Human Pulmonary Adenocarcinoma A549 Cells Research Articles

Role of integrin β1 in sensitivity to chemotherapy of pulmonary adenocarcinoma A549

Objective The aim of our study was to investigate the effect of integrin β1 on influencing the sensitivity to chemotherapy of human pulmonary adenocarcinoma A549 cells.

Enhancement of Recombinant Human Endostatin on the Radiosensitivity of Human Pulmonary Adenocarcinoma A549 Cells and Its Mechanism

We observed the effects of endostar on the radiosensitivity of pulmonary adenocarcinoma A549 cells and found that endostar inhibited A549 cell growth under normoxia and hypoxia in time and dose-dependent manners; the D0 and Dq values in control and endostar groups were (1.36 and 1.30) versus (1.019 and 1.015) under normoxia and (1.693 and 1.39) versus (2.453 and 1.026) under hypoxia, respectively; SER was 1.04 under normoxia and 1.22 under hypoxia in endostar group; under normoxia, the apoptosis rates in control, radiotherapy, endostar and combination groups were 15.9 ± 0.57%, 42.7 ± 0.37%, 19.9 ± 0.48%, and 41.5 ± 0.38%, respectively, with no significant difference between combination and radiotherapy groups; there was significant difference in G2/M phase cells between combination and radiotherapy groups (P = 0.028); under hypoxia, the apoptosis rates in the four groups were 16.7 ± 0.67%, 30.1 ± 0.95%, 26.7 ± 0.62%, and 36.3 ± 0.71%, respectively, with significant difference between combination and radiotherapy groups; G2/M phase cells were higher in combination group than radiotherapy group (P = 0.000); G2/M phase cells were higher in hypoxic combination group than in normoxic combination group (P = 0.003). Based on these results, we conclude that under hypoxia, endostar can enhance the radiosensitivity of A549 cells through G2/M arrest.

Cell fixation in zinc salt solution is compatible with DNA damage response detection by phospho‐specific antibodies

By virtue of superior preservation of proteins and nucleic acids the zinc salt-based fixatives (ZBF) has been proposed as an alternative to precipitants and cross-linking fixatives in histopathology. It was recently reported that ZBF is compatible with analysis of cell surface immunophenotype and detection of intracellular epitopes by flow cytometry. The aim of this study was to explore whether ZBF is also compatible with the detection of DNA damage response assessed by phospho-specific antibodies (Abs) detecting phosphorylation of the key proteins of that pathway. DNA damage in human pulmonary adenocarcinoma A549 cells was induced by treatment with the DNA topoisomerase I inhibitor camptothecin and phosphorylation of histone H2AX on Ser139 (γH2AX) and of ATM on Ser1981 was detected with phospho-specific Abs; cellular fluorescence was measured by laser scanning cytometry (LSC). The sensitivity and accuracy of detection of H2AX and ATM phosphorylation concurrent with the detection of DNA replication by EdU incorporation and "click chemistry" was found in ZBF fixed cells to be comparable to that of cell fixed in formaldehyde. The accuracy of DNA content measurement as evident from the resolution of DNA content frequency histograms of cells stained with DAPI was somewhat better in ZBF- than in formaldehyde-fixed cells. The pattern of chromatin condensation revealed by the intensity of maximal pixel of DAPI that allows one to identify mitotic and immediately post-mitotic cells by LSC was preserved after ZBF fixation. ZBF fixation was also compatible with the detection of γH2AX foci considered to be the hallmarks of induction of DNA double-strand breaks. Analysis of cells by flow cytometry revealed that ZBF fixation of lymphoblastoid TK6 cells led to about 60 and 33% higher intensity of the side and forward light scatter, respectively, compared to formaldehyde fixed cells.

