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Sample records for adenocarcinoma a549 cells

  1. Interleukin 23 Promotes Lung Adenocarcinoma A549 Cell Migration and Invasion

    Sen ZHANG

    2012-05-01

    Full Text Available Background and objective Interleukin 23 (IL-23 is a pro-inflammatory cytokine that plays an important role in inflammatory disease and tumor microenvironment. The IL-23 receptor is expressed in colon, lung, and oral carcinomas. We performed this study to investigate whether IL-23 promotes directly carcinoma cell migration and invasion as well as further explore its mechanism. Methods The migration and invasion of human lung adenocarcinoma A549 cells induced by IL-23 were detected by a scratch test and Transwell experiment. MMP-9 expression of the mRNA and protein levels of A549 cells cultured with and without IL-23 was respectively detected by Real-time PCR and ELISA. The effect of IL-23 on A549 cells was further verified using anti-IL-23p19 neutralization antibody (Ab IL-23p19 to eliminate IL-23. Results IL-23 remarkably promoted A549 cell migration and invasion. MMP-9 expression in A549 cells was upregulated by IL-23 stimulation. In addition, the effect of IL-23 on the migration and invasion of A549, as well as the MMP-9 expression in A549 cells induced by IL-23, was eliminated by Ab IL-23p19. Conclusion IL-23 promotes the migration and invasion of A549 cells by inducing MMP-9 expression.

  2. Phospholipid flippase associates with cisplatin resistance in plasma membrane of lung adenocarcinoma A549 cells

    2001-01-01

    The fusion of the liposomes containing N-(7-nitro-2, 1, 3-benzoxadiazol-4-yl)-i ,2-hexadecanoylSn-glycero-3-1abeled phosphatidylethanolamine (NBD-PE) with A549 and A549/DDP cells was performed, and the activity of the phospholipid flippase in the plasma membrane of the cells was measured by fluorescence intensity change of NBDPE in the outer membrane. When A549 or A549/DDP cells containing N BD-PE were incubated at 37 C for 0, 30, 60 and 90 min, the fluorescence intensities in the outer membrane of the cells were 0%, 1.4%, 2.9% and 7.8% for A59cells, and 0%, 10.5 %, 15. 5 % and 18.3 % for A549/DDP cells respectively, demonstrating that the phospholipid flippase was distributed in the plasma membrane of As49 cells, but its activity in the drug-resistant A549/DDP cells was much higher than that in the A549 cells. When the A549/DDP cells were incubated with a multidrug resistance reverse agent, verapamil, for 60 min at 37C, the results showed that the NBD-PE in outer membrane decreased by 25.0% compared with the control's. Furthermore, when A549/DDP cells were incubated with 25 μmol/L cisplatin, which is a specific anticancer drug, the flippase activity decreased by 31.6%, and it further decreased with the increase of cisplatin concentration, suggesting that phospholipid flippase in the membrane might be related to the cisplatin-resistance of human lung adenocarcinoma cancer cells.

  3. Hypoxia Upregulates the Expression of Annexin A1 in Lung Adenocarcinoma A549 Cells

    Zhenhong HU

    2012-05-01

    Full Text Available Background and objective The growth of tumor often faced up with lackness of blood and oxygen, and it has been reported that Annexin A1 may be involved in tumor. The aim of this investigation is to explore the characteristics of expression of Annexin A1 in lung adenocarcinoma A549 cells after hypoxia. Methods A549 cells were exposured to either normoxia (21%O2 or hypoxia (1%O2 condition for 4 h, 12 h, 24 h. The expressions of Annexin A1 mRNA levels were measured by RT-PCR. The expressions of Annexin 1 protein were investigaged by Western blot. The relative content of reactive oxygen species (ROS were assayed by special kit. The expressions of nuclear translocation of NF-κB was assayed by Western blot; After been treated with ROS scavenger NAC and PDTC, the levels of Annexin 1 protein of A549 cells were measured by Western blot. Results Compared with normoxia group, the Annexin A1 mRNA in hypoxia group increased after 4 h, and then decreased gradually; Moreover, Annexin 1 protein levels of A549 cells were also increased when treated with hypoxia. An increaing of ROS production in cells exprosed to hypoxia was detected. NAC and PDTC inhibited hypoxia-induced Annexin A1 increase. Conclusion Hypoxia upregulates the expression of Annexin A1 in lung adenocarcinoma A549 cells, in which process ROS-NF-κB may paticipate in.

  4. Transcription Activity of Ectogenic Human Carcinoembryonic Antigen Promoter in Lung Adenocarcinoma Cells A549

    XIONG Weining; FANG Huijuan; XU Yongjian; XIONG Shendao; CAO Yong; SONG Qingfeng; ZENG Daxiong; ZHANG Huilan

    2006-01-01

    The transcription activity of ectogenic human carcinoembryonic antigen (CEA) promoter in lung adenocarcinoma cells A549 was investigated for the further gene-targeting therapy. The reporter gene green fluorescent protein (GFP) driven by CEA promoter and human cytomegalovirus (CMV) promoter were relatively constructed and named plasmid pCEA-EGFP and pCMV-GFP respectively. The intensity of fluorescence was detected by fluorescence microscope and flow cytometry analysis after the pCEA-GFP and pSNAV-GFP plasmids were transfected into A549 cells through liposome respectively. The results showed (4.08±0.63) % of the A549 cells transfected with pCEA-AFP plasmid expressed, significantly lower than that of the A549 cells transfected with pCMV-GFP [(43.27±3.54) %]. It was suggested that ectogenic human CEA promoter in lung adenocarcinoma cells A549 was weakly expressed. The distinct specificity of CEA promoter in CEA high expression cells was regarded as a tool in selective gene therapy, but the transcription activity of ectogenic human CEA promoter was needed to increase in the future.

  5. Chemosensitivity of irradiated resistant cells of multicellular spheroids in A549 lung adenocarcinoma

    Objective: To investigate the chemosensitivity of irradiated resistant cells of multicellular spheroids in A549 lung adenocarcinoma. Methods: The A549 irradiated resistant cells were the 10th regrowth generations after irradiated with 2.5 Gy of 6 MV X-ray, the control groups were A549 parent cells and MCFY/VCR resistant cells. The 6 kinds of chemotherapeutic drugs were DDP, VDS, 5-FU, HCP, MMC and ADM respectively, with verapamil (VPL) as reverse agent. The treatment effect was compared with MTT assay, and the multidrug resistant gene expressions of mdrl and MRP were measured with RT-PCR method. Results: A549 cells and irradiated resistant cells were resistant to DDP, but sensitivity to VDS,5-FU, HCP, MMC and ADM. The inhibitory rates of VPL to the above two cells were 98% and 25% respectively(P2-MG and MRP/β2-MG of all A549 cells were about 0 and 0.7 respectively, and those of MCFT/VCR cells were 35 and 4.36. Conclusion: The chemosensitivity of A549 irradiated resistant cells had not changed markedly, the decreased sensitivity to VPL could not be explained by the gene expression of mdrl and MRP. It is conferred that some kinds of changes in the cell membrane and decreased regrowth ability to result in resistance. Unlike multidrug resistance induced by chemotherapy, VPL may be not an ideal reverser to irradiated resistant cells. The new kinds of biological preparation should be sought to combine chemotherapy to treat recurring tumor with irradiated resistance. (authors)

  6. Effects of EPO Gene on Growth and Apoptosis of Lung Adenocarcinoma Cell Line A549

    Jianqing WU

    2009-09-01

    Full Text Available Background and objective Published data on the association between erythropoietin (EPO and cancer cell are inconclusive. The aim of this study is to investigate the effect of erythropoietin (EPO on the growth and survival of lung adenocarcinoma cell line A549. Methods The recombinant plasmid pcDNA3.1(--hEPO was constructed and transfected into A549 cells by liposome protoco1. The Levels of EPO in culture supernatant were detected by ELISA. Effects of EPO gene on growth and survival of the transfected cells were evaluated by MTT assay and flow cytometry (FCM . Levels of vascular endothelial growth factor (VEGF were also evaluated by ELISA. Results The recombinant eukaryotic expression vector pcDNA3.1(--hEPO was successfully constructed. The growth of cells in hEPO transfected cells was significantly inhibited after transfection (P < 0.01. More cells were blocked in S phase in hEPO transfected group compared with control group (P < 0.05, and the apoptotic rate were also significantly higher than those of their controls (P < 0.01. Levels of VEGF in hEPO transfected cells were significantly lower than controls (P < 0.01. Conclusion Exogenous EPO gene expression in A549 cells can induce cell growth inhibition and apoptosis of A549 cells, and expression of VEGF can also be inhibited.

