Treatment Of A549 Cells Research Articles

Targeted interfering DEP domain containing 1 protein induces apoptosis in A549 lung adenocarcinoma cells through the NF-κB signaling pathway.

Ectopic expression of DEP domain containing 1 (DEPDC1) in lung adenocarcinomas is associated with poor prognosis, but its role and the underlying mechanism remain unknown. In this study, DEPDC1 expression in lung cancer cell lines was examined with Western blot assay, and DEPDC1-positive cell A549 was selected for further experiments. DEPDC1 inhibitor miR-130a was overexpressed in A549 cells, and the proliferation and apoptosis of these cells were analyzed with cell counting and flow cytometry assay. Interfering peptide 11R-DEP:611–628 and JNK inhibitor SP600125 were used alone or in combination to treat A549 cells, and the cell proliferation and apoptosis were assessed by flow cytometry assay; caspase 3 and cleaved caspase 3, phosphor-JNK, and total JNK were detected by Western blotting; and nuclear factor kappa B (NF-κB) localization was determined by immunofluorescence staining. We found that miR-130a and 11R-DEP:611–628 peptides (5 μM) both inhibited A549 proliferation and induced apoptosis. We observed that 11R-DEP:611–628 peptide treatment resulted in elevated A20 expression, dramatically reduced nuclear NF-κB, and increased phosphor-JNK. These findings indicate that DEPDC1 inhibits apoptosis of A549 cell by suppressing A20 expression to regulate NF-κB activity, and that JNK plays a protective role upon 11R-DEP:611–628 peptide treatment. In conclusion, DEPDC1 might be a novel therapeutic target for lung cancer, and the 11R-DEP:611–628 peptide is a potent apoptosis inducer in A549 cells.

Pharmacokinetic studies and anticancer activity of curcumin-loaded nanostructured lipid carriers

In order to investigate the potential of nanostructured lipid carriers for efficient and targeted delivery of curcumin, the pharmacokinetic parameters of curcumin-loaded nanostructured lipid carriers (Cur-NLC) were evaluated in rats after a single intraperitoneal dose of Cur-NLC. In addition, the anticancer activity of Cur-NLC against human lung adenocarcinoma A549 cells was verified by a cellular uptake study, and a cytotoxicity and apoptosis assay. Bioavailability of Cur-NLC was better than that of native curcumin (p > 0.01), as seen from the area under the plasma concentration-time curve (AUC), maximum plasma concentration (Cmax), mean residence time (MRT) and total plasma clearance (CLz/F). Cur-NLC has a more obvious lung-targeting property in comparison with native curcumin. Cur-NLC showed higher anticancer activity in vitro against A549 cells than native curcumin (IC50 value of 5.66 vs. 9.81 mg L-1, respectively). Meanwhile, Cur-NLC treated A549 cells showed a higher apoptosis rate compared to that of native curcumin. These results indicate that NLC is a promising system for the delivery of curcumin in the treatment of lung adenocarcinoma.

Inflammation-mediated SOD-2 upregulation contributes to epithelial-mesenchymal transition and migration of tumor cells in aflatoxin G1-induced lung adenocarcinoma

Tumor-associated inflammation plays a critical role in facilitating tumor growth, invasion and metastasis. Our previous study showed Aflatoxin G1 (AFG1) could induce lung adenocarcinoma in mice. Chronic lung inflammation associated with superoxide dismutase (SOD)-2 upregulation was found in the lung carcinogenesis. However, it is unclear whether tumor-associated inflammation mediates SOD-2 to contribute to cell invasion in AFG1-induced lung adenocarcinoma. Here, we found increased SOD-2 expression associated with vimentin, α-SMA, Twist1, and MMP upregulation in AFG1-induced lung adenocarcinoma. Tumor-associated inflammatory microenvironment was also elicited, which may be related to SOD-2 upregulation and EMT in cancer cells. To mimic an AFG1-induced tumor-associated inflammatory microenvironment in vitro, we treated A549 cells and human macrophage THP-1 (MΦ-THP-1) cells with AFG1, TNF-α and/or IL-6 respectively. We found AFG1 did not promote SOD-2 expression and EMT in cancer cells, but enhanced TNF-α and SOD-2 expression in MΦ-THP-1 cells. Furthermore, TNF-α could upregulate SOD-2 expression in A549 cells through NF-κB pathway. Blocking of SOD-2 by siRNA partly inhibited TNF-α-mediated E-cadherin and vimentin alteration, and reversed EMT and cell migration in A549 cells. Thus, we suggest that tumor-associated inflammation mediates SOD-2 upregulation through NF-κB pathway, which may contribute to EMT and cell migration in AFG1-induced lung adenocarcinoma.Introduction.