Magnetic Resonance Imaging Contrast Agent: cRGD-Ferric Oxide Nanometer Particle and Its Role in the Diagnosis of Tumor

To construct tumor-targeted nanometer particles as a negative magnetic resonance imaging (MRI) contrast agent. Ultra-small superparamagnetic iron oxide (USPIO) nanometer particles were prepared by one-step chemical precipitation. The covalent bond between cyclic RGD (cRGD) containing an Arg-Gly-Asp sequence targeting integrin-alphavbeta3, and USPIO was conducted by chemical crosslinking. The physico-chemical property of cRGD-USPIO was detected. Prussian blue staining was applied to detect the specific binding capacity of cRGD-USPIO and USPIO to human pulmonary adenocarcinoma A549 cells and human umbilical vein endothelial cells. Subsequently, A549 xenografts in nude mice were established, and intravenous injections of USPIO and cRGD-USPIO into the vena caudalis were performed. The enhancement of cRGD-USPIO against tumor MRI signal was evaluated. The mean hydrodynamic diameter of cRGD-USPIO was 43.97 +/- 10.10 nm and the size of the ferric oxide core was 5-10 nm. The specific saturation magnetization was 59.94 A x m2 x Kg(-1). The cell conjugation assay results indicated that the positive staining of the cRGD-USPIO group was significantly enhanced. The in vivo MRI diagnosis indicated that the cRGD-USPIO tumor signal was significantly reduced compared to that of the USPIO group (P < 0.01). The targeted superparamagnetic iron oxide nanometer particle can be a novel MRI negative contrast agent for more specific tumor early diagnosis.

Curcumin Inhibits the Proteinase-Activated Receptor-2–Triggered Prostaglandin E2 Production by Suppressing Cyclooxygenase-2 Upregulation and Akt-Dependent Activation of Nuclear Factor-κB in Human Lung Epithelial Cells

We performed this study to determine if curcumin affects pro-inflammatory responses to activation of proteinase-activated receptor-2 (PAR2) in human pulmonary adenocarcinoma A549 cells. Curcumin completely inhibited the PAR2-triggered prostaglandin E2 (PGE2) production, but notably not interleukin-8 release. Cyclooxygenase-2 (COX-2) upregulation, but not its upstream activation of mitogen-activated protein kinases, caused by PAR2 stimulation was partially inhibited by curcumin. Curcumin inhibited the PAR2-triggered phosphorylation of I-κB, an indicator for nuclear factor-κB (NF-κB) activation, and also its upstream signal Akt, which is known to contribute to PAR2-triggered PGE2 formation, but not COX-2 upregulation. Collectively, curcumin inhibits the PAR2-triggered PGE2 production by suppressing COX-2 upregulation and Akt/NF-κB signals in A549 cells.

Pyrogallol inhibits the growth of human pulmonary adenocarcinoma A549 cells by arresting cell cycle and triggering apoptosis

Pyrogallol (PG) is a polyphenol compound and has been known to be an O(2)(*-) generator. We evaluated the effects of PG on the growth of human pulmonary A549 cells in relation to the cell cycle and apoptosis. Treatment with 50 or 100 microM PG significantly inhibited the cell growth of A549 for 72 h. DNA flow cytometric analysis indicated that PG slightly induced a G1 phase arrest of the cell cycle at 24 or 48 h, but did not induce the specific cell cycle arrest at 72 h. Intracellular GSH depletion was observed in PG-treated cells. PG induced apoptosis in A549 cells, as evidenced by sub-G1 cells, annexin V staining cells, and the loss of mitochondrial membrane potential (DeltaPsi(m)). The intracellular ROS (reactive oxygen species) level including O(2)(*-) increased in PG-treated A549 cells at 24 and 48 h, and persisted at 72 h. The changes in GSH as well as ROS levels by PG affected the cell viability in A549 cells. In conclusion, PG inhibited the growth of human pulmonary A549 cells by inducing cell cycle arrest as well as triggering apoptosis.

Effect of hypoxia on migration, invasion and adhesion to endothelium of human pulmonary adenocarcinoma A549 cells

Objective To evaluate the effect of hypoxia on migration, invasion and adhesion to endothelium of human pulmonary adenocarcinoma A549 cells.