  7. Anti-tumor activity of CrTX in human lung adenocarcinoma cell line A549

    Bin YE; Yan XIE; Zheng-hong QIN; Jun-chao WU; Rong HAN; Jing-kang HE

    2011-01-01

    Aim:To assess the cytotoxic effect of crotoxin (CrTX),a potent neurotoxin extracted from the venom of the pit viper Crotalus durissus terrificus,in human lung adenocarcinoma A549 cells and investigated the underlying mechanisms.Methods:A549 cells were treated with gradient concentrations of CrTX,and the cell cycle and apoptosis were analyzed using a flow cytometric assay.The changes of cellular effectors p53,caspase-3 and cleaved caspase-3,total P38MAPK and pP38MAPK were investigated using Western blot assays.A549 xenograft model was used to examine the inhibition of CrTX on tumor growth in vivo.Results:Treatment of A549 cells with CrTX (25-200 μg/mL) for 48 h significantly inhibited the cell growth in a dose-dependent manner (IC50=78 μg/mL).Treatment with CrTX (25 iJg/mL) for 24 h caused G1 arrest and induced cell apoptosis.CrTX (25 μg/mL) significantly increased the expression of wt p53,cleaved caspase-3 and phospho-P38MAPK.Pretreatment with the specific P38MAPK inhibitor SB203580 (5 μmol/L) significantly reduced CrTX-induced apoptosis and cleaved caspase-3 level,but G1 arrest remained unchanged and highly expressed p53 sustained.Intraperitoneal injection of CrTX (10 μg/kg,twice a week for 4 weeks) significantly inhibited A549 tumor xenograft growth,and decreased MVD and VEGF levels.Conclusion:CrTX produced significant anti-tumor effects by inducing cell apoptosis probably due to activation of P38MAPK and caspase-3,and by cell cycle arrest mediated by increased wt p53 expression.In addition,CrTX displayed anti-angiogenic effects in vivo.

  8. Effect of ionizing radiation on invasiveness of pulmonary adenocarcinoma cells A549 and its mechanism

    Objective: To investigate the effect of ionizing radiation on the invasion of the pulmonary adenocarcinoma cell line A549. Methods: The invasiveness of A549 cells irradiated with 2 and 4 Gy doses of γ-rays was detected by using transwell invasion assay. The expression levels of matrix metalloproteinase (MMP)-2 mRNA and protein and phosphorylated signal transducers and activators of transcription 3 (STAT3) protein were detected by reverse transcription PCR and Western blot. Results: After irradiation with 2 or 4 Gy, the invasiveness of A549 cells increased by 200.0% (F=111.7, P<0.01) and 390.9% (F=593.7, P<0.01), respectively, compared with that in untreated A549 cells.Furthermore, the transcription and protein expression of MMP-2 24 h after irradiation and the phosphorylation of STAT3 12 h after irradiation were promoted. The irradiation-induced elevation of MMP-2 protein expression was suppressed using STAT3 phosphorylation specific inhibitor (AG490). Moreover, compared with 4 Gy of irradiation alone, treatment with 4 Gy of irradiation plus AG490 decreased the number of invasive cells by 76.1% (F=555.9, P<0.01), and the number of invasive cells in 4 Gy of irradiation plus AG490 group made up only 117.8% of that in untreated group (F=3.6, P>0.05). Conclusions: Ionizing radiation could activate STAT3, which triggers the transcription of MMP-2, and then promote the invasiveness of A549 cells. (authors)

  9. Chemosensitivity of radioresistant cells in the multicellular spheroids of A549 lung adenocarcinoma

    Huang Gang

    2009-06-01

    Full Text Available Abstract Background The relapse of cancer after radiotherapy is a clinical knotty problem. Previous studies have demonstrated that the elevation of several factors is likely in some way to lead to the development of treatment tolerance, so it is necessary to further explore the problem of re-proliferated radioresistant cells to chemotherapeutic agents. In the present study, we aimed to investigate the chemosensitivity of radioresistant cells originated from the multicellular spheroids of A549 lung adenocarcinoma. Methods After irradiated with 25 Gy of 6 MV X-ray to A549 multicellular spheroids, whose 10th re-proliferated generations were employed as radioresistant cells, and the control groups were A549 parental cells and MCF7/VCR resistant cells. The chemo-sensitivity test was made by six kinds of chemotherapeutic drugs which were DDP, VDS, 5-Fu, HCP, MMC and ADM respectively, while verapamil (VPL was used as the reversal agent. Then the treatment effect was evaluated by MTT assay, and the multidrug resistant gene expressions of mdr1 and MRP were measured by RT-PCR. Results Both A549 parental cells and A549 derived radioresistant cells were resistant to DDP, but sensitive to VDS, 5-Fu, HCP, MMC and ADM. The inhibitory rates of VPL to these two types of cell were 98% and 25% respectively (P Mdr1/β2-MG and MRP/β2-MG of all A549 cells were about 0 and 0.7 respectively, and those of MCF7/VCR cells were 35 and 4.36. Conclusion The chemosensitivity of A549 radioresistant cells had not changed markedly, and the decreased sensitivity to VPL could not be explained by the gene expression of mdr1 and MRP. It is possible that the changes in the cell membrane and decreased proliferate ability might be attributed to the resistance. Unlike multidrug resistance induced by chemotherapy, VPL may be not an ideal reverser to radioresistant cells. Therefore, the new biological strategy needs to be developed to treat recurring radioresistant tumor in combination

  10. Melatonin inhibits the migration of human lung adenocarcinoma A549 cell lines involving JNK/MAPK pathway.

    Qiaoyun Zhou

    Full Text Available OBJECTIVE: Melatonin, an indolamine produced and secreted predominately by the pineal gland, exhibits a variety of physiological functions, possesses antioxidant and antitumor properties. But, the mechanisms for the anti-cancer effects are unknown. The present study explored the effects of melatonin on the migration of human lung adenocarcinoma A549 cells and its mechanism. METHODS: MTT assay was employed to measure the viability of A549 cells treated with different concentrations of melatonin. The effect of melatonin on the migration of A549 cells was analyzed by wound healing assay. Occludin location was observed by immunofluorescence. The expression of occludin, osteopontin (OPN, myosin light chain kinase (MLCK and phosphorylation of myosin light chain (MLC, JNK were detected by western blots. RESULTS: After A549 cells were treated with melatonin, the viability and migration of the cells were inhibited significantly. The relative migration rate of A549 cells treated with melatonin was only about 20% at 24 h. The expression level of OPN, MLCK and phosphorylation of MLC of A549 cells were reduced, while the expression of occludin was conversely elevated, and occludin located on the cell surface was obviously increased. The phosphorylation status of JNK in A549 cells was also reduced when cells were treated by melatonin. CONCLUSIONS: Melatonin significantly inhibits the migration of A549 cells, and this may be associated with the down-regulation of the expression of OPN, MLCK, phosphorylation of MLC, and up-regulation of the expression of occludin involving JNK/MAPK pathway.

