.

Recently, we discovered that Aflatoxin G1 (AFG1 ) induces chronic lung inflammatory responses, which may contribute to lung tumorigenesis in Balb/C mice. The cancer cells originate from alveolar type II cells (AT-II cells). The activated AT-II cells express high levels of MHC-II and COX-2, may exhibit altered phenotypes, and likely inhibit antitumor immunity by triggering regulatory T cells (Tregs). However, the mechanism underlying phenotypic alterations of AT-II cells caused by AFG1 -induced inflammation remains unknown. In this study, increased MHC-II expression in alveolar epithelium was observed and associated with enhanced Treg infiltration in mouse lung tissues with AFG1 -induced inflammation. This provides a link between phenotypically altered AT-II cells and Treg activity in the AFG1 -induced inflammatory microenvironment. AFG1 -activated AT-II cells underwent phenotypic maturation since AFG1 upregulated MHC-II expression on A549 cells and primary human AT-II cells in vitro. However, mature AT-II cells may exhibit insufficient antigen presentation, which is necessary to activate effector T cells, due to the absence of CD80 and CD86. Furthermore, we treated A549 cells with AFG1 and TNF-α together to mimic an AFG1 -induced inflammatory response in vitro, and we found that TNF-α and AFG1 coordinately enhanced MHC-II, CD54, COX-2, IL-10, and TGF-β expression levels in A549 cells compared to AFG1 alone. The phenotypic alterations of A549 cells in response to the combination of TNF-α and AFG1 were mainly regulated by TNF-α-mediated induction of the NF-κB pathway. Thus, enhanced phenotypic alterations of AT-II cells were induced in response to AFG1 -induced inflammation. Thus, AT-II cells are likely to suppress anti-tumor immunity by triggering Treg activity.

MicroRNA-381 Negatively Regulates TLR4 Signaling in A549 Cells in Response to LPS Stimulation.

It is widely reported that miR-381 is dysregulated in various tumors. However, the specific role of miR-381 in respiratory infections has not been reported. To probe this role, A549 cells were pretreated with 1 μg/mL LPS for 24 h. The level of miR-381 was detected using RT-qPCR. The expression of proinflammatory cytokines was determined using an ELISA kit and western blotting. Bioinformatics analysis was used to predict the target genes of miR-381, and a luciferase reporter assay was used to validate the expression of the target genes. miR-381 expression was increased in A549 cells treated with LPS, which is a ligand of TLRs. Further study revealed that the overexpression of miR-381 increased the activity of NF-κB signaling, thereby increasing the expression of IL-6, TNFα, and COX-2. Further study revealed that IκBα was a target gene of miR-381. The upregulation of miR-381 under LPS stimulation contributes to respiratory infections mainly by targeting IκBα.

Systematic identification of transcriptional and post-transcriptional regulations in human respiratory epithelial cells during influenza A virus infection.

BackgroundRespiratory epithelial cells are the primary target of influenza virus infection in human. However, the molecular mechanisms of airway epithelial cell responses to viral infection are not fully understood. Revealing genome-wide transcriptional and post-transcriptional regulatory relationships can further advance our understanding of this problem, which motivates the development of novel and more efficient computational methods to simultaneously infer the transcriptional and post-transcriptional regulatory networks.ResultsHere we propose a novel framework named SITPR to investigate the interactions among transcription factors (TFs), microRNAs (miRNAs) and target genes. Briefly, a background regulatory network on a genome-wide scale (~23,000 nodes and ~370,000 potential interactions) is constructed from curated knowledge and algorithm predictions, to which the identification of transcriptional and post-transcriptional regulatory relationships is anchored. To reduce the dimension of the associated computing problem down to an affordable size, several topological and data-based approaches are used. Furthermore, we propose the constrained LASSO formulation and combine it with the dynamic Bayesian network (DBN) model to identify the activated regulatory relationships from time-course expression data. Our simulation studies on networks of different sizes suggest that the proposed framework can effectively determine the genuine regulations among TFs, miRNAs and target genes; also, we compare SITPR with several selected state-of-the-art algorithms to further evaluate its performance. By applying the SITPR framework to mRNA and miRNA expression data generated from human lung epithelial A549 cells in response to A/Mexico/InDRE4487/2009 (H1N1) virus infection, we are able to detect the activated transcriptional and post-transcriptional regulatory relationships as well as the significant regulatory motifs.ConclusionCompared with other representative state-of-the-art algorithms, the proposed SITPR framework can more effectively identify the activated transcriptional and post-transcriptional regulations simultaneously from a given background network. The idea of SITPR is generally applicable to the analysis of gene regulatory networks in human cells. The results obtained for human respiratory epithelial cells suggest the importance of the transcriptional, post-transcriptional regulations as well as their synergies in the innate immune responses against IAV infection.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2105-15-336) contains supplementary material, which is available to authorized users.