ATM activation accompanies histone H2AX phosphorylation in A549 cells upon exposure to tobacco smoke

BackgroundIn response to DNA damage or structural alterations of chromatin, histone H2AX may be phosphorylated on Ser139 by phosphoinositide 3-kinase related protein kinases (PIKKs) such as ataxia telangiectasia mutated (ATM), ATM-and Rad-3 related (ATR) kinase, or by DNA dependent protein kinase (DNA-PKcs). When DNA damage primarily involves formation of DNA double-strand breaks (DSBs), H2AX is preferentially phosphorylated by ATM rather than by the other PIKKs. We have recently reported that brief exposure of human pulmonary adenocarcinoma A549 cells or normal human bronchial epithelial cells (NHBE) to cigarette smoke (CS) induced phosphorylation of H2AX.ResultsWe report here that H2AX phosphorylation in A549 cells induced by CS was accompanied by activation of ATM, as revealed by ATM phosphorylation on Ser1981 (ATM-S1981P) detected immunocytochemically and by Western blotting. No cell cycle-phase specific differences in kinetics of ATM activation and H2AX phosphorylation were observed. When cells were exposed to CS from cigarettes with different tobacco and filter combinations, the expression levels of ATM-S1981P correlated well with the increase in expression of phosphorylated H2AX (γH2AX) (R = 0.89). In addition, we note that while CS-induced γH2AX expression was localized within discrete foci, the activated ATM was distributed throughout the nucleoplasm.ConclusionThese data implicate ATM as the PIKK that phosphorylates H2AX in response to DNA damage caused by CS. Based on current understanding of ATM activation, expression and localization, these data would suggest that, in addition to inducing potentially carcinogenic DSB lesions, CS may also trigger other types of DNA lesions and cause chromatin alterations. As checkpoint kinase (Chk) 1, Chk2 and the p53 tumor suppressor gene are known to be phosphorylated by ATM, the present data indicate that exposure to CS may lead to their phosphorylation, with the downstream consequences related to the halt in cell cycle progression and increased propensity to undergo apoptosis. Defining the nature and temporal sequence of molecular events that are disrupted by CS through activation and eventual dysregulation of normal defense mechanisms such as ATM and its downstream effectors may allow a more precise understanding of how CS promotes cancer development.

Salvia miltiorrhizaInhibits Tumor Cell Growth in Association with Rb Dephosphorylation through Up-regulation of p21 Via a p53-dependent Pathway

Background: Salvia miltiorrhiza (SM), a traditional oriental medicine, has been reported to have anti-tumor properties, but its exact mechanism remains to be elucidated. In this study, we investigated several of the molecular events that occur in human breast carcinoma MCF-7 cells and human pulmonary adenocarcinoma A549 cells. Methods: For this purpose, we evaluated the growth-inhibitory effect of SM in association with the expressions of p53, p21, cyclin D1, and pRb, which are known to be involved in cell cycle arrest. The extent of thymidine incorporation was also examined to assess G1/S phase cell cycle arrest in both cells by H-thymidine incorporation. Results: Our results show that SM inhibits the growth and the proliferation of MCF-7 and A549 cells. Furthermore, we also observed increased expression of p21 via a p53-dependent pathway in both cell lines after treating with SM. In addition, treatment with SM for 24 hours caused the suppression of hyperphosphorylated retinoblastoma protein (pRb) expression and the dephosphorylation of pRb. Conclusion: These findings suggest that the growth inhibitory and the anti-proliferation effects of SM on MCF-7 cells and A549 cells are mediated via the decreased expression and dephosphorylation of pRB by p21 up-regulation in a p53-dependent manner. To the best of our knowledge, this study is the first to report upon the molecular mechanisms involved in SM-induced tumor cell growth inhibition. (Immune Network 2002;2(1):19-24)

Apoptosis and epithelial phagocytosis in mitomycin C-treated human pulmonary adenocarcinoma A549 cells

Fate of human cancer cells damaged by mitomycin C was investigated by electron microscopy. In a monolayer culture of pulmonary adenocarcinoma A549 cells, the antitumor drug mitomycin C induced dilation of the smooth endoplasmic reticulum and Golgi apparatus. Simultaneously, the myelin figures, autophagosomes and dense tonofilament bundles were formed in the cytosol. Nuclear changes also included nucleolar condensation, as well as the disappearance of the karyosomes and thinned marginal heterochromatins. These nuclei came to be rigid and densely chromatic, finally resulting in apoptosis. There was no alteration in the mitochondria or rough endoplasmic reticulum. Meanwhile, some remaining intact A549 cells extended their cytoplasm towarddetached cells and engulfed them. These results indicate that mitomycin C induces apoptosis in A549 cells and concurrently stimulates epithelial phagocytotic activity against their own damaged cells.