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

    Ghosh, Somnath [Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400 085 (India); Narang, Himanshi, E-mail: himinarang@gmail.com [Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400 085 (India); Sarma, Asitikantha [Radiation Biology Laboratory, Inter University Accelerator Centre, Aruna Asaf Ali Marg, New Delhi 110 067 (India); Krishna, Malini [Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400 085 (India)

    2011-11-01

    Carbon beams (5.16 MeV/u, LET = 290 keV/{mu}m) 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 {gamma}-rays and carbon ion-irradiation. A549 cells were irradiated with 1 Gy carbon or {gamma}-rays. Carbon beam was found to be three times more cytotoxic than {gamma}-rays despite the fact that the numbers of {gamma}-H2AX foci were same. Percentage of cells showing ATM/ATR foci were more with {gamma}-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 {gamma}-rays irradiated cells and prosurvival ERK pathway was activated after {gamma}-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.

  12. Screening radiosensitizing-related genes mediated by elemene in lung adenocarcinoma A549 cells by using gene chip

    Objective: To screen radiosensitizing-related genes mediated by elemene in lung adenocarcinoma A549 cells by using gene chip. Methods: MTT test was used to calculate the IC50 of elemene. (1) The effect of radiosensitivity was detected by colony forming assay. A549 cells were divided into 2 groups: radiation group and radiation + elemene group. Oligonucleotide chip was used to screen the gene expression changes of A549 cells from these 2 groups. The up-regulated gene Egr-1 and the down-regulated gene CyclinD1 were selected to undergo RT-PCR so as to confirm the reliability of the result. Results: MTT test showed the elemene inhibited the proliferation of the A549 cells dose-dependently. The IC50 value of elemene on the A549 cells was 120 mg/L. (2) 10 mg/L elemene had radiosensitising effect on A549 cells.The values of SERD0 and SERDq obtained from the survival curve were (1.54±0. 20) and (1.43±0.15) respectively. Gene chip screened 122 differentially-expressed genes, including 89 up-regulated genes and 33 down-regulated genes. (3) These altered genes could be related to cell structure, substance metabolism,cell proliferation, cell differentiation, signal transduction, material transport, DNA repair, apoptosis, immune response and so forth. The RT-PCR results of Egr-1 and Cyclin D1 were consistent with the gene chip analysis. Conclusions: The mechanism of elemene enhancing the radiosensitivity of lung adenocarcinoma A549 cells is the result of participation and collaboration of multiple genes. Further study of the newly-discovered differentially-expressed gene helps find out new radiosensitizational targets of elemene. (authors)

  13. Role of Rad52 in fractionated irradiation induced signaling in A549 lung adenocarcinoma cells

    Ghosh, Somnath, E-mail: ghosh.barc@gmail.com [Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400085 (India); Krishna, Malini, E-mail: malinik00@gmail.com [Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400085 (India)

    2012-01-03

    The effect of fractionated doses of {gamma}-irradiation (2 Gy per fraction over 5 days), as delivered in cancer radiotherapy, was compared with acute doses of 10 and 2 Gy, in A549 cells. A549 cells were found to be relatively more radioresistant if the 10 Gy dose was delivered as a fractionated regimen. Microarray analysis showed upregulation of DNA repair and cell cycle arrest genes in the cells exposed to fractionated irradiation. There was intense activation of DNA repair pathway-associated genes (DNA-PK, ATM, Rad52, MLH1 and BRCA1), efficient DNA repair and phospho-p53 was found to be translocated to the nucleus of A549 cells exposed to fractionated irradiation. MCF-7 cells responded differently in fractionated regimen. Silencing of the Rad52 gene in fractionated group of A549 cells made the cells radiosensitive. The above result indicated increased radioresistance in A549 cells due to the activation of Rad52 gene.

  14. Role of Rad52 in fractionated irradiation induced signaling in A549 lung adenocarcinoma cells.

    Ghosh, Somnath; Krishna, Malini

    2012-01-01

    The effect of fractionated doses of γ-irradiation (2Gy per fraction over 5 days), as delivered in cancer radiotherapy, was compared with acute doses of 10 and 2Gy, in A549 cells. A549 cells were found to be relatively more radioresistant if the 10Gy dose was delivered as a fractionated regimen. Microarray analysis showed upregulation of DNA repair and cell cycle arrest genes in the cells exposed to fractionated irradiation. There was intense activation of DNA repair pathway-associated genes (DNA-PK, ATM, Rad52, MLH1 and BRCA1), efficient DNA repair and phospho-p53 was found to be translocated to the nucleus of A549 cells exposed to fractionated irradiation. MCF-7 cells responded differently in fractionated regimen. Silencing of the Rad52 gene in fractionated group of A549 cells made the cells radiosensitive. The above result indicated increased radioresistance in A549 cells due to the activation of Rad52 gene. PMID:22001234

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

    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)

  16. Effect of staurosporine on the mobility and invasiveness of lung adenocarcinoma A549 cells: an in vitro study

    Lung cancer is one of the most malignant tumors, representing a significant threat to human health. Lung cancer patients often exhibit tumor cell invasion and metastasis before diagnosis which often render current treatments ineffective. Here, we investigated the effect of staurosporine, a potent protein kinase C (PKC) inhibitor on the mobility and invasiveness of human lung adenocarcinoma A549 cells. All experiments were conducted using human lung adenocarcinoma A549 cells that were either untreated or treated with 1 nmol/L, 10 nmol/L, or 100 nmol/L staurosporine. Electron microscopy analyses were performed to study ultrastructural differences between untreated A549 cells and A549 cells treated with staurosporine. The effect of staurosporine on the mobility and invasiveness of A549 was tested using Transwell chambers. Western blot analyses were performed to study the effect of staurosporine on the levels of PKC-α, integrin β1, E-cadherin, and LnR. Changes in MMP-9 and uPA levels were identified by fluorescence microscopy. We demonstrated that treatment of A549 cells with staurosporine caused alterations in the cell shape and morphology. Untreated cells were primarily short spindle- and triangle-shaped in contrast to staurosporine treated cells which were retracted and round-shaped. The latter showed signs of apoptosis, including vacuole fragmentation, chromatin degeneration, and a decrease in the number of microvilli at the surface of the cells. The A549 cell adhesion, mobility, and invasiveness significantly decreased with higher staurosporine concentrations. E-cadherin, integrin β1, and LnR levels changed by a factor of 1.5, 0.74, and 0.73, respectively compared to untreated cells. In addition, the levels of MMP-9 and uPA decreased in cells treated with staurosporine. In summary, this study demonstrates that staurosporine inhibits cell adhesion, mobility, and invasion of A549 cells. The staurosporine-mediated inhibition of PKC-α, induction of E

  17. Effect of fucoidan from Turbinaria conoides on human lung adenocarcinoma epithelial (A549) cells.

    Alwarsamy, Madhavarani; Gooneratne, Ravi; Ravichandran, Ramanibai

    2016-11-01

    Fucoidan was purified from seaweed, Turbinaria conoides. Isolated fragments were characterized with NMR ((13)C, (1)H), Gas Chromatography-Mass Spectronomy (GC-MS) and HPLC analysis. The autohydrolysate of fucoidans consisted of sulfated fuco-oligosaccharides having the backbone of α-(1, 3)-linked fuco-pyranose derivatives and minor components of galactose, glucose, mannose and xylose sugars. Fucoidan induced a dose-dependent reduction in cell survival of lung cancer A549 cells by MTT assay (GI50, 75μg/mL). However, it was not cytotoxic to a non-tumorigenic human keratinocyte cell line of skin tissue (HaCaT) (GI50>1.0mg/mL). The apoptotic cells in fucoidan-treated A549 cells were visualized by laser confocal microscopy and cell cycle analysis showed induction of G0/G1 phase arrest of the cell progression cycle. Further, CFSE labeling and flow cytometry highlighted that fucoidan significantly (P<0.05) inhibited the proliferation rate of A549 cells by up to 2-fold compared with the control cells. It is concluded that fucoidan has the potential to act as an anti-proliferative agent on lung carcinoma (A549) cells. PMID:27516266

