Ghrelin ameliorates the human alveolar epithelial A549 cell apoptosis induced by lipopolysaccharide

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Huang, Chunrong; Zheng, Haichong; He, Wanmei; Lu, Guifang; Li, Xia [Department of Medical Intensive Care Unit, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou 510080 (China); Deng, Yubin, E-mail: dengyub@mail.sysu.edu.cn [Research Center of Translational Medicine, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou 510080 (China); Zeng, Mian, E-mail: zengmian2004@163.com [Department of Medical Intensive Care Unit, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou 510080 (China)

2016-05-20

Ghrelin is a gastric acyl-peptide that plays an inhibitory role in cell apoptosis. Herein we investigate the protective effects of ghrelin in LPS-induced apoptosis of human alveolar epithelial A549 cells, along with the possible molecular mechanisms. LPS exposure impaired cell viability and increased apoptosis of A549 cells significantly in concentration- and time-dependent manners embodied in increased Bax and cleaved caspase-3 production, coupled with decreased Bcl-2 levels. Simultaneously, LPS remarkably decreased the expression of phosphatidylinositol 3 kinase/protein kinase B (PI3K/Akt) and extracellular signal-regulated kinas (ERK) in A549 cells. However, ghrelin'pretreatment ameliorated LPS-caused alterations in the ratio of Bax/Bcl-2 and cleaved caspase-3 expression, whereas activated the PI3K/Akt and ERK signaling. These results demonstrate that ghrelin lightens LPS-induced apoptosis of human alveolar epithelial cells partly through activating the PI3K/Akt and ERK pathway and thereby might benefit alleviating septic ALI. -- Graphical abstract: Ghrelin ameliorates the human alveolar epithelial A549 cells apoptosis induced by lipopolysaccharide partly through activating the PI3K/Akt and ERK pathway. Display Omitted -- Highlights: •It has been observed that LPS insult significantly increased apoptosis in A549 cells. •Both Akt and ERK signaling are critical adapter molecules to mediate the ghrelin-mediated proliferative effect. •Ghrelin may have a therapeutic effect in the prevention of LPS-induced apoptosis.

Analysis of A549 cell proteome alteration in response to recombinant influenza A virus nucleoprotein and its interaction with cellular proteins, a preliminary study.

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Kumar, D; Tiwari, K; Rajala, M S

Influenza A virus undergoes frequent changes of antigenicity and contributes to seasonal epidemics or unpredictable pandemics. Nucleoprotein, encoded by gene segment 5, is an internal protein of the virus and is conserved among strains of different host origins. In the current study, we analyzed the differentially expressed proteins in A549 cells transiently transfected with the recombinant nucleoprotein of influenza A virus by 2D gel electrophoresis. The resolved protein spots on gel were identified by MALDI-TOF/Mass spectrometry analysis. The majority of the host proteins detected to be differentially abundant in recombinant nucleoprotein-expressing cells as compared to vector-transfected cells are the proteins of metabolic pathways, glycolytic enzymes, molecular chaperones and cytoskeletal proteins. We further demonstrated the interaction of virus nucleoprotein with some of the identified host cellular proteins. In vitro binding assay carried out using the purified recombinant nucleoprotein (pET29a+NP-His) and A549 cell lysate confirmed the interaction between nucleoprotein and host proteins, such as alpha enolase 1, pyruvate kinase and β-actin. The preliminary data of our study provides the information on virus nucleoprotein interaction with proteins involved in glycolysis. However, studies are ongoing to understand the significance of these interactions in modulating the host factors during virus replication.

Curcumin inhibits interferon-α induced NF-κB and COX-2 in human A549 non-small cell lung cancer cells

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Lee, Jeeyun; Im, Young-Hyuck; Jung, Hae Hyun; Kim, Joo Hyun; Park, Joon Oh; Kim, Kihyun; Kim, Won Seog; Ahn, Jin Seok; Jung, Chul Won; Park, Young Suk; Kang, Won Ki; Park, Keunchil

2005-01-01

The A549 cells, non-small cell lung cancer cell line from human, were resistant to interferon (IFN)-α treatment. The IFN-α-treated A549 cells showed increase in protein expression levels of NF-κB and COX-2. IFN-α induced NF-κB binding activity within 30 min and this increased binding activity was markedly suppressed with inclusion of curcumin. Curcumin also inhibited IFN-α-induced COX-2 expression in A549 cells. Within 10 min, IFN-α rapidly induced the binding activity of a γ- 32 P-labeled consensus GAS oligonucleotide probe, which was profoundly reversed by curcumin. Taken together, IFN-α-induced activations of NF-κB and COX-2 were inhibited by the addition of curcumin in A549 cells

Effects of TGF-β signaling blockade on human A549 lung adenocarcinoma cell lines.

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Xu, Cheng-Cheng; Wu, Lei-Ming; Sun, Wei; Zhang, Ni; Chen, Wen-Shu; Fu, Xiang-Ning

2011-01-01

Transforming growth factor β (TGF-β) is overexpressed in a wide variety of cancer types including lung adenocarcinoma (LAC), and the TGF-β signaling pathway plays an important role in tumor development. To determine whether blockade of the TGF-β signaling pathway can inhibit the malignant biological behavior of LAC, RNA interference (RNAi) technology was used to silence the expression of TGF-β receptor, type II (TGFβRII) in the LAC cell line, A549, and its effects on cell proliferation, invasion and metastasis were examined. Three specific small interfering RNAs (siRNAs) designed for targeting human TGFβRII were transfected into A549 cells. The expression of TGFβRII was detected by Western blot analysis. Cell proliferation was measured by MTT and clonogenic assays. Cell apoptosis was assessed by flow cytometry. The invasion and metastasis of A549 cells were investigated using the wound healing and Matrigel invasion assays. The expression of PI3K, phosphorylated Smad2, Smad4, Akt, Erk1/2, P38 and MMPs was detected by Western blot analysis. The TGFβRII siRNA significantly reduced the expression of TGFβRII in A549 cells. The knockdown of TGFβRII in A549 cells resulted in the suppression of cell proliferation, invasion and metastasis and induced cell apoptosis. In addition to the Smad-dependent pathway, independent pathways including the Erk MAPK, PI3K/Akt and p38 MAPK pathways, as well as the expression of MMPs and VEGF, were inhibited. In conclusion, TGF-β signaling is required for LAC progression. Therefore, the blockade of this signaling pathway by the down-regulation of TGFβRII using SiRNA may provide a potential gene therapy for LAC.

Seleno-short-chain chitosan induces apoptosis in human non-small-cell lung cancer A549 cells through ROS-mediated mitochondrial pathway.

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Zhao, Yana; Zhang, Shaojing; Wang, Pengfei; Fu, Shengnan; Wu, Di; Liu, Anjun

2017-12-01

Seleno-short-chain chitosan (SSCC) is a synthesized chitosan derivative. In this study, antitumor activity and underlying mechanism of SSCC on human non-small-cell lung cancer A549 cells were investigated in vitro. The MTT assay showed that SSCC could inhibit cell viability in a dose- and time-dependent manner, and 200 μg/ml SSCC exhibited significantly toxic effects on A549 cells. The cell cycle assay showed that SSCC triggered S phase cell cycle arrest in a dose- and time-dependent manner, which was related to a downregulation of S phase associated cyclin A. The DAPI staining and Annexin V-FITC/PI double staining identified that the SSCC could induce A549 cells apoptosis. Further studies found that SSCC led to the generation of reactive oxygen species (ROS) and the disruption of mitochondrial membrane potential (MMP) by DCFH-DA and Rhodamin 123 staining, respectively. Meanwhile, free radical scavengers N-acetyl-L-cysteine (NAC) pretreatment confirmed that SSCC-induced A549 cells apoptosis was associated with ROS generation. Furthermore, real-time PCR and western blot assay showed that SSCC up-regulated Bax and down-regulated Bcl-2, subsequently incited the release of cytochrome c from mitochondria to cytoplasm, activated the increase of cleaved-caspase 3 and finally induced A549 cells apoptosis in vitro. In general, the present study demonstrated that SSCC induced A549 cells apoptosis via ROS-mediated mitochondrial apoptosis pathway.

Expression of cyclin A in A549 cell line after treatment with arsenic trioxide

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Agnieszka Żuryń

2015-12-01

Full Text Available Background: Arsenic trioxide (ATO is an effective drug used in acute promyelocytic leukemia (AML. Many reports suggest that ATO can also be applied as an anticancer agent for solid tumors in the future. The influence of arsenic trioxide on the expression of different cell cycle regulators is poorly recognized. The purpose of the current study is to investigate how arsenic trioxide affects cyclin A expression and localization in the A549 cell line.Materials and methods: Morphological and ultrastructural changes in A549 cells were observed using light and transmission electron microscopes. Cyclin A localization was determined by immunofluorescence. Image-based cytometry was applied to evaluate the effect of arsenic trioxide on apoptosis and the cell cycle. Expression of cyclin A mRNA was quantified by real-time PCR.Results: After treatment with arsenic trioxide, increased numbers of cells with cytoplasmic localization of cyclin A were observed. The doses of 10 and 15 μM ATO slightly reduced expression of cyclin A mRNA. The apoptotic phenotype of cells was poorly represented, and the Tali imagebased cytometry analysis showed low percentages of apoptotic cells. The A549 population displayed an enriched fraction of cells in G0/G1 phase in the presence of 5μM ATO, whereas starting from the higher concentrations of the drug, i.e. 10 and 15 μM ATO, the G2/M fraction was on the increase.Discussion: Low expression of cyclin A in the A549 cell line may constitute a potential factor determining arsenic trioxide resistance. It could be hypothesized that the observed alterations in cyclin A expression/distribution may correlate well with changes in cell cycle regulation in our model, which in turn determines the outcome of the treatment.

MG132 as a proteasome inhibitor induces cell growth inhibition and cell death in A549 lung cancer cells via influencing reactive oxygen species and GSH level.

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Han, Yong Hwan; Park, Woo Hyun

2010-07-01

Carbobenzoxy-Leu-Leu-leucinal (MG132) as a proteasome inhibitor has been shown to induce apoptotic cell death through formation of reactive oxygen species (ROS). In the present study, we evaluated the effects of MG132 on the growth of A549 lung cancer cells in relation to cell growth, ROS and glutathione (GSH) levels. Treatment with MG132 inhibited the growth of A549 cells with an IC(50) of approximately 20 microM at 24 hours. DNA flow cytometric analysis indicated that 0.5 approximately 30 microM MG132 induced a G1 phase arrest of the cell cycle in A549 cells. Treatment with 10 or 30 microM MG132 also induced apoptosis, as evidenced by sub-G1 cells and annexin V staining cells. This was accompanied by the loss of mitochondrial membrane potential (MMP; Delta psi m). The intracellular ROS levels including O(2) (*-) were strongly increased in 10 or 30 microM MG132-treated A549 cells but were down-regulated in 0.1, 0.5 or 1 microM MG132-treated cells. Furthermore, 10 or 30 microM MG132 increased mitochondrial O(2) (*- ) level but 0.1, 0.5 or 1 microM MG132 decreased that. In addition, 10 or 30 microM MG132 induced GSH depletion in A549 cells. In conclusion, MG132 inhibited the growth of human A549 cells via inducing the cell cycle arrest as well as triggering apoptosis, which was in part correlated with the changes of ROS and GSH levels. Our present data provide important information on the anti-growth mechanisms of MG132 in A549 lung cancer cells in relation to ROS and GSH.

Construction of a CD147 Lentiviral Expression Vector and Establishment of Its Stably Transfected A549 Cell Line

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Shaoxing YANG

2012-12-01

Full Text Available Background and objective CD147, a type of transmembrane glycoprotein embedded on the surface of tumor cells, can promote tumor invasion and metastasis. This aim of this study is to construct a CD147 lentiviral expression vector, establish its stably transfected A549 cell line, and observe the effect of CD147 on MMP-9 proliferation as well as on the invasive ability of human lung adenocarcinoma cells. Methods Full-length CD147 gene was amplified by real-time polymerase chain reaction (RT-PCR, inserted into a pEGFP vector to construct pEGFP-CD147 and pEGFP vectors, and then transfected into 293FT cells to precede the lentivirus equipment package. Subsequently, we collected the lentivirus venom to infect the A549 cells and establish a stable, overexpressed cell line named A549-CD147. The mRNA expression of MMP-9 was examined by RT-PCR. The proliferation and invasive ability of the human lung cancer cells before and after transfection were examined by the CCK-8 and Transwell methods. Results A CD147 lentiviral expression vector (pEGFP-CD147 was successfully constructed by restrictive enzyme digestion and plasmid sequencing. RT-PCR and Western blot analyses revealed increased mRNA and protein expression of CD147 gene in cells transfected with pEGFP-CD147 compared with the control groups. Therefore, the A549-CD147 cell line was successfully established through the experiment. The mRNA expression of MMP-9 also significantly increased after the upregulation of CD147 expression. Meanwhile, CCK-8 and Transwell assays indicated that the proliferation and invasive ability significantly increased in the A549-CD147 cells. Conclusion A lentiviral CD147 expression vector and its A549 cell line (A549-CD14 were successfully constructed. CD147 overexpression upregulated the protein expression of MMP-9, and strengthened the proliferation and invasive ability of human lung adenocarcinoma cells.

Down-regulated βIII-tubulin Expression Can Reverse Paclitaxel Resistance in A549/Taxol Cells Lines

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Yinling ZHUO

2014-08-01

Full Text Available Background and objective Chemotherapy drug resistance is the primary causes of death in patients with pulmonary carcinoma which make tumor recurrence or metastasis. β-tubulin is the main cell targets of anti-microtubule drug. Increased expression of βIII-tubulin has been implicated in non-small cell lung cancer (NSCLC cell lines. To explore the relationship among the expression level of βIII-tubulin and the sensitivity of A549/Taxolcell lines to Taxol and cell cycles and cell apoptosis by RNA interference-mediated inhibition of βIII-tubulin in A549/Taxol cells. Methods Three pairs of siRNA targetd βIII-tubulin were designed and prepared, which were transfected into A549/Taxol cells using LipofectamineTM 2000. We detected the expression of βIII-tubulin mRNA using Real-time fluorescence qRT-PCR. Tedhen we selected the most efficient siRNA by the expression of βIII-tubulin mRNA in transfected group. βIII-tubulin protein level were mesured by Western blot. The taxol sensitivity in transfected group were evaluated by MTT assay. And the cell apoptosis and cell cycles were determined by flow cytometry. Results βIII-tubulin mRNA levels in A549/Taxol cells were significantly decreased in transfected grop by Real-time qRT-PCR than control groups. And βIII-tubulin siRNA-1 sequence showed the highest transfection efficiency, which was (87.73±4.87% (P<0.01; Western blot results showed that the expressional level of BIII tublin protein was significantly down-reulated in the transfectant cells than thant in the control cells. By MTT assay, we showed that the inhibition ratio of Taxol to A549/Taxol cells transfeced was higher than that of control group (51.77±4.60% (P<0.01. The early apoptosis rate of A549/Taxol cells in transfected group were significantly higher than that of control group (P<0.01; G2-M content in taxol group obviously increased than untreated samples by the cell cycle (P<0.05. Conclusion βIII-tubulin down-regulated significantly

Effects of miR-424 on Proliferation and Migration Abilities in Non-small Cell Lung Cancer A549 Cells and Its Molecular Mechanism

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Hongmin LI

2016-09-01

Full Text Available Background and objective The inhibitory ability of miR-424 on the proliferation of renal carcinoma cell and the migration and invasion of cancer cells has been widely explored and demonstrated. However, the effects of miR-424 on non-small cell lung cancer (NSCLC have not been systematically examined. In this study, detected the growth and invasion effect of miR-424 in NSCLC A549 cell. The migration and molecular mechanism of this cell are also detected. Methods NSCLC A549 cell was transfected with miR-424 and its inhibitor. After transfection, the proliferation ability of A549 cell was detectedby CCK8 assay. Then, the migration ability in A549 cell was detected by migration assays. Furthermore, the expression level of MMP2 and MMP9 in A549 was detected by Western blot and immune fluorescence. The 3'UTR of E2F6 was cloned into luciferase reporter vector and its enzymatic activitywas detected to verify whether miR-424 can target E2F6. The expression level of E2F6 in a549 cell after transfecing with miR-424 was detected by Western blot. Results After transfection of miR-424, the proliferation and migration abilities were remarkably decreased and the expression level of MMP-2 and MMP-9 were down-regulated in A549. Moreover, MiR-424 inhibited the enzymatic activity of luviferase reporter vector of E2F6. Specifically, the expression level of E2F6 was down-regulated in A549. Conclusion miR-424 can inhibit the proliferation and migration abilities of A549 by negatively regulating the expression of E2F6.

Cold stress increases reactive oxygen species formation via TRPA1 activation in A549 cells.

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Sun, Wenwu; Wang, Zhonghua; Cao, Jianping; Cui, Haiyang; Ma, Zhuang

2016-03-01

Reactive oxygen species (ROS) are responsible for lung damage during inhalation of cold air. However, the mechanism of the ROS production induced by cold stress in the lung is still unclear. In this work, we measured the changes of ROS and the cytosolic Ca(2+) concentration ([Ca(2+)]c) in A549 cell. We observed that cold stress (from 20 to 5 °C) exposure of A549 cell resulted in an increase of ROS and [Ca(2+)]c, which was completely attenuated by removing Ca(2+) from medium. Further experiments showed that cold-sensing transient receptor potential subfamily member 1 (TRPA1) agonist (allyl isothiocyanate, AITC) increased the production of ROS and the level of [Ca(2+)]c in A549 cell. Moreover, HC-030031, a TRPA1 selective antagonist, significantly inhibited the enhanced ROS and [Ca(2+)]c induced by AITC or cold stimulation, respectively. Taken together, these data demonstrated that TRPA1 activation played an important role in the enhanced production of ROS induced by cold stress in A549 cell.

[Effects of 17-AAG on the proliferation and apoptosis of human lung cancer A549 and H446 cells].

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Niu, Ben; Lin, Jingshuang; Feng, Tao

2015-04-01

To observe the effect of 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) on the apoptosis of human lung cancer cell lines A549 and H446, and to investigate the potential mechanisms. Proliferation inhibition and apoptosis assays, and the cell cycles were detected by MTT and flow cytometry respectively. Western blot was used to determine the expression level of proteins such as Hsp90, Hsp70, AKt, Her-2, Bcl-2 and Bax. After treated with 17-AAG, the proliferation of both A549 and H446 cells was inhibited significantly in a dose-dependent manner; as the concentration of 17-AAG was from 50 to 500 nmol/L, the IC₅₀ values to A549 and H446 cell lines were (222 ± 13) nmol/L and (189 ± 7) nmol/L respectively at 48 h. Cell cycle assays showed that 17-AAG was able to arrest cell cycles of A549 and H446 cell lines at the G₂/M phase. Apoptosis assay showed that 17-AAG was capable of inducing apoptosis in A549 and H446 cell lines. After treated with 17-AAG for 48 h, there were significant differences between the 400 nmol/L groups(46.3% for A549 cell line and 56.9% for H446 cell line) and the control group (11.9% for A549 cell line and 6.9% for H446 cell line, P AAG treatment: Akt and Her-2 decreased significantly while the expression of Hsp70 increased. Meanwhile, the expression of Bcl-2 decreased but that of Bax increased, indicating that 17-AAG was able to promote apoptosis mode in A549 and H446 cells. 17-AAG can regulate the expression level of apoptosis-related proteins such as Bax and Bcl-2 by Hsp90 signaling pathway in A549 and H446 cells, and ultimately inhibit cell proliferation and induce apoptosis.

Antimony trichloride induces a loss of cell viability via reactive oxygen species-dependent autophagy in A549 cells.

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Zhao, Xinyuan; Xing, Fengjun; Cong, Yewen; Zhuang, Yin; Han, Muxi; Wu, Zhiqiang; Yu, Shali; Wei, Haiyan; Wang, Xiaoke; Chen, Gang

2017-12-01

Antimony (Sb) is one of the most prevalent heavy metals and frequently leads to biological toxicity. Although autophagy is believed to be involved in metal-associated cytotoxicity, there is no evidence of its involvement following exposure. Moreover, the underlying mechanism of autophagy remains unclear. In this study, treatment with antimony trichloride caused autophagy in a dose- and time-dependent manner in A549 cells but did not affect the level of Atg5 or Atg7 mRNA expression. Furthermore, Sb enhanced autophagic flux while upregulating p62 gene and protein levels. The classic mechanistic target of rapamycin (mTOR) pathway is not involved in Sb-induced autophagy. However, Sb-induced autophagy and the upregulation of p62 were inhibited by treatment with the antioxidant N-acetylcysteine (NAC). Subsequent analyses demonstrated that the inhibition of autophagy protected A549 cells from a loss of cell viability, while the activation of autophagy by rapamycin had the opposite effect. These data suggest that reactive oxygen species-dependent autophagy mediates Sb-stimulated cell viability loss in A549 cells. Copyright © 2017 Elsevier Ltd. All rights reserved.

Green tea extract induces protective autophagy in A549 non-small lung cancer cell line.

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Izdebska, Magdalena; Klimaszewska-Wiśniewska, Anna; Hałas, Marta; Gagat, Maciej; Grzanka, Alina

2015-12-31

For many decades, polyphenols, including green tea extract catechins, have been reported to exert multiple anti-tumor activities. However, to date the mechanisms of their action have not been completely elucidated. Thus, the aim of this study was to assess the effect of green tea extract on non-small lung cancer A549 cells. A549 cells following treatment with GTE were analyzed using the inverted light and fluorescence microscope. In order to evaluate cell sensitivity and cell death, the MTT assay and Tali image-based cytometer were used, respectively. Ultrastructural alterations were assessed using a transmission electron microscope. The obtained data suggested that GTE, even at the highest dose employed (150 μM), was not toxic to A549 cells. Likewise, the treatment with GTE resulted in only a very small dose-dependent increase in the population of apoptotic cells. However, enhanced accumulation of vacuole-like structures in response to GTE was seen at the light and electron microscopic level. Furthermore, an increase in the acidic vesicular organelles and LC3-II puncta formation was observed under the fluorescence microscope, following GTE treatment. The analysis of the functional status of autophagy revealed that GTE-induced autophagy may provide self-protection against its own cytotoxicity, since we observed that the blockage of autophagy by bafilomycin A1 decreased the viability of A549 cells and potentiated necrotic cell death induction in response to GTE treatment. Collectively, our results revealed that A549 cells are insensitive to both low and high concentrations of the green tea extract, probably due to the induction of cytoprotective autophagy. These data suggest that a potential utility of GTE in lung cancer therapy may lie in its synergistic combinations with drugs or small molecules that target autophagy, rather than in monotherapy.

Green tea extract induces protective autophagy in A549 non-small lung cancer cell line

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Magdalena Izdebska

2015-12-01

Full Text Available Background and objectives: For many decades, polyphenols, including green tea extract catechins, have been reported to exert multiple anti-tumor activities. However, to date the mechanisms of their action have not been completely elucidated. Thus, the aim of this study was to assess the effect of green tea extract on non-small lung cancer A549 cells. Material and methods: A549 cells following treatment with GTE were analyzed using the inverted light and fluorescence microscope. In order to evaluate cell sensitivity and cell death, the MTT assay and Tali image-based cytometer were used, respectively. Ultrastructural alterations were assessed using a transmission electron microscope.Results: The obtained data suggested that GTE, even at the highest dose employed (150 μM, was not toxic to A549 cells. Likewise, the treatment with GTE resulted in only a very small dose-dependent increase in the population of apoptotic cells. However, enhanced accumulation of vacuole-like structures in response to GTE was seen at the light and electron microscopic level. Furthermore, an increase in the acidic vesicular organelles and LC3-II puncta formation was observed under the fluorescence microscope, following GTE treatment. The analysis of the functional status of autophagy revealed that GTE-induced autophagy may provide self-protection against its own cytotoxicity, since we observed that the blockage of autophagy by bafilomycin A1 decreased the viability of A549 cells and potentiated necrotic cell death induction in response to GTE treatment.Conclusion: Collectively, our results revealed that A549 cells are insensitive to both low and high concentrations of the green tea extract, probably due to the induction of cytoprotective autophagy. These data suggest that a potential utility of GTE in lung cancer therapy may lie in its synergistic combinations with drugs or small molecules that target autophagy, rather than in monotherapy.

[Nickel exposure to A549 cell damage and L-ascorbic acid interference effect].

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Fu, Yao; Wang, Yue; Dan, Han; Zhang, Lin; Ma, Wenhan; Pan, Yulin; Wu, Yonghui

2015-05-01

Studying different concentrations of nickel smelting smoke subjects of human lung adenocarcinoma cells (A549) carcinogenic effects, discusses the influence of L-ascorbic acid protection. The A549 cells were divided into experimental and L-ascorbic acid in the intervention group. Plus exposure group concentration of nickel refining dusts were formulated 0.00, 6.25, 12.50, 25.00, 50.00, 100.00 µg/ml suspension, the intervention group on the basis of the added exposure group containing L-ascorbic acid (100 mmol/L), contact 24 h. Detection of cell viability by MTT assay. When the test substance concentration select 0.00, 25.00, 50.00, 100.00 µg/ml experiment for internal Flou-3 fluorescent probe to detect cell Ca²⁺ concentration, within DCFH-DA detect intracellular reactive oxygen (ROS) content, real-time quantitative PCR (real time, in the RT-PCR) was used to detect cell HIF-1α gene expression. With the increase of concentration, subjects increased cell growth inhibition rate, intracellular Ca²⁺ concentration increases, ROS content increased, HIF-1α gene expression increased, differences were statistically significant (P nickel exposure damage to cells. With subjects following exposure to nickel concentration increased, its effect on A549 cell damage increases, L-ascorbic acid cell damage caused by nickel has certain protective effect.

TXNIP mediates the differential responses of A549 cells to sodium butyrate and sodium 4-phenylbutyrate treatment.

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Jin, Xuefang; Wu, Nana; Dai, Juji; Li, Qiuxia; Xiao, XiaoQiang

2017-02-01

Sodium butyrate (NaBu) and sodium 4-phenylbutyrate (4PBA) have promising futures in cancer treatment; however, their underlying molecular mechanisms are not clearly understood. Here, we show A549 cell death induced by NaBu and 4PBA are not the same. NaBu treatment induces a significantly higher level of A549 cell death than 4PBA. A gene expression microarray identified more than 5000 transcripts that were altered (>1.5-fold) in NaBu-treated A549 cells, but fewer than 2000 transcripts that were altered in 4PBA. Moreover, more than 100 cell cycle-associated genes were greatly repressed by NaBu, but slightly repressed by 4PBA; few genes were significantly upregulated only in 4PBA-treated cells. Gene expression was further validated by other experiments. Additionally, A549 cells that were treated with these showed changes in glucose consumption, caspase 3/7 activation and histone modifications, as well as enhanced mitochondrial superoxide production. TXNIP was strongly induced by NaBu (30- to 40-fold mRNA) but was only slightly induced by 4PBA (two to fivefold) in A549 cells. TXNIP knockdown by shRNA in A549 cells significantly attenuated caspase 3/7 activation and restored cell viability, while TXNIP overexpression significantly increased caspase 3/7 activation and cell death only in NaBu-treated cells. Moreover, TXNIP also regulated NaBu- but not 4PBA-induced H4K5 acetylation and H3K4 trimethylation, possibly by increasing WDR5 expression. Finally, we demonstrated that 4PBA induced a mitochondrial superoxide-associated cell death, while NaBu did so mainly through a TXNIP-mediated pathway. The above data might benefit the future clinic application. © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

The Effect of dcEFs on migration behavior of A549 cells and Integrin beta1 expression

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Yunjie WANG

2008-04-01

Full Text Available Background and objective The effect of direct-current electric fields (dcEFs on cells attracted extensive attention. Moreover the metastasis and its potential are considered to be related to dcEFs. The aim is to study the effect of dcEFs on migration behavior of A549 cells, Integrin ?1 and its signal pathways. Methods According to exposure to 5 V/cm dcEFs or not and the time of exposure, the A549 cells were divided into 4 groups. Images were taken per 5 min within 2 h to recode the migration of the cells. The data of results were analyzed statistically. Results Most of A549cells exposed to the dcEFs aligned and elongated perpendicularly to the electric field lines and migrated to the cathode continually during 2 h. On the contrary, cells unexposed to dcEFs showed slightly random movements. Immunofluorescence showed that Integrin ?1 on plasma membrane polarized to the cathode of the dcEFs. Western blot showed that Integrin beta1 downstream signal pathways p-FAK and p-ERK were overexpressed in the dcEFs. Conclusion A549 cells have a galvanotatic feature of cathodal directed migration while exposed to the dcEFs. The polarization of Integrin beta1 and the promotion of its downstream signal pathways may play an important roles in the galvanotaxis of A549 cells.

Curcumin promotes apoptosis in A549/DDP multidrug-resistant human lung adenocarcinoma cells through an miRNA signaling pathway

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Zhang, Jian, E-mail: zhangjian197011@yahoo.com [Department of Respiratory Medicine, Xijing Hospital, The Fourth Military Medical University, Xi' an 710032 (China); Zhang, Tao [Department of Thoracic Surgery, Tangdu Hospital, The Fourth Military Medical University, Xi' an 710038 (China); Ti, Xinyu; Shi, Jieran; Wu, Changgui; Ren, Xinling [Department of Respiratory Medicine, Xijing Hospital, The Fourth Military Medical University, Xi' an 710032 (China); Yin, Hong, E-mail: yinnhong@yahoo.com [The Medical Image Center, Xijing Hospital, The Fourth Military Medical University, Xi' an 710032 (China)

2010-08-13

Research highlights: {yields} Curcumin had anti-cancer effects on A549/DDP multidrug-resistant human lung adenocarcinoma cells {yields} Curcumin promotes apoptosis in A549/DDP cells through a miRNA signaling pathway {yields} Curcumin induces A549/DDP cell apoptosis by downregulating miR-186* {yields} miR-186* may serve as a potential gene therapy target for refractory lung cancer that is sensitive to curcumin -- Abstract: Curcumin extracted from the rhizomes of Curcuma longa L. has been shown to have inhibitory effects on cancers through its anti-proliferative and pro-apoptotic activities. Emerging evidence demonstrates that curcumin can overcome drug resistance to classical chemotherapies. Thus, the mechanisms underlying the anti-tumor activities of curcumin require further study. In our study, we first demonstrated that curcumin had anti-cancer effects on A549/DDP multidrug-resistant human lung adenocarcinoma cells. Further studies showed that curcumin altered miRNA expression; in particular, significantly downregulated the expression of miR-186* in A549/DDP. In addition, transfection of cells with a miR-186* inhibitor promoted A549/DDP apoptosis, and overexpression of miR-186* significantly inhibited curcumin-induced apoptosis in A549/DDP cells. These observations suggest that miR-186* may serve as a potential gene therapy target for refractory lung cancer that is sensitive to curcumin.

Curcumin promotes apoptosis in A549/DDP multidrug-resistant human lung adenocarcinoma cells through an miRNA signaling pathway

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Zhang, Jian; Zhang, Tao; Ti, Xinyu; Shi, Jieran; Wu, Changgui; Ren, Xinling; Yin, Hong

2010-01-01

Research highlights: → Curcumin had anti-cancer effects on A549/DDP multidrug-resistant human lung adenocarcinoma cells → Curcumin promotes apoptosis in A549/DDP cells through a miRNA signaling pathway → Curcumin induces A549/DDP cell apoptosis by downregulating miR-186* → miR-186* may serve as a potential gene therapy target for refractory lung cancer that is sensitive to curcumin -- Abstract: Curcumin extracted from the rhizomes of Curcuma longa L. has been shown to have inhibitory effects on cancers through its anti-proliferative and pro-apoptotic activities. Emerging evidence demonstrates that curcumin can overcome drug resistance to classical chemotherapies. Thus, the mechanisms underlying the anti-tumor activities of curcumin require further study. In our study, we first demonstrated that curcumin had anti-cancer effects on A549/DDP multidrug-resistant human lung adenocarcinoma cells. Further studies showed that curcumin altered miRNA expression; in particular, significantly downregulated the expression of miR-186* in A549/DDP. In addition, transfection of cells with a miR-186* inhibitor promoted A549/DDP apoptosis, and overexpression of miR-186* significantly inhibited curcumin-induced apoptosis in A549/DDP cells. These observations suggest that miR-186* may serve as a potential gene therapy target for refractory lung cancer that is sensitive to curcumin.

Salvianolic acid A reverses cisplatin resistance in lung cancer A549 cells by targeting c-met and attenuating Akt/mTOR pathway

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Xia-li Tang

2017-09-01

Full Text Available Drug resistance is one of the leading causes of chemotherapy failure in non-small cell lung cancer (NSCLC treatment. The purpose of this study was to investigate the role of c-met in human lung cancer cisplatin resistance cell line (A549/DDP and the reversal mechanism of salvianolic acid A (SAA, a phenolic active compound extracted from Salvia miltiorrhiza. In this study, we found that A549/DDP cells exert up-regulation of c-met by activating the Akt/mTOR signaling pathway. We also show that SAA could increase the chemotherapeutic efficacy of cisplatin, suggesting a synergistic effect of SAA and cisplatin. Moreover, we revealed that SAA enhanced sensitivity to cisplatin in A549/DDP cells mainly through suppression of the c-met/AKT/mTOR signaling pathway. Knockdown of c-met revealed similar effects as that of SAA in A549/DDP cells. In addition, SAA effectively prevented multidrug resistance associated protein1 (MDR1 up-regulation in A549/DDP cells. Taken together, our results indicated that SAA suppressed c-met expression and enhanced the sensitivity of lung adenocarcinoma A549 cells to cisplatin through AKT/mTOR signaling pathway.

SOCS3 inhibiting migration of A549 cells correlates with PYK2 signaling in vitro

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Zhang Qingfu

2008-05-01

Full Text Available Abstract Background Suppressor of cytokine signaling 3 (SOCS3 is considered to inhibit cytokine responses and play a negative role in migration of various cells. Proline-rich tyrosine kinase 2 (PYK2 is a non-receptor kinase and has been found crucial to cell motility. However, little is known about whether SOCS3 could regulate PYK2 pro-migratory function in lung cancer. Methods The methylation status of SOCS3 was investigated in HBE and A549 cell lines by methylation-specific PCR. A549 cells were either treated with a demethylation agent 5-aza-2'-deoxycytidine or transfected with three SOCS3 mutants with various functional domains deleted. Besides, cells were pretreated with a proteasome inhibitor β-lactacystin where indicated. The effects of SOCS3 up-regulation on PYK2 expression, PYK2 and ERK1/2 phosphorylations were assessed by western blot using indicated antibodies. RT-PCR was used to estimate PYK2 mRNA levels. Transwell experiments were performed to evaluate cell migration. Results SOCS3 expression was found impaired in A549 cells and higher PYK2 activity was correlated with enhanced cell migration. We identified that SOCS3 was aberrantly methylated in the exon 2, and 5-aza-2'-deoxycytidine restored SOCS3 expression. Reactivation of SOCS3 attenuated PYK2 expression and phosphorylation, cell migration was inhibited as well. Transfection studies indicated that exogenous SOCS3 interacted with PYK2, and both the Src homology 2 (SH2 and the kinase inhibitory region (KIR domains of SOCS3 contributed to PYK2 binding. Furthermore, SOCS3 was found to inhibit PYK2-associated ERK1/2 activity in A549 cells. SOCS3 possibly promoted degradation of PYK2 in a SOCS-box-dependent manner and interfered with PYK2-related signaling events, such as cell migration. Conclusion These data indicate that SOCS3 negatively regulates cell motility and decreased SOCS3 induced by methylation may confer a migration advantage to A549 cells. These results also suggest a

Effects of X-ray irradiation on expression of Pokemon gene in A549 cells of human lung adenocarcinoma

International Nuclear Information System (INIS)

Wang Lu; Zou Yue; Jiang Qisheng; Li Wei; Song Xiujun; Zhou Xiangyan; Wang Cuilan

2011-01-01

Objective: To study the dose-and time-effects of X-ray irradiation on the expression of Pokemon gene in A549 cells of human lung adenocarcinoma. Methods: A549 cells were cultured in vitro and exposed to X-rays with the doses of 2, 4, 6 and 8 Gy, respectively. Untreated A549 cells were used as control group. The relative levels of Pokemon mRNA expression in the cells were detected by using quantitative real-time PCR at 2, 4, 8, 12, 24, 48 and 72 h after irradiation. Results: The Pokemon mRNA expression levels decreased in the early period after irradiation (except 2 and 4 h after irradiation in 2 Gy group) and then increased in the later stage (48 h after irradiation) with significant statistical differences at the most time points in comparison with the control group (t=3.40-154.76, P<0.05). Conclusions: Higher doses of X-rays may degrade the expression of Pokemon mRNA in the human A549 cells and induce apoptosis in the early period, hut also may upgrade its expression in the later period, which might be correlated with the cell cycle regulation and DNA damage repair in the A549 cells. (authors)

Anti-inflammatory effects of embelin in A549 cells and human asthmatic airway epithelial tissues.

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Lee, In-Seung; Cho, Dong-Hyuk; Kim, Ki-Suk; Kim, Kang-Hoon; Park, Jiyoung; Kim, Yumi; Jung, Ji Hoon; Kim, Kwanil; Jung, Hee-Jae; Jang, Hyeung-Jin

2018-02-01

Allergic asthma is the most common type in asthma, which is defined as a chronic inflammatory disease of the lung. In this study, we investigated whether embelin (Emb), the major component of Ardisia japonica BL. (AJB), exhibits anti-inflammatory effects on allergic asthma via inhibition of NF-κB activity using A549 cells and asthmatic airway epithelial tissues. Inflammation was induced in A549 cells, a human airway epithelial cell line, by IL-1β (10 ng/ml) treatment for 4 h. The effects of Emb on NF-κB activity and COX-2 protein expression in inflamed airway epithelial cells and human asthmatic airway epithelial tissues were analyzed via western blot. The secretion levels of NF-κB-mediated cytokines/chemokines, including IL-4, 6, 9, 13, TNF-α and eotaxin, were measured by a multiplex assay. Emb significantly blocked NF-κB activity in IL-1β-treated A549 cells and human asthmatic airway epithelial tissues. COX-2 expression was also reduced in both IL-1β-treated A549 cells and asthmatic tissues Emb application. Emb significantly reduced the secretion of IL-4, IL-6 and eotaxin in human asthmatic airway epithelial tissues by inhibiting activity of NF-κB. The results of this study suggest that Emb may be used as an anti-inflammatory agent via inhibition of NF-κB and related cytokines.

Salvianolic acid A reverses cisplatin resistance in lung cancer A549 cells by targeting c-met and attenuating Akt/mTOR pathway.

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Tang, Xia-Li; Yan, Li; Zhu, Ling; Jiao, De-Min; Chen, Jun; Chen, Qing-Yong

2017-09-01

Drug resistance is one of the leading causes of chemotherapy failure in non-small cell lung cancer (NSCLC) treatment. The purpose of this study was to investigate the role of c-met in human lung cancer cisplatin resistance cell line (A549/DDP) and the reversal mechanism of salvianolic acid A (SAA), a phenolic active compound extracted from Salvia miltiorrhiza. In this study, we found that A549/DDP cells exert up-regulation of c-met by activating the Akt/mTOR signaling pathway. We also show that SAA could increase the chemotherapeutic efficacy of cisplatin, suggesting a synergistic effect of SAA and cisplatin. Moreover, we revealed that SAA enhanced sensitivity to cisplatin in A549/DDP cells mainly through suppression of the c-met/AKT/mTOR signaling pathway. Knockdown of c-met revealed similar effects as that of SAA in A549/DDP cells. In addition, SAA effectively prevented multidrug resistance associated protein1 (MDR1) up-regulation in A549/DDP cells. Taken together, our results indicated that SAA suppressed c-met expression and enhanced the sensitivity of lung adenocarcinoma A549 cells to cisplatin through AKT/mTOR signaling pathway. Copyright © 2017 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

iTRAQ-Based and Label-Free Proteomics Approaches for Studies of Human Adenovirus Infections

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Trinh, Hung V.; Grossmann, Jonas; Gehrig, Peter; Roschitzki, Bernd; Schlapbach, Ralph; Greber, Urs F.; Hemmi, Silvio

2013-01-01

Both isobaric tags for relative and absolute quantitation (iTRAQ) and label-free methods are widely used for quantitative proteomics. Here, we provide a detailed evaluation of these proteomics approaches based on large datasets from biological samples. iTRAQ-label-based and label-free quantitations were compared using protein lysate samples from noninfected human lung epithelial A549 cells and from cells infected for 24 h with human adenovirus type 3 or type 5. Either iTRAQ-label-based or lab...

8-aminoadenosine enhances radiation-induced cell death in human lung carcinoma A549 cells

International Nuclear Information System (INIS)

Meike, Shunsuke; Yamamori, Tohru; Yasui, Hironobu; Eitaki, Masato; Inanami, Osamu; Matsuda, Akira

2011-01-01

The combination of a chemotherapeutic agent and radiation is widely applied to enhance cell death in solid tumor cells in cancer treatment. The purine analogue 8-aminoadenosine (8-NH 2 -Ado) is known to be a transcription inhibitor that has proved very effective in multiple myeloma cell lines and primary indolent leukemia cells. In this report, to examine whether 8-NH 2 -Ado had the ability to enhance the radiation-induced cell killing in solid tumor cells, human lung adenocarcinoma A549 cells were irradiated in the presence and absence of 8-NH 2 -Ado. 8-NH 2 -Ado significantly increased reproductive cell death and apoptosis in A549 cells exposed to X-rays. When peptide inhibitors against caspase-3, -8, and -9 were utilized to evaluate the involvement of caspases, all inhibitors suppressed the enhancement of radiation-induced apoptosis, suggesting that not only mitochondria-mediated apoptotic signal transduction pathways but also death receptor-mediated pathways were involved in this enhancement of apoptosis. In addition, in the cells exposed to the treatment combining X-irradiation and 8-NH 2 -Ado, reduction of the intracellular ATP concentration was essential for survival, and down-regulation of the expression of antiapoptotic proteins such as survivin and X-linked inhibitor of apoptosis protein (XIAP) was observed. These results indicate that 8-NH 2 -Ado has potential not only as an anti-tumor drug for leukemia and lymphoma but also as a radiosensitizing agent for solid tumors. (author)

Integrating cell biology and proteomic approaches in plants.

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Takáč, Tomáš; Šamajová, Olga; Šamaj, Jozef

2017-10-03

Significant improvements of protein extraction, separation, mass spectrometry and bioinformatics nurtured advancements of proteomics during the past years. The usefulness of proteomics in the investigation of biological problems can be enhanced by integration with other experimental methods from cell biology, genetics, biochemistry, pharmacology, molecular biology and other omics approaches including transcriptomics and metabolomics. This review aims to summarize current trends integrating cell biology and proteomics in plant science. Cell biology approaches are most frequently used in proteomic studies investigating subcellular and developmental proteomes, however, they were also employed in proteomic studies exploring abiotic and biotic stress responses, vesicular transport, cytoskeleton and protein posttranslational modifications. They are used either for detailed cellular or ultrastructural characterization of the object subjected to proteomic study, validation of proteomic results or to expand proteomic data. In this respect, a broad spectrum of methods is employed to support proteomic studies including ultrastructural electron microscopy studies, histochemical staining, immunochemical localization, in vivo imaging of fluorescently tagged proteins and visualization of protein-protein interactions. Thus, cell biological observations on fixed or living cell compartments, cells, tissues and organs are feasible, and in some cases fundamental for the validation and complementation of proteomic data. Validation of proteomic data by independent experimental methods requires development of new complementary approaches. Benefits of cell biology methods and techniques are not sufficiently highlighted in current proteomic studies. This encouraged us to review most popular cell biology methods used in proteomic studies and to evaluate their relevance and potential for proteomic data validation and enrichment of purely proteomic analyses. We also provide examples of

The Effect of 5-FU and Radiation on A549 Cells In Vitro

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Lee, Myung Za [Hanyang University College of Medicine, Seoul (Korea, Republic of); Chun, Ha Chong [Medical College of Virgina, Richmond (United States); Lee, Won Young [Yonsei University College of Medicine, Seoul (Korea, Republic of)

1989-06-15

Effects of ionizing radiation alone and combined with chemotherapy on tumor growth and it clonal specificity Monitored by changes in distribution of chromosome number were studies in A549 cell line originated from human adenocarcinoma of the lung. Radiation (300 rad, 600 rad and 900 rad) were delivered with or without 5-FU. Forty eight hours later, 57.5% of growth inhibition of cell was Seen in cells treated with 5-FU concentration of 0.47g/ml for 24 hr exposure. Cell survival carves after radiation with and without 5-FU were made. Chromosomal analysis of cells in metaphase in control, and in cells treated with 300 rad of radiation, or 0.47g/ml of 5-FU treatment, and combined treatment of cloth were 77ne to examine the changes in ploidy and number of chromosome. Radiation combined with 5-FU enhanced growth inhibition of A549 cells. However, no evidence of synergetic effects in growth inhibition was observed in the cells treated with the combination therapy. Pattern of chromosomal distribution of survived cells were shifted from hyperploidy to hypoploidy by single dose of radiation(300 rad). As radiation dose increased a large number of hypoploidy cells were observed. Following treatment of cells with 5-FU, chomosomal distribution of survived cells were also shifted to hypodiploidy, which were seen in cells treated with radiation. The cell treated with 5-FU and followed by radiation within 24 hrs had cell with increased number of hypodiploidy cells. Almost same type of chromosomal changes were reproduced in cells treated with combined treatment with radiation and 5-FU. Minor differences were that cells with fewer number of chromosome were more frequent in cells treated with combined therapy. Further increase in cells of hypoploidy(93%) having 1-10 chromosome were induced by additional radiation. Therefore, the enhanced therapeutic effect of 5-FU combined with radiation of A549 cells appeared to be additive rather than synergistic.

Induction of apoptosis in non-small cell lung carcinoma A549 cells by PGD₂ metabolite, 15d-PGJ₂.

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Wang, Jun-Jie; Mak, Oi-Tong

2011-11-01

PGD2 (prostaglandin D2) is a mediator in various pathophysiological processes, including inflammation and tumorigenesis. PGD2 can be converted into active metabolites and is known to activate two distinct receptors, DP (PGD2 receptor) and CRTH2/DP2 (chemoattractant receptor-homologous molecule expressed on Th2 cells). In the past, PGD2 was thought to be involved principally in the process of inflammation. However, in recent years, several studies have shown that PGD2 has anti-proliferative ability against tumorigenesis and can induce cellular apoptosis via activation of the caspase-dependent pathway in human colorectal cancer cells, leukaemia cells and eosinophils. In the lung, where PGD2 is highly released when sensitized mast cells are challenged with allergen, the mechanism of PGD2-induced apoptosis is unclear. In the present study, A549 cells, a type of NSCLC (non-small cell lung carcinoma), were treated with PGD2 under various conditions, including while blocking DP and CRTH2/DP2 with the selective antagonists BWA868C and ramatroban respectively. We report here that PGD2 induces A549 cell death through the intrinsic apoptotic pathway, although the process does not appear to involve either DP or CRTH2/DP2. Similar results were also found with H2199 cells, another type of NSCLC. We found that PGD2 metabolites induce apoptosis effectively and that 15d-PGJ2 (15-deoxy-Δ12,14-prostaglandin J2) is a likely candidate for the principal apoptotic inducer in PGD2-induced apoptosis in NSCLC A549 cells.

Garcinol from Garcinia indica Downregulates Cancer Stem-like Cell Biomarker ALDH1A1 in Nonsmall Cell Lung Cancer A549 Cells through DDIT3 Activation.

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Wang, Jinhan; Wang, Liwen; Ho, Chi-Tang; Zhang, Kunsheng; Liu, Qiang; Zhao, Hui

2017-05-10

Nonsmall cell lung cancer (NSCLC) is the predominant type of lung cancer. Patients with NSCLC show high mortality rates because of failure to clean up cancer stem cells (CSCs). The anticancer activity of phytochemical garcinol has been identified in various cancer cell models. However, the effect of garcinol on NSCLC cell lines is still lacking. Of the NSCLC cell lines we tested, A549 cells were the most sensitive to garcinol. Interestingly, Aldehyde Dehydrogenase 1 Family Member A1 (ALDH1A1) was preferentially expressed in A549 cells and downregulated by the addition of garcinol. We also found that garcinol enriched DNA damage-inducible transcript 3 (DDIT3) and then altered DDIT3-CCAAT-enhancer-binding proteins beta (C/EBPβ) interaction resulting in a decreased binding of C/EBPβ to the endogenous ALDH1A1 promoter. Furthermore, garcinol's inhibition of ALDH1A1 was identified in a xenograft mice model. Garcinol repressed ALDH1A1 transcription in A549 cells through alterations in the interaction between DDIT3 and C/EBPβ. Garcinol could be a potential dietary phytochemical candidate for NSCLCs patients whose tumors harbored high ALDH1A1 expression.