High-Throughput Quantitative Proteomic Analysis of Dengue Virus Type 2 Infected A549 Cells

Disease caused by dengue virus is a global health concern with up to 390 million individuals infected annually worldwide. There are no vaccines or antiviral compounds available to either prevent or treat dengue disease which may be fatal. To increase our understanding of the interaction of dengue virus with the host cell, we analyzed changes in the proteome of human A549 cells in response to dengue virus type 2 infection using stable isotope labelling in cell culture (SILAC) in combination with high-throughput mass spectrometry (MS). Mock and infected A549 cells were fractionated into nuclear and cytoplasmic extracts before analysis to identify proteins that redistribute between cellular compartments during infection and reduce the complexity of the analysis. We identified and quantified 3098 and 2115 proteins in the cytoplasmic and nuclear fractions respectively. Proteins that showed a significant alteration in amount during infection were examined using gene enrichment, pathway and network analysis tools. The analyses revealed that dengue virus infection modulated the amounts of proteins involved in the interferon and unfolded protein responses, lipid metabolism and the cell cycle. The SILAC-MS results were validated for a select number of proteins over a time course of infection by Western blotting and immunofluorescence microscopy. Our study demonstrates for the first time the power of SILAC-MS for identifying and quantifying novel changes in cellular protein amounts in response to dengue virus infection.

Part II—mechanism of adaptation: A549 cells adapt to high concentration of nitric oxide through bypass of cell cycle checkpoints

Previous work has shown enhanced survival capacity in high nitric oxide (HNO)-adapted tumor cells. In Part I of this series of manuscripts, we have shown that A549-HNO cells demonstrate an improved growth profile under UV and X-ray radiation treatment. These cells exhibit increased expression of proteins involved in DNA damage recognition and repair pathway, both the non-homologous end joining pathway and homologous recombination. These include Ku80, DNA-PK, XLF ligase and MRN complex proteins. Further, the A549-HNO cells show high levels of ATM, ATR, Chk1 and Chk2, and phospho-p53. Activation of these molecules may lead to cell cycle arrest and apoptosis due to DNA damage. This is observed in parent A549 cells in response to NO donor treatment; however, the A549-HNO cells proliferate and inhibit apoptosis. Cell cycle analysis showed slowed progression through S phase which will allow time for DNA repair. Thus, to better understand the increased growth rate in A549-HNO when compared to the parent cell line A549, we studied molecular mechanisms involved in cell cycle regulation in A549-HNO cells. During the initial time period of NO donor treatment, we observe high levels of cyclin/Cdk complexes involved in regulating various stages of the cell cycle. This would lead to bypass of G1-S and G2-M checkpoints. The HNO cells also show much higher expression of Cdc25A. Cdc25A activates Cdk molecules involved in different phases of the cell cycle. In addition, there is enhanced phosphorylation of the Rb protein in HNO cells. This leads to inactivation of Rb/E2F checkpoint regulating G1-S transition. This may lead to faster progression in S phase. Thus, all of these perturbations in HNO cells lead to accelerated cell cycle progression and a higher growth rate. We also assessed expression of cell cycle inhibitors in HNO cells. Interestingly, the HNO cells show a significant decline in p21CIP1 at initial time points, but with prolonged exposure, the levels were much higher than those of the parent cells. This suggests an initial bypass of cell cycle checkpoints as p21CIP1 can inhibit the activity of all cyclin/Cdk complexes. p21CIP1 is also known to inhibit p53-induced apoptosis. This could be important during later phases of the cell cycle to allow time for repair of damaged DNA and thus better survival of HNO cells.