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

    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 A549 cells. Methods: Eukaryotic expression plasmid containing ATM small interfering RNA (siRNA) (pSilencer2.1-ATM), as well as pSilencer2.1-nonspecific, was constructed.Lung adenocarcinoma A549 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 (D0 ratios) for the positive group and negative group were 1.50 and 1.01, respectively. The flow cytometry revealed that the proportions of A549 cells in G1 and G2/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 A549 cells, probably by affecting the cell cycle and promoting cell apoptosis. (authors)

  19. Effects of paclitaxel on cell proliferation and apoptosis and its mechanism in human lung adenocarcinoma A549 cells

    Baoan Gao; Chunling Du; Wenbo Ding; Shixiong Chen; Jun Yang

    2006-01-01

    Objective: To investigate the effect of paclitaxel on cell proliferation and apoptosis of human lung adenocarcinoma A549 cells line and its mechanism in vitro. Methods: Cell growth inhibition of paclitaxel on A549 cells was analyzed by MTT assay. Cell apoptosis was detected by DNA cytofluorometry, Hoechst33258 staining when treated with paclitaxel for 48hours. Meanwhile, Cell cycle and apoptotic rate were analyzed by flow cytometry. The protein expressions of Bax and Bcl-2 were studied by Western Blot. Results: Paclitaxel inhibited the proliferation of A549 cells in a time-and dose-dependant manner.Hoechst33258 staining indicated that apoptosis was induced by paclitaxel. After treated for 48 hours, cell apoptosis rates of 25nmol/L, 50 nmol/L and 100 nmol/L paclitaxel groups were 11.52 ± 1.94% ,17.73 ± 2.53%, and 29.32 ± 5.51% respectively,which were significantly higher than those of control group 5.88 ± 1.07%(all P < 0.01 ), and apoptosis rate increased in dose-dependant manner. Meanwhile, G2/M stage cell percentage of 25 nmol/L, 50 nmol/L and 100 nmol/L paclitaxel groups were 42.52± 6.25%, 40.46 ± 5.81%, and 35.34 ± 6.17% respectively,which were significantly higher than that of control group 22.32 ±3.30%(all P < 0.01 ); Western blot showed that paclitaxel increased the expression of Bax and decreased the expression of Bcl-2 in dose-dependant manner. Conclusion: Paclitaxel can inhibit A549 cell proliferation in a time- and dose-dependant manner. Its mechanism may be related to arresting cell cycle in G2/M stage and induce cell apoptosis by up-modulating Bax expression and down-modulating Bcl-2 expression.

  20. Effects of herpes simplex virus thymidine kinase/acyclovir system on growth of human pulmonary adenocarcinoma A549 cell line in vitro and in vivo

    HE Xiang-liang; HE Dong-hua; GUO Xian-jian; QIAN Gui-sheng; HUANG Gui-jun; CHEN Wei-zhong; LI Shu-ping

    2002-01-01

    Objective: To observe the effect of anciclovir (ACV) treatment on tumors induced by inoculation of TK gene-transfected human pulmonary adenocarcinoma A549 cells in nude mice. Methods: A recombinant plasmid containing TK gene was constructed and transfected into A549 cells by electroporation. The sensitivity of the transgenic cells (A549-TK) to ACV was examined by MTT assay in vitro and for in vivo observation, inoculation of A549-TK and A-549 cells into nude mice was separately performed to induce tumor growth, the response of which to ACV treatment was observed, and the tumor tissues were pathologically examined. Results: A recombinant plasmid containing TK gene was successfully constructed and transfected into A549 cells. The sensitivity of A549-TK cells to ACV was 43 times higher than that of A549 cells. The tumors induced by A549-TK cells showed no significant increase in size after ACV treatment (P>0. 05), and light microscopy revealed local tissue necrosis, karyoklasis, and nuclei disappearance. Conclusion: A549-TK cells acquires sensitivity to ACV both in vitro and in vivo, and ACV can inhibit the growth of tumors induced by A549-TK cell inoculation in nude mice.

  1. Antineoplastic effects of deoxyelephantopin,a sesquiterpene lactone from Elephantopus scaber, on lung adenocarcinoma (A549) cells

    Farha A.Kabeer; Geetha B.Sreedevi; Mangalam S.Nair; Dhanya S.Rajalekshmi; Latha P.Gopalakrishnan; Sujathan Kunjuraman; Remani Prathapan

    2013-01-01

    OBJECTIVE:Deoxyelephantopin,a sesquiterpene lactone from Elephantopus scaber,showed inhibition of the growth of various tumor cells in vitro.In the present study,we investigated the cytotoxicity and apoptosis-inducing capacity of deoxyelephantopin on lung adenocarcinoma (A549) cells.METHODS:The cytotoxic effect of deoxyelephantopin on A549 cells and normal lymphocytes was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and 50% inhibitory concentration (IC50) value was determined.The self-renewal and proliferating potential of A549 cells after treatment with deoxyelephantopin were examined by colony formation assay.Cellular morphology of deoxyelephantopin-treated cells was observed using phasecontrast microscopy.The induction of apoptosis was evaluated using acridine orange and ethidium bromide staining,Hoechst 33342 staining,terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end-labeling (TUNEL) assay,DNA fragmentation analysis and Annexin V-fluorescein isothiocyanate staining by flow cytometry.Activation of caspases was detected using fluorogenic substrate specific to caspases 2,3,8 and 9 and flow cytometric analysis.The total cellular DNA content and expression of cleaved poly (ADP-ribose) polymerase was also analyzed.RESULTS:Deoxyelephantopin exhibited cytotoxicity to A549 cells (IC5o =12.287 μg/mL),however,there was no toxicity towards normal human lymphocytes.Deoxyelephantopin suppressed the colony-forming ability of A549 cells in a dose-dependent manner.Acridine orange,ethidium bromide and Hoechst 33342 staining showed cell shrinkage,chromosomal condensation and nuclear fragmentation,indicating induction of apoptosis.Deoxyelephantopin increased apoptosis of A549 cells,as evidenced by more TUNEL-positive cells.DNA fragmentation and Annexin V staining revealed late-stage apoptotic cell population.Deoxyelephantopin inhibited A549 cell growth by cell cycle arrest at G2/M phase and induced apoptosis through

  2. 莪术油对人肺腺癌细胞A549增殖的影响%Effect of Zedoary Turmeric Oil on Proliferation in Human Lung Adenocarcinoma Cell Line A549

    王晓波; 牛建昭; 崔巍; 刘飒; 杨长福; 赵丕文; 唐炳华

    2011-01-01

    目的 探讨莪术油对人肺腺癌细胞A549增殖的抑制作用.方法 体外培养肺腺癌细胞A549,MTT比色法测定莪术油对A549细胞作用24、48、72 h后抑制率;流式细胞术分析莪术油对A549细胞作用24 h后细胞周期的变化;Annexin V-FITC/PI双染检测莪术油对A549细胞作用24 h后细胞凋亡与坏死情况.结果 莪术油对A549细胞增殖的抑制率随时间延长明显升高,随药物浓度增加抑制作用增强;莪术油对A549细胞作用24 h后,细胞周期停滞在G0/G1期,阻止其进入S期;细胞的早期凋亡、晚期凋亡和坏死比例随着莪术油浓度的增加而增加,且坏死细胞的比例高于凋亡细胞.结论 莪术油对A549细胞的增殖具有抑制作用,并呈时间、浓度依赖,其作用是通过阻滞细胞周期及诱导凋亡和坏死采实现的.%Objective To explore the inhibiting effect of Zedoary turmeric oil on the proliferation of A549 cell line. Methods Lung adenocarcinoma cell line A549 was cultured in vitro. The inhibition rate of Zedoary turmeric oil on the proliferation of lung adenocarcinoma cell line A549 for 24, 48, 72 h were determined by MTT colorimetric assay. The cell cycle of lung adenocarcinoma cell line A549 stimulated by Zedoary turmeric oil for 24 h was analyzed by flow cytometry. The apoptosis and necrosis of lung adenocarcinoma cell line A549 stimulated by Zedoary turmeric oil for 24 h was tested by Annexin V-FITC/PI assay. Results MTT assay indicated that the inhibition rate of Zedoary turmeric oil on the proliferation of lung adenocarcinoma cell line A549 increased significantly with the growing of time and concentration. Further analysis by flow cytometry indicated that Zedoary turmeric oil stimulating the A549 cells for 24 h led to Go/Gi phase arrest and blocked S phase entry. Meanwhile cells in early apoptosis, late apoptosis and necrosis were increased, and the percentage of necrotic cells was more than apoptotic cells with the increase of