Inhibition of aldehyde dehydrogenase 1 enhances the cytotoxic effect of retinaldehyde on A549 cancer cells.

We hypothesized that aldehyde dehydrogenase1 (ALDH1) protects cancer cells from retinaldehyde-induced cytotoxicity, and that targeting this enzyme would enhance the therapeutic effect of retinaldehyde. ALDEFLUOR™ assays showed high ALDH activity in A549 and H522 cancer cells and low activity in H1666 and T47D cancer cells. Immunoblots showed that expression of ALDH1A1 and ALDH1A3 was high in A549 and H522 cells, but low in H1666 cells. HPLC confirmed that N, N-diethylaminobenzaldehyde (DEAB) inhibits ALDH-mediated disposal of retinaldehyde in A549 cells and lysates. Treatment of A549 cells with retinaldehyde in the presence of DEAB augmented reactive oxygen species production and decreased glucose uptake and oxygen consumption. Importantly, DEAB substantially potentiated the ability of retinaldehyde to dose-dependently suppress the survival of A549 and H522 cells, whereas the added effect of DEAB was minor in H1666 and T47D cells. Gene silencing with specific siRNA revealed that ALDH1A1 contributed to protection of A549 cells against retinaldehyde toxicity. These results demonstrate that ALDH1 confers protection against retinaldehyde toxicity in cancer cells.

Metabolomics analysis reveals that benzo[a]pyrene, a component of PM2.5, promotes pulmonary injury by modifying lipid metabolism in a phospholipase A2-dependent manner in vivo and in vitro

Particulate matter with an aerodynamic diameter less than 2.5μM (PM2.5) is one of the major environmental pollutants in China. In this study, we carried out a metabolomics profile study on PM2.5-induced inflammation. PM2.5 from Beijing, China, was collected and given to rats through intra-tracheal instillation in vivo. Acute pulmonary injury were observed by pulmonary function assessment and H.E. staining. The lipid metabolic profile was also altered with increased phospholipid and sphingolipid metabolites in broncho-alveolar lavage fluid (BALF) after PM2.5 instillation. Organic component analysis revealed that benzo[a]pyrene (BaP) is one of the most abundant and toxic components in the PM2.5 collected on the fiber filter. In vitro, BaP was used to treat A549 cells, an alveolar type II cell line. BaP (4μM, 24h) induced inflammation in the cells. Metabolomics analysis revealed that BaP (4μM, 6h) treatment altered the cellular lipid metabolic profile with increased phospholipid metabolites and reduced sphingolipid metabolites and free fatty acids (FFAs). The proportion of ω–3 polyunsaturated fatty acid (PUFA) was also decreased. Mechanically, BaP (4μM) increased the phospholipase A2 (PLA2) activity at 4h as well as the mRNA level of Pla2g2a at 12h. The pro-inflammatory effect of BaP was reversed by the cytosolic PLA2 (cPLA2) inhibitor and chelator of intracellular Ca2+. This study revealed that BaP, as a component of PM2.5, induces pulmonary injury by activating PLA2 and elevating lysophosphatidylcholine (LPC) in a Ca2+-dependent manner in the alveolar type II cells.