Contribution of the Proinflammatory Cytokine IL‐18 in the Formation of Human Nasal Polyps

Nasal polyposis is a chronic inflammatory disease of the nasal mucosa. The etiology and the mechanisms of formation of nasal polyps are still not clear. Interleukin (IL)-18 is a novel proinflammatory cytokine that plays important roles in regulating immune inflammatory responses. However, the presence of IL-18 in human nasal mucosa and its roles in the inflammatory process of nasal polyps has not been studied yet. In this study, it was the first time to investigate the expression of IL-18 in human nasal mucosa and nasal polyps, and its potential function in the formation of nasal polyps. Surgical samples were analyzed by Western blot and immunohistochemistry to evaluate the expression and location of IL-18, and its correlated cytokines, IL-4, and IFN-γ. Furthermore, the airway epithelial cell line, A549, was used to investigate the mutual regulation of IFN-γ, IL-4, and IL-18. IFN-γ, IL-4, and IL-18 were all highly expressed in the epithelial cells, submucosal glands, and infiltrating inflammatory cells in the nasal polyp tissues, comparing with the control samples. Especially, the expression of IL-18 was upregulated significantly in nasal polyp tissues compared with control tissues. In addition, IL-18 was expressed in A549 cells in response to lipopolysaccharide and IL-4. Our data suggest that nasal epithelial cells are involved in the pathogenesis of nasal polyps formation and potentially via the secretion of IL-18, which is likely to play important roles in the formation of nasal polyps.

Abstract 4996: The VEGF quadruplex-forming sequence inhibits lung cancer cell growth

Abstract Vascular endothelial growth factor (VEGF), commonly overexpressed in a variety of malignancies including lung cancer, is a key regulator of angiogenesis promoting tumor survival, growth, and metastasis. The promoters of several cancer-related genes, including VEGF, contain disproportionate sequences within nuclease hypersensitivity regions capable of forming quadruplex (four-stranded) DNA. The VEGF quadruplex-forming sequence (VEGF q), a polyG/polyC tract located in the proximal promoter region upstream of the transcription initiation site, has been implicated in the regulation of both basal and inducible VEGF expression. However, the biological role of this sequence remains unclear. We hypothesize that treatment of A549 non-small lung cancer cells with an oligonucleotide encoding VEGF q inhibits cell growth by decreasing VEGF expression and subsequent signaling through VEGF receptor 2 (FLK-1). To determine the biological role of VEGF on lung cancer cell proliferation, A549 cells were treated with VEGF q or the corresponding mutant sequence (MutVEGF), which lacks runs of two or more guanines. Our results demonstrate that VEGF q formed a parallel quadruplex structure and showed remarkable serum and intracellular stability. Confocal and flow cytometry analysis of cells treated with FITC-labeled VEGF q showed prominent uptake and nuclear/cytoplasmic localization in contrast to MutVEGF. Treatment of A549 cells with VEGF q caused a significant dose and time-dependent decrease in cell proliferation after 3 and 6 days (IC50<5µM) as determined by MTT assay, which was not a result of cell cycle arrest. Matrigel analysis also showed a significant reduction in tumor invasiveness after 24 h with VEGF q treatment. Although uptake of VEGF q occurred in non-transformed Hs27 human fibroblast cells, no change in cell growth was evident, indicating that the anti-proliferative effects of VEGF q are specific for malignant cells. Inhibition of cell proliferation and invasion corresponded with decreased VEGF protein expression resulting in decreased FLK-1 signaling and phosphorylation of the target proteins ERK 1/2 and AKT. These results demonstrate striking growth inhibition and decreased invasion of A549 cells in response to VEGF q related to inhibition of VEGF expression and signaling through FLK-1 and have significant therapeutic implications for the treatment of non-small cell lung cancer. 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 4996. doi:10.1158/1538-7445.AM2011-4996

Abstract 2949: The VEGF quadruplex-forming sequence inhibits cell growth of lung cancer