The total flavonoids of Clerodendrum bungei suppress A549 cells proliferation, migration, and invasion by impacting Wnt/β-Catenin signaling

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Na Yu

2017-01-01

Full Text Available Objectives: The objective of this study is to evaluate the effect of the total flavonoids of Clerodendrum bungei (TFCB on the proliferation, invasion, and metastasis of A549 lung cancer cells through the Wnt signaling pathway. Materials and Methods: A549 cells were transfected with a β-catenin overexpression plasmid and the empty vector pcDNA3.1. The A549 cells were divided into six groups: normal A549 cell group, normal A549 cells with TFCB group, vector control group, vector with TFCB group, β-catenin overexpression group, and β-catenin with TFCB group. We used the 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide assay to detect cell proliferation, a scratch test was used to observe cell migration, and a transwell experiment was employed to evaluate cell invasion. Proteins related to the Wnt pathway were detected with Western blot analysis, including β-catenin, GSK-3 β, P-GSK-3 β, c-Myc, and CyclinD1. Results: The proliferation, invasion, and metastasis of A549 cells were significantly enhanced after being transfected with the β-catenin overexpression plasmid (P < 0.05 or 0.01, accompanied by increased expression of β-catenin, C-Myc, CyclinD1 and reduced expression of Gsk-3 β and P-GSK-3 β. Treatment of cells with TFCB resulted in inhibition of cell proliferation, migration, and invasion; downregulated expression of β-catenin, C-Myc, and CyclinD1; and upregulated expression of GSK-3 β and P-GSK-3 β, especially in the β-catenin overexpression group. Conclusion: TFCB has the potential to inhibit the Wnt/β-catenin pathway by prohibiting the overexpression of β-catenin and regulating its downstream factors.

Mechanisms underlying regulation of cell cycle and apoptosis by hnRNP B1 in human lung adenocarcinoma A549 cells.

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Han, Juan; Tang, Feng-ming; Pu, Dan; Xu, Dan; Wang, Tao; Li, Weimin

2014-01-01

Overexpression of heterogeneous nuclear ribonucleoprotein B1 (hnRNP B1), a nuclear RNA binding protein, has been reported to occur in early-stage lung cancer and in premalignant lesions. DNA-dependent protein kinase (DNA-PK) is known to be involved in the repair of double-strand DNA breaks. Reduced capacity to repair DNA has been associated with the risk of lung cancer. We investigated a link between hnRNP B1 and DNA-PK and their effects on proliferation, cell cycle, and apoptosis in the human lung adenocarcinoma cell line A549. We found that hnRNP B1 and DNA-PK interact with each other in a complex fashion. Reducing hnRNP B1 expression in A549 cells with the use of RNAi led to upregulation of p53 activity through upregulation of DNA-PK activity but without inducing p53 expression. Further, suppression of hnRNP B1 in A549 cells slowed cell proliferation, promoted apoptosis, and induced cell cycle arrest at the G1 stage. The presence of NU7026 reduced the arrest of cells at the G1 stage and reduced the apoptosis rate while promoting cell growth. Taken together, our results demonstrate that by regulating DNA-PK activity, hnRNP B1 can affect p53-mediated cell cycle progression and apoptosis, resulting in greater cell survival and subsequent proliferation.

Dioscorin protects tight junction protein expression in A549 human airway epithelium cells from dust mite damage.

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Fu, Lin Shien; Ko, Ying Hsien; Lin, Kuo Wei; Hsu, Jeng Yuan; Chu, Jao Jia; Chi, Chin Shiang

2009-12-01

In addition to being an allergen, the trypsin activity of dust mite extract also destroys the tight junctions of bronchial epithelium. Such damage can lead to airway leakage, which increases airway exposure to allergens, irritants, and other pathogens. Dioscorin, the storage protein of yam, demonstrates anti-trypsin activity, as well as other potential anti-inflammatory effects. This study investigated the protective role of dioscorin for tight junctions. The immunofluorescence stains of zonula occludens (ZO-1), E-cadherin (EC) and desmoplakin (DP) proteins were compared. A cultured A549 cell line was used as a control and A549 cells were incubated with mite extract 100 mg/mL for 16 h, with or without dioscorin 100 mg/mL pretreatment for 8 h and with dioscorin 100 mg/mL alone for 16 h. Western blot was performed to detect changes in ZO-1, EC, and DP in the treated A549 cell lines. Loss of tight junction protein expression (ZO-1, EC, DP) was demonstrated after 16-h mite extract incubation. The defect could be restored if cells were pretreated with dioscorin for 8 h. In addition, dioscorin did not cause damage to the A549 cell lines in terms of cell survival or morphology. Western blot showed no change in the amount of tight junction protein under various conditions. Dioscorin is a potential protector of airway damage caused by mite extract.

ROS and NF-κB are involved in upregulation of IL-8 in A549 cells exposed to multi-walled carbon nanotubes

International Nuclear Information System (INIS)

Ye Shefang; Wu Yihui; Hou Zhenqing; Zhang Qiqing

2009-01-01

Carbon nanotubes (CNTs) have potential applications in biosensors, tissue engineering, and biomedical devices because of their unique physico-chemical, electronic and mechanical properties. However, there is limited literature data available concerning the biological properties and toxicity of CNTs. This study aimed to assess the toxicity exhibited by multi-walled CNTs (MWCNTs) and to elucidate possible molecular mechanisms underlying the biological effects of MWCNTs in A549 cells. Exposing A549 cells to MWCNTs led to cell death, changes in cell size and complexity, reactive oxygen species (ROS) production, interleukin-8 (IL-8) gene expression and nuclear factor (NF)-κB activation. Treatment of A549 cells with antioxidants prior to adding MWCNTs decreased ROS production and abrogated expression of IL-8 mRNA. Pretreatment of A549 cells with NF-κB inhibitors suppressed MWCNTs-induced IL-8 mRNA expression. These results indicate that MWCNTs are able to induce expression of IL-8 in A549 cells, at least in part, mediated by oxidative stress and NF-κB activation.

Protocol for Lipid-Mediated Transient Transfection in A549 Epithelial Lung Cell Line.

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Marcos-Vadillo, Elena; García-Sánchez, Asunción

2016-01-01

Trials of transfection in eukaryotic cells are essential tools for the study of gene and protein function. They have been used in a wide range of research fields. In this chapter, a method of transient transfection of the A549 cell line, human lung cells of alveolar epithelium, with an expression plasmid is described. In addition, the fundamental characteristics of this experimental procedure are addressed.

Apoptosis of human lung adenocarcinoma A549 cells induced by prodigiosin analogue obtained from an entomopathogenic bacterium Serratia marcescens.

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Zhou, Wei; Jin, Zhi-Xiong; Wan, Yong-Ji

2010-12-01

An entomopathogenic bacterial strain SCQ1 was isolated from silkworm (Bombyx mori) and identified as Serratia marcescens via 16S rRNA gene analysis. This strain produces a red pigment that causes acute septicemia of silkworm. The red pigment of strain SCQ1 was identified as prodigiosin analogue (PGA) with various reported biological activities. In this study, we found that low concentration of PGA showed significant anticancer activity in human lung adenocarcinoma A549 cells, but has little effect in human bone marrow stem cells, in vitro. By exposure to different concentrations of PGA for 24 h, morphological changes and the MTT assay showed that A549 cell line was very sensitive to PGA, with IC(50) value about 2.2 mg/L. Early stage of apoptosis was detected by flow cytometry while A549 cells were treated with PGA for 4 and 12 h, respectively. The proportion of dead cells was increased with treatment time or the concentrations of PGA, but it was inversely proportional to that of apoptotic cells. These results indicate that PGA obtained from strain SCQ1 induces apoptosis in A549 cells, but the molecular mechanisms of cell death are complicated, and the S. marcescens strain SCQ1 may serve as a source of the anticancer compound, PGA.

Oxidative stress and inflammatory response to printer toner particles in human epithelial A549 lung cells.

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Könczöl, Mathias; Weiß, Adilka; Gminski, Richard; Merfort, Irmgard; Mersch-Sundermann, Volker

2013-02-04

Reports on adverse health effects related to occupational exposure to toner powder are still inconclusive. Therefore, we have previously conducted an in vitro-study to characterize the genotoxic potential of three commercially available black printer toner powders in A549 lung cells. In these cell-based assays it was clearly demonstrated that the tested toner powders damage DNA and induce micronucleus (MN) formation. Here, we have studied the cytotoxic and proinflammatory potential of these three types of printer toner particles and the influence of ROS and NF-κB induction in order to unravel the underlying mechanisms. A549 cells were exposed to various concentrations of printer toner particle suspensions for 24 h. The toner particles were observed to exert significant cytotoxic effects in the WST-1 and neutral red (NR)-assays, although to a varying extent. Caspase 3/7 activity increased, while the mitochondrial membrane potential (MMP) was not affected. Particles of all three printer toner powders induced concentration-dependent formation of reactive oxygen species (ROS), as measured in the DCFH-DA assay. Furthermore, toner particle exposure enhanced interleukin-6 and interleukin-8 production, which is in agreement with activation of the transcription factor NF-κB in A549 cells shown by the electrophoretic mobility shift assay (EMSA). Therefore, it can be concluded that exposure of A549 lung cells to three selected printer toner powders caused oxidative stress through induction of ROS. Increased ROS formation may trigger genotoxic effects and activate proinflammatory pathways. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Curcumin Promoted the Apoptosis of Cisplain-resistant Human Lung Carcinoma Cells A549/DDP through Down-regulating miR-186*

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Jian ZHANG

2010-04-01

Full Text Available Background and objective Curcumin, a natural compound, is derived from the rthizom of Curcuma longa. In vitro and in vivo preclinical studies have shown its anti-inflammatory, antioxidant, anticancer activities and so on. miR-186*, which was found by microarray technology, was highly expressed in lung carcinoma cells A549/DDP. The aim of this study is to illustrate whether Curcumin could promote the apoptosis of A549/DDP cells through regulating the expression of miR-186*. Methods An oligonucleotide microarray chip was used to profile microRNA (miRNA expressions in A549/DDP cells treated with and without Curcumin. The significantly differentially expressed miRNA, which was selected from microarray chip, validated by quantitative real-time PCR. Ultimately, the remarkably expressed miRNA modulated the apoptosis assaying by flow cytometry expriments and the survival rate was measured by MTT method. Results The microarray chip results demonstrated: Curcumin altered the expression level of miRNAs compared with untreated control in A549/DDP cell line, miR-186* was significantly down-regulated after Curcumin treatment, which confirmed by quantitative real-time PCR. Downregulation of miR-186* expression by curcumin elevated the apoptosis, and the survival rate of A549/DDP cells decreased; but up-regulation of miR-186* expression by transfection its mimics restrained the apoptosis, the survival rate of A549/DDP cells increased, which were assayed by flow cytometry expriments and MTT method. Conclusion Modulation of miRNAs expression may be an important mechanism underlying the biological roles of Curcumin.

Heat-modified citrus pectin induces apoptosis-like cell death and autophagy in HepG2 and A549 cancer cells.

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Leclere, Lionel; Fransolet, Maude; Cote, Francois; Cambier, Pierre; Arnould, Thierry; Van Cutsem, Pierre; Michiels, Carine

2015-01-01

Cancer is still one of the leading causes of death worldwide, and finding new treatments remains a major challenge. Previous studies showed that modified forms of pectin, a complex polysaccharide present in the primary plant cell wall, possess anticancer properties. Nevertheless, the mechanism of action of modified pectin and the pathways involved are unclear. Here, we show that citrus pectin modified by heat treatment induced cell death in HepG2 and A549 cells. The induced cell death differs from classical apoptosis because no DNA cleavage was observed. In addition, Z-VAD-fmk, a pan-caspase inhibitor, did not influence the observed cell death in HepG2 cells but appeared to be partly protective in A549 cells, indicating that heat-modified citrus pectin might induce caspase-independent cell death. An increase in the abundance of the phosphatidylethanolamine-conjugated Light Chain 3 (LC3) protein and a decrease in p62 protein abundance were observed in both cell types when incubated in the presence of heat-modified citrus pectin. These results indicate the activation of autophagy. To our knowledge, this is the first time that autophagy has been revealed in cells incubated in the presence of a modified form of pectin. This autophagy activation appears to be protective, at least for A549 cells, because its inhibition with 3-methyladenine increased the observed modified pectin-induced cytotoxicity. This study confirms the potential of modified pectin to improve chemotherapeutic cancer treatments.

Heat-modified citrus pectin induces apoptosis-like cell death and autophagy in HepG2 and A549 cancer cells.

Directory of Open Access Journals (Sweden)

Lionel Leclere

Full Text Available Cancer is still one of the leading causes of death worldwide, and finding new treatments remains a major challenge. Previous studies showed that modified forms of pectin, a complex polysaccharide present in the primary plant cell wall, possess anticancer properties. Nevertheless, the mechanism of action of modified pectin and the pathways involved are unclear. Here, we show that citrus pectin modified by heat treatment induced cell death in HepG2 and A549 cells. The induced cell death differs from classical apoptosis because no DNA cleavage was observed. In addition, Z-VAD-fmk, a pan-caspase inhibitor, did not influence the observed cell death in HepG2 cells but appeared to be partly protective in A549 cells, indicating that heat-modified citrus pectin might induce caspase-independent cell death. An increase in the abundance of the phosphatidylethanolamine-conjugated Light Chain 3 (LC3 protein and a decrease in p62 protein abundance were observed in both cell types when incubated in the presence of heat-modified citrus pectin. These results indicate the activation of autophagy. To our knowledge, this is the first time that autophagy has been revealed in cells incubated in the presence of a modified form of pectin. This autophagy activation appears to be protective, at least for A549 cells, because its inhibition with 3-methyladenine increased the observed modified pectin-induced cytotoxicity. This study confirms the potential of modified pectin to improve chemotherapeutic cancer treatments.

LW6, a hypoxia-inducible factor 1 inhibitor, selectively induces apoptosis in hypoxic cells through depolarization of mitochondria in A549 human lung cancer cells.

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Sato, Mariko; Hirose, Katsumi; Kashiwakura, Ikuo; Aoki, Masahiko; Kawaguchi, Hideo; Hatayama, Yoshiomi; Akimoto, Hiroyoshi; Narita, Yuichiro; Takai, Yoshihiro

2015-09-01

Hypoxia‑inducible factor 1 (HIF‑1) activates the transcription of genes that act upon the adaptation of cancer cells to hypoxia. LW6, an HIF‑1 inhibitor, was hypothesized to improve resistance to cancer therapy in hypoxic tumors by inhibiting the accumulation of HIF‑1α. A clear anti‑tumor effect under low oxygen conditions would indicate that LW6 may be an improved treatment strategy for cancer in hypoxia. In the present study, the HIF‑1 inhibition potential of LW6 on the growth and apoptosis of A549 lung cancer cells in association with oxygen availability was evaluated. LW6 was observed to inhibit the expression of HIF‑1α induced by hypoxia in A549 cells at 20 mM, independently of the von Hippel‑Lindau protein. In addition, at this concentration, LW6 induced hypoxia‑selective apoptosis together with a reduction in the mitochondrial membrane potential. The intracellular reactive oxygen species levels increased in LW6‑treated hypoxic A549 cells and LW6 induced a hypoxia‑selective increase of mitochondrial O2•‑. In conclusion, LW6 inhibited the growth of hypoxic A549 cells by affecting the mitochondria. The inhibition of the mitochondrial respiratory chain is suggested as a potentially effective strategy to target apoptosis in cancer cells.

Proteomic Analysis of Chinese Hamster Ovary Cells

DEFF Research Database (Denmark)

Baycin-Hizal, Deniz; Tabb, David L.; Chaerkady, Raghothama

2012-01-01

To complement the recent genomic sequencing of Chinese hamster ovary (CHO) cells, proteomic analysis was performed on CHO cells including the cellular proteome, secretome, and glycoproteome using tandem mass spectrometry (MS/MS) of multiple fractions obtained from gel electrophoresis, multidimens......To complement the recent genomic sequencing of Chinese hamster ovary (CHO) cells, proteomic analysis was performed on CHO cells including the cellular proteome, secretome, and glycoproteome using tandem mass spectrometry (MS/MS) of multiple fractions obtained from gel electrophoresis...

Portulaca oleracea Seed Oil Exerts Cytotoxic Effects on Human Liver Cancer (HepG2) and Human Lung Cancer (A-549) Cell Lines.

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Al-Sheddi, Ebtesam Saad; Farshori, Nida Nayyar; Al-Oqail, Mai Mohammad; Musarrat, Javed; Al-Khedhairy, Abdulaziz Ali; Siddiqui, Maqsood Ahmed

2015-01-01

Portulaca oleracea (Family: Portulacaceae), is well known for its anti-inflammatory, antioxidative, anti- bacterial, and anti-tumor activities. However, cytotoxic effects of seed oil of Portulaca oleracea against human liver cancer (HepG2) and human lung cancer (A-549) cell lines have not been studied previously. Therefore, the present study was designed to investigate the cytotoxic effects of Portulaca oleracea seed oil on HepG2 and A-549 cell lines. Both cell lines were exposed to various concentrations of Portulaca oleracea seed oil for 24h. After the exposure, percentage cell viability was studied by (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT), neutral red uptake (NRU) assays, and cellular morphology by phase contrast inverted microscopy. The results showed a concentration-dependent significant reduction in the percentage cell viability and an alteration in the cellular morphology of HepG2 and A-549 cells. The percentage cell viability was recorded as 73%, 63%, and 54% by MTT assay and 76%, 61%, and 50% by NRU assay at 250, 500, and 1000 μg/ml, respectively in HepG2 cells. Percentage cell viability was recorded as 82%, 72%, and 64% by MTT assay and 83%, 68%, and 56% by NRU assay at 250, 500, and 1000 μg/ml, respectively in A-549 cells. The 100 μg/ml and lower concentrations were found to be non cytotoxic to A-549 cells, whereas decrease of 14% and 12% were recorded by MTT and NRU assay, respectively in HepG2 cells. Both HepG2 and A-549 cell lines exposed to 250, 500, and 1000 μg/ ml of Portulaca oleracea seed oil lost their normal morphology, cell adhesion capacity, become rounded, and appeared smaller in size. The data from this study showed that exposure to seed oil of Portulaca oleracea resulted in significant cytotoxicity and inhibition of growth of the human liver cancer (HepG2) and human lung cancer (A-549) cell lines.

Inhibition of lung cancer cells A549 and H460 by curcuminoid extracts and nanoemulsions prepared from Curcuma longa Linnaeus.

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Chang, Hong-Bin; Chen, Bing-Huei

2015-01-01

The objectives of this study were to explore the inhibition mechanism of lung cancer cells A549 and H460 by curcuminoid extracts and nanoemulsions prepared from Curcuma longa Linnaeus. In addition, human bronchus epithelial cell line BEAS-2B (normal cell) was selected for comparison. A high-performance liquid chromatography (HPLC) method was developed to separate and quantify the various curcuminoids in C. longa extract, including curcumin (1,714.5 μg/mL), demethoxycurcumin (1,147.4 μg/mL), and bisdemethoxycurcumin (190.2 μg/mL). A high-stability nanoemulsion composed of Tween 80, water, and curcuminoid extract was prepared, with mean particle size being 12.6 nm. The cell cycle was retarded at G2/M for both the curcuminoid extract and nanoemulsion treatments; however, the inhibition pathway may be different. H460 cells were more susceptible to apoptosis than A549 cells for both curcuminoid extract and nanoemulsion treatments. Growth of BEAS-2B remained unaffected for both the curcuminoid extract and nanoemulsion treatments, with a concentration range from 1 to 4 μg/mL. Also, the activities of caspase-3, caspase-8, and caspase-9 followed a dose-dependent increase for both A549 and H460 cells for both the treatments, accompanied by a dose-dependent increase in cytochrome C expression and a dose-dependent decrease in CDK1 expression. Interestingly, a dose-dependent increase in cyclin B expression was shown for A549 cells for both the treatments, while a reversed trend was found for H460 cells. Both mitochondria and death receptor pathways may be responsible for apoptosis of both A549 and H460 cells.

Factors involved in depletion of glutathione from A549 human lung carcinoma cells: implications for radiotherapy

International Nuclear Information System (INIS)

Biaglow, J.E.; Varnes, M.E.; Epp, E.R.; Clark, E.P.

1984-01-01

The rate of GSH resynthesis has been measured in plateau phase cultures of A549 human lung carcinoma cells subjected to a fresh medium change. Buthionine sulfoximine (BSO) blocks this resynthesis. Diethyl maleate (DEM) causes a decrease in accumulation of GSH. If DEM is added concurrently with BSO there is a rapid decline in GSH that is maximal in the presence of 0.5 mM DEM. GSH depletion rapidly occurs when BSO is added to log phase cultures which initially are higher in GSH content. Twenty-four hr treatment of A549 cells with BSO results in cells that are more radiosensitive in air and show a slight hypoxic radiation response. A 2 hr treatment with DEM results in some hypoxic sensitization and little increase in the aerobic radiation response. Cells treated simultaneously with BSO + DEM show little increase in the hypoxic radiation response, compared to DEM alone, but are more sensitive under aerobic conditions. Decreased cell survival for aerobically irradiated log phase A549 cells occurs within minutes after addition of a mixture of BSO + DEM. The authors suggest that the enhanced aerobic radiation response is related to an inability of GSH depleted cells to inactivate either peroxy radicals or hydroperoxides that may be produced during irradiation of BSO treated cells. Furthermore, enhancement of the aerobic radiation response may be useful in vivo if normal tissue responses are not also increased

Depleted aldehyde dehydrogenase 1A1 (ALDH1A1) reverses cisplatin resistance of human lung adenocarcinoma cell A549/DDP.

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Wei, Yunyan; Wu, Shuangshuang; Xu, Wei; Liang, Yan; Li, Yue; Zhao, Weihong; Wu, Jianqing

2017-01-01

Cisplatin is the standard first-line chemotherapeutic agent for the treatment of non-small cell lung cancer (NSCLC). However, resistance to chemotherapy has been a major obstacle in the management of NSCLC. Aldehyde dehydrogenase 1A1 (ALDH1A1) overexpression has been observed in a variety of cancers, including lung cancer. The purpose of this study was to investigate the effect of ALDH1A1 expression on cisplatin resistance and explore the mechanism responsible. Reverse transcriptase-PCR was applied to measure the messenger RNA expression of ALDH1A1, while Western blot assay was employed to evaluate the protein expression of ALDH1A1, B-cell lymphoma 2, Bcl-2-like protein 4, phospho-protein kinase B (p-AKT) and AKT. A short hairpin RNA was used to knockdown ALDH1A1 expression. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to determine the effect of ALDH1A1 decrease on cell viability. The cell apoptotic rate was tested using flow cytometry assay. ALDH1A1 is overexpressed in cisplatin resistant cell line A549/DDP, compared with A549. ALDH1A1 depletion significantly decreased A549/DDP proliferation, increased apoptosis, and reduced cisplatin resistance. In addition, the phosphoinositide 3-kinase (PI3K) / AKT pathway is activated in A549/DDP, and ALDH1A1 knockdown reduced the phosphorylation level of AKT. Moreover, the combination of ALDH1A1-short hairpin RNA and PI3K/AKT pathway inhibitor LY294002 markedly inhibited cell viability, enhanced apoptotic cell death, and increased cisplatin sensitivity. These results suggest that ALDH1A1 depletion could reverse cisplatin resistance in human lung cancer cell line A549/DDP, and may act as a potential target for the treatment of lung cancers resistant to cisplatin. © 2016 The Authors. Thoracic Cancer published by China Lung Oncology Group and John Wiley & Sons Australia, Ltd.

Effects of exogenous IL-37 on the biological characteristics of human lung adenocarcinoma A549 cells and the chemotaxis of regulatory T cells.

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Chen, Yu-Hua; Zhou, Bi-Yun; Wu, Guo-Cai; Liao, De-Quan; Li, Jing; Liang, Si-Si; Wu, Xian-Jin; Xu, Jun-Fa; Chen, Yong-Hua; Di, Xiao-Qing; Lin, Qiong-Yan

2018-02-14

This study aims to investigate the effects of exogenous interleukin (IL)-37 on the biological characteristics of human lung adenocarcinoma A549 cells and the chemotaxis of regulatory T (Treg) cells. After isolating the CD4+ CD25+ Treg cells from the peripheral blood, flow cytometry was used to detect the purity of the Treg cells. A549 cells were divided into blank (no transfection), empty plasmid (transfection with pIRES2-EGFP empty plasmid) or IL-37 group (transfection with pIRES2-EGFP-IL-37 plasmid). RT-PCR was used to detect mRNA expression of IL-37 and ELISA to determine IL-37 and MMP-9 expressions. Western blotting was applied to detect the protein expressions of PCNA, Ki-67, Cyclin D1, CDK4, cleaved caspase-3 and cleaved caspase-9. MTT assay, flow cytometry, scratch test and transwell assay were performed to detect cell proliferation, cycle, apoptosis, migration and invasion. Effect of exogenous IL-37 on the chemotaxis of Treg cells was measured through transwell assay. Xenograft models in nude mice were eastablished to detect the impact of IL-37 on A549 cells. The IL-37 group had a higher IL-37 expression, cell apoptosis in the early stage and percentage of cells in the G0/G1 phase than the blank and empty plasmid groups. The IL-37 group had a lower MMP-9 expression, optical density (OD), percentage of cells in the S and G2/M phases, migration, invasion and chemotaxis of CD4+CD25+ Foxp3+ Treg cells. The xenograft volume and weight of nude mice in the IL-37 group were lower than those in the blank and empty plasmid groups. Compared with the blank and empty plasmid groups, the IL-37 group had significantly reduced expression of PCNA, Ki-67, Cyclin D1 and CDK4 but elevated expression of cleaved caspase-3 and cleaved caspase-9. Therefore, exogenous IL-37 inhibits the proliferation, migration and invasion of human lung adenocarcinoma A549 cells as well as the chemotaxis of Treg cells while promoting the apoptosis of A549 cells.

The role of reactive oxygen species (ROS) production on diallyl disulfide (DADS) induced apoptosis and cell cycle arrest in human A549 lung carcinoma cells

International Nuclear Information System (INIS)

Wu Xinjiang; Kassie, Fekadu; Mersch-Sundermann, Volker

2005-01-01

Diallyl disulfide (DADS), an oil soluble constituent of garlic (Allium sativum), has been reported to cause antimutagentic and anticarcinogenic effects in vitro and in vivo by modulating phases I and II enzyme activities. In recent years, several studies suggested that the chemopreventive effects of DADS can also be attributed to induction of cell cycle arrest and apoptosis in cancer cells. In the present study, we reported that DADS-induced cell cycle arrest at G2/M and apoptosis in human A549 lung cancer cells in a time- and dose-dependent manner. Additionally, a significant increase of intracellular reactive oxygen species (ROS) was induced in A549 cells less than 0.5 h after DADS treatment, indicating that ROS may be an early event in DADS-modulated apoptosis. Treatment of A549 cells with N-acetyl cysteine (NAC) completely abrogated DADS-induced cell cycle arrest and apoptosis. The result indicated that oxidative stress modulates cell proliferation and cell death induced by DADS

The role of reactive oxygen species (ROS) production on diallyl disulfide (DADS) induced apoptosis and cell cycle arrest in human A549 lung carcinoma cells

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Wu Xinjiang [Institute of Indoor and Environmental Toxicology, Faculty of Medicine, Justus-Liebig-University of Giessen, Aulweg 123, D-35385 Giessen (Germany); Kassie, Fekadu [Institute of Indoor and Environmental Toxicology, Faculty of Medicine, Justus-Liebig-University of Giessen, Aulweg 123, D-35385 Giessen (Germany); Mersch-Sundermann, Volker [Institute of Indoor and Environmental Toxicology, Faculty of Medicine, Justus-Liebig-University of Giessen, Aulweg 123, D-35385 Giessen (Germany)]. E-mail: Volker.mersch-sundermann@uniklinikum-giessen.de

2005-11-11

Diallyl disulfide (DADS), an oil soluble constituent of garlic (Allium sativum), has been reported to cause antimutagentic and anticarcinogenic effects in vitro and in vivo by modulating phases I and II enzyme activities. In recent years, several studies suggested that the chemopreventive effects of DADS can also be attributed to induction of cell cycle arrest and apoptosis in cancer cells. In the present study, we reported that DADS-induced cell cycle arrest at G2/M and apoptosis in human A549 lung cancer cells in a time- and dose-dependent manner. Additionally, a significant increase of intracellular reactive oxygen species (ROS) was induced in A549 cells less than 0.5 h after DADS treatment, indicating that ROS may be an early event in DADS-modulated apoptosis. Treatment of A549 cells with N-acetyl cysteine (NAC) completely abrogated DADS-induced cell cycle arrest and apoptosis. The result indicated that oxidative stress modulates cell proliferation and cell death induced by DADS.

Effects of X-rays on CC-chemokine receptor 7 expression in human lung cancer A549 cells

International Nuclear Information System (INIS)

Wang Cuilan; Jiang Qisheng; Zou Yue; Li Fengsheng; Li Wei; Song Xiujun; He Rui; Wang Lu

2011-01-01

Objective: To study the effects of X-ray radiation on CC-chemokine receptor 7 (CCR7) expression in human non-small cell lung cancer (NSCLC) cells. Methods: Human adenocarcinoma cells of the line A549 were cultured and irradiated by X-ray at the absorbed doses of 2, 4, 6, and 8 Gy respectively by linear accelerator (with the source skin distance of 100 cm and dose rate of 442.89 cGy/min). The relative levels of CCR7 mRNA and protein expression in the A549 cells were respectively detected by real time-PCR and Western blotting 4, 12, 24, 48, and 72 h after radiation.Untreated A549 cells were used as control group. Results: The expression levels of CCR7 mRNA and protein in the A549 cells began to increase since 4 h after radiation and then decreased gradually after they reached the peak. The CCR7 mRNA expression levels 72 h after radiation of the 6 and 8 Gy groups were still significantly higher than those of the control group (t=6.75-7.26, both P<0.01), and the CCR7 protein expression levels of the 2 and 6 Gy group were still significantly higher than those of the control group (t=11.13-14.17, both P<0.01). Then the CCR7 protein expression levels of the 4 and 8 Gy groups decreased to the control group level 48 and 72 h after radiation respectively. Conclusions: The CCR7 mRNA and protein expression levels in the NSCLC cells increase after X-ray irradiation,which may be correlated with the promotion of proliferation and metastasis of NSCLC cells by X-ray irradiation at a certain dose. (authors)

Enhanced production of nitric oxide in A549 cells through activation of TRPA1 ion channel by cold stress.

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Sun, Wenwu; Wang, Zhonghua; Cao, Jianping; Wang, Xu; Han, Yaling; Ma, Zhuang

2014-08-31

The respiratory epithelium is exposed to the external environment, and inhalation of cold air is common during the season of winter. In addition, the lung is a major source of nitric oxide (NO). However, the effect of cold stress on the production of NO is still unclear. In the present work, We measured the change of NO in single cell with DACF-DA and the change in cytosolic Ca(2+) concentration ([Ca(2+)]c) in A549 cell. We observed that cold stress (from 20 °C to 5 °C) induced an increase of NO in A549 cell, which was completely abolished by applying an extracellular Ca(2+) free medium. Further experiments showed that cold-sensing transient receptor potential subfamily member 1 (TRPA1) channel agonist (allyl isothiocyanate, AITC) increased the production of NO and the level of [Ca(2+)]c in A549 cell. Additionally, TRPA1 inhibitor, Ruthenium red (RR) and camphor, significantly blocked the enhanced production of NO and the rise of [Ca(2+)]c induced by AITC or cold stimulation, respectively. Taken together, these data indicated that cold-induced TRPA1 activation was responsible for the enhanced production of NO in A549 cell. Copyright © 2014 Elsevier Inc. All rights reserved.

Ghrelin promotes human non-small cell lung cancer A549 cell proliferation through PI3K/Akt/mTOR/P70S6K and ERK signaling pathways.

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Zhu, Jianhua; Yao, Jianfeng; Huang, Rongfu; Wang, Yueqin; Jia, Min; Huang, Yan

2018-04-06

Ghrelin is a gastric acyl-peptide that plays an important role in cell proliferation. In the present study, we explored the role of ghrelin in A549 cell proliferation and the possible molecular mechanisms. We found that ghrelin promotes A549 cell proliferation, knockdown of the growth hormone secretagogue receptor (GHSR) attenuated A549 cell proliferation caused by ghrelin. Ghrelin induced the rapid phosphorylation of phosphatidylinositol 3-kinase (PI3K), Akt, ERK, mammalian target of rapamycin (mTOR) and P70S6K. PI3K inhibitor (LY 294002), ERK inhibitor (PD98059) and mTOR inhibitor (Rapamycin) inhibited ghrelin-induced A549 cell proliferation. Moreover, GHSR siRNA inhibited phosphorylation of PI3K, Akt, ERK, mTOR and P70S6K induced by ghrelin. Akt and mTOR/P70S6K phosphorylation was inhibited by LY 294002 but not by PD98059. These results indicate that ghrelin promotes A549 cell proliferation via GHSR-dependent PI3K/Akt/mTOR/P70S6K and ERK signaling pathways. Copyright © 2018 Elsevier Inc. All rights reserved.

Andrographolide down-regulates hypoxia-inducible factor-1{alpha} in human non-small cell lung cancer A549 cells

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Lin, Hui-Hsuan [School of Medical Laboratory and Biotechnology, Chung Shan Medical University, Taichung, Taiwan (China); Department of Medical Research, Chung Shan Medical University Hospital, Taichung, Taiwan (China); Tsai, Chia-Wen [Department of Nutrition, China Medical University, Taichung, Taiwan (China); Chou, Fen-Pi [Institute of Biochemistry and Biotechnology, Chung Shan Medical University, Taichung, Taiwan (China); Wang, Chau-Jong [Department of Medical Research, Chung Shan Medical University Hospital, Taichung, Taiwan (China); Institute of Biochemistry and Biotechnology, Chung Shan Medical University, Taichung, Taiwan (China); Hsuan, Shu-Wen [Department of Medical Laboratory Science and Biotechnology, College of Medicine and Life Science, Chung Hwa University of Medical Technology, No.89, Wen Hwa 1st St., Rende Shiang, Tainan County 717, Taiwan (China); Wang, Cheng-Kun [E-Chyun Dermatology Clinic, No.70, Sec. 3, Jhonghua E. Rd., East District, Tainan, Taiwan (China); Chen, Jing-Hsien [Department of Medical Laboratory Science and Biotechnology, College of Medicine and Life Science, Chung Hwa University of Medical Technology, No.89, Wen Hwa 1st St., Rende Shiang, Tainan County 717, Taiwan (China)

2011-02-01

Andrographolide (Andro), a diterpenoid lactone isolated from a traditional herbal medicine Andrographis paniculata, is known to possess multiple pharmacological activities. In our previous study, Andro had been shown to inhibit non-small cell lung cancer (NSCLC) A549 cell migration and invasion via down-regulation of phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. Here we demonstrated that Andro inhibited the expression of hypoxia-inducible factor-1{alpha} (HIF-1{alpha}) in A549 cells. HIF-1{alpha} plays an important role in tumor growth, angiogenesis and lymph node metastasis of NSCLC. The Andro-induced decrease of cellular protein level of HIF-1{alpha} was correlated with a rapid ubiquitin-dependent degradation of HIF-1{alpha}, and was accompanied by increased expressions of hydroxyl-HIF-1{alpha} and prolyl hydroxylase (PHD2), and a later decrease of vascular endothelial growth factor (VEGF) upon the treatment of Andro. The Andro-inhibited VEGF expression appeared to be a consequence of HIF-1{alpha} inactivation, because its DNA binding activity was suppressed by Andro. Molecular data showed that all these effects of Andro might be mediated via TGF{beta}1/PHD2/HIF-1{alpha} pathway, as demonstrated by the transfection of TGF{beta}1 overexpression vector and PHD2 siRNA, and the usage of a pharmacological MG132 inhibitor. Furthermore, we elucidated the involvement of Andro in HIF-1{alpha} transduced VEGF expression in A549 cells and other NSCLC cell lines. In conclusion, these results highlighted the potential effects of Andro, which may be developed as a chemotherapeutic or an anti-angiogenesis agent for NSCLC in the future.

Andrographolide down-regulates hypoxia-inducible factor-1α in human non-small cell lung cancer A549 cells

International Nuclear Information System (INIS)

Lin, Hui-Hsuan; Tsai, Chia-Wen; Chou, Fen-Pi; Wang, Chau-Jong; Hsuan, Shu-Wen; Wang, Cheng-Kun; Chen, Jing-Hsien

2011-01-01

Andrographolide (Andro), a diterpenoid lactone isolated from a traditional herbal medicine Andrographis paniculata, is known to possess multiple pharmacological activities. In our previous study, Andro had been shown to inhibit non-small cell lung cancer (NSCLC) A549 cell migration and invasion via down-regulation of phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. Here we demonstrated that Andro inhibited the expression of hypoxia-inducible factor-1α (HIF-1α) in A549 cells. HIF-1α plays an important role in tumor growth, angiogenesis and lymph node metastasis of NSCLC. The Andro-induced decrease of cellular protein level of HIF-1α was correlated with a rapid ubiquitin-dependent degradation of HIF-1α, and was accompanied by increased expressions of hydroxyl-HIF-1α and prolyl hydroxylase (PHD2), and a later decrease of vascular endothelial growth factor (VEGF) upon the treatment of Andro. The Andro-inhibited VEGF expression appeared to be a consequence of HIF-1α inactivation, because its DNA binding activity was suppressed by Andro. Molecular data showed that all these effects of Andro might be mediated via TGFβ1/PHD2/HIF-1α pathway, as demonstrated by the transfection of TGFβ1 overexpression vector and PHD2 siRNA, and the usage of a pharmacological MG132 inhibitor. Furthermore, we elucidated the involvement of Andro in HIF-1α transduced VEGF expression in A549 cells and other NSCLC cell lines. In conclusion, these results highlighted the potential effects of Andro, which may be developed as a chemotherapeutic or an anti-angiogenesis agent for NSCLC in the future.

TrkB is highly expressed in NSCLC and mediates BDNF-induced the activation of Pyk2 signaling and the invasion of A549 cells

International Nuclear Information System (INIS)

Zhang, Siyang; Guo, Dawei; Luo, Wenting; Zhang, Qingfu; Zhang, Ying; Li, Chunyan; Lu, Yao; Cui, Zeshi; Qiu, Xueshan

2010-01-01

Aberrant regulation in the invasion of cancer cells is closely associated with their metastatic potentials. TrkB functions as a receptor tyrosine kinase and is considered to facilitate tumor metastasis. Pyk2 is a non-receptor tyrosine kinase and integrates signals in cell invasion. However, little is known about the expression of TrkB in NSCLC and whether Pyk2 is involved in TrkB-mediated invasion of A549 cells. The expression of TrkB was investigated in NSCLC by immunohistochemical staining. Both HBE and A549 cells were treated with BDNF. The expression of TrkB, Pyk2 and ERK phosphorylations were assessed by western blot. Besides, A549 cells were transfected with TrkB-siRNA or Pyk2-siRNA, or treated with ERK inhibitor where indicated. Transwell assay was performed to evaluate cell invasion. 40 cases (66.7%) of NSCLC were found higher expression of TrkB and patients with more TrkB expression had significant metastatic lymph nodes (p = 0.028). BDNF facilitated the invasion of A549 cells and the activations of Pyk2 in Tyr402 and ERK. However, the effects of BDNF were not observed in HBE cells with lower expression of TrkB. In addition, the increased Pyk2 and ERK activities induced by BDNF were significantly inhibited by blocking TrkB expression, so was the invasion of A549 cells. Knockdown studies revealed the essential role of Pyk2 for BDNF-induced cell invasion, since the invasion of A549 cells was abolished by Pyk2-siRNA. The application of ERK inhibitor also showed the suppressed ERK phosphorylation and cell invasion. These data indicated that higher expression of TrkB in NSCLC was closely correlated with lymph node metastasis, and BDNF probably via TrkB/Pyk2/ERK promoted the invasion of A549 cells

A polysaccharide fraction of adlay seed (Coixlachryma-jobi L.) induces apoptosis in human non-small cell lung cancer A549 cells

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Lu, Xiangyi; Liu, Wei; Wu, Junhua; Li, Mengxian [Key Laboratory of Food Nutrition and Safety, Ministry of Education, School of Food Engineering and Biotechnology, Tianjin University of Science and Technology, Tianjin 300457 (China); Wang, Juncheng; Wu, Jihui [School of Life Science, University of Science and Technology of China, Hefei 230022 (China); Luo, Cheng, E-mail: Luo58@yahoo.com [Key Laboratory of Food Nutrition and Safety, Ministry of Education, School of Food Engineering and Biotechnology, Tianjin University of Science and Technology, Tianjin 300457 (China)

2013-01-11

Highlights: Black-Right-Pointing-Pointer A polysaccharide from adlay seed, its molecular mass, optical rotation and sugars was determined. Black-Right-Pointing-Pointer We demonstrated that a polysaccharide from adlay can induce apoptosis in cancer cells. Black-Right-Pointing-Pointer The polysaccharide inhibited the metabolism and proliferation of NSCLC A549 cells. Black-Right-Pointing-Pointer The polysaccharide may trigger apoptosis via the mitochondria-dependent pathway. -- Abstract: Different seed extracts from Coix lachryma-jobi (adlay seed) have been used for the treatment of various cancers in China, and clinical data support the use of these extracts for cancer therapy; however, their underlying molecular mechanisms have not been well defined. A polysaccharide fraction, designated as CP-1, was extracted from the C.lachryma-jobi L. var. using the ethanol subsiding method. CP-1 induced apoptosis in A549 cells in a dose-dependent manner, as determined by MTT assay. Apoptotic bodies were observed in the cells by scanning electronic microscopy. Apoptosis and DNA accumulation during S-phase of the cell cycle were determined by annexin V-FITC and PI staining, respectively, and measured by flow cytometry. CP-1 also extended the comet tail length on single cell gel electrophoresis, and disrupted the mitochondrial membrane potential. Further analysis by western blotting showed that the expression of caspase-3 and caspase-9 proteins was increased. Taken together, our results demonstrate that CP-1 is capable of inhibiting A549 cell proliferation and inducing apoptosis via a mechanism primarily involving the activation of the intrinsic mitochondrial pathway. The assay data suggest that in addition to its nutritional properties, CP-1 is a very promising candidate polysaccharide for the development of anti-cancer medicines.

A polysaccharide fraction of adlay seed (Coixlachryma-jobi L.) induces apoptosis in human non-small cell lung cancer A549 cells

International Nuclear Information System (INIS)

Lu, Xiangyi; Liu, Wei; Wu, Junhua; Li, Mengxian; Wang, Juncheng; Wu, Jihui; Luo, Cheng

2013-01-01

Highlights: ► A polysaccharide from adlay seed, its molecular mass, optical rotation and sugars was determined. ► We demonstrated that a polysaccharide from adlay can induce apoptosis in cancer cells. ► The polysaccharide inhibited the metabolism and proliferation of NSCLC A549 cells. ► The polysaccharide may trigger apoptosis via the mitochondria-dependent pathway. -- Abstract: Different seed extracts from Coix lachryma-jobi (adlay seed) have been used for the treatment of various cancers in China, and clinical data support the use of these extracts for cancer therapy; however, their underlying molecular mechanisms have not been well defined. A polysaccharide fraction, designated as CP-1, was extracted from the C.lachryma-jobi L. var. using the ethanol subsiding method. CP-1 induced apoptosis in A549 cells in a dose-dependent manner, as determined by MTT assay. Apoptotic bodies were observed in the cells by scanning electronic microscopy. Apoptosis and DNA accumulation during S-phase of the cell cycle were determined by annexin V-FITC and PI staining, respectively, and measured by flow cytometry. CP-1 also extended the comet tail length on single cell gel electrophoresis, and disrupted the mitochondrial membrane potential. Further analysis by western blotting showed that the expression of caspase-3 and caspase-9 proteins was increased. Taken together, our results demonstrate that CP-1 is capable of inhibiting A549 cell proliferation and inducing apoptosis via a mechanism primarily involving the activation of the intrinsic mitochondrial pathway. The assay data suggest that in addition to its nutritional properties, CP-1 is a very promising candidate polysaccharide for the development of anti-cancer medicines.

A proteomic view at T cell costimulation.

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Rudolf Lichtenfels

Full Text Available The "two-signal paradigm" in T cell activation predicts that the cooperation of "signal 1," provided by the T cell receptor (TCR through engagement of major histocompatility complex (MHC-presented peptide, with "signal 2″ provided by costimulatory molecules, the prototype of which is CD28, is required to induce T cell effector functions. While the individual signalling pathways are well understood, little is known about global changes in the proteome pattern during TCR/CD28-mediated activation. Therefore, comparative 2-DE-based proteome analyses of CD3(+ CD69(- resting T cells versus cells incubated with (i the agonistic anti-CD3 antibody OKT3 mimicking signal 1 in absence or presence of IL-2 and/or with (ii the agonistic antibody 15E8 triggering CD28-mediated signaling were performed. Differentially regulated spots were defined leading to the identification of proteins involved in the regulation of the metabolism, shaping and maintenance of the cytoskeleton and signal transduction. Representative members of the differentially expressed protein families, such as calmodulin (CALM, glyceraldehyde-3-phosphate dehydrogenase (GAPDH, L-lactate dehydrogenase (LDH, Rho GDP-dissociation inhibitor 2 (GDIR2, and platelet basic protein (CXCL7, were independently verified by flow cytometry. Data provide a detailed map of individual protein alterations at the global proteome level in response to TCR/CD28-mediated T cell activation.

Middle infrared radiation induces G2/M cell cycle arrest in A549 lung cancer cells.

Science.gov (United States)

Chang, Hsin-Yi; Shih, Meng-Her; Huang, Hsuan-Cheng; Tsai, Shang-Ru; Juan, Hsueh-Fen; Lee, Si-Chen

2013-01-01

There were studies investigating the effects of broadband infrared radiation (IR) on cancer cell, while the influences of middle-infrared radiation (MIR) are still unknown. In this study, a MIR emitter with emission wavelength band in the 3-5 µm region was developed to irradiate A549 lung adenocarcinoma cells. It was found that MIR exposure inhibited cell proliferation and induced morphological changes by altering the cellular distribution of cytoskeletal components. Using quantitative PCR, we found that MIR promoted the expression levels of ATM (ataxia telangiectasia mutated), ATR (ataxia-telangiectasia and Rad3-related and Rad3-related), TP53 (tumor protein p53), p21 (CDKN1A, cyclin-dependent kinase inhibitor 1A) and GADD45 (growth arrest and DNA-damage inducible), but decreased the expression levels of cyclin B coding genes, CCNB1 and CCNB2, as well as CDK1 (Cyclin-dependent kinase 1). The reduction of protein expression levels of CDC25C, cyclin B1 and the phosphorylation of CDK1 at Thr-161 altogether suggest G(2)/M arrest occurred in A549 cells by MIR. DNA repair foci formation of DNA double-strand breaks (DSB) marker γ-H2AX and sensor 53BP1 was induced by MIR treatment, it implies the MIR induced G(2)/M cell cycle arrest resulted from DSB. This study illustrates a potential role for the use of MIR in lung cancer therapy by initiating DSB and blocking cell cycle progression.