Blockage of glycolysis by targeting PFKFB3 alleviates sepsis-related acute lung injury via suppressing inflammation and apoptosis of alveolar epithelial cells

Sepsis-related acute lung injury (ALI) is characterized by excessive lung inflammation and apoptosis of alveolar epithelial cells resulting in acute hypoxemic respiratory failure. Recent studies indicated that anaerobic glycolysis play an important role in sepsis. However, whether inhibition of aerobic glycolysis exhibits beneficial effect on sepsis-induced ALI is not known. In vivo, a cecal ligation and puncture (CLP)-induced ALI mouse model was set up and mice treated with glycolytic inhibitor 3PO after CLP. The mice treated with the 3PO ameliorated the survival rate, histopathological changes, lung inflammation, lactate increased and lung apoptosis of mice with CLP-induced sepsis. In vitro, the exposure of human alveolar epithelial A549 cells to lipopolysaccharide (LPS) resulted in cell apoptosis, inflammatory cytokine production, enhanced glycolytic flux and reactive oxygen species (ROS) increased. While these changes were attenuated by 3PO treatment. Sequentially, treatment of A549 cells with lactate caused cell apoptosis and enhancement of ROS. Pretreatment with N-acetylcysteine (NAC) significantly lowered LPS and lactate-induced the generation of ROS and cell apoptosis in A549 cells. Therefore, these results indicate that anaerobic glycolysis may be an important contributor in cell apoptosis of sepsis-related ALI. Moreover, LPS specifically induces apoptotic insults to A549 cell through lactate-mediated enhancement of ROS.

EVIDENCE FOR ACE‐2 UBIQUITINATION AND PROTEASOMAL PROCESSING IN LUNG EPITHELIUM

Background and AimThe angiotensin converting enzyme 2 (ACE‐2) was shown to play a protective role in the fibrogenesis and inflammation of many organs including liver and lung. Previous studies from our lab showed that ACE‐2 is downregulated in fibrotic conditions of the adult and neonatal human lung. The exact mechanisms of ACE‐2 downregulation and protein degradation in lung cells still remain unknown. This study aims to investigate whether ACE‐2 is regulated by ubiquitination/proteasome degradation pathways in the lung.MethodsHuman lung epithelial cell line (A549) and mouse lung epithelial cell line (MLE12) were cultured and cell lysates were used for immunoprecipitation using ACE‐2 antibodies. The immunoprecipitate was used to detect ubiquitination. Several proteasome inhibitors (Lactacystin, clasto‐Lactacystin β‐lactone, MG132, HMB‐Val‐Ser‐Leu‐VE, and Epoxomicin) were used on MLE12 cell culture to detect the effects on ACE‐2 protein bands by western blotting and to immunoprecipitate ACE‐2 and detect the effect on the ubiquitination of ACE‐2. Several dosing of the proteasome inhibitor MG132 on MLE12 cells and of clasto‐Lactacystin β‐lactone on A549 cells were used to detect changes in ACE‐2 bands by western blotting.ResultsImmunoprecipitation of ACE‐2 revealed ubiquitination of 3 major bands (150, 45, 40 kDa) detected by anti‐ubiquitin antibodies in MLE12 cells, while ubiquitination of ACE2 was markedly noted in 50 kDa band of A549 cells. On MLE12 cells, all proteasome inhibitors resulted in various degrees of increased total ubiquitinated protein levels. Lactacystin and clasto‐Lactacystin β‐lactone showed increased ACE‐2 isoform band at 70 kDa and were associated with the most increase in total ubiquitinated protein levels, more noted in clasto‐Lactacystin β‐lactone treatment than Lactacystin. Additionally, increasing doses of MG132 treatment on MLE12 cells resulted in a dose dependent increase of the 70 kDa ACE‐2 band with a corresponding decrease in 40 kDa band. Similarly, treatment of A549 cells with increasing doses of clasto‐Lactacystin β‐lactone resulted in a dose dependent decrease in the 50 kDa band.ConclusionThese data present the first evidence suggesting ubiquitination and proteasome dependent degradation of ACE‐2 in lung cells in a species‐dependent pattern. Future work will be directed to detect the roles of this ubiquitination/proteasome degradation in fibrotic lung diseases.Support or Funding InformationPHS HL‐45136; Department of Pediatrics, Michigan State University

The naturally occurring xanthone α-mangostin induces ROS-mediated cytotoxicity in non-small scale lung cancer cells.