  3. Enrichment and identification of lung adenocarcinoma initiating cells from A 549%A549肺腺癌始动细胞的富集和鉴定

    林盛; 张振华; 饶明月; 吴敬波

    2013-01-01

    Objective To obtain the lung adenocarcinoma initiating cells from the A 549 cell line based on paclitaxel treatment combination with serum-free cultivation and to validate spared cells can represent tumor initiating cells (TICs) .Methods After dis-sociated by trypsogen ,about 106 /mL cells were suspended in serum-free medium supplemented with 0 .4% bovine serum albumin (BSA) ,insulin ,basic fibroblast growth factor (bFGF) ,human recombinant epidermal growth factor (EGF) and obtained spheroid cells .At the second passage ,paclitaxel was added at a concentration of 100 nmol/L for 48 h and then replaced with completely fresh medium once or twice per week until new spheroids emerged .Results The subpopulation of cells that survived serum-free cultiva-tion and paclitaxel treatment could highly express the cluster of differentiation 133/cluster of differentiation (CD133/CD326) mo-lecular markers and have features of stemness including differentiation ,high expression of cancer stem cells (CSCs)-associated genes and stronger capability of tumorigenesis .Conclusion The survived subpopulation that highly express the CD 133/CD326 molecu-lar markers presenting the characteristics of stemness in vitro and in vivo ,and could be used in future researches of biological functions .%目的:利用紫杉醇联合无血清培养完成对 A549肺腺癌始动细胞的富集并鉴定富集亚群的干细胞特性。方法对数生长期的 A549细胞经胰酶消化,干细胞培养基重悬,得到成球状生长的细胞;传至第2代时加入紫杉醇作用48 h ,离心去除死细胞和紫杉醇,换新鲜干细胞培养基培养,至存活细胞恢复克隆生长后鉴定其干细胞相关特性。结果紫杉醇联合无血清培养方式成功从 A549细胞中富集得到肿瘤干细胞,该群细胞高表达分化抗原簇蛋白133/人上皮细胞黏附分子(CD133/CD326),具有多向分化潜能、高表达干细胞相关基因及更强的致瘤能力,具备

  4. 低氧对人肺腺癌A549细胞迁移和黏附的影响%Effect of hypoxia on migration, invasion and adhesion to endothelium of human pulmonary adenocarcinoma A549 cells

    Weigan Shen; Jun Zhu; Zhiyong Yu; Qingyu Xue

    2008-01-01

    Objective:To evaluate the effect of hypoxia on migration,invasion and adhesion to endothelium of human pulmonary adenocarcinoma A549 cells.Methods:Wound-healing and Transwell invasion assays were performed to study the effect of hypoxia on migration and invasion of A549 cells,and A549 cells were added to a monolayer of human umbilical vein endothelial cells (HUVECs) to test the ability to adhere to endothelium.Immunofluorescence assay and luciferase reporter gene assay were also used to evaluate the effect of hypoxia on distribution of E-cadherin,β-catenin,and actin,and hypoxia-inducible factor-1 (HIF-1)-dependent transcription,respectively.Results:Hypoxia facilitated A549 cell migration,invasion,and A549 cell-endothelial cells adhesion,and modulated the distribution of E-cadherin and β-catenin,and actin cytoskeleton rearrangement,and up-regulated HIF-1-dependent reporter gene expression in A549 cells.Conclusion:Promotion of A549 cell migration,invasion,and adhesion on endothelium by hypoxia might be modulated through its up-regulating HIF-l-dependent gene expression,which then induced the redistribution of E-cadherin and β-catenin,and the actin cytoskeletal reorganization.

  5. Enhancement of radiosensitivity by topoisomerase II inhibitor, amrubicin and amrubicinol, in human lung adenocarcinoma A549 cells and kinetics of apoptosis and necrosis induction

    Hayashi, Sachiko; Hatashita, Masanori; Matsumoto, Hideki; Shioura, Hiroki; KITAI, Ryuhei; Kano, Eiichi

    2006-01-01

    The effects of amrubicin (AMR) and its activemetabolite, amrubicinol (AMROH), on the sensitivity ofhuman lung adenocarcinoma A549 cells to ionizing radiationwere investigated in vitro. Further, the kinetics of apoptosisand necrosis induction were also analyzed. The cytocidalefftcts of X-ray irradiation on A549 cells resulted in a lowlevel of radiosensitivity with a D value of 12 Gy. The slopesof the survival curves in the exponential phase were plottedon semilogarithmic paper for radiation co...

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

    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.

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

    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.

  8. Okadaic acid inhibits cell multiplication and induces apoptosis in a549 cells, a human lung adenocarcinoma cell line

    Wang, Renjun; Lv, Lili; Zhao, Yunfeng; Yang, Nana

    2014-01-01

    This essay aims to research the effect of okadaic acid (OA) on A549 cell multiplication, and cell apoptosis induced by OA was observed by cell morphology. MTT assay, trypan blue exclusion test (TBET), Giemsa staining method and acridine orange (AO) fluorescence staining assay were applied. The results of cell survival evaluated by TBET and colorimetric assay with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) showed: The number of A549 cells was decreased in a dose-depende...

  9. Influence of Tamoxifen or the combination of Tamoxifen and Cisplatin on the growth of human lung adenocarcinoma A549 cells

    Yuxuan Che; Xiuhua Sun; Chaomei Huang; Jinbo Zhao 

    2014-01-01

    Objective:The experiment aims to investigate the influence of Tamoxifen and the combination of Tamoxifen and Cisplatin (DDP) on the growth of human lung adenocarcinoma A549 cel s. Methods:We treated human lung adenocarcinoma A549 cel s with dif erent concentrations of Tamoxifen, DDP and combination of DDP and Tamoxifen with non-toxicity for 72 h. Then we calculated the inhibition rate through MTT approach and detected the apoptosis rate by flow cytometry. The statistical analysis was performed with SPSS 13.0 software and statistical dif erences were determined by one-way ANOVA. The data were expressed as the mean ± standard deviation and al experiments were performed in three times. The value of P0.05). 2. As the increase concentration of Tamoxifen, the S stage and G2/M of the A549 cel s decreased while the G0/G1 increased. The apoptosis rate of Tamoxifen with 0 µmol/L, 0.1 µmol/L, 1 µmol/L and 10 µmol/L on the A549 cel s were 6.51%, 8.91%, 17.97%and 42.7%, respectively. 3. The inhibition rates of combination of Tamoxifen with 1 µmol/L and DDP with 1.25 µg/mL, 2.5 µg/mL, 5 µg/mL, 10 µg/mL and 20 µg/mL on the A549 cel s were 40.4%, 54.4%, 72.9%, 86.1%and 92.4%, respectively (P<0.05). Conclusion:Tamoxifen can inhibit the proliferation of human lung adenocarcinoma A549 cel s and induce the apoptosis of the A549 cel s. The combination of Tamoxifen with non-toxicity and DDP can improve the sensitivity of chemotherapy on the A549 cel s.