Effect of silencing of ATM expression by siRNA on radiosensitivity of human lung adenocarcinoma A549 cells

International Nuclear Information System (INIS)

Liu Xiaoqun; Qiao Tiankui

2014-01-01

Objective: To investigate the effect of silencing of ataxia-telangiectasia mutated (ATM) expression by plasmid-mediated RNA interference on the radiosensitivity of human lung adenocarcinoma A 549 cells. Methods: Eukaryotic expression plasmid containing ATM small interfering RNA (siRNA) (pSilencer2.1-ATM), as well as pSilencer2.1-nonspecific, was constructed.Lung adenocarcinoma A 549 cells were divided into positive group, negative group,and control group to be transfected with pSilencer2.1-ATM, pSilencer2.1-nonspecific, and no plasmid, respectively. The mRNA and protein expression of ATM was measured by RT-PCR and Western blot, respectively. The change in cell radiosensitivity was observed by colony-forming assay. Cell cycle and cell apoptosis were analyzed by flow cytometry. Results: The eukaryotic expression plasmid containing ATM siRNA was successfully constructed. The RT-PCR and Western blot demonstrated that the expression of ATM was down-regulated in the positive group. The sensitization enhancement ratios (D 0 ratios) for the positive group and negative group were 1.50 and 1.01, respectively. The flow cytometry revealed that the proportions of A 549 cells in G 1 and G 2 /M phases were significantly lower in the positive group than in the control group (51.27% vs 61.85%, P = 0.012; 6.34% vs 10.91%, P = 0.008) and that the apoptosis rate was significantly higher in the positive group than in the control group and negative group (49.31% vs 13.58%, P = 0.000; 49.31% vs 13.17%, P = 0.000). Conclusions: Silencing of ATM expression may increase the radiosensitivity of human lung adenocarcinoma A 549 cells, probably by affecting the cell cycle and promoting cell apoptosis. (authors)

Picfeltarraenin IA inhibits lipopolysaccharide-induced inflammatory cytokine production by the nuclear factor-κB pathway in human pulmonary epithelial A549 cells.

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Shi, Rong; Wang, Qing; Ouyang, Yang; Wang, Qian; Xiong, Xudong

2016-02-01

The present study aimed to investigate the effect of picfeltarraenin IA (IA) on respiratory inflammation by analyzing its effect on interleukin (IL)-8 and prostaglandin E2 (PGE2) production. The expression of cyclooxygenase 2 (COX2) in human pulmonary adenocarcinoma epithelial A549 cells in culture was also examined. Human pulmonary epithelial A549 cells and the human monocytic leukemia THP-1 cell line were used in the current study. Cell viability was measured using a methylthiazol tetrazolium assay. The production of IL-8 and PGE2 was investigated using an enzyme-linked immunosorbent assay. The expression of COX2 and nuclear factor-κB (NF-κB)-p65 was examined using western blot analysis. Treatment with lipopolysaccharide (LPS; 10 µg/ml) resulted in the increased production of IL-8 and PGE2, and the increased expression of COX2 in the A549 cells. Furthermore, IA (0.1-10 µmol/l) significantly inhibited PGE2 production and COX2 expression in cells with LPS-induced IL-8, in a concentration-dependent manner. The results suggested that IA downregulates LPS-induced COX2 expression, and inhibits IL-8 and PGE2 production in pulmonary epithelial cells. Additionally, IA was observed to suppress the expression of COX2 in THP-1 cells, and also to regulate the expression of COX2 via the NF-κB pathway in the A549 cells, but not in the THP-1 cells. These results indicate that IA regulates LPS-induced cytokine release in A549 cells via the NF-κB pathway.

The Effects of Davallic Acid from Davallia divaricata Blume on Apoptosis Induction in A549 Lung Cancer Cells

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Tsu-Liang Chang

2012-11-01

Full Text Available Traditional or folk medicinal herbs continue to be prescribed in the treatment of various diseases and conditions in many cultures. Recent scientific efforts have focused on the potential roles of extracts of traditional herbs as alternative and complementary medications for cancer treatment. In Taiwan, Davallia divaricata Blume has been traditionally employed in folk medicine for therapy of lung cancer, davallic acid being the major active compound of D. divaricata Blume. In this study, we investigated the inhibitory activity of davallic acid on the proliferation of A549 lung cancer cells. Davallic acid was extracted from D. divaricata Blume, and its effects on cell viability, cell cycle distribution, ROS level, and apoptotic protein expression in A549 cells were determined. Davallic acid significantly induced reactive oxygen species (ROS generation as well as caspase-3, -8, and -9 activation, thereby repressing A549 cell growth and elevating apoptotic activity. Since lung cancer has a high incidence of recurrence, these results indicate that davallic acid may have the potential to be a natural anti-lung cancer compound, and may provide a basis for further study of its use in combating cancer.

Inhibition of lung cancer cells A549 and H460 by curcuminoid extracts and nanoemulsions prepared from Curcuma longa Linnaeus

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Chang HB

2015-08-01

Full Text Available Hong-Bin Chang,1 Bing-Huei Chen1,21Department of Food Science, 2Graduate Institute of Medicine, Fu Jen Catholic University, Taipei, TaiwanAbstract: The objectives of this study were to explore the inhibition mechanism of lung cancer cells A549 and H460 by curcuminoid extracts and nanoemulsions prepared from Curcuma longa Linnaeus. In addition, human bronchus epithelial cell line BEAS-2B (normal cell was selected for comparison. A high-performance liquid chromatography (HPLC method was developed to separate and quantify the various curcuminoids in C. longa extract, including curcumin (1,714.5 µg/mL, demethoxycurcumin (1,147.4 µg/mL, and bisdemethoxycurcumin (190.2 µg/mL. A high-stability nanoemulsion composed of Tween 80, water, and curcuminoid extract was prepared, with mean particle size being 12.6 nm. The cell cycle was retarded at G2/M for both the curcuminoid extract and nanoemulsion treatments; however, the inhibition pathway may be different. H460 cells were more susceptible to apoptosis than A549 cells for both curcuminoid extract and nanoemulsion treatments. Growth of BEAS-2B remained unaffected for both the curcuminoid extract and nanoemulsion treatments, with a concentration range from 1 to 4 µg/mL. Also, the activities of caspase-3, caspase-8, and caspase-9 followed a dose-dependent increase for both A549 and H460 cells for both the treatments, accompanied by a dose-dependent increase in cytochrome C expression and a dose-dependent decrease in CDK1 expression. Interestingly, a dose-dependent increase in cyclin B expression was shown for A549 cells for both the treatments, while a reversed trend was found for H460 cells. Both mitochondria and death receptor pathways may be responsible for apoptosis of both A549 and H460 cells.Keywords: curcuminoid extract, curcuminoid nanoemulsion, Curcuma longa Linnaeus, lung cancer cell, cell cycle, apoptosis mechanism

Formoxanthone C, isolated from Cratoxylum formosum ssp. pruniflorum, reverses anticancer drug resistance by inducing both apoptosis and autophagy in human A549 lung cancer cells.

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Kaewpiboon, Chutima; Boonnak, Nawong; Kaowinn, Sirichat; Chung, Young-Hwa

2018-02-15

Multidrug resistance (MDR) cancer toward cancer chemotherapy is one of the obstacles in cancer therapy. Therefore, it is of interested to use formoxanthone C (1,3,5,6-tetraoxygenated xanthone; XanX), a natural compound, which showed cytotoxicity against MDR human A549 lung cancer (A549RT-eto). The treatment with XanX induced not only apoptosis- in A549RT-eto cells, but also autophagy-cell death. Inhibition of apoptosis did not block XanX-induced autophagy in A549RT-eto cells. Furthermore, suppression of autophagy by beclin-1 small interfering RNAs (siRNAs) did not interrupt XanX-induced apoptosis, indicating that XanX can separately induce apoptosis and autophagy. Of interest, XanX treatment reduced levels of histone deacetylase 4 (HDAC4) protein overexpressed in A549RT-etocells. The co-treatment with XanX and HDAC4 siRNA accelerated both autophagy and apoptosis more than that by XanX treatment alone, suggesting survival of HDAC4 in A549RT-eto cells. XanX reverses etoposide resistance in A549RT-eto cells by induction of both autophagy and apoptosis, and confers cytotoxicity through down-regulation of HDAC4. Copyright © 2017. Published by Elsevier Ltd.

A novel small molecule, Rosline, inhibits growth and induces caspase-dependent apoptosis in human lung cancer cells A549 through a reactive oxygen species-dependent mechanism.

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Zhao, Ting; Feng, Yang; Jin, Wenling; Pan, Hui; Li, Haizhou; Zhao, Yang

2016-06-01

Chemical screening using synthetic small molecule libraries has provided a huge amount of novel active molecules. It generates lead compound for drug development and brings focus on molecules for mechanistic investigations on many otherwise intangible biological processes. In this study, using non-small cell lung cancer cell A549 to screen against a structurally novel and diverse synthetic small molecule library of 2,400 compounds, we identified a molecule named rosline that has strong anti-proliferation activity on A549 cells with a 50% cell growth inhibitory concentration (IC50 ) of 2.87 ± 0.39 µM. We showed that rosline treatment increased the number of Annexin V-positive staining cell, as well as G2/M arrest in their cell cycle progression. Further, we have demonstrated that rosline induces a decrease of mitochondrial membrane potential (Δφm ) and an increase of caspases 3/7 and 9 activities in A549 cells, although having no effect on the activity of caspase 8. Moreover, we found that rosline could induce the production of reactive oxygen species (ROS) and inhibit the phosphorylation of signaling molecule Akt in A549 cells. Alternatively, an antioxidant N-acetyl-L-cysteine (NAC) significantly attenuated rosline's effects on the mitochondrial membrane potential, caspases 3/7 and 9 activities, cell viabilities and the phosphorylation of Akt. Our results demonstrated that ROS played an important role in the apoptosis of A549 cells induced by rosline. © 2016 International Federation for Cell Biology.

Chrysophanol-induced cell death (necrosis) in human lung cancer A549 cells is mediated through increasing reactive oxygen species and decreasing the level of mitochondrial membrane potential.

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Ni, Chien-Hang; Yu, Chun-Shu; Lu, Hsu-Feng; Yang, Jai-Sing; Huang, Hui-Ying; Chen, Po-Yuan; Wu, Shin-Hwar; Ip, Siu-Wan; Chiang, Su-Yin; Lin, Jaung-Geng; Chung, Jing-Gung

2014-05-01

Chrysophanol (1,8-dihydroxy-3-methylanthraquinone) is one of the anthraquinone compounds, and it has been shown to induce cell death in different types of cancer cells. The effects of chrysophanol on human lung cancer cell death have not been well studied. The purpose of this study is to examine chrysophanol-induced cytotoxic effects and also to investigate such influences that involved apoptosis or necrosis in A549 human lung cancer cells in vitro. Our results indicated that chrysophanol decreased the viable A549 cells in a dose- and time-dependent manner. Chrysophanol also promoted the release of reactive oxygen species (ROS) and Ca(2+) and decreased the levels of mitochondria membrane potential (ΔΨm ) and adenosine triphosphate in A549 cells. Furthermore, chrysophanol triggered DNA damage by using Comet assay and DAPI staining. Importantly, chrysophanol only stimulated the cytocheome c release, but it did not activate other apoptosis-associated protein levels including caspase-3, caspase-8, Apaf-1, and AIF. In conclusion, human lung cancer A549 cells treated with chrysophanol exhibited a cellular pattern associated with necrotic cell death and not apoptosis in vitro. © 2012 Wiley Periodicals, Inc. Environ Toxicol 29: 740-749, 2014. Copyright © 2012 Wiley Periodicals, Inc., a Wiley company.

Enhancement of cell death by TNF α-related apoptosis-inducing ligand (TRAIL) in human lung carcinoma A549 cells exposed to X rays under hypoxia

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Takahashi, Momoko; Inanami, Osamu; Yasui, Hironobu; Ogura, Aki; Kuwabara, Mikinori; Kubota, Nobuo; Tsujitani, Michihiko

2007-01-01

Our previous study showed that ionizing radiation induced the expression of death receptor DR5 on the cell surface in tumor cell lines and that the death receptor of the TNF α-related apoptosis-inducing ligand TRAIL enhanced the apoptotic pathway (Hamasu et al., (2005) Journal of Radiation Research, 46:103-110). The present experiments were performed to examine whether treatment with TRAIL enhanced the cell killing in tumor cells exposed to ionizing radiation under hypoxia, since the presence of radioresistant cells in hypoxic regions of solid tumors is a serious problem in radiation therapy for tumors. When human lung carcinoma A549 cells were irradiated under normoxia and hypoxia, respectively, radiation-induced enhancement of expression of DR5 was observed under both conditions. Incubation in the presence of TRAIL enhanced the caspase-dependent and chymotrypsin-like-protease-dependent apoptotic cell death in A549 cells exposed to X rays. Furthermore, it was shown that treatment with TRAIL enhanced apoptotic cell death and loss of clonogenic ability in A549 cells exposed to X rays not only under normoxia but also under hypoxia, suggesting that combination treatment with TRAIL and X irradiation is effective for hypoxic tumor cells. (author)

Microarray-based apoptosis gene screening technique in trichostatin A-induced drug-resisted lung cancer A549/CDDP cells

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Ya-jun WANG

2016-09-01

Full Text Available Objective 　To detect the expression profile changes of apoptosis-related genes in trichostatin A (TSA-induced drug-resisted lung cancer cells A549/CDDP by microarray, in order to screen the target genes in TSA treating cisplatin-resisted lung cancer. Methods 　A549/CDDP cells were treated by TSA for 24 hours. Total RNA was extracted and reversely transcribed into cDNA. Gene expression levels were detected by the NimbleGen whole genome microarray. Differences of expression profiles between TSA-treated and control group were measured by NimbleScan 2.5 software and GO analysis. Apoptosis and proliferation related genes were screened from the expression changed genes. Results 　Compared with the control group, 85 apoptosis-related genes were up-regulated and 43 growth or proliferation related genes were down-regulated in the TSA-treated group. GO analysis showed that the functions of these genes are mainly regulating apoptosis, cell resistance to chem ical stimuli protein, as well as regulating cell growth, proliferation and the biological process of maintaining the cell biological quality. TSA-activated not only the mitochondrial apoptotic pathways, but also the death receptor related apoptosis pathway, and down-regulated the drug resistance related genes BAG3 and ABCC2. Conclusion 　TSA may cause the expression changes of apoptotic and proliferation genes in A549/CDDP cells, these genes may play a role in TSA treating cisplatin-resisted lung cancer. DOI: 10.11855/j.issn.0577-7402.2016.08.07

4-methoxychalcone enhances cisplatin-induced oxidative stress and cytotoxicity by inhibiting the Nrf2/ARE-mediated defense mechanism in A549 lung cancer cells.

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Lim, Juhee; Lee, Sung Ho; Cho, Sera; Lee, Ik-Soo; Kang, Bok Yun; Choi, Hyun Jin

2013-10-01

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key transcriptional regulator for the protection of cells against oxidative and xenobiotic stresses. Recent studies have demonstrated that high constitutive expression of Nrf2 is observed in many types of cancer cells showing resistance to anti-cancer drugs, suggesting that the suppression of overexpressed Nrf2 could be an attractive therapeutic strategy to overcome cancer drug resistance. In the present study, we aimed to find small molecule compounds that enhance the sensitivity of tumor cells to cisplatin induced cytotoxicity by suppressing Nrf2-mediated defense mechanism. A549 lung cancer cells were shown to be more resistant to the anti-cancer drug cisplatin than HEK293 cells, with higher Nrf2 signaling activity; constitutively high amounts of Nrf2-downstream target proteins were observed in A549 cells. Among the three chalcone derivatives 4-methoxy-chalcone (4-MC), hesperidin methylchalcone, and neohesperidin dihydrochalcone, 4-MC was found to suppress transcriptional activity of Nrf2 in A549 cells but to activate it in HEK293 cells. 4-MC was also shown to down-regulate expression of Nrf2 and the downstream phase II detoxifying enzyme NQO1 in A549 cells. The PI3K/Akt pathway was found to be involved in the 4-MC-induced inhibition of Nrf2/ARE activity in A549 cells. This inhibition of Nrf2 signaling results in the accelerated generation of reactive oxygen species and exacerbation of cytotoxicity in cisplatin-treated A549 cells. Taken together, these results suggest that the small molecule compound 4-MC could be used to enhance the sensitivity of tumor cells to the therapeutic effect of cisplatin through the regulation of Nrf2/ARE signaling.

Red blood cell (RBC) membrane proteomics--Part I: Proteomics and RBC physiology.

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Pasini, Erica M; Lutz, Hans U; Mann, Matthias; Thomas, Alan W

2010-01-03

Membrane proteomics is concerned with accurately and sensitively identifying molecules involved in cell compartmentalisation, including those controlling the interface between the cell and the outside world. The high lipid content of the environment in which these proteins are found often causes a particular set of problems that must be overcome when isolating the required material before effective HPLC-MS approaches can be performed. The membrane is an unusually dynamic cellular structure since it interacts with an ever changing environment. A full understanding of this critical cell component will ultimately require, in addition to proteomics, lipidomics, glycomics, interactomics and study of post-translational modifications. Devoid of nucleus and organelles in mammalian species other than camelids, and constantly in motion in the blood stream, red blood cells (RBCs) are the sole mammalian oxygen transporter. The fact that mature mammalian RBCs have no internal membrane-bound organelles, somewhat simplifies proteomics analysis of the plasma membrane and the fact that it has no nucleus disqualifies microarray based methods. Proteomics has the potential to provide a better understanding of this critical interface, and thereby assist in identifying new approaches to diseases. (c) 2009 Elsevier B.V. All rights reserved.

Effects of radioactive 125I seeds on A549 cell line and human embryonic lung diploid cell line 2BS cultivated in vitro and assessment of its clinical safety dose

International Nuclear Information System (INIS)

Bian Wenchao; Qi Liangchen

2012-01-01

Objective: To observe the cell count changes of A549 cell line and human embryonic lung diploid cell line 2BS after irradiated by 125 I seeds with different doses, and to study the growth inhibition of 125 I on this two kinds of cell lines, and to determine its clinical safety dose in treatment of non-small cell lung. Methods: 125 I seeds with different doses (low dose: 0.2 mCi, mediate dose: 0.4 mCi, high dose: 0.8 mCi) were chosen and put into A549 cells and human embryonic lung diploid cell line 2BS in vitro, the cells on the 2nd, 4th, 6th and 8th days after irradiation were collected, the alive cells were counted by cells dyeing experiments, then the growth curves were drawn, and the IC 50 of the radioactive 125 I seeds to both two cell lines were calculated. Results: Compared with blank and control groups, the cell proliferation trend of A549 cells in low dose group was not significantly influenced (P>0.05), but the growth of A549 cells in mediate and high dose groups were inhibited in a time-dependent manner, there were significant differences (P<0.05), the most obvious change was on the 6th day. The IC 50 of the radioactive 125 I seeds to A549 cells was about .04 mCi. While the growth inhibition of 125 I 2BS had no statistically significant differences between various dose groups (P>0.05), and the IC 50 of the radioactive 125 I seeds to 2BS cell line was about 1.65 mCi. Conclusion: 0.4 mCi of radioactive 125 I seeds has already had the obvious damage effect on A549 cell, 0.8 mCi of radioactive 125 I seeds has the stronger effect. The IC 50 of the radioactive 125 I seeds to 2BS cells is about 1.65 mCi, so the clinical safety dosage is 0.4-0.8 mCi. (authors)

Nanoparticle abraxane possesses impaired proliferation in A549 cells due to the underexpression of glucosamine 6-phosphate N-acetyltransferase 1 (GNPNAT1/GNA1

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Zhao MZ

2017-03-01

Full Text Available Minzhi Zhao,* Haiyun Li,* Yan Ma, He Gong, Shu Yang, Qiaojun Fang, Zhiyuan Hu Chinese Academy of Sciences Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology, Beijing, People’s Republic of China *These authors contributed equally to this work Abstract: Abraxane (Abr, a US Food and Drug Administration-approved albumin-bound nanoparticle applied for the treatment of non-small-cell lung cancer, has been reported to be more effective than paclitaxel (PTX. To further understand the molecular mechanisms that produce this superior drug efficacy of Abr, a quantitative proteomic approach has been applied to investigate the global protein expression profiles of lung cancer cell A549 treated with Abr and PTX. Only one protein, namely, glucosamine 6-phosphate N-acetyltransferase 1 (GNA1, showed significant differential expression (P<0.05 in the cutoff of 2.0 fold, suggesting that Abr can be used safely as a substitute for PTX. GNA1 is a key enzyme in the biosynthesis of uridine diphosphate-N-acetylglucosamine, which is an important donor substrate for N-linked glycosylation and has several important functions such as embryonic development and growth. Albumin plays a major role in the regulation of this protein. In summary, this study first shows that the superior drug effect of Abr is mainly due to the downregulation of GNA1, which causes proliferative delay and cell adhesion defect. It is also noteworthy that the deficiency of GNA1 might reduce insulin secretion which correlates with type 2 diabetes. Keywords: quantitative proteomics, nano-drug, drug efficacy, lung cancer, molecular mechanisms, abraxane

Toxic response of nickel nanoparticles in human lung epithelial A549 cells.

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Ahamed, Maqusood

2011-06-01

Nickel nanoparticle (Ni NP) is increasingly used in modern industries such as catalysts, sensors and electronic applications. Due to wide-spread industrial applications the inhalation is the primary source of exposure to Ni NPs. However, data demonstrating the effect of Ni NPs on the pulmonary system remain scarce. The present study was designed to examine the toxic effect of human lung epithelial A549 cells treated with well characterized Ni NPs at the concentrations of 0, 1, 2, 5, 10 and 25 μg/ml for 24 and 48 h. Mitochondrial function (MTT assay), membrane leakage of lactate dehydrogenase (LDH assay), reduced glutathione (GSH), reactive oxygen species (ROS), membrane lipid peroxidation (LPO) and caspase-3 activity were assessed as toxicity end points. Results showed that Ni NPs reduced mitochondrial function and induced the leakage of LDH in dose and time-dependent manner. Ni NPs were also found to induce oxidative stress in dose and time-dependent manner indicated by depletion of GSH and induction of ROS and LPO. Further, activity of caspase-3 enzyme, marker of apoptosis was significantly higher in treated cells with time and Ni NPs dosage. The results exhibited significant toxicity of Ni NPs in human lung epithelial A549 cells which is likely to be mediated through oxidative stress. This study warrants more careful assessment of Ni NPs before their industrial applications. Copyright © 2011 Elsevier Ltd. All rights reserved.

Genistein enhances the effect of trichostatin A on inhibition of A549 cell growth by increasing expression of TNF receptor-1

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Wu, Tzu-Chin; Yang, Ying-Chihi; Huang, Pei-Ru; Wen, Yu-Der; Yeh, Shu-Lan

2012-01-01

Our previous study has shown that genistein enhances apoptosis in A549 lung cancer cells induced by trichostatin A (TSA). The precise molecular mechanism underlying the effect of genistein, however, remains unclear. In the present study, we investigated whether genistein enhances the anti-cancer effect of TSA through up-regulation of TNF receptor-1 (TNFR-1) death receptor signaling. We incubated A549 cells with TSA (50 ng/mL) alone or in combination with genistein and then determined the mRNA and protein expression of TNFR-1 as well as the activation of downstream caspases. Genistein at 5 and 10 μM significantly enhanced the TSA-induced decrease in cell number and apoptosis in a dose-dependent manner. The combined treatment significantly increased mRNA and protein expression of TNFR-1 at 6 and 12 h, respectively, compared with that of the control group; while TSA alone had no effect. TSA in combination with 10 μM of genistein increased TNFR-1 mRNA and protein expression by about 70% and 40%, respectively. The underlying mechanism for this effect of genistein may be partly associated with the estrogen receptor pathway. The combined treatment also increased the activation of caspase-3 and ‐10 as well as p53 protein expression in A549 cells. The enhancing effects of genistein on the TSA-induced decrease in cell number and on the expression of caspase-3 in A549 cells were suppressed by silencing TNFR-1 expression. These data demonstrated that the upregulation of TNFR-1 death receptor signaling plays an important role, at least in part, in the enhancing effect of genistein on TSA-induced apoptosis in A549 cells. -- Highlights: ► TSA combined with genistein rather than TSA alone increases the expression of TNFR-1. ► Genistein may exert such an effect partly through estrogen receptor pathway. ► The combined treatment increases the activation of caspase-10 and caspase-3. ► The combined treatment also increases the expression of p53 protein. ► TNFR-1 si

Cell Surface Proteome of Dental Pulp Stem Cells Identified by Label-Free Mass Spectrometry.

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Christian Niehage

Full Text Available Multipotent mesenchymal stromal cells (MSCs are promising tools for regenerative medicine. They can be isolated from different sources based on their plastic-adherence property. The identification of reliable cell surface markers thus becomes the Holy Grail for their prospective isolation. Here, we determine the cell surface proteomes of human dental pulp-derived MSCs isolated from single donors after culture expansion in low (2% or high (10% serum-containing media. Cell surface proteins were tagged on intact cells using cell impermeable, cleavable sulfo-NHS-SS-biotin, which allows their enrichment by streptavidin pull-down. For the proteomic analyses, we first compared label-free methods to analyze cell surface proteomes i.e. composition, enrichment and proteomic differences, and we developed a new mathematical model to determine cell surface protein enrichment using a combinatorial gene ontology query. Using this workflow, we identified 101 cluster of differentiation (CD markers and 286 non-CD cell surface proteins. Based on this proteome profiling, we identified 14 cell surface proteins, which varied consistently in abundance when cells were cultured under low or high serum conditions. Collectively, our analytical methods provide a basis for identifying the cell surface proteome of dental pulp stem cells isolated from single donors and its evolution during culture or differentiation. Our data provide a comprehensive cell surface proteome for the precise identification of dental pulp-derived MSC populations and their isolation for potential therapeutic intervention.

Preliminary Study on the Effect of Adipocytes on the Biological Behaviors of
Lung Adenocarcinoma A549 Cells in Tumor Microenvironment

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Hang ZHANG

2018-05-01

Full Text Available Background and objective Adipocytes in the tumor microenvironment may provide the metabolic fuel or signal transduction through media and other means to promote a variety of malignant proliferation and invasion, of tumor cells, but their role in lung cancer progression is still unclear. The purpose of this study was to investigate the effect of adipocytes on lung cancer cell biology. Methods 3T3-L1 pre-adipocytes were induced into mature adipocytes. The cell morphology was observed by microscopy and Oil Red O staining. MTT assay, colony formation assay, wound-healing and Transwell methods were used to detect lung cancer cell proliferation, migration and invasion ability. The content of triglyceride in cells was determined by colorimetry. Results The morphology of lung adenocarcinoma A549 cells became more slender after co-culture with mature adipocytes, and the proliferation and cloning ability were significantly enhanced (P<0.05. In addition, mature adipocytes can also promote the migration ability (P<0.05, invasion ability (P<0.01 and accumulation of intracellular lipid (P<0.05 of A549 cells. Conclusion These findings suggested that adipocytes in tumor microenvironment can promote the proliferation, migration and invasion of lung adenocarcinoma A549 cells, which may be related to lipid metabolism.

Cytotoxicity and gene expression profiling of polyhexamethylene guanidine hydrochloride in human alveolar A549 cells.

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Jung, Ha-Na; Zerin, Tamanna; Podder, Biswajit; Song, Ho-Yeon; Kim, Yong-Sik

2014-06-01

In Korea, lung disease of children and pregnant women associated with humidifier disinfectant use has become a major concern. A common sterilizer is polyhexamethylene guanidine (PHMG), a member of the guanidine family of antiseptics. This study was done to elucidate the putative cytotoxic effect of PHMG and the PHMG-mediated altered gene expression in human alveolar epithelial A549 cells in vitro. Cell viability analyses revealed the potent cytotoxicity of PHMG, with cell death evident at as low as 5 μg/mL. Death was dose- and time-dependent, and was associated with formation of intracellular reactive oxygen species, and apoptosis significantly, at even 2 μg/mL concentration. The gene expression profile in A549 cells following 24 h exposure to 5 μg/mL of PHMG was investigated using DNA microarray analysis. Changes in gene expression relevant to the progression of cell death included induction of genes related to apoptosis, autophagy, fibrosis, and cell cycle. However, the expressions of genes encoding antioxidant and detoxifying enzymes were down-regulated or not affected. The altered expression of selected genes was confirmed by quantitative reverse transcription-polymerase chain reaction and Western blot analyses. The collective data suggest that PHMG confers cellular toxicity through the generation of intracellular reactive oxygen species and alteration of gene expression. Copyright © 2014 Elsevier Ltd. All rights reserved.

Differential Regulation of Gene Expression of Alveolar Epithelial Cell Markers in Human Lung Adenocarcinoma-Derived A549 Clones

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Hiroshi Kondo

2015-01-01

Full Text Available Stem cell therapy appears to be promising for restoring damaged or irreparable lung tissue. However, establishing a simple and reproducible protocol for preparing lung progenitor populations is difficult because the molecular basis for alveolar epithelial cell differentiation is not fully understood. We investigated an in vitro system to analyze the regulatory mechanisms of alveolus-specific gene expression using a human alveolar epithelial type II (ATII cell line, A549. After cloning A549 subpopulations, each clone was classified into five groups according to cell morphology and marker gene expression. Two clones (B7 and H12 were further analyzed. Under serum-free culture conditions, surfactant protein C (SPC, an ATII marker, was upregulated in both H12 and B7. Aquaporin 5 (AQP5, an ATI marker, was upregulated in H12 and significantly induced in B7. When the RAS/MAPK pathway was inhibited, SPC and thyroid transcription factor-1 (TTF-1 expression levels were enhanced. After treatment with dexamethasone (DEX, 8-bromoadenosine 3′5′-cyclic monophosphate (8-Br-cAMP, 3-isobutyl-1-methylxanthine (IBMX, and keratinocyte growth factor (KGF, surfactant protein B and TTF-1 expression levels were enhanced. We found that A549-derived clones have plasticity in gene expression of alveolar epithelial differentiation markers and could be useful in studying ATII maintenance and differentiation.

A flavonoid isolated from Streptomyces sp. (ERINLG-4) induces apoptosis in human lung cancer A549 cells through p53 and cytochrome c release caspase dependant pathway.

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Balachandran, C; Sangeetha, B; Duraipandiyan, V; Raj, M Karunai; Ignacimuthu, S; Al-Dhabi, N A; Balakrishna, K; Parthasarathy, K; Arulmozhi, N M; Arasu, M Valan

2014-12-05

The aim of this study was to investigate the anticancer activity of a flavonoid type of compound isolated from soil derived filamentous bacterium Streptomyces sp. (ERINLG-4) and to explore the molecular mechanisms of action. Cytotoxic properties of ethyl acetate extract was carried out against A549 lung cancer cell line using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Cytotoxic properties of isolated compound were investigated in A549 lung cancer cell line, COLO320DM cancer cell line and Vero cells. The compound showed potent cytotoxic properties against A549 lung cancer cell line and moderate cytotoxic properties against COLO320DM cancer cell line. Isolated compound showed no toxicity up to 2000 μg/mL in Vero cells. So we have chosen the A549 lung cancer cell line for further anticancer studies. Intracellular visualization was done by using a laser scanning confocal microscope. Apoptosis was measured using DNA fragmentation technique. Treatment of the A549 cancer cells with isolated compound significantly reduced cell proliferation, increased formation of fragmented DNA and apoptotic body. Activation of caspase-9 and caspase-3 indicated that compound may be inducing intrinsic and extrinsic apoptosis pathways. Bcl-2, p53, pro-caspases, caspase-3, caspase-9 and cytochrome c release were detected by western blotting analysis after compound treatment (123 and 164 μM). The activities of pro-caspases-3, caspase-9 cleaved to caspase-3 and caspase-9 gradually increased after the addition of isolated compound. But Bcl-2 protein was down regulated after treatment with isolated compound. Molecular docking studies showed that the compound bound stably to the active sites of caspase-3 and caspase-9. These results strongly suggest that the isolated compound induces apoptosis in A549 cancer cells via caspase activation through cytochrome c release from mitochondria. The present results might provide helpful suggestions for the design of

Preprocessing with Photoshop Software on Microscopic Images of A549 Cells in Epithelial-Mesenchymal Transition.

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Ren, Zhou-Xin; Yu, Hai-Bin; Shen, Jun-Ling; Li, Ya; Li, Jian-Sheng

2015-06-01

To establish a preprocessing method for cell morphometry in microscopic images of A549 cells in epithelial-mesenchymal transition (EMT). Adobe Photoshop CS2 (Adobe Systems, Inc.) was used for preprocessing the images. First, all images were processed for size uniformity and high distinguishability between the cell and background area. Then, a blank image with the same size and grids was established and cross points of the grids were added into a distinct color. The blank image was merged into a processed image. In the merged images, the cells with 1 or more cross points were chosen, and then the cell areas were enclosed and were replaced in a distinct color. Except for chosen cellular areas, all areas were changed into a unique hue. Three observers quantified roundness of cells in images with the image preprocess (IPP) or without the method (Controls), respectively. Furthermore, 1 observer measured the roundness 3 times with the 2 methods, respectively. The results between IPPs and Controls were compared for repeatability and reproducibility. As compared with the Control method, among 3 observers, use of the IPP method resulted in a higher number and a higher percentage of same-chosen cells in an image. The relative average deviation values of roundness, either for 3 observers or 1 observer, were significantly higher in Controls than in IPPs (p Photoshop, a chosen cell from an image was more objective, regular, and accurate, creating an increase of reproducibility and repeatability on morphometry of A549 cells in epithelial to mesenchymal transition.

Unique proteomic signatures distinguish macrophages and dendritic cells.

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Lev Becker

Full Text Available Monocytes differentiate into heterogeneous populations of tissue macrophages and dendritic cells (DCs that regulate inflammation and immunity. Identifying specific populations of myeloid cells in vivo is problematic, however, because only a limited number of proteins have been used to assign cellular phenotype. Using mass spectrometry and bone marrow-derived cells, we provided a global view of the proteomes of M-CSF-derived macrophages, classically and alternatively activated macrophages, and GM-CSF-derived DCs. Remarkably, the expression levels of half the plasma membrane proteins differed significantly in the various populations of cells derived in vitro. Moreover, the membrane proteomes of macrophages and DCs were more distinct than those of classically and alternatively activated macrophages. Hierarchical cluster and dual statistical analyses demonstrated that each cell type exhibited a robust proteomic signature that was unique. To interrogate the phenotype of myeloid cells in vivo, we subjected elicited peritoneal macrophages harvested from wild-type and GM-CSF-deficient mice to mass spectrometric and functional analysis. Unexpectedly, we found that peritoneal macrophages exhibited many features of the DCs generated in vitro. These findings demonstrate that global analysis of the membrane proteome can help define immune cell phenotypes in vivo.

Neferine augments therapeutic efficacy of cisplatin through ROS- mediated non-canonical autophagy in human lung adenocarcinoma (A549 cells).

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Kalai Selvi, Sivalingam; Vinoth, Amirthalingam; Varadharajan, Thiyagarajan; Weng, Ching Feng; Vijaya Padma, Viswanadha

2017-05-01

Combination of dietary components with chemotherapy drugs is an emerging new strategy for cancer therapy to increase antitumor responses. Neferine, major bisbenzylisoquinoline alkaloid isolated from the seed embryo of Nelumbo nucifera (Lotus). In the present study, we investigated the efficacy of the combinatorial regimen of neferine and cisplatin compared to cisplatin high dose in human lung adenocarcinoma (A549) cells. Co-treatment with neferine enhanced cisplatin-induced autophagy in A549 cells was accompanied by Acidic vesicular accumulation (AVO), enhanced generation of reactive oxygen species (ROS) and depletion of intracellular glutathione (GSH), down regulation of PI3K/AKT/mTOR pathway, conversion of LC3B-I to LC3B-II. This enhanced autophagy developed via a non-canonical mechanism that did not require Beclin-1, PI3KCIII. In conclusion, these results suggest that neferine enhances cisplatin -induced autophagic cancer cell death through downregulation of PI3K/Akt/mTOR signaling pro-survival pathway and ROS- mediated Beclin-1 and PI3K CIII independent autophagy in human lung adenocarcinoma (A549 cells). Copyright © 2017 Elsevier Ltd. All rights reserved.

Proteomics of a new esophageal cancer cell line established from Persian patient.

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Moghanibashi, Mehdi; Jazii, Ferdous Rastgar; Soheili, Zahra-Soheila; Zare, Maryam; Karkhane, Aliasghar; Parivar, Kazem; Mohamadynejad, Parisa

2012-05-25

Although the highest incidence of esophageal squamous cell carcinoma (ESCC) has repeatedly been reported from Persia (Iran), nevertheless the so far proteomic published reports were limited to one study on tissue specimens. Here we report the proteome of a newly established cell line from Persian ESCC patients and compare it with the normal primary cell proteome. Among polypeptides, whose expression was different in cell line sixteen polypeptides were identified by MALDI/TOF/TOF spectrometry. S100-A8 protein, annexin A1, annexin A2, regulatory subunit of calpain, subunit alpha type-3 of proteasome and glutamate dehydrogenase 1 were proteins down-regulated in cell line while peroxiredoxin-5, non-muscle myosin light polypeptide 6, keratin 1, annexin A4, keratin 8, tropomyosin 3, stress-induced-phosphoprotein 1 and albumin were found to be subject of up-regulation in cell line compared to the primary normal cells. The proteomic results were further verified by western blotting and RT-PCR on annexin A1 and keratin 8. In addition, among the aforementioned proteins, glutamate dehydrogenase 1, regulatory subunit of calpain, subunit alpha of type-3 proteasome and annexin A4 are proteins whose deregulation in ESCC is reported for the first time by this study. Copyright © 2012 Elsevier B.V. All rights reserved.

Copper doping enhanced the oxidative stress-mediated cytotoxicity of TiO2 nanoparticles in A549 cells.

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Ahmad, J; Siddiqui, M A; Akhtar, M J; Alhadlaq, H A; Alshamsan, A; Khan, S T; Wahab, R; Al-Khedhairy, A A; Al-Salim, A; Musarrat, J; Saquib, Q; Fareed, M; Ahamed, M

2018-05-01

Physicochemical properties of titanium dioxide nanoparticles (TiO 2 NPs) can be tuned by doping with metals or nonmetals. Copper (Cu) doping improved the photocatalytic behavior of TiO 2 NPs that can be applied in various fields such as environmental remediation and nanomedicine. However, interaction of Cu-doped TiO 2 NPs with human cells is scarce. This study was designed to explore the role of Cu doping in cytotoxic response of TiO 2 NPs in human lung epithelial (A549) cells. Characterization data demonstrated the presence of both TiO 2 and Cu in Cu-doped TiO 2 NPs with high-quality lattice fringes without any distortion. The size of Cu-doped TiO 2 NPs (24 nm) was lower than pure TiO 2 NPs (30 nm). Biological results showed that both pure and Cu-doped TiO 2 NPs induced cytotoxicity and oxidative stress in a dose-dependent manner. Low mitochondrial membrane potential and higher caspase-3 enzyme (apoptotic markers) activity were also observed in A549 cells exposed to pure and Cu-doped TiO 2 NPs. We further observed that cytotoxicity caused by Cu-doped TiO 2 NPs was higher than pure TiO 2 NPs. Moreover, antioxidant N-acetyl cysteine effectively prevented the reactive oxygen species generation, glutathione depletion, and cell viability reduction caused by Cu-doped TiO 2 NPs. This is the first report showing that Cu-doped TiO 2 NPs induced cytotoxicity and oxidative stress in A549 cells. This study warranted further research to explore the role of Cu doping in toxicity mechanisms of TiO 2 NPs.

Phosphorylation of p53 at serine 15 in A549 pulmonary epithelial cells exposed to vanadate: Involvement of ATM pathway

International Nuclear Information System (INIS)

Suzuki, Katsura; Inageda, Kiyoshi; Nishitai, Gen; Matsuoka, Masato

2007-01-01

When A549 cells were exposed to sodium metavanadate (NaVO 3 ), the pentavalent species of vanadium (vanadate), phosphorylation of p53 protein at Ser15 was found in a time (8-48 h)- and dose (10-200 μM)-dependent manner. After the incubation with 50 or 100 μM NaVO 3 for 48 h, accumulation of p53 protein was accompanied with Ser15 phosphorylation. Among serines in p53 protein immunoprecipitated from A549 cells treated with 100 μM NaVO 3 for 48 h, only Ser15 was markedly phosphorylated. Treatment with other vanadate compounds, sodium orthovanadate (Na 3 VO 4 ) and ammonium metavanadate (NH 4 VO 3 ), also induced Ser15 phosphorylation and accumulation of p53 protein. While phosphorylation of extracellular signal-regulated protein kinase (ERK) was found in cells treated with NaVO 3 , treatment with U0126 did not suppress Ser15 phosphorylation. On the other hand, treatment with wortmannin or caffeine, the inhibitors to phosphatidylinositol 3-kinase related kinases (PIKKs), suppressed both NaVO 3 -induced Ser15 phosphorylation and accumulation of p53 protein. The silencing of ataxia telangiectasia mutated (ATM) expression using short-interference RNA resulted in the marked suppression of Ser15 phosphorylation in A549 cells exposed to NaVO 3 . However, treatment with antioxidants such as catalase and N-acetylcysteine did not suppress NaVO 3 -induced Ser15 phosphorylation. Transcriptional activation of p53 and DNA fragmentation in A549 cells treated with NaVO 3 were suppressed only slightly by S15A mutation, suggesting that Ser15 phosphorylation is not essential for these responses. The present results showed that vanadate induces the phosphorylation of p53 at Ser15 depending on ATM, one of the members of PIKK family, in this human pulmonary epithelial cell line

SU-F-T-677: Synergistic Effect(s) of Clotrimazole On Radiation Cell Survival of A549 Lung Cancer Cells in Glucose Vs. Galactose Media

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Boss, G; Tambasco, M; Garakani, M [San Diego State University, San Diego, CA (United States)

2016-06-15

Purpose: In order to determine the synergistic effect of clotrimazole on radiosensitivity of A549 lung cancer cells, and the effect of oxidative pathways on modulating radiosensitivity, we studied how these cells survived under varying amounts of radiation and clotrimazole as well ass when glucose was switched for galactose media. Methods: The glucose media was used to determine the presence of any synergistic effect of clotrimazole on radiation using values of radiation and clotrimazole concentrations, varying from 0 – 8 Gy and 0 – 20 µM, respectively. As a galactose diet is known to activate oxidative pathways, which do not rely on hexokinase II (HK2), all trials were repeated using galactose media to determine the extent that HK2 unbinding from the mitochondrial membrane plays a role in modulating the observed radiosensitivity. An apoptosis vs. necrosis assay was implemented to find out the modality by which cell death occurred. An intracellular lactate assay was performed to exhibit the extent of anaerobic glycolysis. Results: After running the primary experiments, it was found that in glucose media, the cancer cells showed higher cell kill when clotrimazole was added to the media, followed by the cells being irradiated. Conclusion: Given the preliminary results it is validated that under higher concentrations of clotrimazole, in glucose media, A549 lung cancer cells exhibit a lower amount of survival. While all results have not yet been gathered. We anticipate that in galactose media the A549 cells will exhibit this effect to a much smaller degree, if at all.

In vitro gene imaging by luciferase to detect the expression and effect of human tumor necrosis factor related apoptosis-inducing ligand in lung cancer A549 cells

International Nuclear Information System (INIS)

Zhao Na; Cui Jianling; Guo Zhiyuan; Guo Zhiping; Sun Yingcai; Liu Jicun

2009-01-01

Objective: To detect the expression and effect of human tumor necrosis factor related apoptosis-inducing ligand(hTRAIL) in vitro by using a novel double expressing adenoviral vector encoding hTRAIL and firefly luciferase (luc) gene (Ad-hTRAIL-luc), in which luc was used as reporter gene. Methods: A549 cells were transduced with the adenoviral vector encoding enhanced green fluorescent protein (EGFP) gene (Ad-EGFP) at variable multiplicity of infection (MOI). Adenoviral transduction efficiency was determined 48 h later. A549 cells were transduced with Ad-hTRAIL-luc at variable MOI, and the following tests were performed 48h later, respectively: the expressive ratio of hTRAIL and the apoptotic ratio of A549 cells were measured by flow cytometer; counts per minute (cpm) of luminescence were measured by scintillation counters. A549 cells were transduced with Ad-luc at variable MOI, and cpm of luminescence was measured by scintillation counters 48 h later. After A549 cells were transduced with Ad-hTRAIL-luc, the expressive ratio of hTRAIL, the apoptotic ratio of A549 cells and cpm of luminescence were analyzed by one-way ANOVA. The positive ratio of EGFP and cpm of luminescence (Ad-luc) were analyzed by nonparametric ANOVA. Results: After A549 cells were transfected with Ad-hTRAIL-luc, the expressive ratio of hTRAIL on the cell membrane of the groups were (2.37±0.04)%, (3.16±0.03)%, (3.64± 0.03)%, (3.96±0.02)%, (4.24±0.02)%, (4.34±0.02)% respectively, which showed significant difference between each other (P<0.01); and the apoptotic ratio of A549 cells were (1.52±0.04)%, (2.93±0.02)%, (3.39±0.02)%, (3.64±0.02)%, (3.86±0.02)%, (4.08±0.02)%, (4.20± 0.02)%, respectively, and it showed significant difference between each other (P<0.01); cpm of luminescence were 465 561 ± 26 801, 1 038 576 ± 29 417, 937 655 ± 23 197, 786 432 ± 20 028, 524 288 ± 16 338, 401 566 ± 15 961, respectively, and it also showed significant difference between each other (P<0

Paclitaxel and the dietary flavonoid fisetin: a synergistic combination that induces mitotic catastrophe and autophagic cell death in A549 non-small cell lung cancer cells.

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Klimaszewska-Wisniewska, Anna; Halas-Wisniewska, Marta; Tadrowski, Tadeusz; Gagat, Maciej; Grzanka, Dariusz; Grzanka, Alina

2016-01-01

The use of the dietary polyphenols as chemosensitizing agents to enhance the efficacy of conventional cytostatic drugs has recently gained the attention of scientists and clinicians as a plausible approach for overcoming the limitations of chemotherapy (e.g. drug resistance and cytotoxicity). The aim of this study was to investigate whether a naturally occurring diet-based flavonoid, fisetin, at physiologically attainable concentrations, could act synergistically with clinically achievable doses of paclitaxel to produce growth inhibitory and/or pro-death effects on A549 non-small cell lung cancer cells, and if it does, what mechanisms might be involved. The drug-drug interactions were analyzed based on the combination index method of Chou and Talalay and the data from MTT assays. To provide some insights into the mechanism underlying the synergistic action of fisetin and paclitaxel, selected morphological, biochemical and molecular parameters were examined, including the morphology of cell nuclei and mitotic spindles, the pattern of LC3-II immunostaining, the formation of autophagic vacuoles at the electron and fluorescence microscopic level, the disruption of cell membrane asymmetry/integrity, cell cycle progression and the expression level of LC3-II, Bax, Bcl-2 and caspase-3 mRNA. Here, we reported the first experimental evidence for the existence of synergism between fisetin and paclitaxel in the in vitro model of non-small cell lung cancer. This synergism was, at least partially, ascribed to the induction of mitotic catastrophe. The switch from the cytoprotective autophagy to the autophagic cell death was also implicated in the mechanism of the synergistic action of fisetin and paclitaxel in the A549 cells. In addition, we revealed that the synergism between fisetin and paclitaxel was cell line-specific as well as that fisetin synergizes with arsenic trioxide, but not with mitoxantrone and methotrexate in the A549 cells. Our results provide rationale for

Proteomic profiling of the human T-cell nucleolus.

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Jarboui, Mohamed Ali; Wynne, Kieran; Elia, Giuliano; Hall, William W; Gautier, Virginie W

2011-12-01

The nucleolus, site of ribosome biogenesis, is a dynamic subnuclear organelle involved in diverse cellular functions. The size, number and organisation of nucleoli are cell-specific and while it remains to be established, the nucleolar protein composition would be expected to reflect lineage-specific transcriptional regulation of rDNA genes and have cell-type functional components. Here, we describe the first characterisation of the human T-cell nucleolar proteome. Using the Jurkat T-cell line and a reproducible organellar proteomic approach, we identified 872 nucleolar proteins. In addition to ribosome biogenesis and RNA processing networks, network modeling and topological analysis of nucleolar proteome revealed distinct macromolecular complexes known to orchestrate chromatin structure and to contribute to the regulation of gene expression, replication, recombination and repair, and chromosome segregation. Furthermore, among our dataset, we identified proteins known to functionally participate in T-cell biology, including RUNX1, ILF3, ILF2, STAT3, LSH, TCF-1, SATB1, CTCF, HMGB3, BCLAF1, FX4L1, ZAP70, TIAM1, RAC2, THEMIS, LCP1, RPL22, TOPK, RETN, IFI-16, MCT-1, ISG15, and 14-3-3τ, which support cell-specific composition of the Jurkat nucleolus. Subsequently, the nucleolar localisation of RUNX1, ILF3, STAT3, ZAP70 and RAC2 was further validated by Western Blot analysis and immunofluorescence microscopy. Overall, our T-cell nucleolar proteome dataset not only further expands the existing repertoire of the human nucleolar proteome but support a cell type-specific composition of the nucleolus in T cell and highlights the potential roles of the nucleoli in lymphocyte biology. Copyright © 2011 Elsevier Ltd. All rights reserved.