Small cell lung cancer (NSCLC) accounts for 85% of total deaths globally, and recent studies indicate the increasing risks of NSCLC in China and South Asian countries. Hence, development of new therapeutics against NSCLC has been a major concern. α-Mangostin, a naturally occurring xanthone, found abundantly in pericarps of mangosteen fruit is well known for its medicinal importance. The anticancer properties of α-mangostin against several types of cancer are also well documented. But the mechanism of action of α-mangostin against lung cancer is not well understood and requires further investigation. Therefore in the present study, we explored the therapeutic potential of α-mangostin against A549 cells. Treatment of A549 cells with α-mangostin resulted in a dose-dependent loss of cell viability, while the non-malignant cells such as hPBMC and WI-38 remained unaffected. Further we observed that the ROS plays an important role in α-mangostin -induced apoptosis in A549 cells, and administration of N-acetyl cysteine significantly abrogates α-mangostin -mediated cytotoxicity in lung cancer cells. Overall, α-mangostin induces ROS-mediated cytotoxicity in NSCLC cells.

Neferine Attenuates Epithelial-Mesenchymal Transition of Alveolar Epithelial Cells via TGF-β Signaling Pathway

Background: Idiopathic pulmonary fibrosis (IPF) is an incurable, progressive, and fatal interstitial lung disease with the characteristics of lung tissue damage and an enhancement in extracellular matrix (ECM). Alveolar epithelial cells (AMs) are major target cells that can directly promote to occurring of pulmonary fibrosis by acquisition of a mesenchymal phenotype through epithelial- mesenchymal transition (EMT). Neferine, a component of Chinese herbs, has been thought to be involved in anti-fibrotic activity in experimental lung fibrosis. However, its mechanism is not clear. In this study, we explore the regulation of neferine in TGF-β-induced EMT in lung fibrosis model and illustrate its mechanism of action. Methods: The alveolar epithelial cell line A549 was stimulated with TGF-β1 with or without Neferine pretreatment in advance. Morphologic variations and expression of EMT-related markers, including E-cadherin, β-catenina-SMA and Vimentin were detected. Expressions of Smad2, p- Smad 2, Smad3 and p-Smad3 were measured. Results: TGF-β1–treated A549 cells were transformed into the mesenchymal morphology with less E-cadherin, β-catenin and more a-SMA, Vimentin expression. The addition of Neferine inhibited the TGF-β1–induced change of the mesenchymal phenotype. Furthermore, Neferine inhibited the TGF-β1–induced increase in the expression of p- Smad2 and p-Smad3. Conclusions: Our study illustrate that Neferine inhibits TGF-β1–induced EMT in lung fibrosis model via TGF-β signaling pathway.

Identification of a diverse synthetic abietane diterpenoid library for anticancer activity

A diverse natural-product-like synthetic abietane diterpenoid library contains about 56 compounds were obtained, and evaluated for their potential in vitro cytotoxic or antitumor activity against A549, PC-3 and SKOV-3 tumor cell lines by SRB assay. Treatment of A549 cells with the most potent compound ketone 19 showed induction of apoptosis, as revealed by JC-1 mitochondrial membrane potential staining, TUNNEL assay, western blotting analysis and flow cytometry assay.

Effects of RNAi-mediated TUSC3 silencing on radiation-induced autophagy and radiation sensitivity of human lung adenocarcinoma cell line A549 under hypoxic condition.