  10. Effect of RNAi targeting survivin gene combined with X-rays radiation on apoptosis of lung adenocarcinoma A549 cells

    Objective: To construct the vector of RNA interference (RNAi) targeting survivin gene and observe its effect combined with X-rays radiation on lung adenocarcinoma A549 cell apoptosis. Methods: One pair of RNAi sequence targeting survivin gene were designed according to its cDNA sequence reported in GenBank, the recombinant RNAi plasmid pGenesil2-survivin was constructed. After identified by enzyme digestion and sequencing, the pGenesil2-survivin plasmid was trasfeced into A549 cells.In the experiment, normal group,pGenesil2 group, pGenesil2-survivin group,5 Gy irradiation group and pGenesil2-survivin + 5 Gy irradiation group were set up.The apoptosis of A549 cells was measured by flow cytometry with PI/Annexin V and TUNEL,the survivin and caspase-3 expressions were measured by Western blotting. Results: Two fragments about 389 bp and 4 206 bp were gotten by Kpn I and EcoR I enzyme digestion, they are the same to expected result, the sequencing result was compared to oligonucleotide chain with DNAssist 2.0, they were equal, these indicated the identification of pGenesil2-survivin vector was right; pGenesil2-survivin was transfected into A549 cells for 48 h, the apoptotic percentage in pGenesil2-survivin and 5 Gy X-rays groups increased obviously (P< 0.05), when the both were combined, the effect was more obvious;the Western blotting results appeared that the survivin gray scale/β-actin gray scale in pGenesil2-survivin group was lower than that in normal group(P< 0.01), and the caspase-3 gray scale/β-actin gray scale was higher than that in normal group,and that ratio in pGenesil2-survivin+5 Gy irradiation group was more high(P< 0.01). Conclusion: RNAi targeting surviving gene could inhibit survivin protein expression,but enhance caspase-3 protein expression, and promote apoptosis. When it is combined with 5 Gy X-rays irradiation, the promotion of apoptosis is enhanced. (authors)

  11. 三种缝线材料对人肺腺癌细胞A549增殖和细胞周期的影响%Effect of three suture lines on the proliferation and cell cycle of lung adenocarcinoma cell A549 in vitro

    Lianhua Ye; Yunchao Huang; Qilin Jin; Feng Hua; Guangqiang Zhao

    2011-01-01

    Objective: The interaction of cell and medical biomaterial is one of the significant factors to affect clinical application of medical biomaterial. This research is to investigate three of suture lines how to affect the proliferation and cell cycle of lung adenocarcinoma cell A549 in vitro. Methods: Three of suture lines were respectively cultivated with lung adenocarcinoma cell A549, after of 72 hours, we detected absorptions of each group by MTT method in order to reflect the proliferation of lung adenocarcinoma cell A549, and also examined percentage of G1 period cells and S period cells of each group by flow cytometry. Results: Different of suture lines had different effects on the proliferation and cell cycle of lung adenocarcinoma cell A549 (P < 0.05). The effect of absorbent suture line was the strongest on the proliferation and cell cycle of lung adenocarcinoma cell A549, the effect of chorda serica chirurgicalis was medium, and the effect of slide wire was poor. Different length of each suture line had different effects on the proliferation and cell cycle of lung adenocarcinoma cell A549 (P < 0.05).Conclusion: Three of suture line materials have different effects on the proliferation and cell cycle of lung adenocarcinoma cell A549, with dose-effect relationship.

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

    da Volta Soares, Mariana; Oliveira, Mainara Rangel; dos Santos, Elisabete Pereira; de Brito Gitirana, Lycia; Barbosa, Gleyce Moreno; Quaresma, Carla Holandino; Ricci-Júnior, Eduardo

    2011-01-01

    In this study, zinc phthalocyanine (ZnPc) was loaded onto poly-ɛ-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 ZnPcloaded NPs and irradiated with red light. ZnPc-loaded PCL NPs are the release systems that promise photodynamic therapy use. PMID:21499420

  13. Effect of antisense transfecting of monocarboxylate transporter gene on biological characteristics of lung adenocarcinoma A549 cells

    ZHANG Gui-zhi; HUANG Gui-jun; GUO Xian-jian; QIAN Gui-sheng

    2002-01-01

    Objective: To study the influence of transfecting antisense expression vector of the first subtype of the monocarboxylate transporter (MCT1) gene into lung cancer cells on pHi regulation, lactate transportation and cell growth, Methods: MCT1 antisense gene recombinant vector was introduced into human lung cancer cell line A549 by electroporation. The transfected A549 cells resistant to G418 were selected. Positive clones were examined by using PCR. The changes of intracellular pH and lactate were examined with spectrophotometric method. Cell growth was studied with cell growth curve. Results: Intracellular pH and lactate were remarkably decreased in the cells transfected pLXSN-MCT1 in comparison with A549 cells without transfection (P<0. 001). The growth of A549 cells transfected pLXSN-MCT1 was also inhibited remarkably. Conclusion: MCT1 gene may play an important role in pHi regulation, lactate transportation and cell growth in tumor cells.

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

    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 <10 nm in deionized water or acetone by pulsed laser ablation, that permits preparation of uncoated and highly stable Gold nanoparticles in pure solvents. Subsequently, we investigated the role of surface chemistry, size, and dispersivity of synthesized Gold nanoparticles in exerting toxicity in a cell model system of deep respiratory tract, representing the main route of 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

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

    Bucchianico, Sebastiano Di [Karolinska Institutet, Institute of Environmental Medicine (Sweden); Migliore, Lucia [University of Pisa, Department of Translational Research and New Technologies in Medicine and Surgery, Division of Medical Genetics (Italy); Marsili, Paolo [Institute of Complex Systems (ISC-CNR) (Italy); Vergari, Chiara [Plasma Diagnostics and Technologies s.r.l. (Italy); Giammanco, Francesco [University of Pisa, Department of Physics “E. Fermi” (Italy); Giorgetti, Emilia, E-mail: emilia.giorgetti@fi.isc.cnr.it [Institute of Complex Systems (ISC-CNR) (Italy)

    2015-05-15

    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 <10 nm in deionized water or acetone by pulsed laser ablation, that permits preparation of uncoated and highly stable Gold nanoparticles in pure solvents. Subsequently, we investigated the role of surface chemistry, size, and dispersivity of synthesized Gold nanoparticles in exerting toxicity in a cell model system of deep respiratory tract, representing the main route of 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.

  16. Modification of radio- and thermo-sensitivity by amrubicin or amrubicinol in human lung adenocarcinoma A549 cells

    Amrubicin (AMR) is a totally synthetic 9-aminoanthracyclin anticancer drug. It is considered that AMR is an inhibitor of DNA topoisomerase II as the case of another anthracyclin anticancer drug, adriamycin (ADM) (1), which has significant antitumor activity against a broad spectra of human malignancies. The antitumor activity of AMR was found superior to that of ADM in experimental therapeutic models of human tumor xenografts (nude mouse). AMR was converted in vivo to major metabolite, amrubicinol (AMROH), which was markedly more effective cytotoxic agent than the mother compound. In the clinical studies currently conducted on malignant lymphoma, non-small or small cell lung carcinoma, the activity of AMR was shown very promising. However, the interactive cytocidal effects of the combined treatment with AMR or AMROH and radiation or hyperthermia are under investigation. In the present study, we examined chemical modification of radio- and thermo-sensitivity by AMR or AMROH in cultured human lung adenocarcinoma A549 cells. Sublethal damage repair (SLDR) was inhibited by the pretreatment with AMR or AMROH followed by X-irradiation. This finding suggests the possibility of the combined treatment of AMR or AMROH and X-irradiation as clinical cancer therapy strategy, since the doses in the routine clinical radiotherapy is ranged with a sublethal dose of 2 Gy. We also found that SLDR was inhibited by the pretreatment with AMR or AMROH followed by hyperthermia. We will discuss about clinical adoption of the combined treatment with AMR or AMROH and radiation or hyperthermia

  17. Mitochondrial transcription factor A regulated ionizing radiation-induced mitochondrial biogenesis in human lung adenocarcinoma A549 cells