Pirfenidone inhibits TGF-β1-induced over-expression of collagen type I and heat shock protein 47 in A549 cells

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Hisatomi Keiko

2012-06-01

Full Text Available Abstract Background Pirfenidone is a novel anti-fibrotic and anti-inflammatory agent that inhibits the progression of fibrosis in animal models and in patients with idiopathic pulmonary fibrosis (IPF. We previously showed that pirfenidone inhibits the over-expression of collagen type I and of heat shock protein (HSP 47, a collagen-specific molecular chaperone, in human lung fibroblasts stimulated with transforming growth factor (TGF-β1 in vitro. The increased numbers of HSP47-positive type II pneumocytes as well as fibroblasts were also diminished by pirfenidone in an animal model of pulmonary fibrosis induced by bleomycin. The present study evaluates the effects of pirfenidone on collagen type I and HSP47 expression in the human alveolar epithelial cell line, A549 cells in vitro. Methods The expression of collagen type I, HSP47 and E-cadherin mRNAs in A549 cells stimulated with TGF-β1 was evaluated by Northern blotting or real-time PCR. The expression of collagen type I, HSP47 and fibronectin proteins was assessed by immunocytochemical staining. Results TGF-β1 stimulated collagen type I and HSP47 mRNA and protein expression in A549 cells, and pirfenidone significantly inhibited this process. Pirfenidone also inhibited over-expression of the fibroblast phenotypic marker fibronectin in A549 cells induced by TGF-β1. Conclusion We concluded that the anti-fibrotic effects of pirfenidone might be mediated not only through the direct inhibition of collagen type I expression but also through the inhibition of HSP47 expression in alveolar epithelial cells, which results in reduced collagen synthesis in lung fibrosis. Furthermore, pirfenidone might partially inhibit the epithelial-mesenchymal transition.

Nickel decreases cellular iron level and converts cytosolic aconitase to iron-regulatory protein 1 in A549 cells

International Nuclear Information System (INIS)

Chen Haobin; Davidson, Todd; Singleton, Steven; Garrick, Michael D.; Costa, Max

2005-01-01

Nickel (Ni) compounds are well-established carcinogens and are known to initiate a hypoxic response in cells via the stabilization and transactivation of hypoxia-inducible factor-1 alpha (HIF-1α). This change may be the consequence of nickel's interference with the function of several Fe(II)-dependent enzymes. In this study, the effects of soluble nickel exposure on cellular iron homeostasis were investigated. Nickel treatment decreased both mitochondrial and cytosolic aconitase (c-aconitase) activity in A549 cells. Cytosolic aconitase was converted to iron-regulatory protein 1, a form critical for the regulation of cellular iron homeostasis. The increased activity of iron-regulatory protein 1 after nickel exposure stabilized and increased transferrin receptor (Tfr) mRNA and antagonized the iron-induced ferritin light chain protein synthesis. The decrease of aconitase activity after nickel treatment reflected neither direct interference with aconitase function nor obstruction of [4Fe-4S] cluster reconstitution by nickel. Exposure of A549 cells to soluble nickel decreased total cellular iron by about 40%, a decrease that likely caused the observed decrease in aconitase activity and the increase of iron-regulatory protein 1 activity. Iron treatment reversed the effect of nickel on cytosolic aconitase and iron-regulatory protein 1. To assess the mechanism for the observed effects, human embryonic kidney (HEK) cells over expressing divalent metal transporter-1 (DMT1) were compared to A549 cells expressing only endogenous transporters for inhibition of iron uptake by nickel. The inhibition data suggest that nickel can enter via DMT1 and compete with iron for entry into the cell. This disturbance of cellular iron homeostasis by nickel may have a great impact on the ability of the cell to regulate a variety of cell functions, as well as create a state of hypoxia in cells under normal oxygen tension. These effects may be very important in how nickel exerts phenotypic

Oleiferoside W from the roots of Camellia oleifera C. Abel, inducing cell cycle arrest and apoptosis in A549 cells.

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Wu, Jiang-Ping; Kang, Nai-Xin; Zhang, Mi-Ya; Gao, Hong-Wei; Li, Xiao-Ran; Liu, Yan-Li; Xu, Qiong-Ming; Yang, Shi-Lin

2017-07-06

Camellia oleifera C. Abel has been widely cultivated in China, and a group of bioactive constituents such as triterpeniod saponin have been isolated from C. oleifera C. Abel. In the current study, a new triterpeniod saponin was isolated from the EtOH extract of the roots of C. oleifera C. Abel, named as oleiferoside W, and the cytotoxic properties of oleiferoside W were evaluated in non-small cell lung cancer A549 cells. At the same time the inducing apoptosis, the depolarization of mitochondrial membrane potential (Δψ), the up-regulation of related pro-apoptotic proteins, such as cleaved-PARP, cleaved-caspase-3, and the down-regulation of anti-apoptotic marker Bcl-2/Bax were measured on oleiferoside W. Furthermore, the function, inducing the generation of reactive oxygen species (ROS) and apoptosis, of oleiferoside W could be reversed by N-acetylcysteine (NAC). In conclusion, our findings showed that oleiferoside W induced apoptosis involving mitochondrial pathway and increasing intracellular ROS production in the A549 cells, suggesting that oleiferoside W may have the possibility to be a useful anticancer agent for therapy in lung cancer.

The antitumor effect of tanshinone IIA on anti-proliferation and decreasing VEGF/VEGFR2 expression on the human non-small cell lung cancer A549 cell line

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Jun Xie

2015-11-01

Full Text Available The effects of tanshinone IIA on the proliferation of the human non-small cell lung cancer cell line A549 and its possible mechanism on the VEGF/VEGFR signal pathway were investigated. The exploration of the interaction between tanshinone IIA and its target proteins provides a feasible platform for studying the anticancer mechanism of active components of herbs. The CCK-8 assay was used to evaluate the proliferative activity of A549 cells treated with tanshinone IIA (2.5−80 μmol/L for 24, 48 and 72 h, respectively. Flow cytometry was used for the detection of cell apoptosis and cell cycle perturbation. VEGF and VEGFR2 expression were studied by Western blotting. The binding mode of tanshinone IIA within the crystal structure of the VEGFR2 protein was evaluated with molecular docking analysis by use of the CDOCKER algorithm in Discovery Studio 2.1. The CCK-8 results showed that tanshinone IIA can significantly inhibit A549 cell proliferation in a dose- and time-dependent manner. Flow cytometry results showed that the apoptosis rate of tested group was higher than the vehicle control, and tanshinone IIA-treated cells accumulated at the S phase, which was higher than the vehicle control. Furthermore, the expression of VEGF and VEGFR2 was decreased in Western blot. Finally, molecular docking analysis revealed that tanshinone IIA could be stably docked into the kinase domain of VEGFR2 protein with its unique modes to form H-bonds with Cys917 and π–π stacking interactions with Val848. In conclusion, tanshinone IIA may suppress A549 proliferation, induce apoptosis and cell cycle arrest at the S phase. This drug may suppress angiogenesis by targeting the protein kinase domains of VEGF/VEGFR2.

ANTITUMOR AND APOPTOTIC EFFECTS OF CUCURBITACIN A IN A-549 LUNG CARCINOMA CELLS IS MEDIATED VIA G2/M CELL CYCLE ARREST AND M-TOR/PI3K/AKT SIGNALLING PATHWAY.

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Wang, Wen-Dong; Liu, Yan; Su, Yuan; Xiong, Xian-Zhi; Shang, Dan; Xu, Juan-Juan; Liu, Hong-Ju

2017-01-01

The main aim of this study was to demonstrate the antitumor potential of cucurbitacin A on A-549 NSCLC (non-small cell lung cancer cells). The effects of Cucurbitacin A on apoptotic induction, cell physic, cell cycle failure and m-TOR/PI3K/Akt signalling pathway were also investigated in the present study. MTT assay and clonogenic assay were carried out to study effects of this compound on cell cytotoxicity and colony forming tendency in A-549 cells. Moreover, phase and fluorescence microscopic techniques were used to examine the effects on cell morphology and induction of apoptosis. The effects on cell cycle phase distribution were investigated by flow cytometry and effects on m-TOR/PI3K/Akt signalling proteins were assessed by western blot analysis. Results showed that cucurbitacin A induced dose-dependent cytotoxic effects along with suppressing the colony forming tendency in these cells. Cucurbitacin A also induced morphological changes in these cells featuring chromatin condensation, cell shrinkage and apoptotic body formation. G2/M phase cell cycle collapse was also induced by Cucurbitacin A along with inhibition of expression levels of m-TOR/PI3K/Akt proteins. In conclusion, cucurbitacin A inhibits cancer growth in A-549 NSCLC cells by inducing apoptosis, targeting m-TOR/PI3K/Akt signalling pathway and G2/M cell cycle.

Effect of X-irradiation on the protein expression of P57kip2 and TGF-β1 in lung cancer cell stain A549

International Nuclear Information System (INIS)

Zou Huawei; Tan Yonggang; Zhang Heying

2008-01-01

Objective: To analyze the effect of X-irradiation on the proteins expression of p57 kip2 and TGF-β1 in lung cancer cell stain A549 and its clinical significance. Methods: Lung cancer cell stain A549 was cultivated and cell, protein was extracted at 6,12,24,36 and 48 hours after X-irradiation by different doses(2,4, 8 and 12 Gy). The expression of p57 kip2 and TGF-β1 proteins were examined by Western blot. Results: The expression of p57 kip2 in lung cancer cell stain A549 was very low before X-irradiation, and increased significantly after irradiation with different doses and reached the peak level at 12 hours after irradiation (P kip2 and TGF-β1 proteins which increased with certain doses, p57 kip2 and TGF-β1 could be used to predict the damage degree of cancer cells by X-ray. (authors)

Effect of Circular RNA UBAP2 Silencing on Proliferation and Invasion of Human Lung Cancer A549 Cells and Its Mechanism

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Yujing YIN

2017-12-01

Full Text Available Background and objective It has been proven that circular RNAs (circRNAs play an important role on the process of many types cancer and circUBAP2 was a cancer-promoting circRNA, however, the role and mechanism in lung cancer was not clear. The aim of this study is to investigate the effects of circUBAP2 on cell proliferation and invasion of human lung cancer A549 cells. Methods CCK-8 assay was employed to detect the effect of circUBAP2 sliencing on cell proliferation of A549 cells. Fow cytometry was applied to detect the impact of circUBAP2 sliencing on cell cycle and cell anoikis, and Transwell invasion assay was applied to determine cell invasion of A549 cells. We also employed Western blot and Real-time PCR to determine the expressions of CDK6, cyclin D1, p27 and c-IAP1, Bcl-2, Survivin, Bax, FAK, Rac1 and MMP2, and the activities of JNK and ERK1/2, luciferase report gene assay was used to detect the targets. Results CCK-8 assay showed that the inhibition of cell proliferation in the circUBAP2-siRNA group compared to untreated group and siRNA control group. Results of cell cycle detected by flow cytometry showed that cell cycle arrestd at G0/G1 after circUBAP2 silencing, cell apoptosis rate increased also. We also found that after circUBAP2 silencing, cell invasion of A549 cells was significantly inhibited. Western blot and Real-time PCR results showed that expression of CDK6, cyclin D1, c-IAP1, Bcl-2, Survivin, FAK, Rac1 and MMP2 were down-regulated, and the expression of p27 and Bax were up-regulated. Moreover, the activities of JNK and ERK1/2 were inhibited because of circUBAP2 silencing, the target genes were miR-339-5p, miR-96-3p and miR-135b-3p. Conclusion CircUBAP2 plays an important role in the proliferation and invasion of human lung cancer. Silencing of circUBAP2 might be a novel target for molecular targeted therapy of patients with lung cancer.

Antitumor and apoptotic effects of cucurbitacin a in A-549 lung ...

African Journals Online (AJOL)

Background: The main aim of this study was to demonstrate the antitumor potential of cucurbitacin A on A-549 NSCLC (non-small cell lung cancer cells). The effects of Cucurbitacin A on apoptotic induction, cell physic, cell cycle failure and m-TOR/PI3K/Akt signalling pathway were also investigated in the present study.

Telmisartan Exerts Anti-Tumor Effects by Activating Peroxisome Proliferator-Activated Receptor-γ in Human Lung Adenocarcinoma A549 Cells

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Juan Li

2014-03-01

Full Text Available Telmisartan, a member of the angiotensin II type 1 receptor blockers, is usually used for cardiovascular diseases. Recent studies have showed that telmisartan has the property of PPARγ activation. Meanwhile, PPARγ is essential for tumor proliferation, invasion and metastasis. In this work we explore whether telmisartan could exert anti-tumor effects through PPARγ activation in A549 cells. MTT and trypan blue exclusion assays were included to determine the survival rates and cell viabilities. RT-PCR and western blotting were used to analyze the expression of ICAM-1, MMP-9 and PPARγ. DNA binding activity of PPARγ was evaluated by EMSA. Our data showed that the survival rates and cell viabilities of A549 cells were all reduced by telmisartan in a time- and concentration-dependent manner. Meanwhile, our results also demonstrated that telmisartan dose-dependently inhibited the expression of ICAM-1 and MMP-9. Moreover, the cytotoxic and anti-proliferative effects, ICAM-1 and MMP-9 inhibitive properties of telmisartan were totally blunted by the PPARγ antagonist GW9662. Our findings also showed that the expression of PPARγ was up-regulated by telmisartan in a dose dependent manner. And, the EMSA results also figured out that DNA binding activity of PPARγ was dose-dependently increased by telmisartan. Additionally, our data also revealed that telmisartan-induced PPARγ activation was abrogated by GW9662. Taken together, our results indicated that telmisartan inhibited the expression of ICAM-1 and MMP-9 in A549 cells, very likely through the up-regulation of PPARγ synthesis.

Andrographolide antagonizes cigarette smoke extract-induced inflammatory response and oxidative stress in human alveolar epithelial A549 cells through induction of microRNA-218.

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Li, Ying-jie; Yu, Chang-hai; Li, Jing-bo; Wu, Xi-ya

2013-12-01

Andrographolide is a major bioactive labdane diterpenoid isolated from Andrographis paniculata and has protective effects against cigarette smoke (CS)-induced lung injury. This study was done to determine whether such protective effects were mediated through modulation of microRNA (miR)-218 expression. Therefore, we exposed human alveolar epithelial A549 cells to cigarette smoke extract (CSE) with or without andrographolide pretreatment and measured the level of glutathione, nuclear factor-kappaB (NF-κB) activation, proinflammatory cytokine production, and miR-218 expression. We found that andrographolide pretreatment significantly restored the glutathione level in CSE-exposed A549 cells, coupled with reduced inhibitor κB (IκB)-α phosphorylation and p65 nuclear translocation and interleukin (IL)-8 and IL-6 secretion. The miR-218 expression was significantly upregulated by andrographolide pretreatment. To determine the biological role of miR-218, we overexpressed and downregulated its expression using miR-218 mimic and anti-miR-218 inhibitor, respectively. We observed that miR-218 overexpression led to a marked reduction in IκB-α phosphorylation, p65 nuclear accumulation, and NF-κB-dependent transcriptional activity in CSE-treated A549 cells. In contrast, miR-218 silencing enhanced IκB-α phosphorylation and p65 nuclear accumulation in cells with andrographolide pretreatment and reversed andrographolide-mediated reduction of IL-6 and IL-8 production. In addition, depletion of miR-218 significantly reversed the upregulation of glutathione levels in A549 cells by andrographolide. Taken together, our results demonstrate that andrographolide mitigates CSE-induced inflammatory response in A549 cells, largely through inhibition of NF-κB activation via upregulation of miR-218, and thus has preventive benefits in CS-induced inflammatory lung diseases.

Direct electric current treatment modifies mitochondrial function and lipid body content in the A549 cancer cell line.

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Holandino, Carla; Teixeira, Cesar Augusto Antunes; de Oliveira, Felipe Alves Gomes; Barbosa, Gleyce Moreno; Siqueira, Camila Monteiro; Messeder, Douglas Jardim; de Aguiar, Fernanda Silva; da Veiga, Venicio Feo; Girard-Dias, Wendell; Miranda, Kildare; Galina, Antonio; Capella, Marcia Alves Marques; Morales, Marcelo Marcos

2016-10-01

Electrochemical therapy (EChT) entails treatment of solid tumors with direct electric current (DC). This work evaluated the specific effects of anodic flow generated by DC on biochemical and metabolic features of the A549 human lung cancer cell line. Apoptosis was evaluated on the basis of caspase-3 activity and mitochondrial transmembrane potential dissipation. Cell morphology was analyzed using transmission electron microscopy, and lipid droplets were studied through morphometric analysis and X-ray qualitative elemental microanalysis. High-resolution respirometry was used to assess mitochondrial respiratory parameters. Results indicated A549 viability decreased in a dose-dependent manner with a prominent drop between 18 and 24h after treatment (ppotential. Furthermore, treated cells demonstrated important ultrastructural mitochondria damage and a three-fold increase in the cytoplasmic lipid bodies' number, quantified by morphometrical analyses. Conversely, 24h after treatment, the cells presented a two-fold increase of residual oxygen consumption, accounting for 45.3% of basal oxygen consumption. These results show remarkable alterations promoted by anodic flow on human lung cancer cells which are possibly involved with the antitumoral effects of EChT. Copyright © 2016 Elsevier B.V. All rights reserved.

Mitotic spindle proteomics in Chinese hamster ovary cells.

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Mary Kate Bonner

Full Text Available Mitosis is a fundamental process in the development of all organisms. The mitotic spindle guides the cell through mitosis as it mediates the segregation of chromosomes, the orientation of the cleavage furrow, and the progression of cell division. Birth defects and tissue-specific cancers often result from abnormalities in mitotic events. Here, we report a proteomic study of the mitotic spindle from Chinese Hamster Ovary (CHO cells. Four different isolations of metaphase spindles were subjected to Multi-dimensional Protein Identification Technology (MudPIT analysis and tandem mass spectrometry. We identified 1155 proteins and used Gene Ontology (GO analysis to categorize proteins into cellular component groups. We then compared our data to the previously published CHO midbody proteome and identified proteins that are unique to the CHO spindle. Our data represent the first mitotic spindle proteome in CHO cells, which augments the list of mitotic spindle components from mammalian cells.

Interleukin-1β-induced iNOS expression in human lung carcinoma A549 cells: involvement of STAT and MAPK pathways

International Nuclear Information System (INIS)

Ravichandran, Kameswaran; Tyagi, Alpna; Deep, Gagan; Agarwal, Chapla; Agarwal, Rajesh

2011-01-01

For understanding of signaling molecules important in lung cancer growth and progression, IL-1β effect was analyzed on iNOS expression and key signaling molecules in human lung carcinoma A549 cells and established the role of specific signaling molecules by using specific chemical inhibitors. IL-1β exposure (10 ng/ml) induced strong iNOS expression in serum starved A549 cells. Detailed molecular analyses showed that IL-1β increased expression of phosphorylated STAT1 (Tyr701 and Ser727) and STAT3 (Tyr705 and Ser727) both in total cell lysates and nuclear lysates. Further, IL-1β exposure strongly activated MAPKs (ERK1/2, JNK1/2 and p38) and Akt as well as increased nuclear levels of NF-κB and HIF-1α in A549 cells. Use of specific chemical inhibitors for JAK1 kinase (piceatannol), JAK2 kinase (AG-490), MEK1/2 (PD98059) and JNK1/2 (SP600125) revealed that IL-1β-induced iNOS expression involved signaling pathways in addition to JAKSTAT and ERK1/2-JNK1/2 activation. Overall, these results suggested that instead of specific pharmacological inhibitors, use of chemopreventive agents with broad spectrum efficacy to inhibit IL-1β-induced signaling cascades and iNOS expression would be a better strategy towards lung cancer prevention and/or treatment. (author)

Silica nanoparticles and biological dispersants: genotoxic effects on A549 lung epithelial cells

International Nuclear Information System (INIS)

Brown, David M.; Varet, Julia; Johnston, Helinor; Chrystie, Alison; Stone, Vicki

2015-01-01

Silica nanoparticle exposure could be intentional (e.g. medical application or food) or accidental (e.g. occupational inhalation). On entering the body, particles become coated with specific proteins depending on the route of entry. The ability of silica particles of different size and charge (non-functionalized 50 and 200 nm and aminated 50 and 200 nm) to cause genotoxic effects in A549 lung epithelial cells was investigated. Using the modified comet assay and the micronucleus assay, we examined the effect of suspending the particles in different dispersion media [RPMI or Hanks’ balanced salt solution (HBSS), supplemented with bovine serum albumin (BSA), lung lining fluid (LLF) or serum] to determine if this influenced the particle’s activity. Particle characterisation suggested that the particles were reasonably well dispersed in the different media, with the exception of aminated 50 nm particles which showed evidence of agglomeration. Plain 50, 200 nm and aminated 50 nm particles caused significant genotoxic effects in the presence of formamidopyrimidine-DNA glycosylase when dispersed in HBSS or LLF. These effects were reduced when the particles were dispersed in BSA and serum. There was no significant micronucleus formation produced by any of the particles when suspended in any of the dispersants. The data suggest that silica particles can produce a significant genotoxic effect according to the comet assay in A549 cells, possibly driven by an oxidative stress-dependent mechanism which may be modified depending on the choice of dispersant employed

Silica nanoparticles and biological dispersants: genotoxic effects on A549 lung epithelial cells

Energy Technology Data Exchange (ETDEWEB)

Brown, David M., E-mail: d.brown@hw.ac.uk [Heriot-Watt University, Nanosafety Research Group, School of Life Sciences (United Kingdom); Varet, Julia, E-mail: julia.varet@IOM-world.org [Institute of Occupational Medicine (United Kingdom); Johnston, Helinor, E-mail: h.johnston@hw.ac.uk; Chrystie, Alison; Stone, Vicki, E-mail: v.stone@hw.ac.uk [Heriot-Watt University, Nanosafety Research Group, School of Life Sciences (United Kingdom)

2015-10-15

Silica nanoparticle exposure could be intentional (e.g. medical application or food) or accidental (e.g. occupational inhalation). On entering the body, particles become coated with specific proteins depending on the route of entry. The ability of silica particles of different size and charge (non-functionalized 50 and 200 nm and aminated 50 and 200 nm) to cause genotoxic effects in A549 lung epithelial cells was investigated. Using the modified comet assay and the micronucleus assay, we examined the effect of suspending the particles in different dispersion media [RPMI or Hanks’ balanced salt solution (HBSS), supplemented with bovine serum albumin (BSA), lung lining fluid (LLF) or serum] to determine if this influenced the particle’s activity. Particle characterisation suggested that the particles were reasonably well dispersed in the different media, with the exception of aminated 50 nm particles which showed evidence of agglomeration. Plain 50, 200 nm and aminated 50 nm particles caused significant genotoxic effects in the presence of formamidopyrimidine-DNA glycosylase when dispersed in HBSS or LLF. These effects were reduced when the particles were dispersed in BSA and serum. There was no significant micronucleus formation produced by any of the particles when suspended in any of the dispersants. The data suggest that silica particles can produce a significant genotoxic effect according to the comet assay in A549 cells, possibly driven by an oxidative stress-dependent mechanism which may be modified depending on the choice of dispersant employed.

Silica nanoparticles and biological dispersants: genotoxic effects on A549 lung epithelial cells

Science.gov (United States)

Brown, David M.; Varet, Julia; Johnston, Helinor; Chrystie, Alison; Stone, Vicki

2015-10-01

Silica nanoparticle exposure could be intentional (e.g. medical application or food) or accidental (e.g. occupational inhalation). On entering the body, particles become coated with specific proteins depending on the route of entry. The ability of silica particles of different size and charge (non-functionalized 50 and 200 nm and aminated 50 and 200 nm) to cause genotoxic effects in A549 lung epithelial cells was investigated. Using the modified comet assay and the micronucleus assay, we examined the effect of suspending the particles in different dispersion media [RPMI or Hanks' balanced salt solution (HBSS), supplemented with bovine serum albumin (BSA), lung lining fluid (LLF) or serum] to determine if this influenced the particle's activity. Particle characterisation suggested that the particles were reasonably well dispersed in the different media, with the exception of aminated 50 nm particles which showed evidence of agglomeration. Plain 50, 200 nm and aminated 50 nm particles caused significant genotoxic effects in the presence of formamidopyrimidine-DNA glycosylase when dispersed in HBSS or LLF. These effects were reduced when the particles were dispersed in BSA and serum. There was no significant micronucleus formation produced by any of the particles when suspended in any of the dispersants. The data suggest that silica particles can produce a significant genotoxic effect according to the comet assay in A549 cells, possibly driven by an oxidative stress-dependent mechanism which may be modified depending on the choice of dispersant employed.

DNA damage response signaling in lung adenocarcinoma A549 cells following gamma and carbon beam irradiation.

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Ghosh, Somnath; Narang, Himanshi; Sarma, Asitikantha; Krishna, Malini

2011-11-01

Carbon beams (5.16MeV/u, LET=290keV/μm) are high linear energy transfer (LET) radiation characterized by higher relative biological effectiveness than low LET radiation. The aim of the current study was to determine the signaling differences between γ-rays and carbon ion-irradiation. A549 cells were irradiated with 1Gy carbon or γ-rays. Carbon beam was found to be three times more cytotoxic than γ-rays despite the fact that the numbers of γ-H2AX foci were same. Percentage of cells showing ATM/ATR foci were more with γ-rays however number of foci per cell were more in case of carbon irradiation. Large BRCA1 foci were found in all carbon irradiated cells unlike γ-rays irradiated cells and prosurvival ERK pathway was activated after γ-rays irradiation but not carbon. The noteworthy finding of this study is the early phase apoptosis induction by carbon ions. In the present study in A549 lung adenocarcinoma, authors conclude that despite activation of same repair molecules such as ATM and BRCA1, differences in low and high LET damage responses might be due to their distinct macromolecular complexes rather than their individual activation and the activation of cytoplasmic pathways such as ERK, whether it applies to all the cell lines need to be further explored. Copyright © 2011 Elsevier B.V. All rights reserved.

Studies on cytotoxic constituents from the leaves of Elaeagnus oldhamii Maxim. in non-small cell lung cancer A549 cells.

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Liao, Chi-Ren; Kuo, Yueh-Hsiung; Ho, Yu-Ling; Wang, Ching-Ying; Yang, Chang-Syun; Lin, Cheng-Wen; Chang, Yuan-Shiun

2014-07-04

Elaeagnus oldhamii Maxim. is a commonly used traditional herbal medicine. In Taiwan the leaves of E. oldhamii Maxim. are mainly used for treating lung disorders. Twenty five compounds were isolated from the leaves of E. oldhamii Maxim. in the present study. These included oleanolic acid (1), 3-O-(Z)-coumaroyl oleanolic acid (2), 3-O-(E)-coumaroyl oleanolic acid (3), 3-O-caffeoyl oleanolic acid (4), ursolic acid (5), 3-O-(Z)-coumaroyl ursolic acid (6), 3-O-(E)-coumaroyl ursolic acid (7), 3-O-caffeoyl ursolic acid (8), 3β, 13β-dihydroxyolean-11-en-28-oic acid (9), 3β, 13β-dihydroxyurs-11-en-28-oic acid (10), uvaol (11), betulin (12), lupeol (13), kaempferol (14), aromadendrin (15), epigallocatechin (16), cis-tiliroside (17), trans-tiliroside (18), isoamericanol B (19), trans-p-coumaric acid (20), protocatechuic acid (21), salicylic acid (22), trans-ferulic acid (23), syringic acid (24) and 3-O-methylgallic acid (25). Of the 25 isolated compounds, 21 compounds were identified for the first time in E. oldhamii Maxim. These included compounds 1, 4, 5 and 8-25. These 25 compounds were evaluated for their inhibitory activity against the growth of non-small cell lung cancer A549 cells by the MTT assay, and the corresponding structure-activity relationships were discussed. Among these 25 compounds, compound 6 displayed the best activity against the A549 cell line in vitro (CC50=8.56±0.57 μg/mL, at 48 h of MTT asssay). Furthermore, compound 2, 4, 8 and 18 exhibited in vitro cytotoxicity against the A549 cell line with the CC50 values of less than 20 μg/mL at 48 h of MTT asssay. These five compounds 2, 4, 6, 8 and 18 exhibited better cytotoxic activity compared with cisplatin (positive control, CC50 value of 14.87±1.94 μg/mL, at 48 h of MTT asssay). The result suggested that the five compounds might be responsible for its clinical anti-lung cancer effect.

Involvement of ERK, Bcl-2 family and caspase 3 in recombinant human activin A-induced apoptosis in A549

International Nuclear Information System (INIS)

Wang Baiding; Feng Yuling; Song Xingbo; Liu Qingqing; Ning Yunye; Ou Xuemei; Yang Jie; Zhang Xiaohong; Wen, Fuqiang

2009-01-01

Background: Activins are members of the transforming growth factor-β (TGF-β) superfamily. Previous studies have shown that activin A may have a central role in regulating both apoptosis and proliferation. However, direct studies of recombination human activin A on human NSCLC A549 cells have not yet been reported. The purpose of this study was to investigate whether activin A could induce apoptosis in A549 cells and the possible mechanisms via which it worked. Methods: Cellular apoptosis induced by activin A was detected by TUNEL assay and the levels of protein expression were detected by western blot. Results: Recombination human activin A induced apoptosis in human NSCLC A549 cells in a concentrate-dependent manner. Activin A-induced A549 apoptosis was accompanied by the up-regulation of Bax, Bad and Bcl-Xs and down-regulation of Bcl-2. Moreover, activin A treatment increased the expression of its typeII receptors, activated ERK and caspase 3 in A549. These results clearly demonstrate that the induction of apoptosis by activin-A involves multiple cellular/molecular pathways and strongly suggest that pro- and anti-apoptotic Bcl-2 family proteins and caspase 3 participate in activin A-induced apoptotic process in A549 cells. On the other hand, activin A treatment had little effect on primary human small airway epithelial cells (SAECs). Conclusion: Recombination human activin A induced apoptosis in A549 cells, at least partially, through ERK and mitochondrial pathway. The result that activin A did not affect the normal SAEC revealed activin A might be considered as a potential anticancer agent and worthy of further studies

TGF-β and Hypoxia/Reoxygenation Promote Radioresistance of A549 Lung Cancer Cells through Activation of Nrf2 and EGFR

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Sae-lo-oom Lee

2016-01-01

Full Text Available Although many studies have examined the roles of hypoxia and transforming growth factor- (TGF- β separately in the tumor microenvironment, the effects of simultaneous treatment with hypoxia/reoxygenation and TGF-β on tumor malignancy are unclear. Here, we investigated the effects of redox signaling and oncogenes on cell proliferation and radioresistance in A549 human lung cancer cells in the presence of TGF-β under hypoxia/reoxygenation conditions. Combined treatment with TGF-β and hypoxia activated epidermal growth factor receptor (EGFR and nuclear factor (erythroid-derived 2-like 2 (Nrf2, a redox-sensitive transcription factor. Interestingly, Nrf2 knockdown suppressed the effects of combined treatment on EGFR phosphorylation. In addition, blockade of EGFR signaling also suppressed induction of Nrf2 following combined treatment with hypoxia and TGF-β, indicating that the combined treatment induced positive crosstalk between Nrf2 and EGFR. TGF-β and hypoxia/reoxygenation increased the accumulation of reactive oxygen species (ROS, while treatment with N-acetyl-L-cysteine abolished the activation of Nrf2 and EGFR. Treatment with TGF-β under hypoxic conditions increased the proliferation of A549 cells compared with that after vehicle treatment. Moreover, cells treated with the combined treatment exhibited resistance to ionizing radiation (IR, and knockdown of Nrf2 increased IR-induced cell death under these conditions. Thus, taken together, our findings suggested that TGF-β and hypoxia/reoxygenation promoted tumor progression and radioresistance of A549 cells through ROS-mediated activation of Nrf2 and EGFR.

β-Sitosterol targets Trx/Trx1 reductase to induce apoptosis in A549 cells via ROS mediated mitochondrial dysregulation and p53 activation.

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Rajavel, Tamilselvam; Packiyaraj, Pandian; Suryanarayanan, Venkatesan; Singh, Sanjeev Kumar; Ruckmani, Kandasamy; Pandima Devi, Kasi

2018-02-01

β-Sitosterol (BS), a major bioactive constituent present in plants and vegetables has shown potent anticancer effect against many human cancer cells, but the underlying mechanism remain elusive on NSCLC cancers. We found that BS significantly inhibited the growth of A549 cells without harming normal human lung and PBMC cells. Further, BS treatment triggered apoptosis via ROS mediated mitochondrial dysregulation as evidenced by caspase-3 & 9 activation, Annexin-V/PI positive cells, PARP inactivation, loss of MMP, Bcl-2-Bax ratio alteration and cytochrome c release. Moreover, generation of ROS species and subsequent DNA stand break were found upon BS treatment which was reversed by addition of ROS scavenger (NAC). Indeed BS treatment increased p53 expression and its phosphorylation at Ser15, while silencing the p53 expression by pifithrin-α, BS induced apoptosis was reduced in A549 cells. Furthermore, BS induced apoptosis was also observed in NCI-H460 cells (p53 wild) but not in the NCI-H23 cells (p53 mutant). Down-regulation of Trx/Trx1 reductase contributed to the BS induced ROS accumulation and mitochondrial mediated apoptotic cell death in A549 and NCI-H460 cells. Taken together, our findings provide evidence for the novel anti-cancer mechanism of BS which could be developed as a promising chemotherapeutic drug against NSCLC cancers.

Development of drug-loaded chitosan hollow nanoparticles for delivery of paclitaxel to human lung cancer A549 cells.

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Jiang, Jie; Liu, Ying; Wu, Chao; Qiu, Yang; Xu, Xiaoyan; Lv, Huiling; Bai, Andi; Liu, Xuan

2017-08-01

In this study, biodegradable chitosan hollow nanospheres (CHN) were fabricated using polystyrene nanospheres (PS) as templates. CHN were applied to increase the solubility of poorly water-soluble drugs. The lung cancer drug paclitaxel (PTX), which is used as a model drug, was loaded into CHN by the adsorption equilibrium method. The drug-loaded sample (PTX-CHN) offered sustained PTX release and good bioavailability. The state characterization of PTX by differential scanning calorimetry (DSC), X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR) showed that the PTX absorbed into CHN existed in an amorphous state. An in vitro toxicity experiment indicated that CHN were nontoxic as carriers of poorly water-soluble drugs. The PTX-CHN produced a marked inhibition of lung cancer A549 cells proliferation and encouraged apoptosis. A cell uptake experiment indicated that PTX-CHN was successfully taken up by lung cancer A549 cells. Furthermore, a degradation experiment revealed that CHN were readily biodegradable. These findings state clearly that CHN can be regarded as promising biomaterials for lung cancer treatment.

Effect of etoposide-induced alteration of the Mdm2-Rb signaling pathway on cellular senescence in A549 lung adenocarcinoma cells.

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Dai, Wenjing; Jiang, Yi; Chen, Kairong; Qiu, Jing; Sun, Jian; Zhang, Wei; Zhou, Xiafei; Huang, Na; Li, Yunhui; Li, Wancheng

2017-10-01

The present study aimed to investigate the effect of various concentrations of etoposide (VP-16) on the E3 ubiquitin-protein ligase Mdm2 (Mdm2)-retinoblastoma (Rb) signaling pathway in the cellular senescence of A549 lung adenocarcinoma cells. A549 cells were randomly divided into the following four groups: Control group (no treatment), group 1 (1 µmol/l VP-16), group 2 (5 µmol/l VP-16) and group 3 (25 µmol/l VP-16). Each group was cultured for 48 h after treatment prior to observation of the alterations to cellular morphology. The cell cycle distribution of each group was also detected by flow cytometry. In addition, the activity of cellular senescence-associated β-galactosidase, and the expression of Mdm2 and phosphorylated (p-) Rb protein, was measured. The percentage of senescent cells was significantly higher following VP-16 treatment compared with the control group. The percentage of G 1 phase cells, and p-Rb protein and Mdm2 protein expression were also significantly different following VP-16 treatment compared with the control group. VP-16 increased the activity of β-galactosidase in the A459 cells. VP-16 also decreased the expression level of Mdm2 and p-Rb protein and inhibited cell cycle progression in G 1 . These results indicate that VP-16 induces the cellular senescence of A549 cells via the Mdm2-Rb signaling pathway. However, further investigations are required to validate the mechanisms underlying these effects of VP-16.

Ameliorative Effects of Dimetylthiourea and N-Acetylcysteine on Nanoparticles Induced Cyto-Genotoxicity in Human Lung Cancer Cells-A549

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Srivastava, Ritesh Kumar; Rahman, Qamar; Kashyap, Mahendra Pratap; Lohani, Mohtashim; Pant, Aditya Bhushan

2011-01-01

We study the ameliorative potential of dimetylthiourea (DMTU), an OH• radical trapper and N-acetylcysteine (NAC), a glutathione precursor/H2O2 scavenger against titanium dioxide nanoparticles (TiO2-NPs) and multi-walled carbon nanotubes (MWCNTs) induced cyto-genotoxicity in cultured human lung cancer cells-A549. Cytogenotoxicity was induced by exposing the cells to selected concentrations (10 and 50 µg/ml) of either of TiO2-NPs or MWCNTs for 24 h. Anti-cytogenotoxicity effects of DMTU and NAC were studied in two groups, i.e., treatment of 30 minutes prior to toxic insult (short term exposure), while the other group received DMTU and NAC treatment during nanoparticles exposure, i.e., 24 h (long term exposure). Investigations were carried out for cell viability, generation of reactive oxygen species (ROS), micronuclei (MN), and expression of markers of oxidative stress (HSP27, CYP2E1), genotoxicity (P53) and CYP2E1 dependent n- nitrosodimethylamine-demethylase (NDMA-d) activity. In general, the treatment of both DMTU and NAC was found to be effective significantly against TiO2-NPs and MWCNTs induced cytogenotoxicity in A549 cells. Long-term treatment of DMTU and NAC during toxic insults has shown better prevention than short-term pretreatment. Although, cells responded significantly to both DMTU and NAC, but responses were chemical specific. In part, TiO2-NPs induced toxic responses were mediated through OH• radicals generation and reduction in the antioxidant defense system. While in the case of MWCNTs, adverse effects were primarily due to altering/hampering the enzymatic antioxidant system. Data indicate the applicability of human lung cancer cells-A549 as a pre-screening tool to identify the target specific prophylactic and therapeutic potential of drugs candidate molecules against nanoparticles induced cellular damages. PMID:21980536

Ameliorative effects of dimetylthiourea and N-acetylcysteine on nanoparticles induced cyto-genotoxicity in human lung cancer cells-A549.

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Ritesh Kumar Srivastava

Full Text Available We study the ameliorative potential of dimetylthiourea (DMTU, an OH• radical trapper and N-acetylcysteine (NAC, a glutathione precursor/H₂O₂ scavenger against titanium dioxide nanoparticles (TiO₂-NPs and multi-walled carbon nanotubes (MWCNTs induced cyto-genotoxicity in cultured human lung cancer cells-A549. Cytogenotoxicity was induced by exposing the cells to selected concentrations (10 and 50 µg/ml of either of TiO₂-NPs or MWCNTs for 24 h. Anti-cytogenotoxicity effects of DMTU and NAC were studied in two groups, i.e., treatment of 30 minutes prior to toxic insult (short term exposure, while the other group received DMTU and NAC treatment during nanoparticles exposure, i.e., 24 h (long term exposure. Investigations were carried out for cell viability, generation of reactive oxygen species (ROS, micronuclei (MN, and expression of markers of oxidative stress (HSP27, CYP2E1, genotoxicity (P⁵³ and CYP2E1 dependent n- nitrosodimethylamine-demethylase (NDMA-d activity. In general, the treatment of both DMTU and NAC was found to be effective significantly against TiO₂-NPs and MWCNTs induced cytogenotoxicity in A549 cells. Long-term treatment of DMTU and NAC during toxic insults has shown better prevention than short-term pretreatment. Although, cells responded significantly to both DMTU and NAC, but responses were chemical specific. In part, TiO₂-NPs induced toxic responses were mediated through OH• radicals generation and reduction in the antioxidant defense system. While in the case of MWCNTs, adverse effects were primarily due to altering/hampering the enzymatic antioxidant system. Data indicate the applicability of human lung cancer cells-A549 as a pre-screening tool to identify the target specific prophylactic and therapeutic potential of drugs candidate molecules against nanoparticles induced cellular damages.

Nanoparticles of Selaginella doederleinii leaf extract inhibit human lung cancer cells A549

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Syaefudin; Juniarti, A.; Rosiyana, L.; Setyani, A.; Khodijah, S.

2016-01-01

The aim of the present study is to evaluate cytotoxicity effect of nanoparticles of Selaginella doederleinii (S. doederleinii) leaves extract. S. doederleinii was extracted by maceration method using 70%(v/v) ethanol as solvent. Phytochemical content was analyzed qualitatively by using Harborne and Thin Layer Chromatography (TLC) methods. Nanoparticle extract was prepared by ionic gelation using chitosan as encapsulant agent. Anticancer activity was performed by using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The results showed that S. doederleinii contains of flavonoids. Nanoparticle of S. doederleinii leaves extract greatly inhibited A549 cells growth (cancer cells), with IC50 of 3% or 1020 μg/ml. These nanoparticles extract also inhibited the growth of Chang cells (normal cells), with IC50 of 4% or 1442 μg/ml. The effective concentration of nanoparticles extract which inhibits cancer cells without harming the normal cells is 0.5% or 167 μg/ml. Further studies are needed to obtain the concentration of nanoparticles extract which can selectively suppress cancer cells.

Nimesulide has a role of radio-sensitizer against lung carcinoma A549 cells

International Nuclear Information System (INIS)

Won, Joo Yoon; Park, Jong Kuk; Hong, Sung Hee

2006-01-01

Cyclooxygenases (COX) are key enzymes in the prostaglandin synthesis. There are two isoforms of the COX enzyme, COX-1 and COX-2. COX-2 expression is associated with carcinogenesis in variety of cancers and to render cells resistant to apoptotic stimuli. Increased expression of COX-2 is shown in non-small cell lung cancer (NSCLC), specifically in adenocarcinomas. Radiotherapy has been the important treatment for NSCLC. In recent studies, newer molecules that target specific pathophysiology or molecular pathways have been tested for the radiation sensitizers. COX-2 inhibitors are shown to enhanced radioresponse of cultured human cancer cell lines and immunodeficient mice. However, little is known about the molecular and biochemical mechanisms how NSAIDs enhance radioresponse of tumor cells. Nimesulide (methanesulfonamide, N-(4-nitro-2- phenoxyphenyl)), selective COX-2 inhibitors, is a drug with anti-inflammatory, anti-pyretic and analgesic properties. Nimesulide has the specific affinity to inhibit the inducible form of cyclooxygenase (COX-2) rather than the constitutive form (COX-1), and is well tolerated by adult, elderly and pediatric patients. Nimesulide was found also to have a chemopreventive activity against colon, urinary bladder, breast, tongue, and liver carcinogenesis. In this study, we examined whether nimesulide can increase radiation induced cell death and its mechanism in NSCLC cells A549

Proteomics in studying cancer stem cell biology.

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Kranenburg, Onno; Emmink, Benjamin L; Knol, Jaco; van Houdt, Winan J; Rinkes, Inne H M Borel; Jimenez, Connie R

2012-06-01

Normal multipotent tissue stem cells (SCs) are the driving force behind tissue turnover and repair. The cancer stem cell theory holds that tumors also contain stem-like cells that drive tumor growth and metastasis formation. However, very little is known about the regulation of SC maintenance pathways in cancer and how these are affected by cancer-specific genetic alterations and by treatment. Proteomics is emerging as a powerful tool to identify the signaling complexes and pathways that control multi- and pluri-potency and SC differentiation. Here, the authors review the novel insights that these studies have provided and present a comprehensive strategy for the use of proteomics in studying cancer SC biology.

Quantitative Proteomics Reveals Temporal Proteomic Changes in Signaling Pathways during BV2 Mouse Microglial Cell Activation.

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Woo, Jongmin; Han, Dohyun; Wang, Joseph Injae; Park, Joonho; Kim, Hyunsoo; Kim, Youngsoo

2017-09-01

The development of systematic proteomic quantification techniques in systems biology research has enabled one to perform an in-depth analysis of cellular systems. We have developed a systematic proteomic approach that encompasses the spectrum from global to targeted analysis on a single platform. We have applied this technique to an activated microglia cell system to examine changes in the intracellular and extracellular proteomes. Microglia become activated when their homeostatic microenvironment is disrupted. There are varying degrees of microglial activation, and we chose to focus on the proinflammatory reactive state that is induced by exposure to such stimuli as lipopolysaccharide (LPS) and interferon-gamma (IFN-γ). Using an improved shotgun proteomics approach, we identified 5497 proteins in the whole-cell proteome and 4938 proteins in the secretome that were associated with the activation of BV2 mouse microglia by LPS or IFN-γ. Of the differentially expressed proteins in stimulated microglia, we classified pathways that were related to immune-inflammatory responses and metabolism. Our label-free parallel reaction monitoring (PRM) approach made it possible to comprehensively measure the hyper-multiplex quantitative value of each protein by high-resolution mass spectrometry. Over 450 peptides that corresponded to pathway proteins and direct or indirect interactors via the STRING database were quantified by label-free PRM in a single run. Moreover, we performed a longitudinal quantification of secreted proteins during microglial activation, in which neurotoxic molecules that mediate neuronal cell loss in the brain are released. These data suggest that latent pathways that are associated with neurodegenerative diseases can be discovered by constructing and analyzing a pathway network model of proteins. Furthermore, this systematic quantification platform has tremendous potential for applications in large-scale targeted analyses. The proteomics data for

Proteomic cornerstones of hematopoietic stem cell differentiation

DEFF Research Database (Denmark)

Klimmeck, Daniel; Hansson, Jenny; Raffel, Simon

2012-01-01

Regenerative tissues such as the skin epidermis, the intestinal mucosa or the hematopoietic system are organized in a hierarchical manner with stem cells building the top of this hierarchy. Somatic stem cells harbor the highest self-renewal activity and generate a series of multipotent progenitors...... which differentiate into lineage committed progenitors and subsequently mature cells. In this report, we applied an in-depth quantitative proteomic approach to analyze and compare the full proteomes of ex vivo isolated and FACS-sorted populations highly enriched for either multipotent hematopoietic stem....../progenitor cells (HSPCs, Lin(neg)Sca-1(+)c-Kit(+)) or myeloid committed precursors (Lin(neg)Sca-1(-)c-Kit(+)). By employing stable isotope dimethyl labeling and high-resolution mass spectrometry, more than 5,000 proteins were quantified. From biological triplicate experiments subjected to rigorous statistical...

Inhibitory Effects of Salinomycin on Cell Survival, Colony Growth, Migration, and Invasion of Human Non-Small Cell Lung Cancer A549 and LNM35: Involvement of NAG-1.

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Kholoud Arafat

Full Text Available A major challenge for oncologists and pharmacologists is to develop more potent and less toxic drugs that will decrease the tumor growth and improve the survival of lung cancer patients. Salinomycin is a polyether antibiotic used to kill gram-positive bacteria including mycobacteria, protozoans such as plasmodium falciparum, and the parasites responsible for the poultry disease coccidiosis. This old agent is now a serious anti-cancer drug candidate that selectively inhibits the growth of cancer stem cells. We investigated the impact of salinomycin on survival, colony growth, migration and invasion of the differentiated human non-small cell lung cancer lines LNM35 and A549. Salinomycin caused concentration- and time-dependent reduction in viability of LNM35 and A549 cells through a caspase 3/7-associated cell death pathway. Similarly, salinomycin (2.5-5 µM for 7 days significantly decreased the growth of LNM35 and A549 colonies in soft agar. Metastasis is the main cause of death related to lung cancer. In this context, salinomycin induced a time- and concentration-dependent inhibition of cell migration and invasion. We also demonstrated for the first time that salinomycin induced a marked increase in the expression of the pro-apoptotic protein NAG-1 leading to the inhibition of lung cancer cell invasion but not cell survival. These findings identify salinomycin as a promising novel therapeutic agent for lung cancer.