Abstract Vascular endothelial growth factor (VEGF), commonly overexpressed in a variety of malignancies including lung cancer, is a key regulator of angiogenesis promoting tumor survival, growth, and metastasis. The promoters of several cancer-related genes, including VEGF, contain disproportionate sequences within nuclease hypersensitivity regions capable of forming quadruplex (four-stranded) DNA. The VEGF quadruplex-forming sequence (VEGFq), a polyG/polyC tract located in the proximal promoter region upstream of the transcription initiation site, has been implicated in the regulation of both basal and inducible VEGF expression. However, the biological role of this sequence remains unclear. We hypothesize that treatment of A549 non-small lung cancer cells with VEGFq induces cell death by inhibiting gene expression. To determine the biological role of VEGF on lung cancer cell proliferation, A549 cells were treated with VEGFq or the corresponding mutant sequence (MutVEGF), which lacks runs of two or more guanines. Changes in cell proliferation were compared to levels of VEGF protein expression. Quadruplex formation was confirmed by circular dichroism spectroscopy. Our results demonstrate that VEGFq formed a stable parallel quadruplex structure, while the MutVEGF sequence did not form a quadruplex. Treatment of A549 cells with VEGFq caused a significant dose and time-dependent decrease in cell proliferation after 3 and 6 days (IC50<5µM) as determined by MTT assay. No change in the growth of nontransformed fibroblast cells occurred in response to VEGFq indicating the effect is specific for malignant cells. Confocal and flow cytometry analysis of cells treated with FITC-labeled VEGFq or MutVEGF showed prominent uptake and nuclear localization of VEGFq not MutVEGF after 72 h. Inhibition of cell proliferation corresponded with decreased VEGF protein expression. No specific change in the cell cycle was measured after 72 h. These results demonstrate striking growth inhibition of A549 cells in response to the VEGF quadruplex-forming sequence related to inhibition of VEGF expression and suggest significant therapeutic implications for the treatment of non-small cell lung cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2949.

MiR-181a and miR-630 Regulate Cisplatin-Induced Cancer Cell Death

MicroRNAs (miRNA) are noncoding RNAs that regulate multiple cellular processes, including proliferation and apoptosis. We used microarray technology to identify miRNAs that were upregulated by non-small cell lung cancer (NSCLC) A549 cells in response to cisplatin (CDDP). The corresponding synthetic miRNA precursors (pre-miRNAs) per se were not lethal when transfected into A549 cells yet affected cell death induction by CDDP, C2-ceramide, cadmium, etoposide, and mitoxantrone in an inducer-specific fashion. Whereas synthetic miRNA inhibitors (anti-miRNAs) targeting miR-181a and miR-630 failed to modulate the response of A549 to CDDP, pre-miR-181a and pre-miR-630 enhanced and reduced CDDP-triggered cell death, respectively. Pre-miR-181a and pre-miR-630 consistently modulated mitochondrial/postmitochondrial steps of the intrinsic pathway of apoptosis, including Bax oligomerization, mitochondrial transmembrane potential dissipation, and the proteolytic maturation of caspase-9 and caspase-3. In addition, pre-miR-630 blocked early manifestations of the DNA damage response, including the phosphorylation of the ataxia-telangiectasia mutated (ATM) kinase and of two ATM substrates, histone H2AX and p53. Pharmacologic and genetic inhibition of p53 corroborated the hypothesis that pre-miR-630 (but not pre-miR-181a) blocks the upstream signaling pathways that are ignited by DNA damage and converge on p53 activation. Pre-miR-630 arrested A549 cells in the G0-G1 phase of the cell cycle, correlating with increased levels of the cell cycle inhibitor p27(Kip1) as well as with reduced proliferation rates and resulting in greatly diminished sensitivity of A549 cells to the late S-G2-M cell cycle arrest mediated by CDDP. Altogether, these results identify miR-181a and miR-630 as novel modulators of the CDDP response in NSCLC.