    Mitochondrial transcription factor A (TFAM), the first well-characterized transcription factor from vertebrate mitochondria, is closely related to mitochondrial DNA (mtDNA) maintenance and repair. Recent evidence has shown that the ratio of mtDNA to nuclearDNA (nDNA) is increased in both human cells and murine tissues after ionizing radiation (IR). However, the underlying mechanism has not as yet been clearly identified. In the present study, we demonstrated that in human lung adenocarcinoma A549 cells, expression of TFAM was upregulated, together with the increase of the relative mtDNA copy number and cytochrome c oxidase (COX) activity after α-particle irradiation. Furthermore, short hairpin RNA (shRNA)-mediated TFAM knockdown inhibited the enhancement of the relative mtDNA copy number and COX activity caused by α-particles. Taken together, our data suggested that TFAM plays a crucial role in regulating mtDNA amplification and mitochondrial biogenesis under IR conditions. (author)

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

    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

  19. X射线对人肺腺癌A549细胞Pokemon基因表达的影响%Effects of X-ray irradiation on expression of Pokemon gene in A549 cells of human lung adenocarcinoma

    王璐; 邹跃; 江其生; 李伟; 宋秀军; 周湘艳; 王翠兰

    2011-01-01

    目的 研究不同剂量X射线照射及照射后不同时间点对人肺腺癌A549细胞Pokemon基因表达的影响.方法 用吸收剂量分别为2、4、6和8 Gy的X射线照射体外堵养的人肺腺癌A549细胞,2、4、8、12、24、48和72 ha,用实时定量PCR技术检测其中的Pokemon mRNA表达水平,以未照射组为对照.结果 在2、4、6、8 Gy X射线照射后的早期(除2 Gy照射后的2和4 h外)Pokemon mRNA的表达降低,但在晚期(48 h以后)呈升高趋势,在大部分时间点实验组与对照组的差异有统计学意义(t=3.40~154.76,P<0.05).结论 较大剂量的X射线在早期可下调A549细胞Pokemon基因mRNA的表达,诱导肿瘤细胞凋亡;但在晚期又可诱导A549细胞高表达PokemonmRNA,这可能与辐射所致A549细胞的DNA损伤修复和细胞周期调控有关.%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.

  20. Effects of Hypoxia on Expression of P-gp and Mutltidrug Resistance Protein in Human Lung Adenocarcinoma A549 Cell Line

    XIA Shu; YU Shiying; YUAN Xianglin

    2005-01-01

    Summary: To study the effects of hypoxia on the expression of P-gp and mutltidrug resistance protein in human lung adenocarcinoma A549 cell line, and to explore the probable mechanism of hypoxia in tumor cell of MDR. The expression of hypoxia inducible factor-1α, P-gp and mutltidrug resistance protein was immunohistochemically detected by culturing human lung adenocarcinoma A549 cell under hypoxia (2 % O2) for 24 h. After interaction with adriamycin or cisplatin under hypoxia (2 % O2) for 24 h, the cell survival rate was detected by MTT. Our results showed that the expression of hypoxia inducible factor-1α, P-gp and mutltidrug resistance protein under hypoxia were higher than the expression under normoxia, and correlations between the expression of HIF-1α and P-gp or multidrug resistance-associated protein was observed (P<0.05). The resistance of adriamycin of A549 cell was enhanced under hypoxia. It is concluded that the resistance of tumor chemotherapy is enhanced in hypoxia. The expression of HIF-1α is obviously correlated with the expression of P-gp and mutltidrug resistance protein.

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

    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.

  2. Inhibitory effect of recombinant vector pEgr1-hsTRAIL induced by radiation on growth of lung adenocarcinoma A549 cells

    Objective: to construct human secreted TRAIL (hsTRAIL) recombinant vector pEgr1-hsTRAIL mediated by Egr1, and to explore the inhibitory effect on the growth of lung adenocarcinoma A549 cells. Methods: The hsTRAIL vet or mediated by Egr1 was constructed by gene recombination technique, the A549 cells were transfected with the plasmid after identification by PCR, restrictive enzyme digestion and sequencing, and irradiated by 6 Gy X-rays. There were control group, pEgr1-hsTRAIL group, 6 Gy X-rays group and pEgr1-hsTRAIL + 6 Gy X-rays group in the experiment. The expression of hsTRAIL in A549 cells was detected by ELISA method, the cell proliferation was detected by MTT assay, the cycle changes of cell cycle the detected by flow cytometry and the apoptosis was measured by TUNEL method. Results: The hsTRAIL recombinant vector pEgr1-hsTRAIL mediated by Egr1 was constructed successfully. The cells were irradiated by 6 Gy X-rays after transfected with plasmid. The hsTRAIL protein expressions in control, 6 Gy and pEgr1-hsTRAIL groups didn't change significantly with the time prolongation, but the expression in pEgr1-hsTRAIL + 6 Gy group was increased significantly with the time prolongation (P<0.05 or P<0.01), and reached to peak value at 8 h. There was no significant difference of A549 cell proliferation ability between control group and pEgr1-hsTRAIL group, but the proliferation abilities in 6 Gy and pEgr1-hsTRAIL + 6 Gy groups were decreased significantly compared with control group, especially in pEgr1-hsTRAIL + 6 Gy group (P<0.05 or P<0.01). Compared with control group, the percentages of A549 cells at different phases in pEgr1-hsTRAIL group didn't change significantly, but the percentages of A549 cells at G0/G1 phase in 6 Gy and pEgr1-hsTRAIL + 6 Gy groups were increased significantly (P<0.05), the percentages of A549 cells at G2/M phase were decreased significantly (P<0.05), the percentages of A549 cells at S phase didn't change significantly. The

  3. Costunolide induces lung adenocarcinoma cell line A549 cells apoptosis through ROS (reactive oxygen species)-mediated endoplasmic reticulum stress.

    Wang, Zhen; Zhao, Xin; Gong, Xingguo

    2016-03-01

    Costunolide is an active sesquiterpene lactone derived from many herbal medicines. It has a broad spectrum of bioactivities, including anti-inflammatory and potential anti-tumor effects. The aims of the present study were to evaluate the inhibitory effects of costunolide on A549 cell growth and to explore the underlying molecular mechanisms. Annexin V-FITC/PI flow cytometry analysis revealed that costunolide induced apoptosis. To study the mechanism, we found that costunolide exposure activated the unfolded protein response (UPR) signaling pathways, as shown by the up-regulation of GRP78 and IRE1α and the activation of ASK1 and JNK. Meanwhile, siRNA knockdown of IRE1α significantly attenuated costunolide-induced apoptosis and partly restored the mitochondrial membrane potential. ER stress-activated JNK phosphorylated Bcl-2 at Ser70, which changes the anti-apoptotic function of Bcl-2, resulting in mitochondrial dysfunction and leading to mitochondrial activation of apoptosis. Furthermore, costunolide induced ROS generation, while the antioxidant N-acetyl cysteine (NAC) effectively blocked ER stress and apoptosis activation, suggesting that ROS acts as an upstream signaling molecule in triggering ER stress and mitochondrial apoptotic pathways. Taken together, our research demonstrates that costunolide exhibits its anti-tumor activity though inducing apoptosis, which is mediated by ER stress. We further confirm that Bcl-2 is a key molecule connecting the ER stress and mitochondrial pathways. PMID:26609913

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

    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

  5. Enhancement of radiosensitivity by topoisomerase II inhibitor, amrubicin and amrubicinol, in human lung adenocarcinoma A549 cells and kinetics of apoptosis and necrosis induction.