Proteome alteration induced by hTERT transfection of human fibroblast cells.

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Mazzucchelli, Gabriel D; Gabelica, Valérie; Smargiasso, Nicolas; Fléron, Maximilien; Ashimwe, Wilson; Rosu, Frédéric; De Pauw-Gillet, Marie-Claire; Riou, Jean-François; De Pauw, Edwin

2008-04-17

Telomerase confers cellular immortality by elongating telomeres, thereby circumventing the Hayflick limit. Extended-life-span cells have been generated by transfection with the human telomerase reverse transcriptase (hTERT) gene. hTERT transfected cell lines may be of outstanding interest to monitor the effect of drugs targeting the telomerase activity. The incidence of hTERT gene transfection at the proteome level is a prerequisite to that purpose. The effect of the transfection has been studied on the proteome of human fibroblast (WI38). Cytosolic and nuclear fractions of WI38 cells, empty vector transfected WI38 (WI38-HPV) and hTERT WI38 cells were submitted to a 2D-DIGE (Two-Dimensional Differential In-Gel Electrophoresis) analysis. Only spots that had a similar abundance in WI38 and WI38-HPV, but were differentially expressed in WI38 hTERT were selected for MS identification. This method directly points to the proteins linked with the hTERT expression. Number of false positive differentially expressed proteins has been excluded by using control WI38-HPV cells. The proteome alteration induced by hTERT WI38 transfection should be taken into account in subsequent use of the cell line for anti-telomerase drugs evaluation. 2D-DIGE experiment shows that 57 spots out of 2246 are significantly differentially expressed in the cytosolic fraction due to hTERT transfection, and 38 were confidently identified. In the nuclear fraction, 44 spots out of 2172 were selected in the differential proteome analysis, and 14 were identified. The results show that, in addition to elongating telomeres, hTERT gene transfection has other physiological roles, among which an enhanced ER capacity and a potent cell protection against apoptosis. We show that the methodology reduces the complexity of the proteome analysis and highlights proteins implicated in other processes than telomere elongation. hTERT induced proteome changes suggest that telomerase expression enhances natural cell repair

Proteome alteration induced by hTERT transfection of human fibroblast cells

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Riou Jean-François

2008-04-01

Full Text Available Abstract Background Telomerase confers cellular immortality by elongating telomeres, thereby circumventing the Hayflick limit. Extended-life-span cells have been generated by transfection with the human telomerase reverse transcriptase (hTERT gene. hTERT transfected cell lines may be of outstanding interest to monitor the effect of drugs targeting the telomerase activity. The incidence of hTERT gene transfection at the proteome level is a prerequisite to that purpose. The effect of the transfection has been studied on the proteome of human fibroblast (WI38. Cytosolic and nuclear fractions of WI38 cells, empty vector transfected WI38 (WI38-HPV and hTERT WI38 cells were submitted to a 2D-DIGE (Two-Dimensional Differential In-Gel Electrophoresis analysis. Only spots that had a similar abundance in WI38 and WI38-HPV, but were differentially expressed in WI38 hTERT were selected for MS identification. This method directly points to the proteins linked with the hTERT expression. Number of false positive differentially expressed proteins has been excluded by using control WI38-HPV cells. The proteome alteration induced by hTERT WI38 transfection should be taken into account in subsequent use of the cell line for anti-telomerase drugs evaluation. Results 2D-DIGE experiment shows that 57 spots out of 2246 are significantly differentially expressed in the cytosolic fraction due to hTERT transfection, and 38 were confidently identified. In the nuclear fraction, 44 spots out of 2172 were selected in the differential proteome analysis, and 14 were identified. The results show that, in addition to elongating telomeres, hTERT gene transfection has other physiological roles, among which an enhanced ER capacity and a potent cell protection against apoptosis. Conclusion We show that the methodology reduces the complexity of the proteome analysis and highlights proteins implicated in other processes than telomere elongation. hTERT induced proteome changes suggest

Detection of Nuclear Protein Profile Changes by Human Metapneumovirus M2-2 Protein Using Quantitative Differential Proteomics

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Yuping Ren

2017-12-01

Full Text Available Human metapneumovirus (hMPV is a leading cause of lower respiratory infection in pediatric populations globally. This study examined proteomic profile changes in A549 cells infected with hMPV and two attenuated mutants with deleted PDZ domain-binding motif(s in the M2-2 protein. These motifs are involved in the interruption of antiviral signaling, namely the interaction between the TNF receptor associated factor (TRAF and mitochondrial antiviral-signaling (MAVS proteins. The aim of this study was to provide insight into the overall and novel impact of M2-2 motifs on cellular responses via an unbiased comparison. Tandem mass tagging, stable isotope labeling, and high-resolution mass spectrometry were used for quantitative proteomic analysis. Using quantitative proteomics and Venn analysis, 1248 common proteins were detected in all infected samples of both technical sets. Hierarchical clustering of the differentiated proteome displayed distinct proteomic signatures that were controlled by the motif(s. Bioinformatics and experimental analysis confirmed the differentiated proteomes, revealed novel cellular biological events, and implicated key pathways controlled by hMPV M2-2 PDZ domain-binding motif(s. This provides further insight for evaluating M2-2 mutants as potent vaccine candidates.

Analysis of gene expression changes in A549 cells induced by organic compounds from respirable air particles

Czech Academy of Sciences Publication Activity Database

Líbalová, Helena; Krčková, S.; Uhlířová, Kateřina; Kléma, J.; Ciganek, M.; Rössner ml., Pavel; Šrám, Radim; Vondráček, J.; Machala, M.; Topinka, Jan

2014-01-01

Roč. 770, DEC 2014 (2014), s. 94-105 ISSN 0027-5107 R&D Projects: GA ČR GAP503/11/0142; GA ČR(CZ) GBP503/12/G147 Institutional support: RVO:68378041 Keywords : Ah receptor * gene expression profile * A549 cells Subject RIV: DN - Health Impact of the Environment Quality Impact factor: 3.680, year: 2014

Cytotoxicity of semiconductor nanoparticles in A549 cells is attributable to their intrinsic oxidant activity

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Escamilla-Rivera, Vicente; Uribe-Ramirez, Marisela [Centro de Investigación y de Estudios Avanzados del IPN (CINVESTAV-IPN), Departamento de Toxicología (Mexico); Gonzalez-Pozos, Sirenia [CINVESTAV-IPN, Unidad de Microscopia Electrónica (LaNSE) (Mexico); Velumani, Subramaniam [CINVESTAV-IPN, Departamento de Ingeniería Eléctrica (Mexico); Arreola-Mendoza, Laura [Centro Interdisciplinario de Investigaciones y Estudios sobre Medio Ambiente y Desarrollo del Instituto Politécnico Nacional (CIIEMAD-IPN), Departamento de Biociencias e Ingeniería (Mexico); Vizcaya-Ruiz, Andrea De, E-mail: avizcaya@cinvestav.mx [Centro de Investigación y de Estudios Avanzados del IPN (CINVESTAV-IPN), Departamento de Toxicología (Mexico)

2016-04-15

Copper indium gallium diselenide (CIGS) and cadmium sulfide (CdS) nanoparticles (NP) are next generation semiconductors used in photovoltaic cells (PV). They possess high quantum efficiency, absorption coefficient, and cheaper manufacturing costs compared to silicon. Due to their potential for an industrial development and the lack of information about the risk associated in their use, we investigated the influence of the physicochemical characteristics of CIGS (9 nm) and CdS (20 nm) in relation to the induction of cytotoxicity in human alveolar A549 cells through ROS generation and mitochondrial dysfunction. CIGS induced cytotoxicity in a dose dependent manner in lower concentrations than CdS; both NP were able to induce ROS in A549. Moreover, CIGS interact directly with mitochondria inducing depolarization that leads to the induction of apoptosis compared to CdS. Antioxidant pretreatment significantly prevented the loss of mitochondrial membrane potential and cytotoxicity, suggesting ROS generation as the main cytotoxic mechanism. These results demonstrate that semiconductor characteristics of NP are crucial for the type and intensity of the cytotoxic effects. Our work provides relevant information that may help guide the production of a safer NP-based PV technologies, and would be a valuable resource on future risk assessment for a safer use of nanotechnology in the development of clean sources of renewable energy.

Effects of Human Parvovirus B19 and Bocavirus VP1 Unique Region on Tight Junction of Human Airway Epithelial A549 Cells

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Chiu, Chun-Ching; Shi, Ya-Fang; Yang, Jiann-Jou; Hsiao, Yuan-Chao; Tzang, Bor-Show; Hsu, Tsai-Ching

2014-01-01

As is widely recognized, human parvovirus B19 (B19) and human bocavirus (HBoV) are important human pathogens. Obviously, both VP1 unique region (VP1u) of B19 and HBoV exhibit the secreted phospholipase A2 (sPLA2)-like enzymatic activity and are recognized to participate in the pathogenesis of lower respiratory tract illnesses. However, exactly how, both VP1u from B19 and HBoV affect tight junction has seldom been addressed. Therefore, this study investigates how B19-VP1u and HBoV-VP1u may affect the tight junction of the airway epithelial A549 cells by examining phospholipase A2 activity and transepithelial electrical resistance (TEER) as well as performing immunoblotting analyses. Experimental results indicate that TEER is more significantly decreased in A549 cells by treatment with TNF-α (10 ng), two dosages of B19-VP1u and BoV-VP1u (400 ng and 4000 ng) or bee venom PLA2 (10 ng) than that of the control. Accordingly, more significantly increased claudin-1 and decreased occludin are detected in A549 cells by treatment with TNF-α or both dosages of HBoV-VP1u than that of the control. Additionally, more significantly decreased Na+/K+ ATPase is observed in A549 cells by treatment with TNF-α, high dosage of B19-VP1u or both dosages of BoV-VP1u than that of the control. Above findings suggest that HBoV-VP1u rather than B19 VP1u likely plays more important roles in the disruption of tight junction in the airway tract. Meanwhile, this discrepancy appears not to be associated with the secreted phospholipase A2 (sPLA2)-like enzymatic activity. PMID:25268969

Social network architecture of human immune cells unveiled by quantitative proteomics.

Science.gov (United States)

Rieckmann, Jan C; Geiger, Roger; Hornburg, Daniel; Wolf, Tobias; Kveler, Ksenya; Jarrossay, David; Sallusto, Federica; Shen-Orr, Shai S; Lanzavecchia, Antonio; Mann, Matthias; Meissner, Felix

2017-05-01

The immune system is unique in its dynamic interplay between numerous cell types. However, a system-wide view of how immune cells communicate to protect against disease has not yet been established. We applied high-resolution mass-spectrometry-based proteomics to characterize 28 primary human hematopoietic cell populations in steady and activated states at a depth of >10,000 proteins in total. Protein copy numbers revealed a specialization of immune cells for ligand and receptor expression, thereby connecting distinct immune functions. By integrating total and secreted proteomes, we discovered fundamental intercellular communication structures and previously unknown connections between cell types. Our publicly accessible (http://www.immprot.org/) proteomic resource provides a framework for the orchestration of cellular interplay and a reference for altered communication associated with pathology.

Digoxin Downregulates NDRG1 and VEGF through the Inhibition of HIF-1α under Hypoxic Conditions in Human Lung Adenocarcinoma A549 Cells

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Dong Wei

2013-04-01

Full Text Available Digoxin, an inhibitor of Na+/K+ ATPase, has been used in the treatment of heart-related diseases (such as congestive heart failure and atrial arrhythmia for decades. Recently, it was reported that digoxin is also an effective HIF-1α inhibitor. We investigated whether digoxin could suppress tumor cell growth through HIF-1α in non-small cell lung cancer cells (A549 cells under hypoxic conditions. An MTT assay was used to measure cell viability. RT-PCR and western blotting were performed to analyze the mRNA and protein expression of VEGF, NDRG1, and HIF-1α. HIF-1α nuclear translocation was then determined by EMSA. Digoxin was found to inhibit the proliferation of A549 cells under hypoxic conditions. Our results showed that hypoxia led to the upregulation of VEGF, NDRG1, and HIF-1α both at the mRNA and protein levels. We also found that the hypoxia-induced overexpression of VEGF, NDRG1, and HIF-1α was suppressed by digoxin in a concentration-dependent manner. As expected, our EMSA results demonstrated that under hypoxic conditions HIF-1α nuclear translocation was also markedly reduced by digoxin in a concentration-dependent manner. Our results suggest that digoxin downregulated hypoxia-induced overexpression of VEGF and NDRG1 at the transcriptional level probably through the inhibition of HIF-1α synthesis in A549 cells.

Toxicity of engineered nanomaterials and their transformation products following wastewater treatment on A549 human lung epithelial cells

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Yanjun Ma

2014-01-01

Full Text Available Here we characterize the toxicity of environmentally-relevant forms of engineered nanomaterials (ENMs, which can transform during wastewater treatment and persist in aqueous effluents and biosolids. In an aerosol exposure scenario, cytotoxicity and genotoxicity of effluents and biosolids from lab-scale sequencing batch reactors (SBRs to A549 human lung epithelial cells were examined. The SBRs were dosed with nanoAg, nano zero-valent iron (NZVI, nanoTiO2 and nanoCeO2 at sequentially increasing concentrations from 0.1 to 20 mg/L. Toxicities were compared to outputs from SBRs dosed with ionic/bulk analogs, undosed SBRs, and pristine ENMs. Pristine nanoAg and NZVI showed significant cytotoxicity to A549 cells in a dose-dependent manner from 1 to 67 μg/mL, while nanoTiO2 and nanoCeO2 only exerted cytotoxicity at 67 μg/mL. Only nanoAg induced a genotoxic response, at 9, 33 and 53 μg/mL. However, no significant cytotoxic or genotoxic effects of the SBR effluents or biosolids containing nanomaterials were observed.

Evaluation of Anti-Metastatic Potential of the Combination of Fisetin with Paclitaxel on A549 Non-Small Cell Lung Cancer Cells.

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Klimaszewska-Wiśniewska, Anna; Hałas-Wiśniewska, Marta; Grzanka, Alina; Grzanka, Dariusz

2018-02-27

The identification and development of new agents with a therapeutic potential as well as novel drug combinations are gaining the attention of scientists and clinicians as a plausible approach to improve therapeutic regimens for chemoresistant tumors. We have recently reported that the flavonoid fisetin (FIS), at physiologically attainable concentrations, acts synergistically with clinically achievable doses of paclitaxel (PTX) to produce growth inhibitory and pro-death effects on A549 human non-small cell lung cancer (NSCLC) cells. To further investigate a potential therapeutic efficacy of the combination of fisetin with paclitaxel, we decided to assess its impact on metastatic capability of A549 cells as well as its toxicity toward normal human lung fibroblast. Cell viability, cell migration, and invasion were measured by thiazolyl blue tetrazolium bromide (MTT) assay, wound healing assay, and Transwell chamber assay, respectively. The expression of metastasis-related genes was assessed with quantitative reverse transcriptase real-time polymerase chain reaction (qRT-PCR). Actin and vimentin filaments were examined under the fluorescence microscope. The combination of FIS and PTX significantly reduced cancer cell migration and invasion, at least partially, through a marked rearrangement of actin and vimentin cytoskeleton and the modulation of metastasis-related genes. Most of these effects of the combination treatment were significantly greater than those of individual agents. Paclitaxel alone was even more toxic to normal cells than the combination of this drug with the flavonoid, suggesting that FIS may provide some protection against PTX-mediated cytotoxicity. The combination of FIS and PTX is expected to have a synergistic anticancer efficacy and a significant potential for the treatment of NSCLC, however, further in vitro and in vivo studies are required to confirm this preliminary evidence.

Evaluation of Anti-Metastatic Potential of the Combination of Fisetin with Paclitaxel on A549 Non-Small Cell Lung Cancer Cells

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Anna Klimaszewska-Wiśniewska

2018-02-01

Full Text Available The identification and development of new agents with a therapeutic potential as well as novel drug combinations are gaining the attention of scientists and clinicians as a plausible approach to improve therapeutic regimens for chemoresistant tumors. We have recently reported that the flavonoid fisetin (FIS, at physiologically attainable concentrations, acts synergistically with clinically achievable doses of paclitaxel (PTX to produce growth inhibitory and pro-death effects on A549 human non-small cell lung cancer (NSCLC cells. To further investigate a potential therapeutic efficacy of the combination of fisetin with paclitaxel, we decided to assess its impact on metastatic capability of A549 cells as well as its toxicity toward normal human lung fibroblast. Cell viability, cell migration, and invasion were measured by thiazolyl blue tetrazolium bromide (MTT assay, wound healing assay, and Transwell chamber assay, respectively. The expression of metastasis-related genes was assessed with quantitative reverse transcriptase real-time polymerase chain reaction (qRT-PCR. Actin and vimentin filaments were examined under the fluorescence microscope. The combination of FIS and PTX significantly reduced cancer cell migration and invasion, at least partially, through a marked rearrangement of actin and vimentin cytoskeleton and the modulation of metastasis-related genes. Most of these effects of the combination treatment were significantly greater than those of individual agents. Paclitaxel alone was even more toxic to normal cells than the combination of this drug with the flavonoid, suggesting that FIS may provide some protection against PTX-mediated cytotoxicity. The combination of FIS and PTX is expected to have a synergistic anticancer efficacy and a significant potential for the treatment of NSCLC, however, further in vitro and in vivo studies are required to confirm this preliminary evidence.

Oxidative Stress Facilitates IFN-γ-Induced Mimic Extracellular Trap Cell Death in A549 Lung Epithelial Cancer Cells.

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Lin, Chiou-Feng; Chen, Chia-Ling; Chien, Shun-Yi; Tseng, Po-Chun; Wang, Yu-Chih; Tsai, Tsung-Ting

2016-01-01

We previously demonstrated that IFN-γ induces an autophagy-regulated mimic extracellular trap cell death (ETosis) in A549 human lung cancer cells. Regarding reactive oxygen species (ROS) are involved in ETosis, this study investigated the role of oxidative stress. After IFN-γ stimulation, a necrosis-like cell death mimic ETosis occurred accompanied by the inhibition of cell growth, aberrant nuclear staining, and nucleosome release. ROS were generated in a time-dependent manner with an increase in NADPH oxidase component protein expression. STAT1-mediated IFN regulatory factor-1 activation was essential for upregulating ROS production. By genetically silencing p47phox, IFN-γ-induced ROS and mimic ETosis were significantly attenuated. This mechanistic study indicated that ROS may mediate DNA damage followed by histone H3 citrullination. Furthermore, ROS promoted IFN-γ-induced mimic ETosis in cooperation with autophagy. These findings further demonstrate that ROS regulates IFN-γ-induced mimic ETosis in lung epithelial malignancy.

Advancing cell biology through proteomics in space and time (PROSPECTS)

DEFF Research Database (Denmark)

Lamond, A.I.; Uhlen, M.; Horning, S.

2012-01-01

a range of sensitive and quantitative approaches for measuring protein structures and dynamics that promise to revolutionize our understanding of cell biology and molecular mechanisms in both human cells and model organisms. The Proteomics Specification in Time and Space (PROSPECTS) Network is a unique EU......-funded project that brings together leading European research groups, spanning from instrumentation to biomedicine, in a collaborative five year initiative to develop new methods and applications for the functional analysis of cellular proteins. This special issue of Molecular and Cellular Proteomics presents 16...... quantification of protein levels. Manuscripts in this issue exemplify approaches for performing quantitative measurements of cell proteomes and for studying their dynamic responses to perturbation, both during normal cellular responses and in disease mechanisms. Here we present a perspective on how...

Proteomic analysis of cell walls of two developmental stages of alfalfa stems

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Julian C Verdonk

2012-12-01

Full Text Available Cell walls are important for the growth and development of all plants. They are also valuable resources for feed and fiber, and more recently as a potential feedstock for bioenergy production. Cell wall proteins comprise only a fraction of the cell wall, but play important roles in establishing the walls and in the chemical interactions (e.g. crosslinking of cell wall components. This crosslinking provides structure, but restricts digestibility of cell wall complex carbohydrates, limiting available energy in animal and bioenergy production systems. Manipulation of cell wall proteins could be a strategy to improve digestibility. An analysis of the cell wall proteome of apical alfalfa stems (less mature, more digestible and basal alfalfa stems (more mature, less digestible was conducted using a recently developed low-salt/density gradient method for the isolation of cell walls. Walls were subsequently subjected to a modified extraction utilizing EGTA to remove pectins, followed by a LiCl extraction to isolate more tightly bound proteins. Recovered proteins were identified using shotgun proteomics. We identified 272 proteins in the alfalfa stem cell wall proteome, 153 of which had not previously been identified in cell wall proteomic analyses. Nearly 70% percent of the identified proteins were predicted to be secreted, as would be expected for most cell wall proteins, an improvement over previously published studies using traditional cell wall isolation methods. A comparison of our and several other cell wall proteomic studies indicates little overlap in identified proteins among them, which may be largely due to differences in the tissues used as well as differences in experimental approach.

Evaluation of cell wall preparations for proteomics: a new procedure for purifying cell walls from Arabidopsis hypocotyls

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Canut Hervé

2006-05-01

Full Text Available Abstract Background The ultimate goal of proteomic analysis of a cell compartment should be the exhaustive identification of resident proteins; excluding proteins from other cell compartments. Reaching such a goal closely depends on the reliability of the isolation procedure for the cell compartment of interest. Plant cell walls possess specific difficulties: (i the lack of a surrounding membrane may result in the loss of cell wall proteins (CWP during the isolation procedure, (ii polysaccharide networks of cellulose, hemicelluloses and pectins form potential traps for contaminants such as intracellular proteins. Several reported procedures to isolate cell walls for proteomic analyses led to the isolation of a high proportion (more than 50% of predicted intracellular proteins. Since isolated cell walls should hold secreted proteins, one can imagine alternative procedures to prepare cell walls containing a lower proportion of contaminant proteins. Results The rationales of several published procedures to isolate cell walls for proteomics were analyzed, with regard to the bioinformatic-predicted subcellular localization of the identified proteins. Critical steps were revealed: (i homogenization in low ionic strength acid buffer to retain CWP, (ii purification through increasing density cushions, (iii extensive washes with a low ionic strength acid buffer to retain CWP while removing as many cytosolic proteins as possible, and (iv absence of detergents. A new procedure was developed to prepare cell walls from etiolated hypocotyls of Arabidopsis thaliana. After salt extraction, a high proportion of proteins predicted to be secreted was released (73%, belonging to the same functional classes as proteins identified using previously described protocols. Finally, removal of intracellular proteins was obtained using detergents, but their amount represented less than 3% in mass of the total protein extract, based on protein quantification. Conclusion The

Study of the Effects of Betaine and/or C-Phycocyanin on the Growth of Lung Cancer A549 Cells In Vitro and In Vivo

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Rea Bingula

2016-01-01

Full Text Available We investigated the effects of betaine, C-phycocyanin (C-PC, and their combined use on the growth of A549 lung cancer both in vitro and in vivo. When cells were coincubated with betaine and C-PC, an up to 60% decrease in viability was observed which is significant compared to betaine (50% or C-PC treatment alone (no decrease. Combined treatment reduced the stimulation of NF-κB expression by TNF-α and increased the amount of the proapoptotic p38 MAPK. Interestingly, combined treatment induced a cell cycle arrest in G2/M phase for ~60% of cells. In vivo studies were performed in pathogen-free male nude rats injected with A549 cells in their right flank. Their daily food was supplemented with either betaine, C-PC, both, or neither. Compared to the control group, tumour weights and volumes were significantly reduced in either betaine- or C-PC-treated groups and no additional decrease was obtained with the combined treatment. This data indicates that C-PC and betaine alone may efficiently inhibit tumour growth in rats. The synergistic activity of betaine and C-PC on A549 cells growth observed in vitro remains to be further confirmed in vivo. The reason behind the nature of their interaction is yet to be sought.

Proteomic Analysis of the Cell Cycle of Procylic Form Trypanosoma brucei.

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Crozier, Thomas W M; Tinti, Michele; Wheeler, Richard J; Ly, Tony; Ferguson, Michael A J; Lamond, Angus I

2018-06-01

We describe a single-step centrifugal elutriation method to produce synchronous Gap1 (G1)-phase procyclic trypanosomes at a scale amenable for proteomic analysis of the cell cycle. Using ten-plex tandem mass tag (TMT) labeling and mass spectrometry (MS)-based proteomics technology, the expression levels of 5325 proteins were quantified across the cell cycle in this parasite. Of these, 384 proteins were classified as cell-cycle regulated and subdivided into nine clusters with distinct temporal regulation. These groups included many known cell cycle regulators in trypanosomes, which validates the approach. In addition, we identify 40 novel cell cycle regulated proteins that are essential for trypanosome survival and thus represent potential future drug targets for the prevention of trypanosomiasis. Through cross-comparison to the TrypTag endogenous tagging microscopy database, we were able to validate the cell-cycle regulated patterns of expression for many of the proteins of unknown function detected in our proteomic analysis. A convenient interface to access and interrogate these data is also presented, providing a useful resource for the scientific community. Data are available via ProteomeXchange with identifier PXD008741 (https://www.ebi.ac.uk/pride/archive/). © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Comparison of the uptakes of Tc-99m MIBI and Tc-99m tetrofosmin in A549, an MRP-expressing cancer cell, in vitro and in vivo

International Nuclear Information System (INIS)

Yoo, Jeong Ah; Jeong, Shin Young; Seo, Myung Rang; Bae, Jin Ho; Ahn, Byeong Cheol; Lee, Kyu Bo; Lee, Jae Tae; Choi, Sang Woon; Lee, Byung Ho

2003-01-01

Uptakes of Tc-99m MIBI (MIBI) and Tc-99m tetrofosmin (tetrofosmin) in human non-small cell lung cancer A549, multidrug-resistance associated protein (MRP) expressing cell, were investigated in vitro and in vivo. Western blot analysis and immunohistochemistry were used for detetion of MRP in A549 cells with anti-MRPr1 antibody. Cellular uptakes of two tracers were evaluated at 100 μM of verapamil (Vrp), 50 μM of cyclosporin A (CsA) and 25 μM of butoxysulfoximide (BSO) after incubation with MIBI and tetrofosmin for 30 and 60 min at 37.deg.C, using single cell suspensions at 1x10 6 cells/ml. Radioactivities in supernatants and pellets were measured with gamma well counter. A549 cells were inoculated in each flanks of 24 nude mice. Group 1 (Gr1) and Gr3 mice were treated with only MIBI or tetrofosmin, and Gr2 and Gr4 mice were treated with 70mg/kg of CsA i.p. for 1 hour before injection of 370KBq of MIBI or tetrofosmin. Mice were sacrificed at 10, 60 and 240 min. Radioactivities of organs and tumors were expressed as percentage injected dose per gram of tissue (%ID/gm). Western blot analysis of the A549 cells detected expression of MRPr1 (190 kDa) and immunohistochemical staining of tumor tissue for MRPr1 revealed brownish staining in cell membrane but not P-gp. Upon incubating A549 cells for 60 min with MIBI and tetrofosmin, cellular uptake of MIBI was higher than that of tetrofosmin. Coincubation with modulators resulted in an increase in cellular uptakes of MIBI and tetrofosmin. Coincubation with modulators resulted in an increase in cellular uptakes of MIBI and tetorfosmin. Percentage increase of MIBI was higher than that of tetrofosmin with Vrp by 623% and 427%, CsA by 753% and 629% and BSO by 219% and 140%, respectively. There was no significant difference in tumoral uptakes of MIBI and tetrofosmin between Gr1 and Gr3. Percentage increases in MIBI (114% at 10 min, 257% at 60 min, 396% at 240 min) and tetrofosmin uptake (110% at 10 min, 205% at 60 min, 410% at

Asiatic Acid (AA) Sensitizes Multidrug-Resistant Human Lung Adenocarcinoma A549/DDP Cells to Cisplatin (DDP) via Downregulation of P-Glycoprotein (MDR1) and Its Targets.

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Cheng, Qilai; Liao, Meixiang; Hu, Haibo; Li, Hongliang; Wu, Longhuo

2018-01-01

P-glycoprotein (P-gp, i.e., MDR1) is associated with the phenotype of multidrug resistance (MDR) and causes chemotherapy failure in the management of cancers. Searching for effective MDR modulators and combining them with anticancer drugs is a promising strategy against MDR. Asiatic acid (AA), a natural triterpene isolated from the plant Centella asiatica, may have an antitumor activity. The present study assessed the reversing effect of AA on MDR and possible molecular mechanisms of AA action in MDR1-overexpressing cisplatin (DDP)-resistant lung cancer cells, A549/DDP. Human lung adenocarcinoma A549/DDP cells were either exposed to different concentrations of AA or treated with DDP, and their viability was measured by the MTT assay. A Rhodamine 123 efflux assay, immunofluorescent staining, ATPase assay, reverse-transcription PCR (RT-PCR), and western blot analysis were conducted to elucidate the mechanisms of action of AA on MDR. Our results showed that AA significantly enhanced the cytotoxicity of DDP toward A549/DDP cells but not its parental A549 cells. Furthermore, AA strongly inhibited P-gp expression by blocking MDR1 gene transcription and increased the intracellular accumulation of the P-gp substrate Rhodamine 123 in A549/DDP cells. Nuclear factor (NF)-kB (p65) activity, IkB degradation, and NF-kB/p65 nuclear translocation were markedly inhibited by pretreatment with AA. Additionally, AA inhibited the MAPK-ERK pathway, as indicated by decreased phosphorylation of ERK1 and -2, AKT, p38, and JNK, thus resulting in reduced activity of the Y-box binding protein 1 (YB1) via blockage of its nuclear translocation. AA reversed P-gp-mediated MDR by inhibition of P-gp expression. This effect was likely related to downregulation of YB1, and this effect was mediated by the NF-kB and MAPK-ERK pathways. AA may be useful as an MDR reversal agent for combination therapy in clinical trials. © 2018 The Author(s). Published by S. Karger AG, Basel.

Tumor-targeting magnetic lipoplex delivery of short hairpin RNA suppresses IGF-1R overexpression of lung adenocarcinoma A549 cells in vitro and in vivo

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Wang, Chunmao; Ding, Chao; Kong, Minjian [Department of Cardiothoracic Surgery, Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310009 (China); Dong, Aiqiang, E-mail: dr_dongaiqiang@sina.com [Department of Cardiothoracic Surgery, Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310009 (China); Qian, Jianfang; Jiang, Daming; Shen, Zhonghua [Department of Cardiothoracic Surgery, Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310009 (China)

2011-07-08

Highlights: {yields} We compared lipofection with magnetofection about difference of transfection efficiency on delivery a therapeutic gene in vitro and in vivo. {yields} We investigated the difference of shRNA induced by magnetofection and lipofection into A549 cell and subcutaneous tumor to knockdown IGF-1R overexpressed in A549 cell and A549 tumor. {yields} We investigated in vivo shRNA silenced IGF-1R overexpression 24, 48, and 72 h after shRNA intravenous injection into tumor-bearing mice by way of magnetofection and lipofection. {yields} Our results showed that magnetofection could achieve therapeutic gene targeted delivery into special site, which contributed to targeted gene therapy of lung cancers. -- Abstract: Liposomal magnetofection potentiates gene transfection by applying a magnetic field to concentrate magnetic lipoplexes onto target cells. Magnetic lipoplexes are self-assembling ternary complexes of cationic lipids with plasmid DNA associated with superparamagnetic iron oxide nanoparticles (SPIONs). Type1insulin-like growth factor receptor (IGF-1R), an important oncogene, is frequently overexpressed in lung cancer and mediates cancer cell proliferation and tumor growth. In this study, we evaluated the transfection efficiency (percentage of transfected cells) and therapeutic potential (potency of IGF-1R knockdown) of liposomal magnetofection of plasmids expressing GFP and shRNAs targeting IGF-1R (pGFPshIGF-1Rs) in A549 cells and in tumor-bearing mice as compared to lipofection using Lipofectamine 2000. Liposomal magnetofection provided a threefold improvement in transgene expression over lipofection and transfected up to 64.1% of A549 cells in vitro. In vitro, IGF-1R specific-shRNA transfected by lipofection inhibited IGF-1R protein by 56.1 {+-} 6% and by liposomal magnetofection by 85.1 {+-} 3%. In vivo delivery efficiency of the pGFPshIGF-1R plasmid into the tumor was significantly higher in the liposomal magnetofection group than in the

Tumor-targeting magnetic lipoplex delivery of short hairpin RNA suppresses IGF-1R overexpression of lung adenocarcinoma A549 cells in vitro and in vivo

International Nuclear Information System (INIS)

Wang, Chunmao; Ding, Chao; Kong, Minjian; Dong, Aiqiang; Qian, Jianfang; Jiang, Daming; Shen, Zhonghua

2011-01-01

Highlights: → We compared lipofection with magnetofection about difference of transfection efficiency on delivery a therapeutic gene in vitro and in vivo. → We investigated the difference of shRNA induced by magnetofection and lipofection into A549 cell and subcutaneous tumor to knockdown IGF-1R overexpressed in A549 cell and A549 tumor. → We investigated in vivo shRNA silenced IGF-1R overexpression 24, 48, and 72 h after shRNA intravenous injection into tumor-bearing mice by way of magnetofection and lipofection. → Our results showed that magnetofection could achieve therapeutic gene targeted delivery into special site, which contributed to targeted gene therapy of lung cancers. -- Abstract: Liposomal magnetofection potentiates gene transfection by applying a magnetic field to concentrate magnetic lipoplexes onto target cells. Magnetic lipoplexes are self-assembling ternary complexes of cationic lipids with plasmid DNA associated with superparamagnetic iron oxide nanoparticles (SPIONs). Type1insulin-like growth factor receptor (IGF-1R), an important oncogene, is frequently overexpressed in lung cancer and mediates cancer cell proliferation and tumor growth. In this study, we evaluated the transfection efficiency (percentage of transfected cells) and therapeutic potential (potency of IGF-1R knockdown) of liposomal magnetofection of plasmids expressing GFP and shRNAs targeting IGF-1R (pGFPshIGF-1Rs) in A549 cells and in tumor-bearing mice as compared to lipofection using Lipofectamine 2000. Liposomal magnetofection provided a threefold improvement in transgene expression over lipofection and transfected up to 64.1% of A549 cells in vitro. In vitro, IGF-1R specific-shRNA transfected by lipofection inhibited IGF-1R protein by 56.1 ± 6% and by liposomal magnetofection by 85.1 ± 3%. In vivo delivery efficiency of the pGFPshIGF-1R plasmid into the tumor was significantly higher in the liposomal magnetofection group than in the lipofection group. In vivo IGF-1R

MiR-509-3-5p causes aberrant mitosis and anti-proliferative effect by suppression of PLK1 in human lung cancer A549 cells

International Nuclear Information System (INIS)

Wang, Xian-Hui; Lu, Yao; Liang, Jing-Jing; Cao, Ji-Xiang; Jin, Ya-Qiong; An, Guo-Shun; Ni, Ju-Hua; Jia, Hong-Ti; Li, Shu-Yan

2016-01-01

MicroRNAs (miRNAs) are potent post-transcriptional regulators of gene expression and play roles in DNA damage response (DDR). PLK1 is identified as a modulator of DNA damage checkpoint. Although down-regulation of PLK1 by certain microRNAs has been reported, little is known about the interplay between PLK1 and miR-509-3-5p in DDR. Here we have demonstrated that miR-509-3-5p repressed PLK1 expression by targeting PLK1 3′-UTR, thereby causing mitotic aberration and growth arrest of human lung cancer A549 cells. Repression of PLK1 by miR-509-3-5p was further evidenced by over-expression of miR-509-3-5p in A549, HepG2 and HCT116p53 −/− cancer cells, in which PLK1 protein was suppressed. Consistently, miR-509-3-5p was stimulated, while PLK1 protein was down-regulated in A549 cells exposed to CIS and ADR, suggesting that suppression of PLK1 by miR-509-3-5p is a component of CIS/ADR-induced DDR pathway. Flow cytometry and immunofluorescence labeling showed that over-expression of miR-509-3-5p in A549 induced G2/M arrest and aberrant mitosis characterized by abnormal bipolar mitotic spindles, condensed chromosomes, lagging DNA and chromosome bridges. In addition, over-expression of miR-509-3-5p markedly blocked A549 cell proliferation and sensitized the cells to CIS and ADR treatment. Taken together, miR-509-3-5p is a feasible suppressor for cancer by targeting PLK1. Our data may provide aid in potential design of combined chemotherapy and in our better understanding of the roles of microRNAs in response to DNA damage. - Highlights: • MiR-509-3-5p represses PLK1 expression by targeting PLK1 3ГЉВ№-UTR. • Expression of miR-509-3-5p is induced and PLK1 repressed upon DNA damage. • Overexpression of miR-509-3-5p induces G2/M arrest and aberrant mitosis. • MiR-509-3-5p inhibits cell proliferation and sensitizes cells to DNA damage agents.

Sulfamic and succinic acid derivatives of 25-OH-PPD and their activities to MCF-7, A-549, HCT-116, and BGC-823 cell lines.

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Zhou, Wu-Xi; Cao, Jia-Qing; Wang, Xu-De; Guo, Jun-Hui; Zhao, Yu-Qing

2017-02-15

In the search for new anti-tumor agents with higher potency than our previously identified compound 1 (25-OH-PPD, 25-hydroxyprotopanaxadiol), 12 novel sulfamic and succinic acid derivatives that could improve water solubility and contribute to good drug potency and pharmacokinetic profiles were designed and synthesized. Their in vitro anti-tumor activities in MCF-7, A-549, HCT-116, and BGC-823 cell lines and one normal cell line were tested by standard MTT assay. Results showed that compared with compound 1, compounds 2, 3, and 7 exhibited higher cytotoxic activity on A-549 and BGC-823 cell lines, together with lower toxicity in the normal cell. In particular, compound 2 exhibited the best anti-tumor activity in the in vitro assays, which may provide valuable data for the research and development of new anti-tumor agents. Copyright © 2016 Elsevier Ltd. All rights reserved.

Proteomic Analysis to Identify Tightly-Bound Cell Wall Protein in Rice Calli

OpenAIRE

Cho, Won Kyong; Hyun, Tae Kyung; Kumar, Dhinesh; Rim, Yeonggil; Chen, Xiong Yan; Jo, Yeonhwa; Kim, Suwha; Lee, Keun Woo; Park, Zee-Yong; Lucas, William J.; Kim, Jae-Yean

2015-01-01

Rice is a model plant widely used for basic and applied research programs. Plant cell wall proteins play key roles in a broad range of biological processes. However, presently, knowledge on the rice cell wall proteome is rudimentary in nature. In the present study, the tightly-bound cell wall proteome of rice callus cultured cells using sequential extraction protocols was developed using mass spectrometry and bioinformatics methods, leading to the identification of 1568 candidate proteins. Ba...

Predictive role of computer simulation in assessing signaling pathways of crizotinib-treated A549 lung cancer cells.

Science.gov (United States)

Xia, Pu; Mou, Fei-Fei; Wang, Li-Wei

2012-01-01

Non-small-cell lung cancer (NSCLC) is a leading cause of cancer deaths worldwide. Crizotinib has been approved by the U.S. Food and Drug Administration for the treatment of patients with advanced NSCLC. However, understanding of mechanisms of action is still limited. In our studies, we confirmed crizotinib-induced apoptosis in A549 lung cancer cells. In order to assess mechanisms, small molecular docking technology was used as a preliminary simulation of signaling pathways. Interesting, our results of experiments were consistent with the results of computer simulation. This indicates that small molecular docking technology should find wide use for its reliability and convenience.

Single-cell proteomics: potential implications for cancer diagnostics.

Science.gov (United States)

Gavasso, Sonia; Gullaksen, Stein-Erik; Skavland, Jørn; Gjertsen, Bjørn T

2016-01-01

Single-cell proteomics in cancer is evolving and promises to provide more accurate diagnoses based on detailed molecular features of cells within tumors. This review focuses on technologies that allow for collection of complex data from single cells, but also highlights methods that are adaptable to routine cancer diagnostics. Current diagnostics rely on histopathological analysis, complemented by mutational detection and clinical imaging. Though crucial, the information gained is often not directly transferable to defined therapeutic strategies, and predicting therapy response in a patient is difficult. In cancer, cellular states revealed through perturbed intracellular signaling pathways can identify functional mutations recurrent in cancer subsets. Single-cell proteomics remains to be validated in clinical trials where serial samples before and during treatment can reveal excessive clonal evolution and therapy failure; its use in clinical trials is anticipated to ignite a diagnostic revolution that will better align diagnostics with the current biological understanding of cancer.

Comparative proteomics analysis of oral cancer cell lines: identification of cancer associated proteins

Science.gov (United States)

2014-01-01

Background A limiting factor in performing proteomics analysis on cancerous cells is the difficulty in obtaining sufficient amounts of starting material. Cell lines can be used as a simplified model system for studying changes that accompany tumorigenesis. This study used two-dimensional gel electrophoresis (2DE) to compare the whole cell proteome of oral cancer cell lines vs normal cells in an attempt to identify cancer associated proteins. Results Three primary cell cultures of normal cells with a limited lifespan without hTERT immortalization have been successfully established. 2DE was used to compare the whole cell proteome of these cells with that of three oral cancer cell lines. Twenty four protein spots were found to have changed in abundance. MALDI TOF/TOF was then used to determine the identity of these proteins. Identified proteins were classified into seven functional categories – structural proteins, enzymes, regulatory proteins, chaperones and others. IPA core analysis predicted that 18 proteins were related to cancer with involvements in hyperplasia, metastasis, invasion, growth and tumorigenesis. The mRNA expressions of two proteins – 14-3-3 protein sigma and Stress-induced-phosphoprotein 1 – were found to correlate with the corresponding proteins’ abundance. Conclusions The outcome of this analysis demonstrated that a comparative study of whole cell proteome of cancer versus normal cell lines can be used to identify cancer associated proteins. PMID:24422745

Region and cell-type resolved quantitative proteomic map of the human heart

DEFF Research Database (Denmark)

Doll, Sophia; Dreßen, Martina; Geyer, Philipp E

2017-01-01

The heart is a central human organ and its diseases are the leading cause of death worldwide, but an in-depth knowledge of the identity and quantity of its constituent proteins is still lacking. Here, we determine the healthy human heart proteome by measuring 16 anatomical regions and three major...... cardiac cell types by high-resolution mass spectrometry-based proteomics. From low microgram sample amounts, we quantify over 10,700 proteins in this high dynamic range tissue. We combine copy numbers per cell with protein organellar assignments to build a model of the heart proteome at the subcellular...

TRAIL overexpression co-regulated by Egr1 and HRE enhances radiosensitivity of hypoxic A549 cells depending on its apoptosis inducing role.

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Yang, Yan-Ming; Fang, Fang; Li, Xin; Yu, Lei; Wang, Zhi-Cheng

2017-01-01

Ionizing radiation can upregulate the expression levels of TRAIL and enhance tumor cell apoptosis. While Early growth response 1 (Egr1) gene promoter has radiation inducible characteristics, the expression for exogenous gene controlled by Egr1 promoter could be enhanced by ionizing radiation, but its efficiency is limited by tissue hypoxia. Hypoxia response elements (HREs) are important hypoxic response regulatory sequences and sensitivity enhancers. Therefore, we chose TRAIL as the gene radiotherapy to observe whether it is regulated by Egr1 and HER and its effects on A549 cells and its mechanism. The pcDNA3.1-Egr1-TRAIL (pc-E-hsT) and pcDNA3.1-HRE/Egr1-TRAIL (pc-H/E-hsT) plasmids containing Egr1-hsTRAIL and HRE/Egr1-hsTRAIL were transfected into A549 cells, the cells were treated by hypoxia and radiation. The TRAIL mRNA in the cells and protein concentration in the culture supernatants were measured by RT-PCR and ELISA, respectively. Mean lethal dose D0 value was evaluated with colony forming assay. The cell apoptotic rates were analyzed by FCM and TUNEL assay. Expression of DR4, DR5 and cleaved caspase-3 proteins were analyzed by western blotting. It showed that TRAIL mRNA expression and TRAIL concentration all significantly increased under hypoxia and/or radiation. D0 value of pc-H/E‑hsT transfected cells under hypoxia was lowest, indicating more high radiosensitivity. Hypoxia could not cause the pc-E-hsT transfected cell apoptotic rate increase, but there were promoting effects in pc-H/E-hsT transfected cells. DR4 had not obvious change in pc-E-hsT and pc-H/E-hsT transfected cells under normoxic and hypoxic condition, otherwise, DR5 and cleaved caspase-3 increased mostly in pc-H/E-hsT transfected cells under hypoxic condition. TRAIL overexpression was co-regulated by Egr1 and HRE. TRAIL might promote hypoxic A549 cell radiosensitivity and induce apoptosis depending on DR5 to caspase-3 pathways.

Proteomic analysis of the response to cell cycle arrests in human myeloid leukemia cells.

Science.gov (United States)

Ly, Tony; Endo, Aki; Lamond, Angus I

2015-01-02

Previously, we analyzed protein abundance changes across a 'minimally perturbed' cell cycle by using centrifugal elutriation to differentially enrich distinct cell cycle phases in human NB4 cells (Ly et al., 2014). In this study, we compare data from elutriated cells with NB4 cells arrested at comparable phases using serum starvation, hydroxyurea, or RO-3306. While elutriated and arrested cells have similar patterns of DNA content and cyclin expression, a large fraction of the proteome changes detected in arrested cells are found to reflect arrest-specific responses (i.e., starvation, DNA damage, CDK1 inhibition), rather than physiological cell cycle regulation. For example, we show most cells arrested in G2 by CDK1 inhibition express abnormally high levels of replication and origin licensing factors and are likely poised for genome re-replication. The protein data are available in the Encyclopedia of Proteome Dynamics (

Screening of Stat3 inhibitory effects of Korean herbal medicines in the A549 human lung cancer cell line.

Science.gov (United States)

Park, Jong-Shik; Bang, Ok-Sun; Kim, Jinhee

2014-06-01

The transcription factor signal transducer and activator of transcription 3 (Stat3) is constitutively activated in many human cancers. It promotes tumor cell proliferation, inhibits apoptosis, induces angiogenesis and metastasis, and suppresses antitumor host immune responses. Therefore, Stat3 has emerged as a promising molecular target for cancer therapies. In this study, we evaluated the Stat3-suppressive activity of 38 herbal medicines traditionally used in Korea. Medicinal herb extracts in 70% ethanol were screened for their ability to suppress Stat3 in the A549 human lung cancer cell line. A Stat3-responsive reporter assay system was used to detect intracellular Stat3 activity in extract-treated cells, and Western blot analyses were performed to measure the expression profiles of Stat3-regulated proteins. Fifty percent of the 38 extracts possessed at least mild Stat3-suppressive activities (i.e., activity less than 75% of the vehicle control). Ethanol extracts of Bupleurum falcatum L., Taraxacum officinale Weber, Solanum nigrum L., Ulmus macrocarpa Hance, Euonymus alatus Sieb., Artemisia capillaris Thunb., and Saururus chinensis (Lour.) Baill inhibited up to 75% of the vehicle control Stat3 activity level. A549 cells treated with these extracts also had reduced Bcl-xL, Survivin, c-Myc, and Mcl-1 expression. Many medicinal herbs traditionally used in Korea contain Stat3 activity-suppressing substances. Because of the therapeutic impact of Stat3 inhibition, these results could be useful when developing novel cancer therapeutics from medicinal herbs.