Z Proteins of New World Arenaviruses Bind RIG-I and Interfere with Type I Interferon Induction

The retinoic acid-inducible gene I product (RIG-I) is a cellular sensor of RNA virus infection that regulates the cellular beta interferon (IFN-beta) response. The nucleoproteins (NP) of arenaviruses are reported to antagonize the IFN response by inhibiting interferon regulatory factor 3 (IRF-3). Here, we demonstrate that the Z proteins of four New World (NW) arenaviruses, Guanarito virus (GTOV), Junin virus (JUNV), Machupo virus (MAVC), and Sabia virus (SABV), bind to RIG-I, resulting in downregulation of the IFN-beta response. We show that expression of the four NW arenavirus Z proteins inhibits IFN-beta mRNA induction in A549 cells in response to RNA bearing 5' phosphates (5'pppRNA). NW arenavirus Z proteins interact with RIG-I in coimmunoprecipitation studies and colocalize with RIG-I. Furthermore, expression of Z proteins interferes with the interaction between RIG-I and MAVS. Z expression also impedes the nuclear factor kappa light chain enhancer of activated B cells (NF-kappaB) and IRF-3 activation. Our results indicate that NW arenavirus Z proteins, but not Z protein of the Old World (OW) arenavirus lymphocytic choriomeningitis virus (LCMV) or Lassa virus, bind to RIG-I and inhibit downstream activation of the RIG-I signaling pathway, preventing the transcriptional induction of IFN-beta.

Adenovirus E1A regulates lung epithelial ICAM-1 expression by interacting with transcriptional regulators at its promoter

We focused on the regulation of inflammatory mediator expression by adenovirus E1A in lung epithelial cells and the role of this viral protein in the pathogenesis of chronic obstructive pulmonary disease (COPD). We previously reported that E1A, a well-known regulator of host genes, increased ICAM-1 expression in human bronchial epithelial (HBE) and A549 cells in response to LPS stimulation. In this report, we clarified the mechanism of this regulation. We found NF-kappaB translocation to the nucleus after LPS stimulation in both E1A-positive and -negative HBE cells. ICAM-1 promoter reporter constructs revealed that a mutation in the proximal NF-kappaB binding site completely inhibited increased transcription, whereas the mutation in a distal site did not. We analyzed the participation of E1A in transcriptional complex formation at this promoter using chromatin immunoprecipitation. In E1A-positive HBE and A549 cells, LPS stimulation increased ICAM-1 promoter immunoprecipitation by NF-kappaB p65 and p300 but not activator protein-1 antibodies with a concomitant increase by the E1A antibody. No increase was found in E1A-negative cells except in HBE cells with p65 antibody. The association of E1A with the increased promoter immunoprecipitation with p300 was also observed after TNF-alpha stimulation of A549 cells. These results suggest that adenovirus E1A regulates the ICAM-1 promoter through its proximal NF-kappaB binding site, most likely by interacting with the transcriptional complex that forms at this site. E1A regulation of the LPS response may play a role in acute exacerbations as a consequence of bacterial infections in COPD.

Down-regulation of Micro-RNA-1 (miR-1) in Lung Cancer: Suppression of Tumorigenic Property of Lung Cancer Cells and Their Sensitization to Doxorubicin-Induced Apoptosis by miR-1

Micro-RNAs are approximately 21-25-nucleotide-long noncoding RNAs that regulate gene expression primarily at the post-transcriptional level in animals. Here, we report that micro-RNA-1 (miR-1), abundant in the cardiac and smooth muscles, is expressed in the lung and is down-regulated in human primary lung cancer tissues and cell lines. In situ hybridization demonstrated localization of miR-1 in bronchial epithelial cells. The tumor suppressor C/EBPalpha, frequently suppressed in lung cancer, reactivated miR-1 expression in the lung cancer cells. Repressed miR-1 was also activated in lung cancer cells upon treatment with a histone deacetylase inhibitor. These observations led us to examine the antitumorigenic potential of miR-1 in lung cancer cells. Expression of miR-1 in nonexpressing A549 and H1299 cells reversed their tumorigenic properties, such as growth, replication potential, motility/migration, clonogenic survival, and tumor formation in nude mice. Exogenous miR-1 significantly reduced expression of oncogenic targets, such as MET, a receptor tyrosine kinase, and Pim-1, a Ser/Thr kinase, frequently up-regulated in lung cancer. Similarly, the levels of two additional targets, FoxP1, a transcription factor with oncogeneic property, and HDAC4 that represses differentiation-promoting genes, were reduced in miR-1-expressing cells. Conversely, depletion of miR-1 facilitated N417 cell growth with concomitant elevation of these targets. Further, ectopic miR-1 induced apoptosis in A549 cells in response to the potent anticancer drug doxorubicin. Enhanced activation of caspases 3 and 7, cleavage of their substrate PARP-1, and depletion of anti-apoptotic Mcl-1 contributed to the sensitivity of miR-1-expressing cells to doxorubicin. Thus, miR-1 has potential therapeutic application against lung cancers.