This study examined the effects of RNAi-mediated TUSC3 silencing on radiation-induced autophagy and radiation sensitivity of human lung adenocarcinoma cell line A549 under hypoxic condition. Different CoCl2 concentrations were used to treat A549 cells and establish a CoCl2-induced hypoxic model of A549 cells. MTT and clone formation assays were used to determine the effects of different concentrations of CoCl2 on the growth and proliferation of A549 cells treated by different doses of X-ray irradiation. The siRNA-expressing vector was transfected by liposomes and for silencing of TUSC3. Flow cytometry was used to measure cell cycle changes and apoptosis rate. Real-time quantitative polymerase chain reaction (qRT-PCR) assay was performed to detect the expression of TUSC3 mRNA. Western blotting was applied to detect the changes of TUSC3, LC3, and p62 proteins under different CoCl2 concentrations and after siRNA silencing of TUSC3. The TUSC3 levels in A549 cells increased under hypoxic conditions in a dose-dependent manner (P<0.05). Hypoxia inhibited the growth and proliferation of A549 cells and promoted apoptosis (P<0.05). With an increasing dose of X-ray irradiation, A549 cells showed significantly increased growth and proliferation and decreased apoptosis (P<0.05). After siRNA-TUSC3 was transfected by liposome, the TUSC3 level was substantially inhibited (P<0.05). Silencing TUSC3 inhibited A549 cell growth and proliferation after radiotherapy under hypoxic condition, promoted apoptosis, increased G0/G1 phase cells, and reduced S phase cells (all P<0.05). Hypoxia and radiation along with different CoCl2 concentrations could induce cell autophagy, which increased with concentration and dose, while silencing the TUSC3 gene inhibited autophagy (all P<0.05). RNAi silencing of TUSC3 inhibited growth and proliferation, while enhanced apoptosis and radiation sensitivity of hypoxic A549 lung adenocarcinoma cells.

Preparation and characterization of novel chitosan-protamine nanoparticles for nucleus-targeted anticancer drug delivery.

It is well known that most anticancer drugs commonly show high toxicity to the DNA of tumor cells and exert effects by combining with the DNA or associated enzymes in the nucleus. Most developed drugs are first delivered into the cytoplasm and then transferred to the nucleus through the membrane pores. Sometimes, the transportation of drugs from cytoplasm to nucleus is not efficient and often results in poor therapeutic effects. In this study, we developed special and novel nanoparticles (NPs) made of chitosan and protamine for targeted nuclear capture of drugs to enhance anticancer effects. The anticancer effects of nuclear targeted-delivery of drugs in NPs were also evaluated by investigating cytotoxicity, cellular uptake mechanism, and cell apoptosis on cells. Chitosan–protamine NPs were characterized by good drug entrapment, sustained release, small average particle size, low polydispersity index, and high encapsulation efficiency; and accomplished the efficient nuclear delivery of fluorouracil (5-Fu). Compared with free 5-Fu and 5-Fu-loaded chitosan NPs, treatment of A549 cells and HeLa cells with 5-Fu-loaded chitosan–protamine NPs showed the highest cytotoxicity and further induced the significant apoptosis of cells. In addition, 5-Fu-loaded chitosan–protamine NPs exhibited the best efficiency in inhibiting tumor growth than the other three formulations. 5-Fu-loaded chitosan–protamine NPs enhanced antitumor efficacy through the targeted nuclear capture of drugs and showed promising potential as a nanodelivery system for quickly locating drugs in the nucleus of cells.

GL-9 peptide regulates gene expression of CD44 cancer marker and pro-inflammatory cytokine TNF-α in human lung epithelial adenocarcinoma cell line (A549).

In this study, we examined cytotoxic effect of GL-9 peptide on A459 cell line through studying the changes in TNF-α and CD44 gene expression and ROS production. Real-time PCR analysis showed that the treated A549 cells highly over expressed TNF-α, which was associated with a significant reduction of CD44 gene expression levels (p < 0.05). ROS production rate was measured through the usage of DCFH-DA primer. Results demonstrated that GL-9 peptide could also induce cell death via ROS production. The effect of GL-9 peptide on human erythrocytes and leukocytes was analyzed. GL-9 peptide showed no significant toxic effect on human blood cells. Our results suggested that the GL-9 peptide as a potent natural agent could modulate gene expression of cancer cell markers.