Cytotoxicity evaluation of nanoclays in human epithelial cell line A549 using high content screening and real-time impedance analysis

Energy Technology Data Exchange (ETDEWEB)

Verma, Navin K. [Trinity College Dublin, Department of Clinical Medicine, Institute of Molecular Medicine (Ireland); Moore, Edward; Blau, Werner [Trinity College Dublin, School of Physics (Ireland); Volkov, Yuri [Trinity College Dublin, Department of Clinical Medicine, Institute of Molecular Medicine (Ireland); Ramesh Babu, P., E-mail: babup@tcd.ie [Trinity College Dublin, Centre for Research on Adaptive Nanostructures and Nanodevices (Ireland)

2012-09-15

Continuously expanding use of products containing nanoclays for wide range of applications have raised public concerns about health and safety. Although the products containing nanoclays may not be toxic, it is possible that nanomaterials may come in contact with humans during handling, manufacture, or disposal, and cause adverse health impact. This necessitates biocompatibility evaluation of the commonly used nanoclays. Here, we investigated the cytotoxic effects of platelet (Bentone MA, ME-100, Cloisite Na{sup +}, Nanomer PGV, and Delite LVF) and tubular (Halloysite, and Halloysite MP1) type nanoclays on cultured human lung epithelial cells A549. For the first time with this aim, we employed a cell-based automated high content screening in combination with real-time impedance sensing. We demonstrate varying degree of dose- and time-dependent cytotoxic effects of both nanoclay types. Overall, platelet structured nanoclays were more cytotoxic than tubular type. A low but significant level of cytotoxicity was observed at 25 {mu}g/mL of the platelet-type nanoclays. A549 cells exposed to high concentration (250 {mu}g/mL) of tubular structured nanoclays showed inhibited cell growth. Confocal microscopy indicated intracellular accumulation of nanoclays with perinuclear localization. Results indicate a potential hazard of nanoclay-containing products at significantly higher concentrations, which warrant their further biohazard assessment on the actual exposure in humans.

Cytotoxicity evaluation of nanoclays in human epithelial cell line A549 using high content screening and real-time impedance analysis

International Nuclear Information System (INIS)

Verma, Navin K.; Moore, Edward; Blau, Werner; Volkov, Yuri; Ramesh Babu, P.

2012-01-01

Continuously expanding use of products containing nanoclays for wide range of applications have raised public concerns about health and safety. Although the products containing nanoclays may not be toxic, it is possible that nanomaterials may come in contact with humans during handling, manufacture, or disposal, and cause adverse health impact. This necessitates biocompatibility evaluation of the commonly used nanoclays. Here, we investigated the cytotoxic effects of platelet (Bentone MA, ME-100, Cloisite Na + , Nanomer PGV, and Delite LVF) and tubular (Halloysite, and Halloysite MP1) type nanoclays on cultured human lung epithelial cells A549. For the first time with this aim, we employed a cell-based automated high content screening in combination with real-time impedance sensing. We demonstrate varying degree of dose- and time-dependent cytotoxic effects of both nanoclay types. Overall, platelet structured nanoclays were more cytotoxic than tubular type. A low but significant level of cytotoxicity was observed at 25 μg/mL of the platelet-type nanoclays. A549 cells exposed to high concentration (250 μg/mL) of tubular structured nanoclays showed inhibited cell growth. Confocal microscopy indicated intracellular accumulation of nanoclays with perinuclear localization. Results indicate a potential hazard of nanoclay-containing products at significantly higher concentrations, which warrant their further biohazard assessment on the actual exposure in humans.

Identification of a Short Cell-Penetrating Peptide from Bovine Lactoferricin for Intracellular Delivery of DNA in Human A549 Cells.

Science.gov (United States)

Liu, Betty R; Huang, Yue-Wern; Aronstam, Robert S; Lee, Han-Jung

2016-01-01

Cell-penetrating peptides (CPPs) have been shown to deliver cargos, including protein, DNA, RNA, and nanomaterials, in fully active forms into live cells. Most of the CPP sequences in use today are based on non-native proteins that may be immunogenic. Here we demonstrate that the L5a CPP (RRWQW) from bovine lactoferricin (LFcin), stably and noncovalently complexed with plasmid DNA and prepared at an optimal nitrogen/phosphate ratio of 12, is able to efficiently enter into human lung cancer A549 cells. The L5a CPP delivered a plasmid containing the enhanced green fluorescent protein (EGFP) coding sequence that was subsequently expressed in cells, as revealed by real-time PCR and fluorescent microscopy at the mRNA and protein levels, respectively. Treatment with calcium chloride increased the level of gene expression, without affecting CPP-mediated transfection efficiency. Zeta-potential analysis revealed that positively electrostatic interactions of CPP/DNA complexes correlated with CPP-mediated transport. The L5a and L5a/DNA complexes were not cytotoxic. This biomimetic LFcin L5a represents one of the shortest effective CPPs and could be a promising lead peptide with less immunogenic for DNA delivery in gene therapy.

Application of a lipid-coated hollow calcium phosphate nanoparticle in synergistic co-delivery of doxorubicin and paclitaxel for the treatment of human lung cancer A549 cells

Directory of Open Access Journals (Sweden)

Wu C

2017-10-01

Full Text Available Chao Wu, Jie Xu, Yanna Hao, Ying Zhao, Yang Qiu, Jie Jiang, Tong Yu, Peng Ji, Ying Liu Pharmacy School, Jinzhou Medical University, Jinzhou, China Abstract: In this study, we developed a lipid-coated hollow calcium phosphate (LCP nanoparticle for the combined application of two chemotherapeutic drugs to human lung cancer A549 cells. Hydrophilic doxorubicin (DOX was incorporated into the hollow structure of hollow calcium phosphate (HCP, and a lipid bilayer containing hydrophobic paclitaxel (PTX was subsequently coated on the surface of HCP. The study on combinational effects demonstrated that the combination of DOX and PTX at a mass ratio of 12:1 showed a synergistic effect against A549 cells. The particle size, zeta potential, and encapsulation efficiency were measured to obtain optimal values: particle size was 335.0 3.2 nm, zeta potential -41.1 mV, and encapsulation efficiency 80.40%±2.24%. An in vitro release study indicated that LCP produced a sustained drug release. A549 cells had a better uptake of LCP with good biocompatibility. Furthermore, in vitro cytotoxicity experiment, apoptosis analysis, in vivo anti-tumor efficacy and protein expression analysis of Bax, Bcl-2, and Caspase-3 demonstrated that the co-delivery system based on LCP had significant synergistic anti-tumor activity. All conclusions suggested that LCP is a promising platform for co-delivery of multiple anti-tumor drugs. Keywords: doxorubicin, paclitaxel, co-delivery, lipid, hollow calcium phosphate, lung cancer cell

Global Proteome Analysis of the NCI-60 Cell Line Panel

Directory of Open Access Journals (Sweden)

Amin Moghaddas Gholami

2013-08-01

Full Text Available The NCI-60 cell line collection is a very widely used panel for the study of cellular mechanisms of cancer in general and in vitro drug action in particular. It is a model system for the tissue types and genetic diversity of human cancers and has been extensively molecularly characterized. Here, we present a quantitative proteome and kinome profile of the NCI-60 panel covering, in total, 10,350 proteins (including 375 protein kinases and including a core cancer proteome of 5,578 proteins that were consistently quantified across all tissue types. Bioinformatic analysis revealed strong cell line clusters according to tissue type and disclosed hundreds of differentially regulated proteins representing potential biomarkers for numerous tumor properties. Integration with public transcriptome data showed considerable similarity between mRNA and protein expression. Modeling of proteome and drug-response profiles for 108 FDA-approved drugs identified known and potential protein markers for drug sensitivity and resistance. To enable community access to this unique resource, we incorporated it into a public database for comparative and integrative analysis (http://wzw.tum.de/proteomics/nci60.

Cu(II Complexes of Isoniazid Schiff Bases: DNA/BSA Binding and Cytotoxicity Studies on A549 Cell Line

Directory of Open Access Journals (Sweden)

Pulipaka Ramadevi

2014-01-01

Full Text Available A series of isonicotinoyl hydrazones have been synthesized via template method and were complexed to Cu(II. The ligands are coordinated to Cu(II ion through the enolic oxygen and azomethine nitrogen resulting in a square planar geometry. The CT-DNA and bovine serum albumin binding propensities of the compounds were determined spectrophotometrically, the results of which indicate good binding propensity of complexes to DNA and BSA with high binding constant values. Furthermore, the compounds have been investigated for their cytotoxicities on A549 human lung cancer cell. Also the mode of cell death was examined employing various staining techniques and was found to be apoptotic.

Release behavior and toxicity profiles towards A549 cell lines of ciprofloxacin from its layered zinc hydroxide intercalation compound.

Science.gov (United States)

Abdul Latip, Ahmad Faiz; Hussein, Mohd Zobir; Stanslas, Johnson; Wong, Charng Choon; Adnan, Rohana

2013-01-01

Layered hydroxides salts (LHS), a layered inorganic compound is gaining attention in a wide range of applications, particularly due to its unique anion exchange properties. In this work, layered zinc hydroxide nitrate (LZH), a family member of LHS was intercalated with anionic ciprofloxacin (CFX), a broad spectrum antibiotic via ion exchange in a mixture solution of water:ethanol. Powder x-ray diffraction (XRD), Fourier transform infrared (FTIR) and thermogravimetric analysis (TGA) confirmed the drug anions were successfully intercalated in the interlayer space of LZH. Specific surface area of the obtained compound was increased compared to that of the host due to the different pore textures between the two materials. CFX anions were slowly released over 80 hours in phosphate-buffered saline (PBS) solution due to strong interactions that occurred between the intercalated anions and the host lattices. The intercalation compound demonstrated enhanced antiproliferative effects towards A549 cancer cells compared to the toxicity of CFX alone. Strong host-guest interactions between the LZH lattice and the CFX anion give rise to a new intercalation compound that demonstrates sustained release mode and enhanced toxicity effects towards A549 cell lines. These findings should serve as foundations towards further developments of the brucite-like host material in drug delivery systems.

Human borna disease virus infection impacts host proteome and histone lysine acetylation in human oligodendroglia cells

Energy Technology Data Exchange (ETDEWEB)

Liu, Xia [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016 (China); Department of Neurology, The Fifth People' s Hospital of Shanghai, School of Medicine, Fudan University, Shanghai, 200240 (China); Zhao, Libo [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016 (China); Department of Neurology, The Third People' s Hospital of Chongqing, 400014 (China); Yang, Yongtao [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016 (China); Chongqing Key Laboratory of Neurobiology, Chongqing Medical University, Chongqing, 400016 (China); Institute of Neuroscience, Chongqing Medical University, Chongqing, 400016 (China); Bode, Liv [Bornavirus Research Group affiliated to the Free University of Berlin, Berlin (Germany); Huang, Hua [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016 (China); Chongqing Key Laboratory of Neurobiology, Chongqing Medical University, Chongqing, 400016 (China); Institute of Neuroscience, Chongqing Medical University, Chongqing, 400016 (China); Liu, Chengyu [Chongqing Key Laboratory of Neurobiology, Chongqing Medical University, Chongqing, 400016 (China); Institute of Neuroscience, Chongqing Medical University, Chongqing, 400016 (China); Huang, Rongzhong [Department of Rehabilitative Medicine, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, 400010 (China); Zhang, Liang [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016 (China); Chongqing Key Laboratory of Neurobiology, Chongqing Medical University, Chongqing, 400016 (China); Institute of Neuroscience, Chongqing Medical University, Chongqing, 400016 (China); and others

2014-09-15

Background: Borna disease virus (BDV) replicates in the nucleus and establishes persistent infections in mammalian hosts. A human BDV strain was used to address the first time, how BDV infection impacts the proteome and histone lysine acetylation (Kac) of human oligodendroglial (OL) cells, thus allowing a better understanding of infection-driven pathophysiology in vitro. Methods: Proteome and histone lysine acetylation were profiled through stable isotope labeling for cell culture (SILAC)-based quantitative proteomics. The quantifiable proteome was annotated using bioinformatics. Histone acetylation changes were validated by biochemistry assays. Results: Post BDV infection, 4383 quantifiable differential proteins were identified and functionally annotated to metabolism pathways, immune response, DNA replication, DNA repair, and transcriptional regulation. Sixteen of the thirty identified Kac sites in core histones presented altered acetylation levels post infection. Conclusions: BDV infection using a human strain impacted the whole proteome and histone lysine acetylation in OL cells. - Highlights: • A human strain of BDV (BDV Hu-H1) was used to infect human oligodendroglial cells (OL cells). • This study is the first to reveal the host proteomic and histone Kac profiles in BDV-infected OL cells. • BDV infection affected the expression of many transcription factors and several HATs and HDACs.

Human borna disease virus infection impacts host proteome and histone lysine acetylation in human oligodendroglia cells

International Nuclear Information System (INIS)

Liu, Xia; Zhao, Libo; Yang, Yongtao; Bode, Liv; Huang, Hua; Liu, Chengyu; Huang, Rongzhong; Zhang, Liang

2014-01-01

Background: Borna disease virus (BDV) replicates in the nucleus and establishes persistent infections in mammalian hosts. A human BDV strain was used to address the first time, how BDV infection impacts the proteome and histone lysine acetylation (Kac) of human oligodendroglial (OL) cells, thus allowing a better understanding of infection-driven pathophysiology in vitro. Methods: Proteome and histone lysine acetylation were profiled through stable isotope labeling for cell culture (SILAC)-based quantitative proteomics. The quantifiable proteome was annotated using bioinformatics. Histone acetylation changes were validated by biochemistry assays. Results: Post BDV infection, 4383 quantifiable differential proteins were identified and functionally annotated to metabolism pathways, immune response, DNA replication, DNA repair, and transcriptional regulation. Sixteen of the thirty identified Kac sites in core histones presented altered acetylation levels post infection. Conclusions: BDV infection using a human strain impacted the whole proteome and histone lysine acetylation in OL cells. - Highlights: • A human strain of BDV (BDV Hu-H1) was used to infect human oligodendroglial cells (OL cells). • This study is the first to reveal the host proteomic and histone Kac profiles in BDV-infected OL cells. • BDV infection affected the expression of many transcription factors and several HATs and HDACs

Differential proteome analysis of chikungunya virus infection on host cells.

Directory of Open Access Journals (Sweden)

Christina Li-Ping Thio

Full Text Available BACKGROUND: Chikungunya virus (CHIKV is an emerging mosquito-borne alphavirus that has caused multiple unprecedented and re-emerging outbreaks in both tropical and temperate countries. Despite ongoing research efforts, the underlying factors involved in facilitating CHIKV replication during early infection remains ill-characterized. The present study serves to identify host proteins modulated in response to early CHIKV infection using a proteomics approach. METHODOLOGY AND PRINCIPAL FINDINGS: The whole cell proteome profiles of CHIKV-infected and mock control WRL-68 cells were compared and analyzed using two-dimensional gel electrophoresis (2-DGE. Fifty-three spots were found to be differentially modulated and 50 were successfully identified by MALDI-TOF/TOF. Eight were significantly up-regulated and 42 were down-regulated. The mRNA expressions of 15 genes were also found to correlate with the corresponding protein expression. STRING network analysis identified several biological processes to be affected, including mRNA processing, translation, energy production and cellular metabolism, ubiquitin-proteasome pathway (UPP and cell cycle regulation. CONCLUSION/SIGNIFICANCE: This study constitutes a first attempt to investigate alteration of the host cellular proteome during early CHIKV infection. Our proteomics data showed that during early infection, CHIKV affected the expression of proteins that are involved in mRNA processing, host metabolic machinery, UPP, and cyclin-dependent kinase 1 (CDK1 regulation (in favour of virus survival, replication and transmission. While results from this study complement the proteomics results obtained from previous late host response studies, functional characterization of these proteins is warranted to reinforce our understanding of their roles during early CHIKV infection in humans.

Nanodroplet processing platform for deep and quantitative proteome profiling of 10-100 mammalian cells.

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Zhu, Ying; Piehowski, Paul D; Zhao, Rui; Chen, Jing; Shen, Yufeng; Moore, Ronald J; Shukla, Anil K; Petyuk, Vladislav A; Campbell-Thompson, Martha; Mathews, Clayton E; Smith, Richard D; Qian, Wei-Jun; Kelly, Ryan T

2018-02-28

Nanoscale or single-cell technologies are critical for biomedical applications. However, current mass spectrometry (MS)-based proteomic approaches require samples comprising a minimum of thousands of cells to provide in-depth profiling. Here, we report the development of a nanoPOTS (nanodroplet processing in one pot for trace samples) platform for small cell population proteomics analysis. NanoPOTS enhances the efficiency and recovery of sample processing by downscaling processing volumes to 3000 proteins are consistently identified from as few as 10 cells. Furthermore, we demonstrate quantification of ~2400 proteins from single human pancreatic islet thin sections from type 1 diabetic and control donors, illustrating the application of nanoPOTS for spatially resolved proteome measurements from clinical tissues.

Suitable parameter choice on quantitative morphology of A549 cell in epithelial–mesenchymal transition

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Ren, Zhou-Xin; Yu, Hai-Bin; Li, Jian-Sheng; Shen, Jun-Ling; Du, Wen-Sen

2015-01-01

Evaluation of morphological changes in cells is an integral part of study on epithelial to mesenchymal transition (EMT), however, only a few papers reported the changes in quantitative parameters and no article compared different parameters for demanding better parameters. In the study, the purpose was to investigate suitable parameters for quantitative evaluation of EMT morphological changes. A549 human lung adenocarcinoma cell line was selected for the study. Some cells were stimulated by transforming growth factor-β1 (TGF-β1) for EMT, and other cells were as control without TGF-β1 stimulation. Subsequently, cells were placed in phase contrast microscope and three arbitrary fields were captured and saved with a personal computer. Using the tools of Photoshop software, some cells in an image were selected, segmented out and exchanged into unique hue, and other part in the image was shifted into another unique hue. The cells were calculated with 29 morphological parameters by Image Pro Plus software. A parameter between cells with or without TGF-β1 stimulation was compared statistically and nine parameters were significantly different between them. Receiver operating characteristic curve (ROC curve) of a parameter was described with SPSS software and F-test was used to compare two areas under the curves (AUCs) in Excel. Among them, roundness and radius ratio were the most AUCs and were significant higher than the other parameters. The results provided a new method with quantitative assessment of cell morphology during EMT, and found out two parameters, roundness and radius ratio, as suitable for quantification. PMID:26182364

A Temporal Proteomic Map of Epstein-Barr Virus Lytic Replication in B Cells

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Ina Ersing

2017-05-01

Full Text Available Epstein-Barr virus (EBV replication contributes to multiple human diseases, including infectious mononucleosis, nasopharyngeal carcinoma, B cell lymphomas, and oral hairy leukoplakia. We performed systematic quantitative analyses of temporal changes in host and EBV proteins during lytic replication to gain insights into virus-host interactions, using conditional Burkitt lymphoma models of type I and II EBV infection. We quantified profiles of >8,000 cellular and 69 EBV proteins, including >500 plasma membrane proteins, providing temporal views of the lytic B cell proteome and EBV virome. Our approach revealed EBV-induced remodeling of cell cycle, innate and adaptive immune pathways, including upregulation of the complement cascade and proteasomal degradation of the B cell receptor complex, conserved between EBV types I and II. Cross-comparison with proteomic analyses of human cytomegalovirus infection and of a Kaposi-sarcoma-associated herpesvirus immunoevasin identified host factors targeted by multiple herpesviruses. Our results provide an important resource for studies of EBV replication.

Arabidopsis Regenerating Protoplast: A Powerful Model System for Combining the Proteomics of Cell Wall Proteins and the Visualization of Cell Wall Dynamics

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Yokoyama, Ryusuke; Kuki, Hiroaki; Kuroha, Takeshi; Nishitani, Kazuhiko

2016-01-01

The development of a range of sub-proteomic approaches to the plant cell wall has identified many of the cell wall proteins. However, it remains difficult to elucidate the precise biological role of each protein and the cell wall dynamics driven by their actions. The plant protoplast provides an excellent means not only for characterizing cell wall proteins, but also for visualizing the dynamics of cell wall regeneration, during which cell wall proteins are secreted. It therefore offers a unique opportunity to investigate the de novo construction process of the cell wall. This review deals with sub-proteomic approaches to the plant cell wall through the use of protoplasts, a methodology that will provide the basis for further exploration of cell wall proteins and cell wall dynamics. PMID:28248244

Identification of a Short Cell-Penetrating Peptide from Bovine Lactoferricin for Intracellular Delivery of DNA in Human A549 Cells.

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Betty R Liu

Full Text Available Cell-penetrating peptides (CPPs have been shown to deliver cargos, including protein, DNA, RNA, and nanomaterials, in fully active forms into live cells. Most of the CPP sequences in use today are based on non-native proteins that may be immunogenic. Here we demonstrate that the L5a CPP (RRWQW from bovine lactoferricin (LFcin, stably and noncovalently complexed with plasmid DNA and prepared at an optimal nitrogen/phosphate ratio of 12, is able to efficiently enter into human lung cancer A549 cells. The L5a CPP delivered a plasmid containing the enhanced green fluorescent protein (EGFP coding sequence that was subsequently expressed in cells, as revealed by real-time PCR and fluorescent microscopy at the mRNA and protein levels, respectively. Treatment with calcium chloride increased the level of gene expression, without affecting CPP-mediated transfection efficiency. Zeta-potential analysis revealed that positively electrostatic interactions of CPP/DNA complexes correlated with CPP-mediated transport. The L5a and L5a/DNA complexes were not cytotoxic. This biomimetic LFcin L5a represents one of the shortest effective CPPs and could be a promising lead peptide with less immunogenic for DNA delivery in gene therapy.

Dioscorin pre-treatment protects A549 human airway epithelial cells from hydrogen peroxide-induced oxidative stress.

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Hsu, Jeng-Yuan; Chu, Jao-Jia; Chou, Ming-Chih; Chen, Ya-Wen

2013-10-01

Hydrogen peroxide (H(2)O(2)) is a highly reactive oxygen species involved in lung and bronchial epithelium injury. Increased H(2)O(2) levels have been reported in expired breath condensates of patients with inflammatory airway diseases such as chronic obstructive pulmonary disease. Protecting airway epithelial cells from oxidative stress is an important task in the prevention and management of airway diseases. Previous studies demonstrate that yam (Dioscorea batatas Decne) has antioxidant and anti-trypsin activities. This study evaluated the validity of dioscorin in vitro. The results showed that dioscorin attenuated the alteration of H(2)O(2) on G2/M cell cycle arrest. This might be associated with the activation of IκB and subsequent inactivation of NF-κB. Furthermore, dioscorin suppressed IL-8 secretion and reduced changes of adhesion molecule expressions in H(2)O(2)-injured A549 cells. These results help in understanding the potential of traditional Chinese herbal medicine as treatment for airway inflammatory diseases.

Proteomic profiling of bone marrow mesenchymal stem cells upon TGF-beta stimulation

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Wang, Daojing; Park, Jennifer S.; Chu, Julia S.F.; Ari, Krakowski; Luo, Kunxin; Chen, David J.; Li, Song

2004-08-08

Bone marrow mesenchymal stem cells (MSCs) can differentiate into different types of cells, and have tremendous potential for cell therapy and tissue engineering. Transforming growth factor {beta}1 (TGF-{beta}) plays an important role in cell differentiation and vascular remodeling. We showed that TGF-{beta} induced cell morphology change and an increase in actin fibers in MSCs. To determine the global effects of TGF-{beta} on MSCs, we employed a proteomic strategy to analyze the effect of TGF-{beta} on the human MSC proteome. By using two-dimensional gel electrophoresis and electrospray ionization coupled to Quadrupole/time-of-flight tandem mass spectrometers, we have generated a proteome reference map of MSCs, and identified {approx}30 proteins with an increase or decrease in expression or phosphorylation in response to TGF-{beta}. The proteins regulated by TGF-{beta} included cytoskeletal proteins, matrix synthesis proteins, membrane proteins, metabolic enzymes, etc. TGF-{beta} increased the expression of smooth muscle (SM) {alpha}-actin and decreased the expression of gelsolin. Over-expression of gelsolin inhibited TGF-{beta}-induced assembly of SM {alpha}-actin; on the other hand, knocking down gelsolin expression enhanced the assembly of {alpha}-actin and actin filaments without significantly affecting {alpha}-actin expression. These results suggest that TGF-{beta} coordinates the increase of {alpha}-actin and the decrease of gelsolin to promote MSC differentiation. This study demonstrates that proteomic tools are valuable in studying stem cell differentiation and elucidating the underlying molecular mechanisms.

Plant subcellular proteomics: Application for exploring optimal cell function in soybean.

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Wang, Xin; Komatsu, Setsuko

2016-06-30

Plants have evolved complicated responses to developmental changes and stressful environmental conditions. Subcellular proteomics has the potential to elucidate localized cellular responses and investigate communications among subcellular compartments during plant development and in response to biotic and abiotic stresses. Soybean, which is a valuable legume crop rich in protein and vegetable oil, can grow in several climatic zones; however, the growth and yield of soybean are markedly decreased under stresses. To date, numerous proteomic studies have been performed in soybean to examine the specific protein profiles of cell wall, plasma membrane, nucleus, mitochondrion, chloroplast, and endoplasmic reticulum. In this review, methods for the purification and purity assessment of subcellular organelles from soybean are summarized. In addition, the findings from subcellular proteomic analyses of soybean during development and under stresses, particularly flooding stress, are presented and the proteins regulated among subcellular compartments are discussed. Continued advances in subcellular proteomics are expected to greatly contribute to the understanding of the responses and interactions that occur within and among subcellular compartments during development and under stressful environmental conditions. Subcellular proteomics has the potential to investigate the cellular events and interactions among subcellular compartments in response to development and stresses in plants. Soybean could grow in several climatic zones; however, the growth and yield of soybean are markedly decreased under stresses. Numerous proteomics of cell wall, plasma membrane, nucleus, mitochondrion, chloroplast, and endoplasmic reticulum was carried out to investigate the respecting proteins and their functions in soybean during development or under stresses. In this review, methods of subcellular-organelle enrichment and purity assessment are summarized. In addition, previous findings of

Contribution of MS-based proteomics to the understanding of Herpes Simplex Virus type 1 interaction with host cells

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Enrique eSantamaría

2012-03-01

Full Text Available Like other DNA viruses, Herpes Simplex Virus type 1 (HSV-1 replicates and proliferates in host cells continuously modulating the host molecular environment. Following a sophisticated temporal expression pattern, HSV-1 encodes at least 89 multifunctional proteins that interplay with and modify the host cell proteome. During the last decade, advances in mass spectrometry applications coupled to the development of proteomic separation methods have allowed to partially monitor the impact of HSV-1 infection in human cells. In this review, we discuss the current use of different proteome fractionation strategies to define HSV-1 targets on two major application areas: i viral protein interactomics to decipher viral protein interactions in host cells and ii differential quantitative proteomics to analyse the virally induced changes in the cellular proteome. Moreover, we will also discuss the potential application of high throughput proteomic approaches to study global proteome dynamics and also post-translational modifications in HSV-1-infected cells, what will greatly improved our molecular knowledge of HSV-1 infection.

Inflammatory effects induced by selected limonene oxidation products: 4-OPA, IPOH, 4-AMCH in human bronchial (16HBE14o-) and alveolar (A549) epithelial cell lines.

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Lipsa, Dorelia; Leva, Paolo; Barrero-Moreno, Josefa; Coelhan, Mehmet

2016-11-16

Limonene, a monoterpene abundantly present in most of the consumer products (due to its pleasant citrus smell), easily undergoes ozonolysis leading to several limonene oxidation products (LOPs) such as 4-acetyl-1-methylcyclohexene (4-AMCH), 4-oxopentanal (4-OPA) and 3-isopropenyl-6-oxoheptanal (IPOH). Toxicological studies have indicated that human exposure to limonene and ozone can cause adverse airway effects. However, little attention has been paid to the potential health impact of specific LOPs, in particular of IPOH, 4-OPA and 4-AMCH. This study evaluates the cytotoxic effects of the selected LOPs on human bronchial epithelial (16HBE14o-) and alveolar epithelial (A549) cell lines by generating concentration-response curves using the neutral red uptake assay and analyzing the inflammatory response with a series of cytokines/chemokines. The cellular viability was mostly reduced by 4-OPA [IC 50 =1.6mM (A549) and 1.45mM (16HBE14o-)] when compared to IPOH [IC 50 =3.5mM (A549) and 3.4mM (16HBE14o-)] and 4-AMCH [IC 50 could not be calculated]. As a result from the inflammatory response, IPOH [50μM] induced an increase of both IL-6 and IL-8 secretion in A549 (1.5-fold change) and in 16HBE14o- (2.8- and 7-fold change respectively). 4-OPA [50μM] treatment of A549 increased IL-6 (1.4-times) and IL-8 (1.3-times) levels, while in 16HBE14o- had an opposite effect. A549 treated with 4-AMCH [50μM] elevate both IL-6 and IL-8 levels by 1.2-times, while in 16HBE14o- had an opposite effect. Based on our results, lung cellular injury characterized by inflammatory cytokine release was observed for both cell lines treated with the selected chemicals at concentrations that did not affect their cellular viability. Copyright © 2016 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

IFN-gamma Impairs Release of IL-8 by IL-1beta-stimulated A549 Lung Carcinoma Cells

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Pfeilschifter Josef

2008-09-01

Full Text Available Abstract Background Production of interferon (IFN-γ is key to efficient anti-tumor immunity. The present study was set out to investigate effects of IFNγ on the release of the potent pro-angiogenic mediator IL-8 by human A549 lung carcinoma cells. Methods A549 cells were cultured and stimulated with interleukin (IL-1β alone or in combination with IFNγ. IL-8 production by these cells was analyzed with enzyme linked immuno sorbent assay (ELISA. mRNA-expression was analyzed by real-time PCR and RNase protection assay (RPA, respectively. Expression of inhibitor-κ Bα, cellular IL-8, and cyclooxygenase-2 was analyzed by Western blot analysis. Results Here we demonstrate that IFNγ efficiently reduced IL-8 secretion under the influence of IL-1β. Surprisingly, real-time PCR analysis and RPA revealed that the inhibitory effect of IFNγ on IL-8 was not associated with significant changes in mRNA levels. These observations concurred with lack of a modulatory activity of IFNγ on IL-1β-induced NF-κB activation as assessed by cellular IκB levels. Moreover, analysis of intracellular IL-8 suggests that IFNγ modulated IL-8 secretion by action on the posttranslational level. In contrast to IL-8, IL-1β-induced cyclooxygenase-2 expression and release of IL-6 were not affected by IFNγ indicating that modulation of IL-1β action by this cytokine displays specificity. Conclusion Data presented herein agree with an angiostatic role of IFNγ as seen in rodent models of solid tumors and suggest that increasing T helper type 1 (Th1-like functions in lung cancer patients e.g. by local delivery of IFNγ may mediate therapeutic benefit via mechanisms that potentially include modulation of pro-angiogenic IL-8.

Direct digestion of proteins in living cells into peptides for proteomic analysis.

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Chen, Qi; Yan, Guoquan; Gao, Mingxia; Zhang, Xiangmin

2015-01-01

To analyze the proteome of an extremely low number of cells or even a single cell, we established a new method of digesting whole cells into mass-spectrometry-identifiable peptides in a single step within 2 h. Our sampling method greatly simplified the processes of cell lysis, protein extraction, protein purification, and overnight digestion, without compromising efficiency. We used our method to digest hundred-scale cells. As far as we know, there is no report of proteome analysis starting directly with as few as 100 cells. We identified an average of 109 proteins from 100 cells, and with three replicates, the number of proteins rose to 204. Good reproducibility was achieved, showing stability and reliability of the method. Gene Ontology analysis revealed that proteins in different cellular compartments were well represented.

Acrolein activates cell survival and apoptotic death responses involving the endoplasmic reticulum in A549 lung cells.

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Tanel, André; Pallepati, Pragathi; Bettaieb, Ahmed; Morin, Patrick; Averill-Bates, Diana A

2014-05-01

Acrolein, a highly reactive α,β-unsaturated aldehyde, is a product of endogenous lipid peroxidation. It is a ubiquitous environmental pollutant that is generated mainly by smoke, overheated cooking oil and vehicle exhaust. Acrolein damages cellular proteins, which could lead to accumulation of aberrantly-folded proteins in the endoplasmic reticulum (ER). This study determines the mechanisms involved in acrolein-induced apoptosis mediated by the ER and possible links with the ER stress response in human A549 lung cells. The exposure of cells to acrolein (15-50μM) for shorter times of 15 to 30min activated several ER stress markers. These included the ER chaperone protein BiP and the three ER sensors: (i) the survival/rescue molecules protein kinase RNA (PKR)-like ER kinase (PERK) and eukaryotic initiation factor 2 alpha (eIF2α) were phosphorylated; (ii) cleavage of activating transcription factor 6 (ATF6) occurred, and (iii) inositol-requiring protein-1 alpha (IRE1α) was phosphorylated. Acrolein (25-50μM) caused apoptotic cell death mediated by the ER after 2h, which was characterised by the induction of CHOP and activation of ER proteases calpain and caspase-4. Calpain and caspase-7 were the initiating factors for caspase-4 activation in acrolein-induced apoptosis. These results increase our knowledge about cellular responses to acrolein in lung cells, which have implications for human health. Copyright © 2014 Elsevier B.V. All rights reserved.

25 CFR 700.549 - Employee organizations.

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2010-04-01

... 25 Indians 2 2010-04-01 2010-04-01 false Employee organizations. 700.549 Section 700.549 Indians... Employee Responsibility and Conduct § 700.549 Employee organizations. An employee may not knowingly be a member of an organization of Government employees that advocates the overthrow of the United States...

High-resolution proteome maps of Bacillus licheniformis cells growing in minimal medium.

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Voigt, Birgit; Albrecht, Dirk; Sievers, Susanne; Becher, Dörte; Bongaerts, Johannes; Evers, Stefan; Schweder, Thomas; Maurer, Karl-Heinz; Hecker, Michael

2015-08-01

Bacillus licheniformis is an important host for the industrial production of enzymes mainly because of its ability to secrete large amounts of protein. We analyzed the proteome of B. licheniformis cells growing in a minimal medium. Beside the cytosolic proteome, the membrane and the extracellular proteome were studied. We could identify 1470 proteins; 1168 proteins were classified as cytosolic proteins, 195 proteins with membrane-spanning domains were classified as membrane proteins, and 107 proteins, with either putative signals peptides or flagellin-like sequences, were classified as secreted proteins. The identified proteins were grouped into functional categories and used to reconstruct cellular functions and metabolic pathways of growing B. licheniformis cells. The largest group was proteins with functions in basic metabolic pathways such as carbon metabolism, amino acid and nucleotide synthesis and synthesis of fatty acids and cofactors. Many proteins detected were involved in DNA replication, transcription, and translation. Furthermore, a high number of proteins employed in the transport of a wide variety of compounds were found to be expressed in the cells. All MS data have been deposited in the ProteomeXchange with identifier PXD000791 (http://proteomecentral.proteomexchange.org/dataset/PXD000791). © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The effect of agglomeration state of silver and titanium dioxide nanoparticles on cellular response of HepG2, A549 and THP-1 cells.

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Lankoff, Anna; Sandberg, Wiggo J; Wegierek-Ciuk, Aneta; Lisowska, Halina; Refsnes, Magne; Sartowska, Bożena; Schwarze, Per E; Meczynska-Wielgosz, Sylwia; Wojewodzka, Maria; Kruszewski, Marcin

2012-02-05

Nanoparticles (NPs) occurring in the environment rapidly agglomerate and form particles of larger diameters. The extent to which this abates the effects of NPs has not been clarified. The motivation of this study was to examine how the agglomeration/aggregation state of silver (20nm and 200nm) and titanium dioxide (21nm) nanoparticles may affect the kinetics of cellular binding/uptake and ability to induce cytotoxic responses in THP1, HepG2 and A549 cells. Cellular binding/uptake, metabolic activation and cell death were assessed by the SSC flow cytometry measurements, the MTT-test and the propidium iodide assay. The three types of particles were efficiently taken up by the cells, decreasing metabolic activation and increasing cell death in all the cell lines. The magnitude of the studied endpoints depended on the agglomeration/aggregation state of particles, their size, time-point and cell type. Among the three cell lines tested, A549 cells were the most sensitive to these particles in relation to cellular binding/uptake. HepG2 cells showed a tendency to be more sensitive in relation to metabolic activation. THP-1 cells were the most resistant to all three types of particles in relation to all endpoints tested. Our findings suggest that particle features such as size and agglomeration status as well as the type of cells may contribute to nanoparticles biological impact. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Mass spectrometry based proteomics in cell biology and signaling research

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Mann, M.; Andersen, J.; Ishihama, Y.; Rappsilber, J.; Ong, S.; Foster, L.; Blagoev, B.; Kratchmarova, I.; Lasonder, E.

2002-01-01

Full text: Proteomics is one of the most powerful post-genomics technologies. Recently accomplishments include large scale protein-protein interaction mapping, large scale mapping of phosphorylation sites and the cloning of key signaling molecules. In this talk, current state of the art of the technology will be reviewed. Applications of proteomics to the mapping of multiprotein complexes will be illustrated with recent work on the spliceosome and the nucleolus. More than 300 proteins have been mapped to each of these complexes. Quantitative techniques are becoming more and more essential in proteomics. They are usually performed by the incorporation of stable isotopes - a light form in cell state 'A' and a heavy form in cell state 'E' - and subsequent comparison of mass spectrometric peak heights. A new technique called, SILAC for Stable isotope Incorporation by Amino acids in Cell culture, has been applied to studying cell differentiation and mapping secreted proteins from adipocytes. A number of known and novel proteins important in adipocyte differentiation have been identified by this technique. Some of these proved to be upregulated at the 1 mRNA level, too, whereas others appear to be regulated post-translationally. We have also applied the SILAC method to protein-protein interaction mapping. For example, we compared immunoprecipitates from stimulated and non-stimulated cells to find binding partners recruited to the bait due to the stimulus. Several novel substrates in the EGF pathway were found in this way. An important application of proteomics in the signaling field is the mapping of post-translational modifications. In particular, there are a number of techniques for phosphotyrosine phosphorylation mapping which have proven very useful. Making use of the mass deficiency of the phosphogroup, 'parent ion scans' con be performed, which selectively reveal phosphotyrosine peptides from complex peptides mixtures. This technique has been used to clone several

Revelation of Different Nanoparticle-Uptake Behavior in Two Standard Cell Lines NIH/3T3 and A549 by Flow Cytometry and Time-Lapse Imaging

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André Jochums

2017-07-01

Full Text Available The uptake of nanomaterials into different cell types is a central pharmacological issue for the determination of nanotoxicity as well as for the development of drug delivery strategies. Most responses of the cells depend on their intracellular interactions with nanoparticles (NPs. Uptake behavior can be precisely investigated in vitro, with sensitive high throughput methods such as flow cytometry. In this study, we investigated two different standard cell lines, human lung carcinoma (A549 and mouse fibroblast (NIH/3T3 cells, regarding their uptake behavior of titanium dioxide NPs. Cells were incubated with different concentrations of TiO2 NPs and samples were taken at certain time points to compare the uptake kinetics of both cell lines. Samples were analyzed with the help of flow cytometry by studying changes in the side and forward scattering signal. To additionally enable a detection via fluorescence, NPs were labeled with the fluorescent dye fluorescein isothiocyanate (FITC and propidium iodide (PI. We found that NIH/3T3 cells take up the studied NPs more efficiently than A549 cells. These findings were supported by time-lapse microscopic imaging of the cells incubated with TiO2 NPs. Our results confirm that the uptake behavior of individual cell types has to be considered before interpreting any results of nanomaterial studies.

Proteome of human stem cells from periodontal ligament and dental pulp.

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Enrica Eleuterio

Full Text Available BACKGROUND: Many adult tissues contain a population of stem cells with the ability to regenerate structures similar to the microenvironments from which they are derived in vivo and represent a promising therapy for the regeneration of complex tissues in the clinical disorder. Human adult stem cells (SCs including bone marrow stem cells (BMSCs, dental pulp stem cells (DPSCs and periodontal ligament stem cells (PDLSCs have been characterized for their high proliferative potential, expression of characteristic SC-associated markers and for the plasticity to differentiate in different lineage in vitro. METHODOLOGY/PRINCIPAL FINDINGS: The aim of this study is to define the molecular features of stem cells from oral tissue by comparing the proteomic profiles obtained with 2-DE followed by MALDI-TOF/TOF of ex-vivo cultured human PDLSCs, DPSCs and BMSCs. Our results showed qualitative similarities in the proteome profiles among the SCs examined including some significant quantitative differences. To enrich the knowledge of oral SCs proteome we performed an analysis in narrow range pH 4-7 and 6-9, and we found that DPSCs vs PDLSCs express differentially regulated proteins that are potentially related to growth, regulation and genesis of neuronal cells, suggesting that SCs derived from oral tissue source populations may possess the potential ability of neuronal differentiation which is very consistent with their neural crest origin. CONCLUSION/SIGNIFICANCE: This study identifies some differentially expressed proteins by using comparative analysis between DPSCs and PDLSCs and BMSCs and suggests that stem cells from oral tissue could have a different cell lineage potency compared to BMSCs.

Nanostructured delivery system for zinc phthalocyanine: preparation, characterization, and phototoxicity study against human lung adenocarcinoma A549 cells

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Mariana da Volta Soares

2011-01-01

Full Text Available Mariana da Volta Soares1, Mainara Rangel Oliveira1, Elisabete Pereira dos Santos1, Lycia de Brito Gitirana2, Gleyce Moreno Barbosa3, Carla Holandino Quaresma3, Eduardo Ricci-Júnior11Department of Medicines, Laboratório de Desenvolvimento Galênico (LADEG, Faculty of Pharmacy, 2Laboratory of Animal and Comparative Histology, Glycobiology Research Program, Institute of Biomedical Science, 3Department of Medicines, Laboratório Multidisciplinar de Ciências Farmacêuticas, Faculty of Pharmacy, Federal University of Rio de Janeiro (UFRJ, Rio de Janeiro, BrazilAbstract: In this study, zinc phthalocyanine (ZnPc was loaded onto poly-ε-caprolactone (PCL nanoparticles (NPs using a solvent emulsification–evaporation method. The process yield and encapsulation efficiency were 74.2% ± 1.2% and 67.1% ± 0.9%, respectively. The NPs had a mean diameter of 187.4 ± 2.1 nm, narrow distribution size with a polydispersity index of 0.096 ± 0.004, zeta potential of -4.85 ± 0.21 mV, and spherical shape. ZnPc has sustained release, following Higuchi’s kinetics. The photobiological activity of the ZnPc-loaded NPs was evaluated on human lung adenocarcinoma A549 cells. Cells were incubated with free ZnPc or ZnPc-loaded NPs for 4 h and then washed with phosphate-buffered saline. Culture medium was added to the wells containing the cells. Finally, the cells were exposed to red light (660 nm with a light dose of 100 J/cm2. The cellular viability was determined after 24 h of incubation. ZnPc-loaded NPs and free photosensitizer eliminated about 95.9% ± 1.8% and 28.7% ± 2.2% of A549 cells, respectively. The phototoxicity was time dependent up to 4 h and concentration dependent at 0–5 µg ZnPc. The cells viability decreased with the increase of the light dose in the range of 10–100 J/cm2. Intense lysis was observed in the cells incubated with the ZnPc-loaded NPs and irradiated with red light. ZnPc-loaded PCL NPs are the release systems that promise photodynamic

Intracellular dynamics and fate of polystyrene nanoparticles in A549 Lung epithelial cells monitored by image (cross-) correlation spectroscopy and single particle tracking.

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Deville, Sarah; Penjweini, Rozhin; Smisdom, Nick; Notelaers, Kristof; Nelissen, Inge; Hooyberghs, Jef; Ameloot, Marcel

2015-10-01

Novel insights in nanoparticle (NP) uptake routes of cells, their intracellular trafficking and subcellular targeting can be obtained through the investigation of their temporal and spatial behavior. In this work, we present the application of image (cross-) correlation spectroscopy (IC(C)S) and single particle tracking (SPT) to monitor the intracellular dynamics of polystyrene (PS) NPs in the human lung carcinoma A549 cell line. The ensemble kinetic behavior of NPs inside the cell was characterized by temporal and spatiotemporal image correlation spectroscopy (TICS and STICS). Moreover, a more direct interpretation of the diffusion and flow detected in the NP motion was obtained by SPT by monitoring individual NPs. Both techniques demonstrate that the PS NP transport in A549 cells is mainly dependent on microtubule-assisted transport. By applying spatiotemporal image cross-correlation spectroscopy (STICCS), the correlated motions of NPs with the early endosomes, late endosomes and lysosomes are identified. PS NPs were equally distributed among the endolysosomal compartment during the time interval of the experiments. The cotransport of the NPs with the lysosomes is significantly larger compared to the other cell organelles. In the present study we show that the complementarity of ICS-based techniques and SPT enables a consistent elaborate model of the complex behavior of NPs inside biological systems. Copyright © 2015 Elsevier B.V. All rights reserved.

Cell wall proteome analysis of Mycobacterium smegmatis strain MC2 155

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De Buck Jeroen

2010-04-01

Full Text Available Abstract Background The usually non-pathogenic soil bacterium Mycobacterium smegmatis is commonly used as a model mycobacterial organism because it is fast growing and shares many features with pathogenic mycobacteria. Proteomic studies of M. smegmatis can shed light on mechanisms of mycobacterial growth, complex lipid metabolism, interactions with the bacterial environment and provide a tractable system for antimycobacterial drug development. The cell wall proteins are particularly interesting in this respect. The aim of this study was to construct a reference protein map for these proteins in M. smegmatis. Results A proteomic analysis approach, based on one dimensional polyacrylamide gel electrophoresis and LC-MS/MS, was used to identify and characterize the cell wall associated proteins of M. smegmatis. An enzymatic cell surface shaving method was used to determine the surface-exposed proteins. As a result, a total of 390 cell wall proteins and 63 surface-exposed proteins were identified. Further analysis of the 390 cell wall proteins provided the theoretical molecular mass and pI distributions and determined that 26 proteins are shared with the surface-exposed proteome. Detailed information about functional classification, signal peptides and number of transmembrane domains are given next to discussing the identified transcriptional regulators, transport proteins and the proteins involved in lipid metabolism and cell division. Conclusion In short, a comprehensive profile of the M. smegmatis cell wall subproteome is reported. The current research may help the identification of some valuable vaccine and drug target candidates and provide foundation for the future design of preventive, diagnostic, and therapeutic strategies against mycobacterial diseases.

Protein C inhibits endocytosis of thrombin-thrombomodulin complexes in A549 lung cancer cells and human umbilical vein endothelial cells

International Nuclear Information System (INIS)

Maruyama, I.; Majerus, P.W.

1987-01-01

We investigated the effect of protein C on the endocytosis of thrombin-thrombomodulin complexes. We previously showed that exposure of umbilical vein endothelial cells to thrombin stimulated the internalization and degradation of thrombin. A similar internalization was stimulated by a monoclonal antithrombomodulin antibody. We have repeated these studies in the presence of protein C and found that endocytosis of 125 I-thrombin-thrombomodulin complexes, but not 125 I-antithrombomodulin-thrombomodulin complexes, is inhibited. Activated protein C did not inhibit endocytosis of thrombin-thrombomodulin complexes. Protein C inhibited both internalization and degradation of 125 I-thrombin and diisopropylphosphoryl (DIP) 125 I-thrombin in human lung cancer cells (A549). These effects were observed at protein C concentrations found in human plasma. Protein S had no effect on the inhibition of endocytosis of thrombin-thrombomodulin complexes by protein C. We propose that protein C may regulate the rate of endocytosis of thrombin-thrombomodulin complexes in vivo and thereby control the capacity for endothelium to activate protein C

The Genomic and Proteomic Content of Cancer Cell-Derived Exosomes

International Nuclear Information System (INIS)

Henderson, Meredith C.; Azorsa, David O.

2012-01-01

Exosomes are secreted membrane vesicles that have been proposed as an effective means to detect a variety of disease states, including cancer. The properties of exosomes, including stability in biological fluids, allow for their efficient isolation and make them an ideal vehicle for studies on early disease detection and evaluation. Much data has been collected over recent years regarding the messenger RNA, microRNA, and protein contents of exosomes. In addition, many studies have described the functional role that exosomes play in disease initiation and progression. Tumor cells have been shown to secrete exosomes, often in increased amounts compared to normal cells, and these exosomes can carry the genomic and proteomic signatures characteristic of the tumor cells from which they were derived. While these unique signatures make exosomes ideal for cancer detection, exosomes derived from cancer cells have also been shown to play a functional role in cancer progression. Here, we review the unique genomic and proteomic contents of exosomes originating from cancer cells as well as their functional effects to promote tumor progression.