Effects of Mechanical Forces on Pulmonary Epithelial Gene Expression and Differentiation • 1738

Fetal breathing movements (FBM) are necessary for normal fetal lung growth and maturation. Absence of FBM or lung constriction results in lung hypoplasia. The molecular mechanisms by which FBM stimulate lung growth are not known. We analyzed fetal rat lungs (E19) cultured ± lung distension and tracheal ligation. We also examined the effects of mechanical stretch on rat fetal lung type II cells, human pulmonary adenocarcinoma A549 cells, and a human pulmonary epithelial cell line (NCI-H441) that shows regulated expression of surfactant proteins SP-A and SP-B. Cells were grown to confluence on matrix-coated silastic membranes and mounted in a FX-3000 Strain Unit (Flexcell). Cyclic deformation simulating FBM was achieved by applying a vacuum of 22 kPa (5-15% radial deformation) at 50 cycles/min for 2-24h. Unstretched cells were used as controls. We separately evaluated the effects of fluid shear forces (without cellular radial elongation) and cell deformation. SP-A and SP-B mRNA abundance was assayed by northern blot. Parathyroid hormone-related protein (PTHrP, a stretch-dependent lung developmental regulator), and cyclin-dependent kinase (cdk) inhibitors were analyzed by RNase protection assay (RPA). Our results indicate that static distension for as brief as 4h decreased steady-state SP-A and SP-B mRNA levels in whole lung to 51% and 31%, respectively, of control levels (n=5-6; p SP-A expression 2-4 fold over controls and PTHrP expression by 30-60%. These effects were not mediated by fluid shear but were partially inhibited by lanthanum (a stretch-activated channel blocker). Cyclic deformation also significantly enhanced 3H-choline incorporation into saturated phosphatidylcholine in the different epithelial culture models. Cyclic stretching for 48h altered cell cycle phase distribution, apparently by inducing a block in SG2 progression, and did not activate apoptosis(assayed by DNA flow cytometry). The cell cycle effects do not appear to be due to transcriptional regulation of cdk inhibitor proteins. Our results in lung organ culture are consistent with observations made in vivo in tracheal ligation models, and suggest that continuous lung distension may interfere with airway epithelial cytodifferentiation. In contrast, dynamic mechanodeformation (distension-relaxation) may be a critical stimulus for fetal lung maturation. The molecular mechanisms by which mechanotransduction alters gene expression and cytodifferentiation are under investigation.

Antimycin A as a mitochondrial electron transport inhibitor prevents the growth of human lung cancer A549 cells

Antimycin A (AMA) inhibits mitochondrial electron transport between cytochromes b and c. We evaluated the effects of AMA on the growth of human pulmonary adenocarcinoma A549 cells in relation to cell cycle and apoptosis. Treatment with 2-100 microM AMA significantly inhibited the cell growth of A549 for 72 h. DNA flow cytometry indicated that AMA slightly induced a G1 phase arrest of the cell cycle for 72 h. Treatment with 50 microM AMA induced apoptosis of approximately 17% in view of annexin V-staining cells. The dose of 50 microM AMA also induced loss of the mitochondrial membrane potential (DeltaPsi(m)) of approximately 38%. The intracellular reactive oxygen species (ROS) levels including O2(.-) were significantly increased in AMA-treated A549 cells. In conclusion, AMA inhibited the growth of A549 cells via inducing cell cycle arrest as well as triggering apoptosis. Growth inhibition in AMA-treated A549 cells was accompanied by an increase in ROS levels.