Synthesis and biological evaluation of new benzimidazole-thiazolidinedione hybrids as potential cytotoxic and apoptosis inducing agents

A series of new benzimidazole-thiazolidinedione hybrids has been synthesized and evaluated for their cytotoxic potential against a selected human cancer cell lines of prostate (PC-3 and DU-145), breast (MDA-MB-231), lung (A549) and a normal breast epithelial cells (MCF10A). Among the tested compounds, 11p exhibited promising cytotoxicity with IC50 value of 11.46 ± 1.46 μM on A549 lung cancer cell line and did not show significant toxicity on normal MCF10A cells. Lung cancer cells (A549) have been used to know the mechanism of cell growth inhibition and apoptosis inducing effect with compound 11p. The treatment of A549 cells with 11p showed typical apoptotic morphology like cell shrinkage, chromatin condensation and horseshoe shaped nuclei formation. Flow-cytometry analysis revealed the G2/M phase of cell cycle arrest in a dose dependent manner. Preliminary mechanistic studies suggested that the cell migration was inhibited through the disruption of F-actin protein. Acridine orange-ethidium bromide (AO-EB), DAPI, annexin V-FITC/propidium iodide, rhodamine-123 and MitoSOX assays suggested the induction of apoptosis in A549 cells by compound 11p.

RETRACTED: Crosstalk between C/EBP homologous protein (CHOP) and glucocorticoid receptor in lung cancer

Loss of homeostasis triggers the endoplasmic reticulum (ER) stress response and activates the unfolded protein response (UPR) resulting in the induction of the CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP). Glucocorticoids (GCs), via the glucocorticoid receptor (GR), regulate numerous physiological processes in an effort to maintain homeostasis. Previous studies demonstrated that glucocorticoids suppress ER stress by enhancing correct folding of secreted proteins and degradation of misfolded proteins. Here, we describe a novel crosstalk between ER-stress and the glucocorticoid receptor signaling. We showed that treatment of wild type mice with Tunicamycin (inducer of ER-stress) increased GR protein levels in the lungs. Treatment of A549 cells (human lung cancer cells) with ER stress inducers modulated the Dexamethasone-induced subcellular localization of GR and the phosphorylated forms of GR (pGRSer211 and pGRSer203) with concomitant changes in the expression of primary GR-target genes. We demonstrated a significant protein-protein interaction between GR and CHOP, (GR-CHOP heterocomplex formation) under ER stress conditions. The functional consequences of ER stress- GR signaling crosstalk were assessed and demonstrated that long time exposure (24–48 h) of A549 cells to dexamethasone (10−6 M) reversed the Tunicamycin-induced cell death, a phenomenon associated with parallel increases in GR protein content, increases in cell survival parameters and decreases in cell apoptosis-related parameters. Our study provides evidence that there is a cross talk between ER-stress and GR signaling, this being associated with mutual functional antagonism between CHOP and GR-mediated pathways in lung cells with important implications in lung cell function.

D-4F, an apolipoprotein A-I mimetic, inhibits TGF-β1 induced epithelial-mesenchymal transition in human alveolar epithelial cell

Emerging evidences support that transforming growth factor β1 (TGF-β1) induced epithelial-mesenchymal transition (EMT) participates in the pathogenesis of pulmonary fibrosis and asthmatic airway remodeling. Recent studies demonstrated that apolipoprotein A-I (Apo A-I) is the only known substance that can resolve established pulmonary fibrotic nodules, and Apo A-I mimetic D-4F (a synthetic polypeptide consisting of 18 amino acids) plays an inhibitory role in murine asthmatic model. However, cellular mechanisms for such therapeutic effects of Apo A-I and D-4F remain to be elucidated. This study evaluated the effects of D-4F on TGF-β1 induced EMT in human type II alveolar epithelial cell line A549. A549 cells treated with 10ng/ml of TGF-β1 manifested distinct EMT, including fibroblastic morphological changes, down-regulation of epithelial marker E-cadherin and up-regulation of mesenchymal marker vimentin. These EMT related changes were all inhibited by D-4F in a concentration dependent manner. Transcriptional investigation demonstrated clearly that D-4F dose-dependently compensated for the reduced E-cadherin mRNA level and the increased vimentin mRNA level in TGF-β1 treated A549 cells. Translational analysis revealed that D-4F significantly reversed the TGF-β1 induced changes of E-cadherin and vimentin levels. These results suggested that D-4F inhibits TGF-β1 induced EMT in human alveolar epithelial cell. Given the functional similarities between D-4F and Apo A-I, it is speculated that D-4F and Apo A-I are able to exert possible anti-fibrotic and anti-asthmatic effects via inhibiting alveolar EMT, and D-4F may possess beneficial clinical potential for patients suffering from pulmonary fibrosis and asthma.