Regulators of Mitotic Arrest and Ceramide Metabolism Are Determinants of Sensitivity to Paclitaxel and Other Chemotherapeutic Drugs

Cytotoxic drug resistance is a major cause of cancer treatment failure. We report an RNA interference screen to identify genes influencing sensitivity of different cancer cell types to chemotherapeutic agents. A set of genes whose targeting leads to resistance to paclitaxel is identified, many of which are involved in the spindle assembly checkpoint. Silencing these genes attenuates paclitaxel-induced mitotic arrest and induces polyploidy in the absence of drug. We also identify a ceramide transport protein, COL4A3BP or CERT, whose downregulation sensitizes cancer cells to multiple cytotoxic agents, potentiating endoplasmic reticulum stress. COL4A3BP expression is increased in drug-resistant cell lines and in residual tumor following paclitaxel treatment of ovarian cancer, suggesting that it could be a target for chemotherapy-resistant cancers.

Potentiation of Kinesin Spindle Protein Inhibitor–Induced Cell Death by Modulation of Mitochondrial and Death Receptor Apoptotic Pathways

Targeting the mitotic motor kinesin kinesin spindle protein (KSP) is a new strategy for cancer therapy. We have examined the molecular events induced by KSP inhibition and explored possible mechanisms of resistance and sensitization of tumor cells to KSP inhibitors. We found that KSP inhibition induced cell death primarily via activation of the mitochondrial death pathway. In HeLa cells, inhibition of KSP by small-molecule inhibitor monastrol resulted in mitotic arrest and rapid caspase activation. BclXL phosphorylation and loss of mitochondrial membrane potential was detected before significant caspase activation, which was required to trigger the subsequent apoptotic pathway. In A549 cells, however, KSP inhibition did not induce mitochondrial damage, significant caspase activity, or cell death. A549 cells aberrantly exited mitosis, following a prolonged drug-induced arrest, and arrested in a G(1)-like state with 4N DNA content in a p53-dependent manner. Overexpression of BclXL provided a protective mechanism, and its depletion rescued the apoptotic response to monastrol. In addition, Fas receptor was up-regulated in A549 cells in response to monastrol. Treatment with Fas receptor agonists sensitized the cells to monastrol-induced cell death, following exit from mitosis. Thus, activation of the death receptor pathway offered another mechanism to enhance KSP inhibitor-induced apoptosis. This study has elucidated cellular responses induced by KSP inhibitors, and the results provide insights for a more effective cancer treatment with these agents.

Polymorphonuclear Cell Transmigration Induced by Pseudomonas aeruginosa Requires the Eicosanoid Hepoxilin A3

Lung inflammation resulting from bacterial infection of the respiratory mucosal surface in diseases such as cystic fibrosis and pneumonia contributes significantly to the pathology. A major consequence of the inflammatory response is the recruitment and accumulation of polymorphonuclear cells (PMNs) at the infection site. It is currently unclear what bacterial factors trigger this response and exactly how PMNs are directed across the epithelial barrier to the airway lumen. An in vitro model consisting of human PMNs and alveolar epithelial cells (A549) grown on inverted Transwell filters was used to determine whether bacteria are capable of inducing PMN migration across these epithelial barriers. A variety of lung pathogenic bacteria, including Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa are indeed capable of inducing PMN migration across A549 monolayers. This phenomenon is not mediated by LPS, but requires live bacteria infecting the apical surface. Bacterial interaction with the apical surface of A549 monolayers results in activation of epithelial responses, including the phosphorylation of ERK1/2 and secretion of the PMN chemokine IL-8. However, secretion of IL-8 in response to bacterial infection is neither necessary nor sufficient to mediate PMN transepithelial migration. Instead, PMN transepithelial migration is mediated by the eicosanoid hepoxilin A3, which is a PMN chemoattractant secreted by A549 cells in response to bacterial infection in a protein kinase C-dependent manner. These data suggest that bacterial-induced hepoxilin A3 secretion may represent a previously unrecognized inflammatory mechanism occurring within the lung epithelium during bacterial infections.