Quantitative proteome changes in Arabidopsis thaliana suspension-cultured cells in response to plant natriuretic peptides

KAUST Repository

Turek, Ilona; Wheeler, Janet I.; Gehring, Christoph A; Irving, Helen R.; Marondedze, Claudius

2015-01-01

Proteome changes in the Arabidopsis thaliana suspension cells in response to the A. thaliana plant natriuretic peptide (PNP), AtPNP-A (At2g18660) were assessed using quantitative proteomics employing tandem mass tag (TMT) labeling and tandem mass spectrometry (LC–MS/MS). In this study, we characterized temporal responses of suspension-cultured cells to 1 nM and 10 pM AtPNP-A at 0, 10 and 30 min post-treatment. Both concentrations we found to yield a distinct differential proteome signature. The data shown in this article are associated with the article “Plant natriuretic peptides induce a specific set of proteins diagnostic for an adaptive response to abiotic stress” by Turek et al. (Front. Plant Sci. 5 (2014) 661) and have been deposited to the ProteomeXchange with identifier PXD001386.

Quantitative proteome changes in Arabidopsis thaliana suspension-cultured cells in response to plant natriuretic peptides

KAUST Repository

Turek, Ilona

2015-06-30

Proteome changes in the Arabidopsis thaliana suspension cells in response to the A. thaliana plant natriuretic peptide (PNP), AtPNP-A (At2g18660) were assessed using quantitative proteomics employing tandem mass tag (TMT) labeling and tandem mass spectrometry (LC–MS/MS). In this study, we characterized temporal responses of suspension-cultured cells to 1 nM and 10 pM AtPNP-A at 0, 10 and 30 min post-treatment. Both concentrations we found to yield a distinct differential proteome signature. The data shown in this article are associated with the article “Plant natriuretic peptides induce a specific set of proteins diagnostic for an adaptive response to abiotic stress” by Turek et al. (Front. Plant Sci. 5 (2014) 661) and have been deposited to the ProteomeXchange with identifier PXD001386.

Proteome screening of pleural effusions identifies IL1A as a diagnostic biomarker for non-small cell lung cancer.

Science.gov (United States)

Li, Yuanyuan; Lian, Hengning; Jia, Qingzhu; Wan, Ying

2015-02-06

Non-small cell lung cancer (NSCLC) is a common malignant disease, and in ~10-20% of patients, pleural effusion is the first symptom. The pleural effusion proteome contains information on pulmonary disease that directly or indirectly reflects pathophysiological status. However, the proteome of pleural effusion in NSCLC patients is not well understood, nor is the variability in protein composition between malignant and benign pleural effusions. Here, we investigated the different proteins in pleural effusions from NSCLC and tuberculosis (TB) patients by using nano-scale liquid chromatography-tandem mass spectrometry (nLC-MS/MS) analysis. In total, 363 proteins were identified in the NSCLC pleural effusion proteome with a low false discovery rate (pleural effusion were involved in cell adhesion, proteolysis, and cell migration. Furthermore, interleukin 1 alpha (IL1A), a protein that regulates tumor growth, angiogenesis, and metastasis, was significantly more abundant in the NSCLC group compared to the TB group, a finding that was validated with an ELISA assay. Copyright © 2014 Elsevier Inc. All rights reserved.

Enhanced sensitivity of A549 cells to the cytotoxic action of anticancer drugs via suppression of Nrf2 by procyanidins from Cinnamomi Cortex extract

Energy Technology Data Exchange (ETDEWEB)

Ohnuma, Tomokazu; Matsumoto, Takashi; Itoi, Ayano; Kawana, Ayako; Nishiyama, Takahito; Ogura, Kenichiro [Department of Drug Metabolism and Molecular Toxicology, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji-shi, Tokyo 192-0392 (Japan); Hiratsuka, Akira, E-mail: hiratuka@toyaku.ac.jp [Department of Drug Metabolism and Molecular Toxicology, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji-shi, Tokyo 192-0392 (Japan)

2011-10-07

Highlights: {yields} We found a novel inhibitor of Nrf2 known as a chemoresistance factor. {yields} Overexpressed Nrf2 in lung cancer cells was suppressed by Cinnamomi Cortex extract. {yields} Cytotoxic action of anticancer drugs in cells treated with the extract was enhanced. {yields} Procyanidin tetramers and pentamers were active components in suppressing Nrf2. -- Abstract: Nuclear factor-E2-related factor 2 (Nrf2) is an important cytoprotective transcription factor because Nrf2-regulated enzymes play a key role in antioxidant and detoxification processes. Recent studies have reported that lung cancer cells overexpressing Nrf2 exhibit increased resistance to chemotherapy. Suppression of overexpressed Nrf2 is needed for a new therapeutic approach against lung cancers. In the present study, we found that Cinnamomi Cortex extract (CCE) has an ability to suppress Nrf2-regulated enzyme activity and Nrf2 expression in human lung cancer A549 cells with high Nrf2 activity. Moreover, we demonstrated that CCE significantly enhances sensitivity of A549 cells to the cytotoxic action of doxorubicin and etoposide as well as increasing the intracellular accumulation of both drugs. These results suggest that CCE might be an effective concomitant agent to reduce anticancer drug resistance derived from Nrf2 overexpression. Bioactivity-guided fractionation revealed that procyanidin tetramers and pentamers contained in CCE were active components in suppressing Nrf2.

The time is right: proteome biology of stem cells.

NARCIS (Netherlands)

Whetton, A.D.; Williamson, A.J.K.; Krijgsveld, J.; Lee, B.H.; Lemischka, I.; Oh, S.; Pera, M.; Mummery, C.L.; Heck, A.J.R.

2008-01-01

In stem cell biology, there is a growing need for advanced technologies that may help to unravel the molecular mechanisms of self-renewal and differentiation. Proteomics, the comprehensive analysis of proteins, is such an emerging technique. To facilitate interactions between specialists in

Pseudomonas aeruginosa cells attached to a surface display a typical proteome early as 20 minutes of incubation.

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Marc Crouzet

Full Text Available Biofilms are present in all environments and often result in negative effects due to properties of the biofilm lifestyle and especially antibiotics resistance. Biofilms are associated with chronic infections. Controlling bacterial attachment, the first step of biofilm formation, is crucial for fighting against biofilm and subsequently preventing the persistence of infection. Thus deciphering the underlying molecular mechanisms involved in attachment could allow discovering molecular targets from it would be possible to develop inhibitors against bacterial colonization and potentiate antibiotherapy. To identify the key components and pathways that aid the opportunistic pathogen Pseudomonas aeruginosa in attachment we performed for the first time a proteomic analysis as early as after 20 minutes of incubation using glass wool fibers as a surface. We compared the protein contents of the attached and unattached bacteria. Using mass spectrometry, 3043 proteins were identified. Our results showed that, as of 20 minutes of incubation, using stringent quantification criteria 616 proteins presented a modification of their abundance in the attached cells compared to their unattached counterparts. The attached cells presented an overall reduced gene expression and characteristics of slow-growing cells. The over-accumulation of outer membrane proteins, periplasmic folding proteins and O-antigen chain length regulators was also observed, indicating a profound modification of the cell envelope. Consistently the sigma factor AlgU required for cell envelope homeostasis was highly over-accumulated in attached cells. In addition our data suggested a role of alarmone (pppGpp and polyphosphate during the early attachment phase. Furthermore, almost 150 proteins of unknown function were differentially accumulated in the attached cells. Our proteomic analysis revealed the existence of distinctive biological features in attached cells as early as 20 minutes of

Phenobarbital Induces Alterations in the Proteome of Hepatocytes and Mesenchymal Cells of Rat Livers

Science.gov (United States)

Klepeisz, Philip; Sagmeister, Sandra; Haudek-Prinz, Verena; Pichlbauer, Melanie; Grasl-Kraupp, Bettina; Gerner, Christopher

2013-01-01

Preceding studies on the mode of action of non-genotoxic hepatocarcinogens (NGCs) have concentrated on alterations induced in hepatocytes (HCs). A potential role of non-parenchymal liver cells (NPCs) in NGC-driven hepatocarcinogenesis has been largely neglected so far. The aim of this study is to characterize NGC-induced alterations in the proteome profiles of HCs as well as NPCs. We chose the prototypic NGC phenobarbital (PB) which was applied to male rats for a period of 14 days. The livers of PB-treated rats were perfused by collagenase and the cell suspensions obtained were subjected to density gradient centrifugation to separate HCs from NPCs. In addition, HCs and NPC isolated from untreated animals were treated with PB in vitro. Proteome profiling was done by CHIP-HPLC and ion trap mass spectrometry. Proteome analyses of the in vivo experiments showed many of the PB effects previously described in HCs by other methods, e.g. induction of phase I and phase II drug metabolising enzymes. In NPCs proteins related to inflammation and immune regulation such as PAI-1 and S100-A10, ADP-ribosyl cyclase 1 and to cell migration such as kinesin-1 heavy chain, myosin regulatory light chain RLC-A and dihydropyrimidinase-related protein 1 were found to be induced, indicating major PB effects on these cells. Remarkably, in vitro treatment of HCs and NPCs with PB hardly reproduced the proteome alterations observed in vivo, indicating differences of NGC induced responses of cells at culture conditions compared to the intact organism. To conclude, the present study clearly demonstrated that PB induces proteome alterations not only in HCs but also in NPCs. Thus, any profound molecular understanding on the mode of action of NGCs has to consider effects on cells of the hepatic mesenchyme. PMID:24204595

Phenobarbital induces alterations in the proteome of hepatocytes and mesenchymal cells of rat livers.

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Philip Klepeisz

Full Text Available Preceding studies on the mode of action of non-genotoxic hepatocarcinogens (NGCs have concentrated on alterations induced in hepatocytes (HCs. A potential role of non-parenchymal liver cells (NPCs in NGC-driven hepatocarcinogenesis has been largely neglected so far. The aim of this study is to characterize NGC-induced alterations in the proteome profiles of HCs as well as NPCs. We chose the prototypic NGC phenobarbital (PB which was applied to male rats for a period of 14 days. The livers of PB-treated rats were perfused by collagenase and the cell suspensions obtained were subjected to density gradient centrifugation to separate HCs from NPCs. In addition, HCs and NPC isolated from untreated animals were treated with PB in vitro. Proteome profiling was done by CHIP-HPLC and ion trap mass spectrometry. Proteome analyses of the in vivo experiments showed many of the PB effects previously described in HCs by other methods, e.g. induction of phase I and phase II drug metabolising enzymes. In NPCs proteins related to inflammation and immune regulation such as PAI-1 and S100-A10, ADP-ribosyl cyclase 1 and to cell migration such as kinesin-1 heavy chain, myosin regulatory light chain RLC-A and dihydropyrimidinase-related protein 1 were found to be induced, indicating major PB effects on these cells. Remarkably, in vitro treatment of HCs and NPCs with PB hardly reproduced the proteome alterations observed in vivo, indicating differences of NGC induced responses of cells at culture conditions compared to the intact organism. To conclude, the present study clearly demonstrated that PB induces proteome alterations not only in HCs but also in NPCs. Thus, any profound molecular understanding on the mode of action of NGCs has to consider effects on cells of the hepatic mesenchyme.

New geranylated flavanones from the fruits of Paulownia catalpifolia Gong Tong with their anti-proliferative activity on lung cancer cells A549.

Science.gov (United States)

Gao, Tian-yang; Jin, Xing; Tang, Wen-zhao; Wang, Xiao-jing; Zhao, Yun-xue

2015-09-01

Three new geranylated flavanones, named as paucatalinone A (1), B (2), and isopaucatalinone B (3), were isolated from the fruits of Paulownia catalpifolia Gong Tong (Scrophulariaceae). Their structures were well determined by means of IR, MS, 1D and 2D NMR, and CD techniques. Paucatalinone A (1) is the first sample as a dimeric geranylated flavanone derivative isolated from natural products. Paucatalinone A (1) displayed good antiproliferative effects on human lung cancer cells A549 and resulted in a clear increase of the percentage of cells in G1 phase and a decrease in the percentage of cells in S and G2/M phases in comparison with control cells. Copyright © 2015. Published by Elsevier Ltd.

A platycoside-rich fraction from the root of Platycodon grandiflorum enhances cell death in A549 human lung carcinoma cells via mainly AMPK/mTOR/AKT signal-mediated autophagy induction.

Science.gov (United States)

Yim, Nam-Hui; Hwang, Youn-Hwan; Liang, Chun; Ma, Jin Yeul

2016-12-24

The root of Platycodon grandiflorum (PG), commonly known as Kilkyong in Korea, Jiegeng in China, and Kikyo in Japan, has been extensively used as a traditional anti-inflammatory medicine in Asia for the treatment of respiratory conditions, such as bronchitis, asthma, and tonsillitis. Platycosides isolated from PG are especially well-known for their anti-cancer effects. We investigated the involvement of autophagic cell death and other potential molecular mechanisms induced by the platycoside-containing butanol fraction of PG (PGB) in human lung carcinoma cells. PGB-induced growth inhibition and cell death were measured using a 5-diphenyl-tetrazolium bromide (MTT) assay. The effects of PGB on autophagy were determined by observing microtubule-associated protein 1 light chain 3 (LC3) redistribution with confocal microscopy. The PGB-mediated regulation of autophagy-associated proteins was investigated using Western blotting analysis. Furthermore, the anti-cancer mechanism of PGB was confirmed using chemical inhibitors. A high-performance liquid chromatography (HPLC)-DAD system was used to analyze the platycosides in PGB. In A549 cells, PGB induced significant autophagic cell death. Specifically, PGB upregulated LC3-II in a time- and dose-dependent manner, and it redistributed LC3 via autophagosome formation in the cytoplasm. PGB treatment increased the phosphorylation of AMP-activated protein kinase (AMPK) and subsequently suppressed the AKT/mammalian target of the rapamycin (mTOR) pathway. Furthermore, PGB inhibited cell proliferation by regulating the mitogen-activated protein kinase (MAPK) pathways. In this study, six types of platycosides were identified in the PGB using HPLC. PGB efficiently induced cancer cell death via autophagy and the modulation of the AMPK/mTOR/AKT and MAPK signaling pathways in A549 cells. Therefore, PGB may be an efficacious herbal anti-cancer therapy. Copyright © 2016. Published by Elsevier Ireland Ltd.

Multilineage potential and proteomic profiling of human dental stem cells derived from a single donor

International Nuclear Information System (INIS)

Patil, Rajreddy; Kumar, B. Mohana; Lee, Won-Jae; Jeon, Ryoung-Hoon; Jang, Si-Jung; Lee, Yeon-Mi; Park, Bong-Wook; Byun, June-Ho; Ahn, Chun-Seob; Kim, Jae-Won; Rho, Gyu-Jin

2014-01-01

Dental tissues provide an alternative autologous source of mesenchymal stem cells (MSCs) for regenerative medicine. In this study, we isolated human dental MSCs of follicle, pulp and papilla tissue from a single donor tooth after impacted third molar extraction by excluding the individual differences. We then compared the morphology, proliferation rate, expression of MSC-specific and pluripotency markers, and in vitro differentiation ability into osteoblasts, adipocytes, chondrocytes and functional hepatocyte-like cells (HLCs). Finally, we analyzed the protein expression profiles of undifferentiated dental MSCs using 2DE coupled with MALDI-TOF-MS. Three types of dental MSCs largely shared similar morphology, proliferation potential, expression of surface markers and pluripotent transcription factors, and differentiation ability into osteoblasts, adipocytes, and chondrocytes. Upon hepatogenic induction, all MSCs were transdifferentiated into functional HLCs, and acquired hepatocyte functions by showing their ability for glycogen storage and urea production. Based on the proteome profiling results, we identified nineteen proteins either found commonly or differentially expressed among the three types of dental MSCs. In conclusion, three kinds of dental MSCs from a single donor tooth possessed largely similar cellular properties and multilineage potential. Further, these dental MSCs had similar proteomic profiles, suggesting their interchangeable applications for basic research and call therapy. - Highlights: • Isolated and characterized three types of human dental MSCs from a single donor. • MSCs of dental follicle, pulp and papilla had largely similar biological properties. • All MSCs were capable of transdifferentiating into functional hepatocyte-like cells. • 2DE proteomics with MALDI-TOF/MS identified 19 proteins in three types of MSCs. • Similar proteomic profiles suggest interchangeable applications of dental MSCs

Multilineage potential and proteomic profiling of human dental stem cells derived from a single donor

Energy Technology Data Exchange (ETDEWEB)

Patil, Rajreddy; Kumar, B. Mohana; Lee, Won-Jae; Jeon, Ryoung-Hoon; Jang, Si-Jung; Lee, Yeon-Mi [Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Gyeongsang National University, Jinju 660-701 (Korea, Republic of); Park, Bong-Wook; Byun, June-Ho [Department of Oral and Maxillofacial Surgery, School of Medicine and Institute of Health Science, Gyeongsang National University, Jinju 660-702 (Korea, Republic of); Ahn, Chun-Seob; Kim, Jae-Won [Department of Microbiology, Division of Life Sciences, Research Institute of Life Science, Gyeongsang National University, Jinju 660-701 (Korea, Republic of); Rho, Gyu-Jin, E-mail: jinrho@gnu.ac.kr [Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Gyeongsang National University, Jinju 660-701 (Korea, Republic of); Research Institute of Life Sciences, Gyeongsang National University, Jinju 660-701 (Korea, Republic of)

2014-01-01

Dental tissues provide an alternative autologous source of mesenchymal stem cells (MSCs) for regenerative medicine. In this study, we isolated human dental MSCs of follicle, pulp and papilla tissue from a single donor tooth after impacted third molar extraction by excluding the individual differences. We then compared the morphology, proliferation rate, expression of MSC-specific and pluripotency markers, and in vitro differentiation ability into osteoblasts, adipocytes, chondrocytes and functional hepatocyte-like cells (HLCs). Finally, we analyzed the protein expression profiles of undifferentiated dental MSCs using 2DE coupled with MALDI-TOF-MS. Three types of dental MSCs largely shared similar morphology, proliferation potential, expression of surface markers and pluripotent transcription factors, and differentiation ability into osteoblasts, adipocytes, and chondrocytes. Upon hepatogenic induction, all MSCs were transdifferentiated into functional HLCs, and acquired hepatocyte functions by showing their ability for glycogen storage and urea production. Based on the proteome profiling results, we identified nineteen proteins either found commonly or differentially expressed among the three types of dental MSCs. In conclusion, three kinds of dental MSCs from a single donor tooth possessed largely similar cellular properties and multilineage potential. Further, these dental MSCs had similar proteomic profiles, suggesting their interchangeable applications for basic research and call therapy. - Highlights: • Isolated and characterized three types of human dental MSCs from a single donor. • MSCs of dental follicle, pulp and papilla had largely similar biological properties. • All MSCs were capable of transdifferentiating into functional hepatocyte-like cells. • 2DE proteomics with MALDI-TOF/MS identified 19 proteins in three types of MSCs. • Similar proteomic profiles suggest interchangeable applications of dental MSCs.

Hypoxia-inducible transcription factor-1α promotes hypoxia-induced A549 apoptosis via a mechanism that involves the glycolysis pathway

International Nuclear Information System (INIS)

Luo, FengMing; Liu, XiaoJing; Yan, NaiHong; Li, ShuangQing; Cao, GuiQun; Cheng, QingYing; Xia, QingJie; Wang, HongJing

2006-01-01

Hypoxia-inducible transcription factor-1α (HIF-1α), which plays an important role in controlling the hypoxia-induced glycolysis pathway, is a 'master' gene in the tissue hypoxia response during tumor development. However, its role in the apoptosis of non-small cell lung cancer remains unknown. Here, we have studied the effects of HIF-1α on apoptosis by modulating HIF-1α gene expression in A549 cells through both siRNA knock-down and over-expression. A549 cells were transfected with a HIF-1α siRNA plasmid or a HIF-1α expression vector. Transfected cells were exposed to a normoxic or hypoxic environment in the presence or absence of 25 mM HEPES and 2-deoxyglucose (2-DG) (5 mM). The expression of three key genes of the glycolysis pathway, glucose transporter type 1(GLUT1), phosphoglycerate kinase 1(PGK1), and hexokinase 1(HK1), were measured using real-time RT-PCR. Glycolysis was monitored by measuring changes of pH and lactate concentration in the culture medium. Apoptosis was detected by TUNEL assay and flow cytometry. Knocking down expression of HIF-1α inhibited the glycolysis pathway, increased the pH of the culture medium, and protected the cells from hypoxia-induced apoptosis. In contrast, over-expression of HIF-1α accelerated glycolysis in A549 cells, decreased the pH of the culture medium, and enhanced hypoxia-induced apoptosis. These effects of HIF-1α on glycolysis, pH of the medium, and apoptosis were reversed by treatment with the glycolytic inhibitor, 2-DG. Apoptosis induced by HIF-1α over-expression was partially inhibited by increasing the buffering capacity of the culture medium by adding HEPES. During hypoxia in A549 cells, HIF-1α promotes activity of the glycolysis pathway and decreases the pH of the culture medium, resulting in increased cellular apoptosis

Biochemistry, proteomics and phosphoproteomics of plant mitochondria from non-photosynthetic cells

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Jesper Foged Havelund

2013-03-01

Full Text Available Mitochondria fulfill some basic roles in all plant cells. They supply the cell with energy in the form of ATP and reducing equivalents (NAD(PH and they provide the cell with intermediates for a range of biosynthetic pathways. In addition to this, mitochondria contribute to a number of specialized functions depending on the tissue and cell type, as well as environmental conditions. We will here review the biochemistry and proteomics of mitochondria from non-green cells and organs, which differ from those of photosynthetic organs in a number of respects. We will briefly cover purification of mitochondria and general biochemical properties such as oxidative phosphorylation. We will then mention a few adaptive properties in response to water stress, seed maturation and germination and the ability to function under hypoxic conditions. The discussion will mainly focus on Arabidopsis cell cultures, etiolated germinating rice seedlings and potato tubers as model plants. It will cover the general proteome as well as the posttranslational modification protein phosphorylation. To date 64 phosphorylated mitochondrial proteins with a total of 103 phosphorylation sites have been identified.

Proteomic Profiling of Ex Vivo Expanded CD34-Positive Haematopoetic Cells Derived from Umbilical Cord Blood

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Heiner Falkenberg

2013-01-01

Full Text Available Ex vivo expansion of haematopoetic cells by application of specific cytokines is one approach to overcome boundaries in cord blood transplantation due to limited numbers of haematopoetic stem cells. While many protocols describe an effective increase of total cell numbers and the amount of CD34-positive cells, it still remains unclear if and how the procedure actually affects the cells’ properties. In the presented publications, CD34-positive cells were isolated from cord blood and expanded for up to 7 days in media supplemented with stem cell factor (SCF, thrombopoietin (THPO, interleukin 6 (IL-6, and fms-related tyrosine kinase 3 ligand (FLT3lg. At days 3 and 7, expanded cells were harvested and analyzed by flow cytometry and quantitative proteomics. 2970 proteins were identified, whereof proteomic analysis showed 440 proteins significantly changed in abundance during ex vivo expansion. Despite the fact that haematopoetic cells still expressed CD34 on the surface after 3 days, major changes in regard to the protein profile were observed, while further expansion showed less effect on the proteome level. Enrichment analysis of biological processes clearly showed a proteomic change toward a protein biosynthesis phenotype already within the first three days of expression.

Characterization of human neural differentiation from pluripotent stem cells using proteomics/PTMomics

DEFF Research Database (Denmark)

Braga, Marcella Nunes de Melo; Meyer, Morten; Zeng, Xianmin

2015-01-01

Stem cells are unspecialized cells capable of self-renewal and to differentiate into the large variety of cells in the body. The possibility to differentiate these cells into neural precursors and neural cells in vitro provides the opportunity to study neural development, nerve cell biology, neur...... differentiation from pluripotent stem cells. Moreover, some of the challenges in stem cell biology, differentiation, and proteomics/PTMomics that are not exclusive to neural development will be discussed.......Stem cells are unspecialized cells capable of self-renewal and to differentiate into the large variety of cells in the body. The possibility to differentiate these cells into neural precursors and neural cells in vitro provides the opportunity to study neural development, nerve cell biology...... the understanding of molecular processes in cells. Substantial advances in PTM enrichment methods and mass spectrometry has allowed the characterization of a subset of PTMs in large-scale studies. This review focuses on the current state-of-the-art of proteomic, as well as PTMomic studies related to human neural...

Paraquat induces extrinsic pathway of apoptosis in A549 cells by induction of DR5 and repression of anti-apoptotic proteins, DDX3 and GSK3 expression.

Science.gov (United States)

Hathaichoti, Sasiphen; Visitnonthachai, Daranee; Ngamsiri, Pronrumpa; Niyomchan, Apichaya; Tsogtbayar, Oyu; Wisessaowapak, Churaibhon; Watcharasit, Piyajit; Satayavivad, Jutamaad

2017-08-01

Paraquat (PQ) is a bipyridyl derivative herbicide known to cause lung toxicity partly through induction of apoptosis. Here we demonstrated that PQ caused apoptosis in A549 cells. PQ increased cleavage of caspase-8 and Bid, indicating caspase-8 activation and truncated Bid, the two key mediators of extrinsic apoptosis. Additionally, PQ treatment caused an increase in DR5 (death receptor-5) and caspase-8 interaction, indicating formation of DISC (death-inducing signaling complex). These results indicate that PQ induces apoptosis through extrinsic pathway in A549 cells. Moreover, PQ drastically increased DR5 expression and membrane localization. Furthermore, PQ caused prominent concentration dependent reductions of DDX3 (the DEAD box protein-3) and GSK3 (glycogen synthase kinase-3) which can associate with DR5 and prevent DISC formation. Additionally, PQ decreased DR5-DDX3 interaction, suggesting a reduction of DDX3/GSK3 anti-apoptotic complex. Inhibition of GSK3, which is known to promote extrinsic apoptosis by its pharmacological inhibitor, BIO accentuated PQ-induced apoptosis. Moreover, GSK3 inhibition caused a further decrease in PQ-reduced DR5-DDX3 interaction. Taken together, these results suggest that PQ may induce extrinsic pathway of apoptosis in A549 cells through upregulation of DR5 and repression of anti-apoptotic proteins, DDX3/GSK3 leading to reduction of anti-apoptotic complex. Copyright © 2017 Elsevier Ltd. All rights reserved.

Proteomic Analysis of Laser Microdissected Melanoma Cells from Skin Organ Cultures

Science.gov (United States)

Hood, Brian L.; Grahovac, Jelena; Flint, Melanie S.; Sun, Mai; Charro, Nuno; Becker, Dorothea; Wells, Alan; Conrads, Thomas P

2010-01-01

Gaining insights into the molecular events that govern the progression from melanoma in situ to advanced melanoma, and understanding how the local microenvironment at the melanoma site influences this progression, are two clinically pivotal aspects that to date are largely unexplored. In an effort to identify key regulators of the crosstalk between melanoma cells and the melanoma-skin microenvironment, primary and metastatic human melanoma cells were seeded into skin organ cultures (SOCs), and grown for two weeks. Melanoma cells were recovered from SOCs by laser microdissection and whole-cell tryptic digests analyzed by nanoflow liquid chromatography-tandem mass spectrometry with an LTQ-Orbitrap. The differential protein abundances were calculated by spectral counting, the results of which provides evidence that cell-matrix and cell-adhesion molecules that are upregulated in the presence of these melanoma cells recapitulate proteomic data obtained from comparative analysis of human biopsies of invasive melanoma and a tissue sample of adjacent, non-involved skin. This concordance demonstrates the value of SOCs for conducting proteomic investigations of the melanoma microenvironment. PMID:20459140

SILAC-Based Comparative Proteomic Analysis of Lysosomes from Mammalian Cells Using LC-MS/MS.

Science.gov (United States)

Thelen, Melanie; Winter, Dominic; Braulke, Thomas; Gieselmann, Volkmar

2017-01-01

Mass spectrometry-based proteomics of lysosomal proteins has led to significant advances in understanding lysosomal function and pathology. The ever-increasing sensitivity and resolution of mass spectrometry in combination with labeling procedures which allow comparative quantitative proteomics can be applied to shed more light on the steadily increasing range of lysosomal functions. In addition, investigation of alterations in lysosomal protein composition in the many lysosomal storage diseases may yield further insights into the molecular pathology of these disorders. Here, we describe a protocol which allows to determine quantitative differences in the lysosomal proteome of cells which are genetically and/or biochemically different or have been exposed to certain stimuli. The method is based on stable isotope labeling of amino acids in cell culture (SILAC). Cells are exposed to superparamagnetic iron oxide particles which are endocytosed and delivered to lysosomes. After homogenization of cells, intact lysosomes are rapidly enriched by passing the cell homogenates over a magnetic column. Lysosomes are eluted after withdrawal of the magnetic field and subjected to mass spectrometry.

Quantitative proteomics of Spodoptera frugiperda cells during growth and baculovirus infection.

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Nuno Carinhas

Full Text Available Baculovirus infection of Spodoptera frugiperda cells is a system of choice to produce a range of recombinant proteins, vaccines and, potentially, gene therapy vectors. While baculovirus genomes are well characterized, the genome of S. frugiperda is not sequenced and the virus-host molecular interplay is sparsely known. Herein, we describe the application of stable isotope labeling by amino acids in cell culture (SILAC to obtain the first comparative proteome quantitation of S. frugiperda cells during growth and early baculovirus infection. The proteome coverage was maximized by compiling a search database with protein annotations from insect species. Of interest were differentially proteins related to energy metabolism, endoplasmic reticulum and oxidative stress, yet not investigated in the scope of baculovirus infection. Further, the reduced expression of key viral-encoded proteins early in the infection cycle is suggested to be related with decreased viral replication at high cell density culture. These findings have implications for virological research and improvement of baculovirus-based bioprocesses.

Variation among Staphylococcus aureus membrane vesicle proteomes affects cytotoxicity of host cells.

Science.gov (United States)

Jeon, Hyejin; Oh, Man Hwan; Jun, So Hyun; Kim, Seung Il; Choi, Chi Won; Kwon, Hyo Il; Na, Seok Hyeon; Kim, Yoo Jeong; Nicholas, Asiimwe; Selasi, Gati Noble; Lee, Je Chul

2016-04-01

Staphylococcus aureus secretes membrane-derived vesicles (MVs), which can deliver virulence factors to host cells and induce cytopathology. However, the cytopathology of host cells induced by MVs derived from different S. aureus strains has not yet been characterized. In the present study, the cytotoxic activity of MVs from different S. aureus isolates on host cells was compared and the proteomes of S. aureus MVs were analyzed. The MVs purified from S. aureus M060 isolated from a patient with staphylococcal scalded skin syndrome showed higher cytotoxic activity toward host cells than that shown by MVs from three other clinical S. aureus isolates. S. aureus M060 MVs induced HEp-2 cell apoptosis in a dose-dependent manner, but the cytotoxic activity of MVs was completely abolished by treatment with proteinase K. In a proteomic analysis, the MVs from three S. aureus isolates not only carry 25 common proteins, but also carry ≥60 strain-specific proteins. All S. aureus MVs contained δ-hemolysin (Hld), γ-hemolysin, leukocidin D, and exfoliative toxin C, but exfoliative toxin A (ETA) was specifically identified in S. aureus M060 MVs. ETA was delivered to HEp-2 cells via S. aureus MVs. Both rETA and rHld induced cytotoxicity in HEp-2 cells. In conclusion, MVs from clinical S. aureus isolates differ with respect to cytotoxic activity in host cells, and these differences may result from differences in the MV proteomes. Further proteogenomic analysis or mutagenesis of specific genes is necessary to identify cytotoxic factors in S. aureus MVs. Copyright © 2016 Elsevier Ltd. All rights reserved.

Induction of Apoptotic Effects of Antiproliferative Protein from the Seeds of Borreria hispida on Lung Cancer (A549 and Cervical Cancer (HeLa Cell Lines

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S. Rupachandra

2014-01-01

Full Text Available A 35 KDa protein referred to as F3 was purified from the seeds of Borreria hispida by precipitation with 80% ammonium sulphate and gel filtration on Sephadex G-100 column. RP-HPLC analysis of protein fraction (F3 on an analytical C-18 column produced a single peak, detected at 220 nm. F3 showed an apparent molecular weight of 35 KDa by SDS PAGE and MALDI-TOF-MS analyses. Peptide mass fingerprinting analysis of F3 showed the closest homology with the sequence of 1-aminocyclopropane-1-carboxylate deaminase of Pyrococcus horikoshii. The protein (F3 exhibited significant cytotoxic activity against lung (A549 and cervical (HeLa cancer cells in a dose-dependent manner at concentrations ranging from 10 µg to 1000 µg/mL, as revealed by the MTT assay. Cell cycle analysis revealed the increased growth of sub-G0 population in both cell lines exposed to a concentration of 1000 µg/mL of protein fraction F3 as examined from flow cytometry. This is the first report of a protein from the seeds of Borreria hispida with antiproliferative and apoptotic activity in lung (A549 and cervical (HeLa cancer cells.

Proteomics analysis of BHK-21 cells infected with a fixed strain of rabies virus.

Science.gov (United States)

Zandi, Fatemeh; Eslami, Naser; Soheili, Masoomeh; Fayaz, Ahmad; Gholami, Alireza; Vaziri, Behrouz

2009-05-01

Rabies is a neurotropic virus that causes a life threatening acute viral encephalitis. The complex relationship of rabies virus (RV) with the host leads to its replication and spreading toward the neural network, where viral pathogenic effects appeared as neuronal dysfunction. In order to better understand the molecular basis of this relationship, a proteomics study on baby hamster kidney cells infected with challenge virus standard strain of RV was performed. This cell line is an in vitro model for rabies infection and is commonly used for viral seed preparation. The direct effect of the virus on cellular protein machinery was investigated by 2-DE proteome mapping of infected versus control cells followed by LC-MS/MS identification. This analysis revealed significant changes in expression of 14 proteins, seven of these proteins were viral and the remaining were host proteins with different known functions: cytoskeletal (capping protein, vimentin), anti-oxidative stress (superoxide dismutase), regulatory (Stathmin), and protein synthesis (P0). Despite of limited changes appeared upon rabies infection, they present a set of interesting biochemical pathways for further investigation on viral-host interaction.

Differential effect of TGFβ on the proteome of cancer associated fibroblasts and cancer epithelial cells in a co-culture approach - a short report.

Science.gov (United States)

Koczorowska, Maria Magdalena; Friedemann, Charlotte; Geiger, Klaus; Follo, Marie; Biniossek, Martin Lothar; Schilling, Oliver

2017-12-01

Solid tumors contain various components that together form the tumor microenvironment. Cancer associated fibroblasts (CAFs) are capable of secreting and responding to signaling molecules and growth factors. Due to their role in tumor development, CAFs are considered as potential therapeutic targets. A prominent tumor-associated signaling molecule is transforming growth factor β (TGFβ), an inducer of epithelial-to-mesenchymal transition (EMT). The differential action of TGFβ on CAFs and ETCs (epithelial tumor cells) has recently gained interest. Here, we aimed to investigate the effects of TGFβ on CAFs and ETCs at the proteomic level. We established a 2D co-culture system of differentially fluorescently labeled CAFs and ETCs and stimulated this co-culture system with TGFβ. The respective cell types were separated using FACS and subjected to quantitative analyses of individual proteomes using mass spectrometry. We found that TGFβ treatment had a strong impact on the proteome composition of CAFs, whereas ETCs responded only marginally to TGFβ. Quantitative proteomic analyses of the different cell types revealed up-regulation of extracellular matrix (ECM) proteins in TGFβ treated CAFs. In addition, we found that the TGFβ treated CAFs exhibited increased N-cadherin levels. From our data we conclude that CAFs respond to TGFβ treatment by changing their proteome composition, while ETCs appear to be rather resilient.

Tumor necrosis factor-α induces MMP-9 expression via p42/p44 MAPK, JNK, and nuclear factor-κB in A549 cells

International Nuclear Information System (INIS)

Lin, C.-C.; Tseng, Hsiao-Wei; Hsieh, Hsi-Lung; Lee, Chiang-Wen; Wu, C.-Y.; Cheng, C.-Y.; Yang, C.-M.

2008-01-01

Matrix metalloproteinases (MMPs), in particular MMP-9, have been shown to be induced by cytokines including tumor necrosis factor-α (TNF-α) and contributes to airway inflammation. However, the mechanisms underlying MMP-9 expression induced by TNF-α in human A549 cells remain unclear. Here, we showed that TNF-α induced production of MMP-9 protein and mRNA is determined by zymographic, Western blotting, RT-PCR and ELISA assay, which were attenuated by inhibitors of MEK1/2 (U0126), JNK (SP600125), and NF-κB (helenalin), and transfection with dominant negative mutants of ERK2 (ΔERK) and JNK (ΔJNK), and siRNAs for MEK1, p42 and JNK2. TNF-α-stimulated phosphorylation of p42/p44 MAPK and JNK were attenuated by pretreatment with the inhibitors U0126 and SP600125 or transfection with dominant negative mutants of ΔERK and ΔJNK. Furthermore, the involvement of NF-κB in TNF-α-induced MMP-9 production was consistent with that TNF-α-stimulated degradation of IκB-α and translocation of NF-κB into the nucleus which were blocked by helenalin, but not by U0126 and SP600125, revealed by immunofluorescence staining. The regulation of MMP-9 gene transcription by MAPKs and NF-κB was further confirmed by gene luciferase activity assay. MMP-9 promoter activity was enhanced by TNF-α in A549 cells transfected with wild-type MMP-9-Luc, which was inhibited by helenalin, U0126, or SP600125. In contrast, TNF-α-stimulated MMP-9 luciferase activity was totally lost in cells transfected with mutant-NF-κB MMP-9-luc. Moreover, pretreatment with actinomycin D and cycloheximide attenuated TNF-α-induced MMP-9 expression. These results suggest that in A549 cells, phosphorylation of p42/p44 MAPK, JNK, and transactivation of NF-κB are essential for TNF-α-induced MMP-9 gene expression

Proteomic analysis of cell lines to identify the irinotecan resistance ...

Indian Academy of Sciences (India)

MADHU

was selected from the wild-type LoVo cell line by chronic exposure to irinotecan ... dose–effect curves of anticancer drugs were drawn on semilogarithm .... alcohol metabolites daunorubicinol (Forrest and Gonzalez. 2000; Mordente et al. ..... Chen L, Huang C and Wei Y 2007 Proteomic analysis of liver cancer cells treated ...

In Vitro Antioxidant Activities of Phenols and Oleanolic Acid from Mango Peel and Their Cytotoxic Effect on A549 Cell Line

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Xuelian Bai

2018-06-01

Full Text Available Mango peel, the main by-product of juice processing, possesses appreciable quantities of bioactive phenolic compounds and is worthy of further utilization. The present work reports for the first time the HPLC analysis and in vitro antioxidant evaluation of mango peel phenols (MPPs and their cytotoxic effect on the A549 lung cancer cell line. These results indicated that mango peel has the total phenolic content of 723.2 ± 0.93 mg·kg−1 dry mango peel (DMP, which consisted mainly of vanillic aldehyde, caffeic acid, chlorogenic acid, gallic acid, procyanidin B2 and oleanolic acid. Antioxidant assays showed that MPPs had strong antioxidant activities, with 92 ± 4.2% of DPPH radical scavenging rate, 79 ± 2.5% of ABTS radical inhibition rate and 4.7 ± 0.5 μM Trolox equivalents per kg−1 DMP of ferric reducing power. Gallic acid possess a stronger antioxidant capacity than other phenols. In vitro cytotoxic tests suggested that mango peel extract (MPE had an IC50 value of 15 mg·mL−1 and MPPs had a stronger inhibitory effect on the A549 cell line. Oleanolic acid exhibited the strongest cytotoxicity, with an IC50 value of 4.7 μM, which was similar with that of the positive control 5-fluorouracil.

Indomethacin-Enhanced Anticancer Effect of Arsenic Trioxide in A549 Cell Line: Involvement of Apoptosis and Phospho-ERK and p38 MAPK Pathways

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Ali Mandegary

2013-01-01

Full Text Available Background. Focusing on novel drug combinations that target different pathways especially apoptosis and MAPK could be a rationale for combination therapy in successful treatment of lung cancer. Concurrent use of cyclooxygenase (COX inhibitors with arsenic trioxide (ATO might be a possible treatment option. Methods. Cytotoxicity of ATO, dexamethasone (Dex, celecoxib (Cel, and Indomethacin (Indo individually or in combination was determined at 24, 48, and 72 hrs in A549 lung cancer cells. The COX-2 gene and protein expression, MAPK pathway proteins, and caspase-3 activity were studied for the most cytotoxic combinations. Results. The IC50s of ATO and Indo were 68.7 μmol/L and 396.5 μmol/L, respectively. Treatment of cells with combinations of clinically relevant concentrations of ATO and Indo resulted in greater growth inhibition and apoptosis induction than did either agent alone. Caspase-3 activity was considerably high in the presence of ATO and Indo but showed no difference in single or combination use. Phosphorylation of p38 and ERK1/2 was remarkable in the concurrent presence of both drugs. Conclusions. Combination therapy with ATO and Indo exerted a very potent in vitro cytotoxic effect against A549 lung cancer cells. Activation of ERK and p38 pathways might be the mechanism of higher cytotoxic effect of ATO-Indo combination.

Detailed Functional and Proteomic Characterization of Fludarabine Resistance in Mantle Cell Lymphoma Cells.

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Lucie Lorkova

Full Text Available Mantle cell lymphoma (MCL is a chronically relapsing aggressive type of B-cell non-Hodgkin lymphoma considered incurable by currently used treatment approaches. Fludarabine is a purine analog clinically still widely used in the therapy of relapsed MCL. Molecular mechanisms of fludarabine resistance have not, however, been studied in the setting of MCL so far. We therefore derived fludarabine-resistant MCL cells (Mino/FR and performed their detailed functional and proteomic characterization compared to the original fludarabine sensitive cells (Mino. We demonstrated that Mino/FR were highly cross-resistant to other antinucleosides (cytarabine, cladribine, gemcitabine and to an inhibitor of Bruton tyrosine kinase (BTK ibrutinib. Sensitivity to other types of anti-lymphoma agents was altered only mildly (methotrexate, doxorubicin, bortezomib or remained unaffacted (cisplatin, bendamustine. The detailed proteomic analysis of Mino/FR compared to Mino cells unveiled over 300 differentially expressed proteins. Mino/FR were characterized by the marked downregulation of deoxycytidine kinase (dCK and BTK (thus explaining the observed crossresistance to antinucleosides and ibrutinib, but also by the upregulation of several enzymes of de novo nucleotide synthesis, as well as the up-regulation of the numerous proteins of DNA repair and replication. The significant upregulation of the key antiapoptotic protein Bcl-2 in Mino/FR cells was associated with the markedly increased sensitivity of the fludarabine-resistant MCL cells to Bcl-2-specific inhibitor ABT199 compared to fludarabine-sensitive cells. Our data thus demonstrate that a detailed molecular analysis of drug-resistant tumor cells can indeed open a way to personalized therapy of resistant malignancies.

A Systematic Analysis of a Deep Mouse Epididymal Sperm Proteome

Energy Technology Data Exchange (ETDEWEB)

Chauvin, Theodore; Xie, Fang; Liu, Tao; Nicora, Carrie D.; Yang, Feng; Camp, David G.; Smith, Richard D.; Roberts, Kenneth P.

2012-12-21

Spermatozoa are highly specialized cells that, when mature, are capable of navigating the female reproductive tract and fertilizing an oocyte. The sperm cell is thought to be largely quiescent in terms of transcriptional and translational activity. As a result, once it has left the male reproductive tract, the sperm cell is essentially operating with a static population of proteins. It is therefore theoretically possible to understand the protein networks contained in a sperm cell and to deduce its cellular function capabilities. To this end we have performed a proteomic analysis of mouse sperm isolated from the cauda epididymis and have confidently identified 2,850 proteins, which is the most comprehensive sperm proteome for any species reported to date. These proteins comprise many complete cellular pathways, including those for energy production via glycolysis, β-oxidation and oxidative phosphorylation, protein folding and transport, and cell signaling systems. This proteome should prove a useful tool for assembly and testing of protein networks important for sperm function.

Quantitative Proteomic Analysis of the Response to Zinc, Magnesium, and Calcium Deficiency in Specific Cell Types of Arabidopsis Roots

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Yoichiro Fukao

2016-01-01

Full Text Available The proteome profiles of specific cell types have recently been investigated using techniques such as fluorescence activated cell sorting and laser capture microdissection. However, quantitative proteomic analysis of specific cell types has not yet been performed. In this study, to investigate the response of the proteome to zinc, magnesium, and calcium deficiency in specific cell types of Arabidopsis thaliana roots, we performed isobaric tags for relative and absolute quantification (iTRAQ-based quantitative proteomics using GFP-expressing protoplasts collected by fluorescence-activated cell sorting. Protoplasts were collected from the pGL2-GFPer and pMGP-GFPer marker lines for epidermis or inner cell lines (pericycle, endodermis, and cortex, respectively. To increase the number of proteins identified, iTRAQ-labeled peptides were separated into 24 fractions by OFFGFEL electrophoresis prior to high-performance liquid chromatography coupled with mass spectrometry analysis. Overall, 1039 and 737 proteins were identified and quantified in the epidermal and inner cell lines, respectively. Interestingly, the expression of many proteins was decreased in the epidermis by mineral deficiency, although a weaker effect was observed in inner cell lines such as the pericycle, endodermis, and cortex. Here, we report for the first time the quantitative proteomics of specific cell types in Arabidopsis roots.

Overexpression of microRNA miR-30a or miR-191 in A549 lung cancer or BEAS-2B normal lung cell lines does not alter phenotype.

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Santosh Kumar Patnaik

Full Text Available BACKGROUND: MicroRNAs (miRNAs are small, noncoding RNAs (ribonucleic acids that regulate translation. Several miRNAs have been shown to be altered in whole cancer tissue compared to normal tissue when quantified by microarray. Based on previous such evidence of differential expression, we chose to study the functional significance of miRNAs miR-30a and -191 alterations in human lung cancer. METHODOLOGY/PRINCIPAL FINDINGS: The functional significance of miRNAs miR-30a and -191 was studied by creating stable transfectants of the lung adenocarcinoma cell line A549 and the immortalized bronchial epithelial cell line BEAS-2B with modest overexpression of miR-30a or -191 using a lentiviral system. When compared to the corresponding controls, both cell lines overexpressing miR-30a or -191 do not demonstrate any significant changes in cell cycle distribution, cell proliferation, adherent colony formation, soft agar colony formation, xenograft formation in a subcutaneous SCID mouse model, and drug sensitivity to doxorubicin and cisplatin. There is a modest increase in cell migration in cell lines overexpressing miR-30a compared to their controls. CONCLUSIONS/SIGNIFICANCE: Overexpression of miR-30a or -191 does not lead to an alteration in cell cycle, proliferation, xenograft formation, and chemosensitivity of A549 and BEAS-2B cell lines. Using microarray data from whole tumors to select specific miRNAs for functional study may be a suboptimal strategy.