Cigarette smoke extract (CSE) induces transient receptor potential ankyrin 1(TRPA1) expression via activation of HIF1αin A549 cells

We previously found that transient receptor potential ankyrin 1 (TRPA1) in guinea pig tracheal epithelial cells was elevated after 14 days of cigarette smoke (CS) exposure. However, the mechanism underlying CS-induced TRPA1 expression remains unknown. Here, we explored whether cigarette smoke extract (CSE)-induced TRPA1 expression is related with modulation of HIF1α in A549 cells. Our results showed that CSE increased TRPA1 expression in A549 cells, decreased Iκ B, PHD2, and HDAC2, and increased ROS release and nuclear translocation of NF-κ B and HIF1α. Moreover, HIF1α siRNA and/or MG132 (a proteasome inhibitor) pretreatment significantly inhibited CSE-induced TRPA1 expression and HIF1α nuclear translocation in A549 cells. However, HIF1α siRNA pretreatment did not affect CSE-induced NF-κ B nuclear translocation, suggesting that CSE-induced TRPA1 expression in A549 cells is directly mediated by HIF1α, but not by NF-κ B. Similar to CSE treatment, treatment of A549 cells with LPS caused significant increases in nuclear translocation of NF-κ B and HIF1α mRNA expression, but did not alter TRPA1 mRNA expression. However, pretreatment with PHD2 siRNA did result in increased TRPA1 mRNA expression in LPS-treated A549 cells; an effect that was inhibited by SN50 (a NF-κ B inhibitor). It suggests a role for NF-κ B to indirectly regulate TRPA1 mRNA expression via modulating HIF1α mRNA transcription. In addition, treatment cells with HDAC2 siRNA plus 2%CSE resulted in increased HIF1α nuclear translocation and TRPA1 expression, which was significantly inhibited by MG132 and HIF1α siRNA. These results suggest that HDAC2 indirectly modulates TRPA1 expression by promoting the DNA-binding activity of HIF1α. These findings show that CSE increases TRPA1 expression in airway epithelial cells by directly activating HIF1α, and that this increase in TRPA1 expression is indirectly regulated via NF-κ B, PHD2 and HDAC2 modulation of HIF1α activity.

Abstract 3655: Suppression of Plk3 expression by nickel may contribute to lung carcinogenesis

Abstract Exposure to nickel (Ni) compounds is known to induce human lung malignancies. Research shows that Ni induces lung cancer mainly through epigenetic and metabolic pathways. Polo-like kinase 3 (Plk3) is a multifunctional protein kinase that involves in tumor initiation and progression. Plk3−/− mice tend to develop highly vascularized lung tumors at an advanced age. Reduced expression of Plk3 was reported in human lung cancers. In this study, we examined the effect of Ni on Plk3 expression in A549 lung carcinoma cells. Western blot showed that NiCl2 reduced the Plk3 protein level in a time- and dose-dependent manner. We then treated A549 cells with cycloheximide for various times to examine the effect of NiCl2 on the half-life of Plk3 protein. We observed a shortened half-life of Plk3 in the presence of NiCl2. A reduced level of Plk3 protein was also found in hypoxia-treated A549 cells. In addition, MG132, a specific proteasome inhibitor, could partially reverse the effect of NiCl2 or hypoxia on the Plk3 protein level. Our cell fractionation experiments showed that majority of the endogenous Plk3 protein is located in the cytoplasm where degradation of Plk3 likely occurs. Transfection of A549 cells with FLAG-tagged ubiquitin (FLAG-Ub) followed by treatment with NiCl2 and/or MG132, immunoprecipitation (IP) with an anti-FLAG antibody, and Western blot with an anti-Plk3 antibody showed that Plk3 was poly-ubiquitinated and that the ubiquitination was significantly enhanced by NiCl2 and/or MG132 treatments. The elevated ubiquitination of Plk3 in response to NiCl2 was further confirmed in 293T cells with co-transfection of Plk3 and FLAG-Ub. Furthermore, overexpression of USP28, a deubiquitinase known to be inhibited by Ni or hypoxia, partially restored the protein level of Plk3 suppressed by NiCl2. Moreover, real-time PCR analysis showed that NiCl2 also reduced the Plk3 mRNA level, in a time- and dose-dependent pattern. Taken together, our data show that NiCl2 suppresses Plk3 expressions by lowering the Plk3 mRNA level and promoting proteasome-mediated degradation of the Plk3 protein. Given the potential role of Plk3 as a tumor suppressor in the lung, suppression of Plk3 expression likely contributes to Ni-induced lung carcinogenesis and tumor progression. Citation Format: Cen Li, Soyoung Park, Wei Dai, Dazhong Xu. Suppression of Plk3 expression by nickel may contribute to lung carcinogenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3655.