RNA interference for HIF-1α inhibits its downstream signalling and affects cellular proliferation

Transcription factor HIF-1 is a key determinant of oxygen-dependent gene regulation. Suppression of HIF-1α is important for exploring HIF-1-dependent processes and for interfering with hypoxia-induced pathophysiological events. This study applied RNA-interference targeting HIF-1α to the human lung A549 cell line. Transfection of HIF-1 α- siRNA reduced HIF-1α synthesis as measured on mRNA and protein level by realtime RT-PCR, Western blot, and immuncytochemistry. A time kinetic for hypoxic stabilization of HIF-1α protein and its inhibition by HIF-1 α- siRNA is included. Hypoxic induction of HIF-1-controlled target genes as heme oxygenase I (HO-1), phosphoglycerate kinase (PGK), and vascular endothelial growth factor (VEGF) was markedly attenuated by HIF-1 α- siRNA treatment. Correspondingly, gene activation via hypoxia-responsive-element, as shown by reporter gene assay, was inhibited by HIF-1 α- siRNA. Moreover, this approach was found to suppress the shift from from S-phase to G 1-phase observed in A549 cells in response to hypoxia, supporting a role of HIF-1α in oxygen-dependent cell cycle regulation.

Monitoring cellular responses of engine-emitted particles by using a direct air–cell interface deposition technique

The impacts of ultrafine airborne particles generated by diesel or gasoline engines on human lung cells have been investigated using a new in vitro cellular exposure technique. This technique enables direct deposition of the gasoline engine exhaust particles (GEP) and diesel engine exhaust particles (DEP) on human lung cells located at the air–cell interface on a transwell membrane in an exposure apparatus. The cellular responses to particle exposure were measured by the levels of IL-8 chemokines produced as a function of exposure time. The findings suggest that GEP and high-sulfur DEP induced the production of similar levels of IL-8 by unprimed A549 cells. The level of IL-8 produced by unprimed A549 cells in response to low-sulfur DEP was found lower than that produced in response to high-sulfur DEP and GEP. When cells were primed, simulating predisposed conditions, significant levels of IL-8 were produced. GEP triggered a much higher level of IL-8 production than DEP did. Furthermore, the time profile of IL-8 production induced by GEP was markedly different from that induced by DEP. The findings indicate that GEP could induce the production of higher levels of chemokines (i.e., IL-8) than DEP did, implying that exposure to GEP could be a greater health risk than exposure to DEP.

Bleomycin-induced apoptosis of alveolar epithelial cells requires angiotensin synthesis de novo.

Primary cultures of rat type II alveolar epithelial cells (AECs) or human AEC-derived A549 cells, when exposed to bleomycin (Bleo), exhibited concentration-dependent apoptosis detected by altered nuclear morphology, fragmentation of DNA, activation of caspase-3, and net cell loss over time. In both cell culture models, exposure to Bleo caused time-dependent increases in angiotensinogen (ANGEN) mRNA. Antisense oligonucleotides against ANGEN mRNA inhibited Bleo-induced apoptosis of rat AEC or A549 cells by 83 and 84%, respectively (P < 0.01 and P < 0.05), and prevented Bleo-induced net cell loss. Apoptosis of rat AECs or A549 cells in response to Bleo was inhibited 91% by the ANG-converting enzyme inhibitor captopril or 82%, respectively, by neutralizing antibodies specific for ANG II (both P < 0.01). Antagonists of ANG receptor AT(1) (losartan, L-158809, or saralasin), but not an AT(2)-selective blocker (PD-123319), inhibited Bleo-induced apoptosis of either rat AECs (79%, P < 0.01) or A549 cells (83%, P < 0.01) and also reduced the activity of caspase-3 by 52% (P < 0.05). These data indicate that Bleo, like Fas(L) or TNF-alpha, induces transactivation of ANG synthesis de novo that is required for AEC apoptosis. They also support the theory that ANG system antagonists have potential for the blockade of AEC apoptosis in situ.