In vitro cytotoxic effects of PM2.5 from the city of Abidjan (Ivory Coast) on human A549 lung cells

International Nuclear Information System (INIS)

Kouassi, Kouakou-Serge; Billet, Sylvain; Garcon, Guillaume; Verdin, Anthony; Courcot, Dominique; Shirali, Pirouz; Diouf, Amadou; Cazier, Fabrice; Djaman, Joseph

2012-01-01

Epidemiological studies associate air pollution, especially particulate, increased morbidity and mortality from respiratory and cardiovascular origin . Africa, which has an urbanization rate among the highest in the world, is particularly exposed. The 'Initiative on the air quality in Sub-Saharan Africa' showed the importance of atmospheric concentrations of certain pollutants such as nitrogen oxides, sulfur dioxide and particulate matter (PM 10 ). Like the great capitals of Africa, Abidjan, economic capital and most industrialized city of Ivory Coast is facing an air pollution from industrial-urban and health consequences for its population of nearly 6 million inhabitants. To better understand the mechanisms of action resulting from pulmonary exposure to particulate atmospheric aerosols, we proposed: (i) to collect atmospheric particles (PM 2.5 ) using high volume cascade impaction in the District of Abidjan in three influences (rural, urban or industrial), (ii) to determine their main physicochemical, (iii) assess their cytotoxicity and their role in the induction of oxidative damage in a model of human lung cells (A549) in culture. The chemical composition of the atmospheric particles revealed their heterogeneity, and many inorganic (e.g. Al, Ca, Fe, Mn, Zn, Ni, Cr, Cu, Pb, Mg) and organic compounds (e.g. paraffins) were quantified at the three sites. Their effect concentrations (EC) to 10 and 50% on the A549 were as follows: influence rural: EC 10 = 5.91 μg/cm 2 and EC 50 29.55 μg/cm 2 , urban influence: EC 10 = 5 .45 μg/cm 2 and EC 50 = 27.23 μg/cm 2 , and industrial influence: EC 10 = 6.86 μg/cm 2 and EC 50 = 34.29 μg/cm 2 . Exposure of A549 cells to Abidjan city's PM samples for 24, 48 or 72 hours to their EC 10 or EC 50 induced oxidative damage, as demonstrated by the formation of malon-dialdehyde, changes in enzyme activity of superoxide dismutase and alteration of glutathione status. (authors)

In vitro cytotoxicity effect and antibacterial performance of human lung epithelial cells A549 activity of Zinc oxide doped TiO2 nanocrystals: Investigation of bio-medical application by chemical method

International Nuclear Information System (INIS)

Kaviyarasu, K.; Geetha, N.; Kanimozhi, K.; Maria Magdalane, C.; Sivaranjani, S.; Ayeshamariam, A.; Kennedy, J.; Maaza, M.

2017-01-01

We report the synthesis of high quality ZnO doped TiO 2 nanocrystals by chemical method at room temperature (RT), it can cause serious oxidative stress and DNA damage to human lung epithelial cells (A549) lines. Our aim in this study, to reduce the cytotoxicity effect of ZnO doped TiO 2 nanocrystals are widely in biological fields. Several studies have been performed to understand the influence of ZnO doped titanium dioxide (TiO 2 -NPs) on cell function; however the effects of nanoparticle against to exposure on the cell membrane have been duly addressed fascinatingly so far. However, In this interaction, which may alter cell metabolism and integrity, it is one of the importance to understand the modifications of the cell membrane, mechanisms of pulmonary A549 cell lines nanoparticles were uptake and the molecular pathway during the initial cell responses are still unclear and much more investigative efforts are need to properly characterize the ZnO doped titanium dioxide nanoparticles were reported successfully. In particular of the epithelial cells, upon particles are exposed human pulmonary epithelial cells (A549) to various concentrations of composition, structure and morphology of the nanocrystals were analyzed by X-ray diffraction (XRD) and high resolution transmission electron microscopy (HRTEM). XRD assessed the crystal structure of the nanocrystals which identified peaks associated with (002), (100) and (101) planes of hexagonal wurtzite-type ZnO with lattice constants of a = b = 3.249 Å and c = 5.219 Å. The IR results showed high purity of products and indicated that the nanocrystals are made up of Ti−O and Zn−O bonds. The Photoluminescence (PL) spectra are dominated by a strong narrow band edge emission tunable in the blue region of the visible spectra indicating a narrow size distribution of ZnO/TiO 2 nanocrystals which exhibits antibacterial activity over a broad range of bacterial species and in particular against Stre. Mut where it out competes

A novel method for sample preparation of fresh lung cancer tissue for proteomics analysis by tumor cell enrichment and removal of blood contaminants

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Orre Lotta

2010-02-01

Full Text Available Abstract Background In-depth proteomics analyses of tumors are frequently biased by the presence of blood components and stromal contamination, which leads to large experimental variation and decreases the proteome coverage. We have established a reproducible method to prepare freshly collected lung tumors for proteomics analysis, aiming at tumor cell enrichment and reduction of plasma protein contamination. We obtained enriched tumor-cell suspensions (ETS from six lung cancer cases (two adenocarcinomas, two squamous-cell carcinomas, two large-cell carcinomas and from two normal lung samples. The cell content of resulting ETS was evaluated with immunocytological stainings and compared with the histologic pattern of the original specimens. By means of a quantitative mass spectrometry-based method we evaluated the reproducibility of the sample preparation protocol and we assessed the proteome coverage by comparing lysates from ETS samples with the direct lysate of corresponding fresh-frozen samples. Results Cytological analyses on cytospin specimens showed that the percentage of tumoral cells in the ETS samples ranged from 20% to 70%. In the normal lung samples the percentage of epithelial cells was less then 10%. The reproducibility of the sample preparation protocol was very good, with coefficient of variation at the peptide level and at the protein level of 13% and 7%, respectively. Proteomics analysis led to the identification of a significantly higher number of proteins in the ETS samples than in the FF samples (244 vs 109, respectively. Albumin and hemoglobin were among the top 5 most abundant proteins identified in the FF samples, showing a high contamination with blood and plasma proteins, whereas ubiquitin and the mitochondrial ATP synthase 5A1 where among the top 5 most abundant proteins in the ETS samples. Conclusion The method is feasible and reproducible. We could obtain a fair enrichment of cells but the major benefit of the method

Proteomic Analysis of Pathogenic Fungi Reveals Highly Expressed Conserved Cell Wall Proteins

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Jackson Champer

2016-01-01

Full Text Available We are presenting a quantitative proteomics tally of the most commonly expressed conserved fungal proteins of the cytosol, the cell wall, and the secretome. It was our goal to identify fungi-typical proteins that do not share significant homology with human proteins. Such fungal proteins are of interest to the development of vaccines or drug targets. Protein samples were derived from 13 fungal species, cultured in rich or in minimal media; these included clinical isolates of Aspergillus, Candida, Mucor, Cryptococcus, and Coccidioides species. Proteomes were analyzed by quantitative MSE (Mass Spectrometry—Elevated Collision Energy. Several thousand proteins were identified and quantified in total across all fractions and culture conditions. The 42 most abundant proteins identified in fungal cell walls or supernatants shared no to very little homology with human proteins. In contrast, all but five of the 50 most abundant cytosolic proteins had human homologs with sequence identity averaging 59%. Proteomic comparisons of the secreted or surface localized fungal proteins highlighted conserved homologs of the Aspergillus fumigatus proteins 1,3-β-glucanosyltransferases (Bgt1, Gel1-4, Crf1, Ecm33, EglC, and others. The fact that Crf1 and Gel1 were previously shown to be promising vaccine candidates, underlines the value of the proteomics data presented here.

Reactive oxygen species mediated DNA damage in human lung alveolar epithelial (A549) cells from exposure to non-cytotoxic MFI-type zeolite nanoparticles.

Science.gov (United States)

Bhattacharya, Kunal; Naha, Pratap C; Naydenova, Izabela; Mintova, Svetlana; Byrne, Hugh J

2012-12-17

Increasing utilization of engineered nanoparticles in the field of electronics and biomedical applications demands an assessment of risk associated with deliberate or accidental exposure. Metal based nanoparticles are potentially most important of all the nanoparticles in terms of health risks. Microporous alumino-silicates and pure silicates named as zeolites and zeo-type materials with variety of structures, chemical compositions, particle sizes and morphologies have a significant number of industrial uses such as in catalysis, sorption and ion-exchange processes. In particular, the nanosized particles due to their unique properties are used in hybrid organic-inorganic materials for photography, photonics, electronics, labeling, imaging, and sensing. The aim of the current study is to investigate pure silica MFI-type zeolites nanoparticles with sizes of 50nm and 100nm (samples MFI-50 and MFI-100) under suspended conditions and their toxicological effects on human lung alveolar (A549) cells under in vitro conditions. Live cell imaging showed that the nanoparticles precipitated from the colloidal suspension of cell culture media as large agglomerates, coming in contact with the cell surface through sedimentation. A cellular proliferative capacity test showed the zeolite nanoparticles to exhibit no significant cytotoxicity below a concentration of 100μg/ml. However, both the MFI-50 and MFI-100 nanoparticles induced high intracellular reactive oxygen species (ROS) generation and elevated mitochondrial membrane potential in the A549 cells over the measured time period of 12h and at concentrations up to ≤50μg/ml. DNA fragmentation analysis using the comet assay showed that the MFI-50 and MFI-100 nanoparticles cause genotoxicity in a concentration dependent manner. Furthermore, the rate at which maximum genomic damage was caused by MFI-100 nanoparticles in the A549 cells was found to be high as compared to the MFI-50 nanoparticles. However, the damage caused by the

Proteomic characterization of golgi membranes enriched from Arabidopsis suspension cell cultures

DEFF Research Database (Denmark)

Hansen, Sara Fasmer; Ebert, Berit; Rautengarten, Carsten

2016-01-01

The plant Golgi apparatus has a central role in the secretory pathway and is the principal site within the cell for the assembly and processing of macromolecules. The stacked membrane structure of the Golgi apparatus along with its interactions with the cytoskeleton and endoplasmic reticulum has...... historically made the isolation and purification of this organelle difficult. Density centrifugation has typically been used to enrich Golgi membranes from plant microsomal preparations, and aside from minor adaptations, the approach is still widely employed. Here we outline the enrichment of Golgi membranes...... from an Arabidopsis cell suspension culture that can be used to investigate the proteome of this organelle. We also provide a useful workflow for the examination of proteomic data as the result of multiple analyses. Finally, we highlight a simple technique to validate the subcellular localization...

Gleditsia Saponin C Induces A549 Cell Apoptosis via Caspase-Dependent Cascade and Suppresses Tumor Growth on Xenografts Tumor Animal Model

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Ye Cheng

2018-01-01

Full Text Available Saponins are natural compounds and possess the most promising anti-cancer function. Here, a saponin gleditsia saponin C (GSC, extracted from gleditsiae fructus abnormalis, could induce apoptosis of lung tumor cell line A549 via caspase dependent cascade and this effect could be prevented by the caspase inhibitors. In addition, GSC induced cell death companied with an increase ratio of Bax:Bcl-2 and inhibition of ERK and Akt signaling pathways. Meanwhile, GSC suppressed TNFα inducing NF-κB activation and increased the susceptibility of lung cancer cell to TNFα induced apoptosis. Furthermore, on mouse xenograft model, GSC significantly suppressed tumor growth and induced cancer cell apoptosis, which validated the anti-tumor effect of GSC. Based on these results, GSC might be a promising drug candidate of anti-lung cancer for its potential clinical applications.

Proteome profile of swine testicular cells infected with porcine transmissible gastroenteritis coronavirus.

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Ruili Ma

Full Text Available The interactions occurring between a virus and a host cell during a viral infection are complex. The purpose of this paper was to analyze altered cellular protein levels in porcine transmissible gastroenteritis coronavirus (TGEV-infected swine testicular (ST cells in order to determine potential virus-host interactions. A proteomic approach using isobaric tags for relative and absolute quantitation (iTRAQ-coupled two-dimensional liquid chromatography-tandem mass spectrometry identification was conducted on the TGEV-infected ST cells. The results showed that the 4-plex iTRAQ-based quantitative approach identified 4,112 proteins, 146 of which showed significant changes in expression 48 h after infection. At 64 h post infection, 219 of these proteins showed significant change, further indicating that a larger number of proteomic changes appear to occur during the later stages of infection. Gene ontology analysis of the altered proteins showed enrichment in multiple biological processes, including cell adhesion, response to stress, generation of precursor metabolites and energy, cell motility, protein complex assembly, growth, developmental maturation, immune system process, extracellular matrix organization, locomotion, cell-cell signaling, neurological system process, and cell junction organization. Changes in the expression levels of transforming growth factor beta 1 (TGF-β1, caspase-8, and heat shock protein 90 alpha (HSP90α were also verified by western blot analysis. To our knowledge, this study is the first time the response profile of ST host cells following TGEV infection has been analyzed using iTRAQ technology, and our description of the late proteomic changes that are occurring after the time of vigorous viral production are novel. Therefore, this study provides a solid foundation for further investigation, and will likely help us to better understand the mechanisms of TGEV infection and pathogenesis.

PKM2 Thr454 phosphorylation increases its nuclear translocation and promotes xenograft tumor growth in A549 human lung cancer cells

Energy Technology Data Exchange (ETDEWEB)

Yu, Zhenhai, E-mail: tomsyu@163.com [Center for Reproductive Medicine, Affiliated Hospital of Weifang Medical University, Weifang, Shandong, 261031 (China); Huang, Liangqian [Institute of Health Sciences, Shanghai Institutes for Biological Sciences (SIBS), Chinese Academy of Sciences (CAS) & Shanghai Jiao Tong University School of Medicine -SJTUSM, Shanghai, 200025 (China); Qiao, Pengyun; Jiang, Aifang; Wang, Li; Yang, Tingting [Center for Reproductive Medicine, Affiliated Hospital of Weifang Medical University, Weifang, Shandong, 261031 (China); Tang, Shengjian; Zhang, Wei [Plastic Surgery Institute of Weifang Medical University, Weifang, Shandong, 261041 (China); Ren, Chune, E-mail: ren@wfmc.edu.cn [Center for Reproductive Medicine, Affiliated Hospital of Weifang Medical University, Weifang, Shandong, 261031 (China)

2016-05-13

Pyruvate kinase M2 (PKM2) is a key enzyme of glycolysis which is highly expressed in many tumor cells, and plays an important role in the Warburg effect. In previous study, we found PIM2 phosphorylates PKM2 at Thr454 residue (Yu, etl 2013). However, the functions of PKM2 Thr454 modification in cancer cells still remain unclear. Here we find PKM2 translocates into the nucleus after Thr454 phosphorylation. Replacement of wild type PKM2 with a mutant (T454A) enhances mitochondrial respiration, decreases pentose phosphate pathway, and enhances chemosensitivity in A549 cells. In addition, the mutant (T454A) PKM2 reduces xenograft tumor growth in nude mice. These findings demonstrate that PKM2 T454 phosphorylation is a potential therapeutic target in lung cancer.

Proteomic profile of acute myeloid leukaemia: A review update

African Journals Online (AJOL)

attention to the progress and advancements in cancer proteomics technology with the aim of simplifying ... hematopoietic cells leading to distinct differences ... procedures like bone marrow and tissue biopsies. [7,8]. .... patients who were subjected to transplantation ..... Boyd RS, Dyer MJ, Cain K. Proteomic analysis of b-cell.

[The effect and mechanism of vinorelbine on cisplatin resistance of human lung cancer cell line A549/DDP].

Science.gov (United States)

Qi, Chunsheng; Gao, Sen; Li, Huiqiang; Gao, Weizhen

2014-02-01

Drug resistance is a major obstacle on lung cancer treatment and Vinorelbine is an effective drug to inhibition of tumor proliferation and metastasis. In this study, we investigated the effect and mechanism of Vinorelbine on reversing the cisplatin resistance of human lung cancer A549/DDP cell line. With 1 μmol/L and 5 μmol/L Vinorelbine treatment, MTS assay was employed to determine the effect of the cisplatin sensitivity of tumor cells, flow cytometry to determine the apoptosis rate and change of Rh-123 content; Western blot to determine the expression of MDR1, Bcl-2, surviving, PTEN, caspase-3/8 and phosphorylation level of Akt (p-Akt); Real-time PCR was to determine the mRNA expression of MDR1, Bcl-2, survivin and PTEN. Finally the transcriptional activities of NF-κB, Twist and Snail were determined by reporter gene system. With 1 μmol/L and 5 μmol/L Vinorelbine treatment, the sensitivity of cancer cells to cisplatin was increased by 1.91- and 2.54- folds respectively, flow cytometry showed that the content of Rh-123 was elevated 1.93- and 2.95- folds and apoptosis rate was increased 2.25- and 3.82- folds, Western blot showed that the expression of multidrug resistance related proteins MDR, Bcl-2 and survivin were downregulated, caspase-3/8 and PTEN was upregulated, phosphorylation of Akt was downregulated as well, real-time assay showed that the mRNA expression of MDR1 was downregulated 43.5% and 25.8%, Bcl-2 was downregulated 57.3% and 34.1%, survivin was downregulated 37.6% and 12.4%, PTEN was upregulated 183.4% and 154.2%, the transcriptional activities of NF-κB was downregulated 53.2% and 34.5%, Twist was downregulated 61.4% and 33.5%, and Snail was downregulated 57.8% and 18.7%. Vinorelbine treatment led to increase of cisplatin sensitivity of A549/DDP cells and the mechanisms included the regulation of PTEN/AKT/NF-κB signal pathway to decreased drug resistance gene expression and increased pro-apoptosis gene expression.

Nitric oxide generated by ionizing radiation and EGF is implicated in EGF receptor phosphorylation in A549 lung carcinoma cells

International Nuclear Information System (INIS)

Park, In Chul; Lee, Hyung Chahn; Rhee, Chang Hun; Hong, Seok Il

2004-01-01

Although it has been demonstrated that ionizing radiation (IR) control various cell functions in a different cell types, the mechanisms of its action via NO are not well understood. NO may potentially affect every type of mammalian cells, owing to its ubiquitous production and participate in the control of cell proliferation in a great variety of cell types. The epidermal growth factor (EGF) receptor is a transmembrane glycoprotein of Mr 170,000. When EGF binds to its receptor, the receptor is dimerized and autophosphorylated at the carboxyl-terminal tyrosine 992, 1608, 1086, 1148 and 1173. This phosphorylated receptor initiates a series of signal tranduction events through interacting proteins of SH2 family including Shc, Grb2 and Sos, which in turn trigger ativation of MAPK cascades. Although the number of signaling events mediated by IR-induced NO is growing, it is still unclear how NO activate cellular signaling events. Thus, we examined the effect of NO on cellular phosphorylation and found that NO was produced by ionizing radiation in A549 lung adenocarcinoma cells and enhances the unique tyrosine phosphorylation on EGF receptor

28 CFR 549.73 - Appealing the fee.

Science.gov (United States)

2010-07-01

... 28 Judicial Administration 2 2010-07-01 2010-07-01 false Appealing the fee. 549.73 Section 549.73 Judicial Administration BUREAU OF PRISONS, DEPARTMENT OF JUSTICE INSTITUTIONAL MANAGEMENT MEDICAL SERVICES Fees for Health Care Services § 549.73 Appealing the fee. You may seek review of issues related to...

Proteomic approach toward molecular backgrounds of drug resistance of osteosarcoma cells in spheroid culture system.

Science.gov (United States)

Arai, Kazuya; Sakamoto, Ruriko; Kubota, Daisuke; Kondo, Tadashi

2013-08-01

Chemoresistance is one of the most critical prognostic factors in osteosarcoma, and elucidation of the molecular backgrounds of chemoresistance may lead to better clinical outcomes. Spheroid cells resemble in vivo cells and are considered an in vitro model for the drug discovery. We found that spheroid cells displayed more chemoresistance than conventional monolayer cells across 11 osteosarcoma cell lines. To investigate the molecular mechanisms underlying the resistance to chemotherapy, we examined the proteomic differences between the monolayer and spheroid cells by 2D-DIGE. Of the 4762 protein species observed, we further investigated 435 species with annotated mass spectra in the public proteome database, Genome Medicine Database of Japan Proteomics. Among the 435 protein species, we found that 17 species exhibited expression level differences when the cells formed spheroids in more than five cell lines and four species out of these 17 were associated with spheroid-formation associated resistance to doxorubicin. We confirmed the upregulation of cathepsin D in spheroid cells by western blotting. Cathepsin D has been implicated in chemoresistance of various malignancies but has not previously been implemented in osteosarcoma. Our study suggested that the spheroid system may be a useful tool to reveal the molecular backgrounds of chemoresistance in osteosarcoma. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Challenges and Strategies for Proteome Analysis of the Interaction of Human Pathogenic Fungi with Host Immune Cells.

Science.gov (United States)

Krüger, Thomas; Luo, Ting; Schmidt, Hella; Shopova, Iordana; Kniemeyer, Olaf

2015-12-14

Opportunistic human pathogenic fungi including the saprotrophic mold Aspergillus fumigatus and the human commensal Candida albicans can cause severe fungal infections in immunocompromised or critically ill patients. The first line of defense against opportunistic fungal pathogens is the innate immune system. Phagocytes such as macrophages, neutrophils and dendritic cells are an important pillar of the innate immune response and have evolved versatile defense strategies against microbial pathogens. On the other hand, human-pathogenic fungi have sophisticated virulence strategies to counteract the innate immune defense. In this context, proteomic approaches can provide deeper insights into the molecular mechanisms of the interaction of host immune cells with fungal pathogens. This is crucial for the identification of both diagnostic biomarkers for fungal infections and therapeutic targets. Studying host-fungal interactions at the protein level is a challenging endeavor, yet there are few studies that have been undertaken. This review draws attention to proteomic techniques and their application to fungal pathogens and to challenges, difficulties, and limitations that may arise in the course of simultaneous dual proteome analysis of host immune cells interacting with diverse morphotypes of fungal pathogens. On this basis, we discuss strategies to overcome these multifaceted experimental and analytical challenges including the viability of immune cells during co-cultivation, the increased and heterogeneous protein complexity of the host proteome dynamically interacting with the fungal proteome, and the demands on normalization strategies in terms of relative quantitative proteome analysis.

28 CFR 549.60 - Purpose and scope.

Science.gov (United States)

2010-07-01

... 28 Judicial Administration 2 2010-07-01 2010-07-01 false Purpose and scope. 549.60 Section 549.60 Judicial Administration BUREAU OF PRISONS, DEPARTMENT OF JUSTICE INSTITUTIONAL MANAGEMENT MEDICAL SERVICES Hunger Strikes, Inmate § 549.60 Purpose and scope. The Bureau of Prisons provides guidelines for the medical and administrative management of...

Effect of glutathione depletion on the aerobic radiation response of A549 human lung carcinoma cells

International Nuclear Information System (INIS)

Biaglow, J.E.; Clark, E.P.; Varnes, M.E.; Tuttle, S.W.; Epp, E.R.

1985-01-01

The authors demonstrated that depletion of glutathione (GSH) from cultured A549 cells to non-detectable levels, using L-buthionine sulfoximine (L-BSO), results in an increased aerobic radiation response. This response can be further increased if dimethylfumarate (DMF) is added concurrently with L-BSO. L-BSO is a relatively slow depletor of GSH compared to DMF, which acts by both spontaneous and enzyme catalysed reactions. The authors have studied: 1. the effect of continuous long-term exposure to 0.1 mM L-BSO on GSH levels and the subsequent radiation response and 2. the effect of GSH depletion on enzymes essential for radical detoxification. The results show an enhanced aerobic radiation response that increases with the time of exposure to L-BSO. For example surviving fraction (S.F.) after 5 Gy for cells exposed to L-BSO for 24 hrs is 0.004 and 0.08 for control cultures. Cells washed free of medium and irradiated in Hanks' show 0.0007 S.F. after 120 hr exposure to L-BSO and S.F. of 0.075 for the control cultures. The relationship between the chronic GSH depleted state, GSH peroxidase, and radiation induced lipid peroxidation is being investigated

In vitro cytotoxicity effect and antibacterial performance of human lung epithelial cells A549 activity of Zinc oxide doped TiO{sub 2} nanocrystals: Investigation of bio-medical application by chemical method

Energy Technology Data Exchange (ETDEWEB)

Kaviyarasu, K., E-mail: kaviyarasuloyolacollege@gmail.com [UNESCO-UNISA Africa Chair in Nanosciences/Nanotechnology Laboratories, College of Graduate Studies, University of South Africa (UNISA), Muckleneuk Ridge, PO Box 392, Pretoria (South Africa); Nanosciences African Network (NANOAFNET), Materials Research Group (MRG), i Themba LABS-National Research Foundation - NRF, 1 Old Faure Road, 7129, PO Box 722, Somerset West, Western Cape Province (South Africa); Geetha, N. [Research and Development Center, Bharathiyar University, Coimbatore 641046 (India); Kanimozhi, K. [PG Research & Department of Chemistry, Auxilium College (Autonomous), Vellore (India); Maria Magdalane, C. [Department of Chemistry, St. Xavier’s College (Autonomous), Tirunelveli 627002 (India); LIFE, Department of Chemistry, Loyola College (Autonomous), Chennai 600034 (India); Sivaranjani, S. [Research and Development Center, Bharathiyar University, Coimbatore 641046 (India); Department of Physics, SBM College of Engineering and Technology, Dindigul -624 005 (India); Ayeshamariam, A. [Research and Development Center, Bharathiyar University, Coimbatore 641046 (India); Department of Physics, Khadir Mohideen College, Adirampattinam 614601 (India); Kennedy, J. [UNESCO-UNISA Africa Chair in Nanosciences/Nanotechnology Laboratories, College of Graduate Studies, University of South Africa (UNISA), Muckleneuk Ridge, PO Box 392, Pretoria (South Africa); National Isotope Centre, GNS Science, PO Box 31312, Lower Hutt 5010 (New Zealand); Maaza, M. [UNESCO-UNISA Africa Chair in Nanosciences/Nanotechnology Laboratories, College of Graduate Studies, University of South Africa (UNISA), Muckleneuk Ridge, PO Box 392, Pretoria (South Africa); Nanosciences African Network (NANOAFNET), Materials Research Group (MRG), i Themba LABS-National Research Foundation - NRF, 1 Old Faure Road, 7129, PO Box 722, Somerset West, Western Cape Province (South Africa)

2017-05-01

We report the synthesis of high quality ZnO doped TiO{sub 2} nanocrystals by chemical method at room temperature (RT), it can cause serious oxidative stress and DNA damage to human lung epithelial cells (A549) lines. Our aim in this study, to reduce the cytotoxicity effect of ZnO doped TiO{sub 2} nanocrystals are widely in biological fields. Several studies have been performed to understand the influence of ZnO doped titanium dioxide (TiO{sub 2}-NPs) on cell function; however the effects of nanoparticle against to exposure on the cell membrane have been duly addressed fascinatingly so far. However, In this interaction, which may alter cell metabolism and integrity, it is one of the importance to understand the modifications of the cell membrane, mechanisms of pulmonary A549 cell lines nanoparticles were uptake and the molecular pathway during the initial cell responses are still unclear and much more investigative efforts are need to properly characterize the ZnO doped titanium dioxide nanoparticles were reported successfully. In particular of the epithelial cells, upon particles are exposed human pulmonary epithelial cells (A549) to various concentrations of composition, structure and morphology of the nanocrystals were analyzed by X-ray diffraction (XRD) and high resolution transmission electron microscopy (HRTEM). XRD assessed the crystal structure of the nanocrystals which identified peaks associated with (002), (100) and (101) planes of hexagonal wurtzite-type ZnO with lattice constants of a = b = 3.249 Å and c = 5.219 Å. The IR results showed high purity of products and indicated that the nanocrystals are made up of Ti−O and Zn−O bonds. The Photoluminescence (PL) spectra are dominated by a strong narrow band edge emission tunable in the blue region of the visible spectra indicating a narrow size distribution of ZnO/TiO{sub 2} nanocrystals which exhibits antibacterial activity over a broad range of bacterial species and in particular against Stre. Mut

Optimized method for identification of the proteomes secreted by cardiac cells

Czech Academy of Sciences Publication Activity Database

Šťastná, Miroslava; Van Eyk, J.E.

2013-01-01

Roč. 1005, č. 1005 (2013), s. 225-235 ISSN 1940 -6029 Institutional support: RVO:68081715 Keywords : cardiac cells * secreted proteins * proteomic technology Subject RIV: CB - Analytical Chemistry, Separation

Optimized method for identification of the proteomes secreted by cardiac cells

Czech Academy of Sciences Publication Activity Database

Šťastná, Miroslava; Van Eyk, J.E.

2013-01-01

Roč. 1005, č. 1005 (2013), s. 225-235 ISSN 1940-6029 Institutional support: RVO:68081715 Keywords : cardiac cells * secreted proteins * proteomic technology Subject RIV: CB - Analytical Chemistry, Separation

Proteomics of cancer cell lines resistant to microtubule-stabilizing agents

DEFF Research Database (Denmark)

Albrethsen, Jakob; Angeletti, Ruth H; Horwitz, Susan Band

2014-01-01

Despite the clinical success of microtubule-interacting agents (MIA), a significant challenge for oncologists is the inability to predict the response of individual patients with cancer to these drugs. In the present study, six cell lines were compared by 2D DIGE proteomics to investigate cellula...

Integration of genomics, proteomics, and imaging for cardiac stem cell therapy

International Nuclear Information System (INIS)

Chun, Hyung J.; Wilson, Kitch O.; Huang, Mei; Wu, Joseph C.

2007-01-01

Cardiac stem cell therapy is beginning to mature as a valid treatment for heart disease. As more clinical trials utilizing stem cells emerge, it is imperative to establish the mechanisms by which stem cells confer benefit in cardiac diseases. In this paper, we review three methods - molecular cellular imaging, gene expression profiling, and proteomic analysis - that can be integrated to provide further insights into the role of this emerging therapy. (orig.)

Cyto- and genotoxicity assessment of Gold nanoparticles obtained by laser ablation in A549 lung adenocarcinoma cells

Science.gov (United States)

Di Bucchianico, Sebastiano; Migliore, Lucia; Marsili, Paolo; Vergari, Chiara; Giammanco, Francesco; Giorgetti, Emilia

2015-05-01

Gold nanoparticles have attracted enormous interest in biomedical applications, based on their unique optical properties. However, their toxicity on human tissues is still an open issue. Beyond the potential intrinsic toxicity of nanostructured gold, a non-negligible contribution of stabilizers or reaction by-products related to current wet chemical synthesis procedures can be expected. Aimed at isolating gold contribution from that of any other contaminant, we produced colloidal suspensions of Gold nanoparticles having average size exposure to NPs, namely adenocarcinoma epithelial A549 cells. Gold nanoparticles prepared in water showed no particular signs of cytotoxicity, cytostasis, and/or genotoxicity as assessed by MTT colorimetric viability test and Cytokinesis-block micronucleus cytome assay up to concentrations of the order of 5 μg/mL. In contrast, Gold nanoparticles produced in pure acetone and then transferred into deionized water showed impaired cell viability, apoptosis responses, micronuclei, and dicentric chromosomes induction as well as nuclear budding, as a function of the amount of surface contaminants like amorphous carbon and enolate ions.

Proteomic analysis of proton beam irradiated human melanoma cells.

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Sylwia Kedracka-Krok

Full Text Available Proton beam irradiation is a form of advanced radiotherapy providing superior distributions of a low LET radiation dose relative to that of photon therapy for the treatment of cancer. Even though this clinical treatment has been developing for several decades, the proton radiobiology critical to the optimization of proton radiotherapy is far from being understood. Proteomic changes were analyzed in human melanoma cells treated with a sublethal dose (3 Gy of proton beam irradiation. The results were compared with untreated cells. Two-dimensional electrophoresis was performed with mass spectrometry to identify the proteins. At the dose of 3 Gy a minimal slowdown in proliferation rate was seen, as well as some DNA damage. After allowing time for damage repair, the proteomic analysis was performed. In total 17 protein levels were found to significantly (more than 1.5 times change: 4 downregulated and 13 upregulated. Functionally, they represent four categories: (i DNA repair and RNA regulation (VCP, MVP, STRAP, FAB-2, Lamine A/C, GAPDH, (ii cell survival and stress response (STRAP, MCM7, Annexin 7, MVP, Caprin-1, PDCD6, VCP, HSP70, (iii cell metabolism (TIM, GAPDH, VCP, and (iv cytoskeleton and motility (Moesin, Actinin 4, FAB-2, Vimentin, Annexin 7, Lamine A/C, Lamine B. A substantial decrease (2.3 x was seen in the level of vimentin, a marker of epithelial to mesenchymal transition and the metastatic properties of melanoma.

Proteomic changes in a childhood acute lymphoblastic leukemia cell line during the adaptation to vincristine.

Science.gov (United States)

Guzmán-Ortiz, Ana Laura; Aparicio-Ozores, Gerardo; Valle-Rios, Ricardo; Medina-Contreras, Oscar; Patiño-López, Genaro; Quezada, Héctor

Relapse occurs in approximately 20% of Mexican patients with childhood acute lymphoblastic leukemia (ALL). In this group, chemoresistance may be one of the biggest challenges. An overview of complex cellular processes like drug tolerance can be achieved with proteomic studies. The B-lineage pediatric ALL cell line CCRF-SB was gradually exposed to the chemotherapeutic vincristine until proliferation was observed at 6nM, control cells were cultured in the absence of vincristine. The proteome from each group was analyzed by nanoHPLC coupled to an ESI-ion trap mass spectrometer. The identified proteins were grouped into overrepresented functional categories with the PANTHER classification system. We found 135 proteins exclusively expressed in the presence of vincristine. The most represented functional categories were: Toll receptor signaling pathway, Ras Pathway, B and T cell activation, CCKR signaling map, cytokine-mediated signaling pathway, and oxidative phosphorylation. Our study indicates that signal transduction and mitochondrial ATP production are essential during adaptation of leukemic cells to vincristine, these processes represent potential therapeutic targets. Copyright © 2017 Hospital Infantil de México Federico Gómez. Publicado por Masson Doyma México S.A. All rights reserved.

Deep coverage mouse red blood cell proteome: a first comparison with the human red blood cell

DEFF Research Database (Denmark)

Pasini, Erica M; Kirkegaard, Morten; Salerno, Doris

2008-01-01

Mice have close genetic/physiological relationships to humans, breed rapidly, and can be genetically modified, making them the most used mammal in biomedical research. Because the red blood cell (RBC) is the sole gas transporter in vertebrates, diseases of the RBC are frequently severe; much...... proteome have been confirmed here. This comparison sheds light on several open issues in RBC biology and provides a departure point for more comprehensive understanding of RBC function....

Gel-aided sample preparation (GASP)--a simplified method for gel-assisted proteomic sample generation from protein extracts and intact cells.

Science.gov (United States)

Fischer, Roman; Kessler, Benedikt M

2015-04-01

We describe a "gel-assisted" proteomic sample preparation method for MS analysis. Solubilized protein extracts or intact cells are copolymerized with acrylamide, facilitating denaturation, reduction, quantitative cysteine alkylation, and matrix formation. Gel-aided sample preparation has been optimized to be highly flexible, scalable, and to allow reproducible sample generation from 50 cells to milligrams of protein extracts. This methodology is fast, sensitive, easy-to-use on a wide range of sample types, and accessible to nonspecialists. © 2014 The Authors. PROTEOMICS published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Green synthesis of zero valent colloidal nanosilver targeting A549 lung cancer cell: In vitro cytotoxicity

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Minakshi Jha

2018-06-01

Full Text Available An eco-friendly green approach was proposed to synthesise stable, cytotoxic colloidal silver nanoparticles (AgNPs using Momordica charantia (M. charantia fruit extract. Bioinspired green method adopted for fabrication of AgNPs because of easy, fast, low-cost and benign bioprocess. Phytocomponents played the crucial role in capping, stabilisation and inherent cytotoxic potential of colloidal nanosilver. The physiochemical, crystalline, optical and morphological properties of AgNPs were characterized using UV-vis, FT-IR, XRD, SEM, TEM, EDX and AFM. FT-IR reveals the presence of carbonyl, methyl, polyphenol (flavonoid, primary and secondary amine (protein, carboxyl group, ester as major functional groups over the surface of nanomaterials. Mechanistic pathway for formation and stabilisation of colloidal nanosilver has been discussed. Average crystalline size of AgNPs was found to be 12.55 nm from XRD. TEM shows AgNPs nanosphere with size range 1–13.85 nm. Consistency in spherical morphology was also confirmed through Atomic Force Microscopy (AFM. AFM measurement provided image Rq value 3.62, image Ra 2.47, roughness Rmax 36.4 nm, skewness 1.99 and kurtosis 9.87. The SRB assay revealed substantial in vitro noticeable anti-cancer activity of colloidal nanosilver on A549 and HOP-62 human lung cancer cells in a dose dependent manner with IC50 value of 51.93 µg/ml and 76.92 µg/ml. In addition, M. charantia capped AgNPs were found to be more biocompatible in comparison to M. charantia FE. Our study demonstrated the integration of green chemistry principle in nanomaterials fabrication and focused on the potential use of M. charantia fruit extract as an efficient precursor for biocompatible AgNPs anodrug formulation with improved cytotoxic applications. Keywords: M. charantia, Silver nanoparticles, TEM, Anticancer activity, A549, HOP-62

Differential replication of avian influenza H9N2 viruses in human alveolar epithelial A549 cells

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Peiris Malik

2010-03-01

Full Text Available Abstract Avian influenza virus H9N2 isolates cause a mild influenza-like illness in humans. However, the pathogenesis of the H9N2 subtypes in human remains to be investigated. Using a human alveolar epithelial cell line A549 as host, we found that A/Quail/Hong Kong/G1/97 (H9N2/G1, which shares 6 viral "internal genes" with the lethal A/Hong Kong/156/97 (H5N1/97 virus, replicates efficiently whereas other H9N2 viruses, A/Duck/Hong Kong/Y280/97 (H9N2/Y280 and A/Chicken/Hong Kong/G9/97 (H9N2/G9, replicate poorly. Interestingly, we found that there is a difference in the translation of viral protein but not in the infectivity or transcription of viral genes of these H9N2 viruses in the infected cells. This difference may possibly be explained by H9N2/G1 being more efficient on viral protein production in specific cell types. These findings suggest that the H9N2/G1 virus like its counterpart H5N1/97 may be better adapted to the human host and replicates efficiently in human alveolar epithelial cells.

Proteomic techniques for characterisation of mesenchymal stem cell secretome.

Czech Academy of Sciences Publication Activity Database

Kupcová Skalníková, Helena

2013-01-01

Roč. 95, č. 12 (2013), s. 2196-2211 ISSN 0300-9084 R&D Projects: GA MŠk ED2.1.00/03.0124; GA TA ČR TA01011466 Institutional support: RVO:67985904 Keywords : mesenchymal stem cells * secretome * exosome * conditioned medium * proteomics Subject RIV: CE - Biochemistry Impact factor: 3.123, year: 2013

Proteomic Analysis of Lipid Raft-Like Detergent-Resistant Membranes of Lens Fiber Cells.

Science.gov (United States)

Wang, Zhen; Schey, Kevin L

2015-12-01

Plasma membranes of lens fiber cells have high levels of long-chain saturated fatty acids, cholesterol, and sphingolipids-key components of lipid rafts. Thus, lipid rafts are expected to constitute a significant portion of fiber cell membranes and play important roles in lens biology. The purpose of this study was to characterize the lens lipid raft proteome. Quantitative proteomics, both label-free and iTRAQ methods, were used to characterize lens fiber cell lipid raft proteins. Detergent-resistant, lipid raft membrane (DRM) fractions were isolated by sucrose gradient centrifugation. To confirm protein localization to lipid rafts, protein sensitivity to cholesterol removal by methyl-β-cyclodextrin was quantified by iTRAQ analysis. A total of 506 proteins were identified in raft-like detergent-resistant membranes. Proteins identified support important functions of raft domains in fiber cells, including trafficking, signal transduction, and cytoskeletal organization. In cholesterol-sensitivity studies, 200 proteins were quantified and 71 proteins were strongly affected by cholesterol removal. Lipid raft markers flotillin-1 and flotillin-2 and a significant fraction of AQP0, MP20, and AQP5 were found in the DRM fraction and were highly sensitive to cholesterol removal. Connexins 46 and 50 were more abundant in nonraft fractions, but a small fraction of each was found in the DRM fraction and was strongly affected by cholesterol removal. Quantification of modified AQP0 confirmed that fatty acylation targeted this protein to membrane raft domains. These data represent the first comprehensive profile of the lipid raft proteome of lens fiber cells and provide information on membrane protein organization in these cells.

Responses of genes involved in cell cycle control to diverse DNA damaging chemicals in human lung adenocarcinoma A549 cells

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Gooderham Nigel J

2005-08-01

Full Text Available Abstract Background Many anticancer agents and carcinogens are DNA damaging chemicals and exposure to such chemicals results in the deregulation of cell cycle progression. The molecular mechanisms of DNA damage-induced cell cycle alteration are not well understood. We have studied the effects of etoposide (an anticancer agent, cryptolepine (CLP, a cytotoxic alkaloid, benzo [a]pyrene (BaP, a carcinogenic polycyclic aromatic hydrocarbon and 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP, a cooked-meat derived carcinogen on the expression of cell cycle regulatory genes to understand the molecular mechanisms of the cell cycle disturbance. Results A549 cells were treated with DMSO or chemicals for up to 72 h and periodically sampled for cell cycle analysis, mRNA and protein expression. DMSO treated cells showed a dominant G1 peak in cell cycle at all times examined. Etoposide and CLP both induced G2/M phase arrest yet the former altered the expression of genes functioning at multiple phases, whilst the latter was more effective in inhibiting the expression of genes in G2-M transition. Both etoposide and CLP induced an accumulation of p53 protein and upregulation of p53 transcriptional target genes. Neither BaP nor PhIP had substantial phase-specific cell cycle effect, however, they induced distinctive changes in gene expression. BaP upregulated the expression of CYP1B1 at 6–24 h and downregulated many cell cycle regulatory genes at 48–72 h. By contrast, PhIP increased the expression of many cell cycle regulatory genes. Changes in the expression of key mRNAs were confirmed at protein level. Conclusion Our experiments show that DNA damaging agents with different mechanisms of action induced distinctive changes in the expression pattern of a panel of cell cycle regulatory genes. We suggest that examining the genomic response to chemical exposure provides an exceptional opportunity to understand the molecular mechanism involved in cellular

Arabidopsis Regenerating Protoplast: A Powerful Model System for Combining the Proteomics of Cell Wall Proteins and the Visualization of Cell Wall Dynamics

OpenAIRE

Yokoyama, Ryusuke; Kuki, Hiroaki; Kuroha, Takeshi; Nishitani, Kazuhiko

2016-01-01

The development of a range of sub-proteomic approaches to the plant cell wall has identified many of the cell wall proteins. However, it remains difficult to elucidate the precise biological role of each protein and the cell wall dynamics driven by their actions. The plant protoplast provides an excellent means not only for characterizing cell wall proteins, but also for visualizing the dynamics of cell wall regeneration, during which cell wall proteins are secreted. It therefore offers a uni...

Proteomic explorations of autism spectrum disorder.

Science.gov (United States)

Szoko, Nicholas; McShane, Adam J; Natowicz, Marvin R

2017-09-01

Proteomics, the large-scale study of protein expression in cells and tissues, is a powerful tool to study the biology of clinical conditions and has provided significant insights in many experimental systems. Herein, we review the basics of proteomic methodology and discuss challenges in using proteomic approaches to study autism. Unlike other experimental approaches, such as genomic approaches, there have been few large-scale studies of proteins in tissues from persons with autism. Most of the proteomic studies on autism used blood or other peripheral tissues; few studies used brain tissue. Some studies found dysregulation of aspects of the immune system or of aspects of lipid metabolism, but no consistent findings were noted. Based on the challenges in using proteomics to study autism, we discuss considerations for future studies. Apart from the complex technical considerations implicit in any proteomic analysis, key nontechnical matters include attention to subject and specimen inclusion/exclusion criteria, having adequate sample size to ensure appropriate powering of the study, attention to the state of specimens prior to proteomic analysis, and the use of a replicate set of specimens, when possible. We conclude by discussing some potentially productive uses of proteomics, potentially coupled with other approaches, for future autism research including: (1) proteomic analysis of banked human brain specimens; (2) proteomic analysis of tissues from animal models of autism; and (3) proteomic analysis of induced pluripotent stem cells that are differentiated into various types of brain cells and neural organoids. Autism Res 2017, 10: 1460-1469. © 2017 International Society for Autism Research, Wiley Periodicals, Inc. © 2017 International Society for Autism Research, Wiley Periodicals, Inc.

Proteomics Insights into Autophagy.

Science.gov (United States)

Cudjoe, Emmanuel K; Saleh, Tareq; Hawkridge, Adam M; Gewirtz, David A

2017-10-01

Autophagy, a conserved cellular process by which cells recycle their contents either to maintain basal homeostasis or in response to external stimuli, has for the past two decades become one of the most studied physiological processes in cell biology. The 2016 Nobel Prize in Medicine and Biology awarded to Dr. Ohsumi Yoshinori, one of the first scientists to characterize this cellular mechanism, attests to its importance. The induction and consequent completion of the process of autophagy results in wide ranging changes to the cellular proteome as well as the secretome. MS-based proteomics affords the ability to measure, in an unbiased manner, the ubiquitous changes that occur when autophagy is initiated and progresses in the cell. The continuous improvements and advances in mass spectrometers, especially relating to ionization sources and detectors, coupled with advances in proteomics experimental design, has made it possible to study autophagy, among other process, in great detail. Innovative labeling strategies and protein separation techniques as well as complementary methods including immuno-capture/blotting/staining have been used in proteomics studies to provide more specific protein identification. In this review, we will discuss recent advances in proteomics studies focused on autophagy. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Quantitative and temporal proteome analysis of butyrate-treated colorectal cancer cells.

Science.gov (United States)

Tan, Hwee Tong; Tan, Sandra; Lin, Qingsong; Lim, Teck Kwang; Hew, Choy Leong; Chung, Maxey C M

2008-06-01

Colorectal cancer is one of the most common cancers in developed countries, and its incidence is negatively associated with high dietary fiber intake. Butyrate, a short-chain fatty acid fermentation by-product of fiber induces cell maturation with the promotion of growth arrest, differentiation, and/or apoptosis of cancer cells. The stimulation of cell maturation by butyrate in colonic cancer cells follows a temporal progression from the early phase of growth arrest to the activation of apoptotic cascades. Previously we performed two-dimensional DIGE to identify differentially expressed proteins induced by 24-h butyrate treatment of HCT-116 colorectal cancer cells. Herein we used quantitative proteomics approaches using iTRAQ (isobaric tags for relative and absolute quantitation), a stable isotope labeling methodology that enables multiplexing of four samples, for a temporal study of HCT-116 cells treated with butyrate. In addition, cleavable ICAT, which selectively tags cysteine-containing proteins, was also used, and the results complemented those obtained from the iTRAQ strategy. Selected protein targets were validated by real time PCR and Western blotting. A model is proposed to illustrate our findings from this temporal analysis of the butyrate-responsive proteome that uncovered several integrated cellular processes and pathways involved in growth arrest, apoptosis, and metastasis. These signature clusters of butyrate-regulated pathways are potential targets for novel chemopreventive and therapeutic drugs for treatment of colorectal cancer.

ATP mediates NADPH oxidase/ROS generation and COX-2/PGE2 expression in A549 cells: role of P2 receptor-dependent STAT3 activation.

Directory of Open Access Journals (Sweden)

Shin-Ei Cheng

Full Text Available BACKGROUND: Up-regulation of cyclooxygenase (COX-2 and its metabolite prostaglandin E(2 (PGE(2 are frequently implicated in lung inflammation. Extracellular nucleotides, such as ATP have been shown to act via activation of P2 purinoceptors, leading to COX-2 expression in various inflammatory diseases, such as lung inflammation. However, the mechanisms underlying ATP-induced COX-2 expression and PGE(2 release remain unclear. PRINCIPAL FINDINGS: Here, we showed that ATPγS induced COX-2 expression in A549 cells revealed by western blot and real-time PCR. Pretreatment with the inhibitors of P2 receptor (PPADS and suramin, PKC (Gö6983, Gö6976, Ro318220, and Rottlerin, ROS (Edaravone, NADPH oxidase [diphenyleneiodonium chloride (DPI and apocynin], Jak2 (AG490, and STAT3 [cucurbitacin E (CBE] and transfection with siRNAs of PKCα, PKCι, PKCμ, p47(phox, Jak2, STAT3, and cPLA(2 markedly reduced ATPγS-induced COX-2 expression and PGE(2 production. In addition, pretreatment with the inhibitors of P2 receptor attenuated PKCs translocation from the cytosol to the membrane in response to ATPγS. Moreover, ATPγS-induced ROS generation and p47(phox translocation was also reduced by pretreatment with the inhibitors of P2 receptor, PKC, and NADPH oxidase. On the other hand, ATPγS stimulated Jak2 and STAT3 activation which were inhibited by pretreatment with PPADS, suramin, Gö6983, Gö6976, Ro318220, GF109203X, Rottlerin, Edaravone, DPI, and apocynin in A549 cells. SIGNIFICANCE: Taken together, these results showed that ATPγS induced COX-2 expression and PGE(2 production via a P2 receptor/PKC/NADPH oxidase/ROS/Jak2/STAT3/cPLA(2 signaling pathway in A549 cells. Increased understanding of signal transduction mechanisms underlying COX-2 gene regulation will create opportunities for the development of anti-inflammation therapeutic strategies.