Role of reactive oxygen species-mediated MAPK and NF-κB activation in polygonatum cyrtonema lectin-induced apoptosis and autophagy in human lung adenocarcinoma A549 cells.

Polygonatum cyrtonema lectin (PCL), a mannose/sialic acid-binding lectin isolated from the rhizomes of Polygonatum cyrtonema Hua, has been reported to possess remarkable anti-tumour effects via inducing apoptosis and autophagy. The aim of this study was to investigate the molecular mechanisms mediating PCL-induced apoptosis and autophagy in A549 cells. Herein, we found that the treatment of A549 cells with PCL caused a remarkable generation of reactive oxygen species (ROS) and ROS scavenger N-acetyl-cysteine (NAC) inhibited PCL-induced apoptosis and autophagy. In addition, PCL treatment activated mitogen-activated protein kinase (MAPK) members extracellular signal-regulated kinase (ERK), JNK and p38, JNK inhibitor and p38 inhibitor partially reduced PCL-induced apoptosis and autophagy. Moreover, PCL administration activated NF-κB survival pathway in A549 cells, NF-κB inhibitor Bay11-7082 promoted PCL-induced apoptosis. Importantly, we found PCL may bind to the cell surface in a mannose-specific manner, and was then internalized and accumulated primarily onto the mitochondria. These findings may provide a new perspective of PCL as a potential anti-tumour drug targeting apoptosis and autophagy pathways for future cancer therapeutics.

Novel CHOP activator LGH00168 induces necroptosis in A549 human lung cancer cells via ROS-mediated ER stress and NF-κB inhibition.

C/EBP homologous protein (CHOP) is a transcription factor that is activated at multiple levels during ER stress and plays an important role in ER stress-induced apoptosis. In this study we identified a novel CHOP activator, and further investigated its potential to be a therapeutic agent for human lung cancer. HEK293-CHOP-luc reporter cells were used in high-throughput screening (HTS) to identify CHOP activators. The cytotoxicity against cancer cells in vitro was measured with MTT assay. The anticancer effects were further examined in A549 human non-small cell lung cancer xenograft mice. The mechanisms underlying CHOP activation were analyzed using luciferase assays, and the anticancer mechanisms were elucidated in A549 cells. From chemical libraries of 50 000 compounds, LGH00168 was identified as a CHOP activator, which showed cytotoxic activities against a panel of 9 cancer cell lines with an average IC50 value of 3.26 μmol/L. Moreover, administration of LGH00168 significantly suppressed tumor growth in A549 xenograft bearing mice. LGH00168 activated CHOP promoter via AARE1 and AP1 elements, increased DR5 expression, decreased Bcl-2 expression, and inhibited the NF-κB pathway. Treatment of A549 cells with LGH00168 (10 μmol/L) did not induce apoptosis, but lead to RIP1-dependent necroptosis, accompanied by cell swelling, plasma membrane rupture, lysosomal membrane permeabilization, MMP collapse and caspase 8 inhibition. Furthermore, LGH00168 (10 and 20 μmol/L) dose-dependently induced mito-ROS production in A549 cells, which was reversed by the ROS scavenger N-acetyl-L-cysteine (NAC, 10 mmol/L). Moreover, NAC significantly diminished LGH00168-induced CHOP activation, NF-κB inhibition and necroptosis in A549 cells. LGH00168 is a CHOP activator that inhibits A549 cell growth in vitro and lung tumor growth in vivo.