    Hayashi, Sachiko; Hatashita, Masanori; Matsumoto, Hideki; Shioura, Hiroki; Kitai, Ryuhei; Kano, Eiichi

    2006-11-01

    The effects of amrubicin (AMR) and its active metabolite, amrubicinol (AMROH), on the sensitivity of human lung adenocarcinoma A549 cells to ionizing radiation were investigated in vitro. Further, the kinetics of apoptosis and necrosis induction were also analyzed. The cytocidal effects of X-ray irradiation on A549 cells resulted in a low level of radiosensitivity with a D0 value of 12 Gy. The slopes of the survival curves in the exponential phase were plotted on semilogarithmic paper for radiation combined with AMR (2.5 microg/ml) and AMROH (0.02 microg/ml) treatment, and were shown to be approximately parallel to treatment with irradiation alone. The initial shoulder-shape portion of the survival curve for radiation alone, indicating the repair of sublethal damage, was reduced as compared to that for sequential combined treatment with AMR or AMROH. Sequential treatments with AMR or AMROH prior to ionizing radiation resulted in an additive radio-enhancement effect that reduced not only survival, but also the shoulder width. Fractionated irradiation with 2 Gy per fraction of A549 cells was carried out in vitro similar to that commonly performed in clinical radiotherapy and the radio-resistance of the cells was shown to be inhibited by AMR and AMROH. Similar to AMR and AMROH, adriamycin and etoposide (VP-16) are DNA topoisomerase II inhibitors. The effects of these 4 agents on cells that received X-ray irradiation were compared and all of the agents exhibited comparable radio-enhancement effects. The induction of apoptosis was investigated at 48 and 72 h after administration of AMROH, radiation or combined treatment, and apoptosis was not significantly induced after any of the treatments. We also examined the induction of necrosis, and found that the incidence of necrosis following combined treatment was approximately 2 times higher than that with either of the single treatments. PMID:17016621

  6. Endostar combined with cryoablation for subcutaneous xenografted tumor model of lung adenocarcinoma cell line A549 in BALB/c nude mice: an experimental study

    Objective: To investigate the inhibitory effect of Endostar combined with cryoablation on Lung adenocarcinoma cell line A549 in BALB/c nude mice, and to discuss its interaction mechanisms. Methods: The lung adenocarcinoma A549 model in BALB/c nude mice were established. When the largest diameter of tumor reached 1.0 cm, a total of 24 mice were randomly and equally divided into 4 groups: control group, Endostar group, cryoablation group and cryoablation plus Endostar group. The largest diameter and the vertical diameter of the tumors were measured at different points of time after treatment. At the 21st day, the mice were sacrificed and the tumors were removed and the rate of tumor cell apoptosis, the microvessel density (MVD) and the expression level of vascular endothelial growth factor (VEGF) were determined by using immunohistochemistry method. The results were statistically analyzed. Results: The tumor growth velocity of the control group, Endostar group, cryoablation group and cryoablation plus Endostar group was (2.36.68±51.23)%, (220.02±30.61)%, (159.46±29.33)% and (103.34±25.50)%, respectively (P<0.01). The rate of apoptosis of the four groups was (21.67±2.34)%, (22.17±1.47)%, (38.33±1.37)% and (49.17±1.72)%, respectively (P<0.01). The MVD and the expression levels of VEGF of the cryoablation plus Endostar group were significantly lower than those of the other three groups (P<0.01). Statistical analysis revealed that a positive correlation existed between the express of VEGF and MVD. Conclusion: Endostar can obviously enhance the therapeutic efficacy of cryoablation on lung adenocarcinoma A549 in BALB/c nude mice. The underlying mechanisms may be the Endostar-inhibited angiogenesis through down-regulating the expression of VEGF, and the cooperative effect of Endostar and cryoablation on the promotion of tumor cell apoptosis. (authors)

  7. CK2 inhibitor CX-4945 blocks TGF-β1-induced epithelial-to-mesenchymal transition in A549 human lung adenocarcinoma cells.

    Jiyeon Kim

    Full Text Available BACKGROUND: The epithelial-to-mesenchymal transition (EMT is a major phenotype of cancer metastasis and invasion. As a druggable cancer target, the inhibition of protein kinase CK2 (formally named to casein kinase 2 has been suggested as a promising therapeutic strategy to treat EMT-controlled cancer metastasis. This study aimed to evaluate the effect of the CK2 inhibitor CX-4945 on the processes of cancer migration and invasion during the EMT in A549 human lung adenocarcinoma cells. MATERIALS AND METHODS: The effect of CX-4945 on TGF-β1-induced EMT was evaluated in A549 cells treated with TGF-β1 (5 ng/ml and CX-4945. The effect of CX-4945 on TGF-β1-induced cadherin switch and activation of key signaling molecules involved in Smad, non-Smad, Wnt and focal adhesion signaling pathways were investigated by Western blot analysis, immunocytochemistry and reporter assay. Additionally, the effect of CX-4945 on TGF-β1-induced migration and invasion was investigated by wound healing assay, Boyden chamber assay, gelatin zymography, and the quantitative real-time PCR. RESULTS: CX-4945 inhibits the TGF-β1-induced cadherin switch and the activation of key signaling molecules involved in Smad (Smad2/3, Twist and Snail, non-Smad (Akt and Erk, Wnt (β-catenin and focal adhesion signaling pathways (FAK, Src and paxillin that cooperatively regulate the overall process of EMT. As a result, CX-4945 inhibits the migration and invasion of A549 cells accompanied with the downregulation of MMP-2 and 9. CONCLUSIONS: Clinical evaluation of CX-4945 in humans as a single agent in solid tumors and multiple myeloma has established its promising pharmacokinetic, pharmacodynamic, and safety profiles. Beyond regression of tumor mass, CX-4945 may be advanced as a new therapy for cancer metastasis and EMT-related disorders.

  8. 去甲斑蝥素对人肺腺癌A549细胞的抑制作用%The inhibition of norcantharidin on human lung adenocarcinoma A549 cells

    崔宝弟; 王敏; 孙震晓

    2015-01-01

    OBJECTIVE:To study the effects of norcantharidin (NCTD) on human lung cancer cells,and investigate the mechanisms.METHODS:The growth inhibition of A549 cells treated with 0-240µmol/L NCTD for 0-72 hours was analyzed by MTT assay. The recovery growth and proliferation of A549 cells treated with 0-120µmol/L NCTD for 24 h was evaluated by MTT assay. The morphological changes of A549 cells treated with 40,50 and 60µmol/L NCTD for 0-72 h were examined under inverted microscope. The apoptosis and cell cycle changes of A549 cells treated with 40-60µmol/L NCTD were detected by flow cytometry.RESULTS:NCTD inhibited the growth of A549 cells in 30-240 µmol/L(P0.05)。结论:40~60µmol/L NCTD主要通过诱导A549细胞G2~M期阻滞而抑制细胞生长。

  9. Radio-sensitization effect of an mTOR inhibitor, temsirolimus, on lung adenocarcinoma A549 cells under normoxic and hypoxic conditions

    The mammalian target of rapamycin (mTOR) correlates with cell survival under hypoxia and regulates hypoxia-inducible factor-1α (HIF-1α), a key protein in hypoxia-related events. However, the role of mTOR in radio-resistance has not been fully investigated. Therefore, the effect of mTOR on the radio-resistance of cancer cells under hypoxia was evaluated using the mTOR inhibitor temsirolimus. Clonogenic survival was examined in the A549 human lung adenocarcinoma cell line under normoxia or hypoxia, with or without temsirolimus. An oxygen enhancement ratio (OER) was calculated using the D10 values, the doses giving 10% survival. Western blotting was performed to investigate the effect of temsirolimus on mTOR and the HIF-1α pathway under normoxia and hypoxia. A549 cells showed a radio-resistance of 5.1 and 14.2 Gy, as indicated by D10 values under normoxia and hypoxia, respectively; the OER was 2.8. The cell survival rates under hypoxia and with temsirolimus remarkably decreased compared with those under normoxia. The D10 values of the cells under normoxia and hypoxia were 4.8 and 5.4 Gy, respectively (OER = 1.1). mTOR expression was suppressed by temsirolimus under both normoxia and hypoxia. HIF-1α expression decreased under hypoxia in the presence of temsirolimus. These results suggest that temsirolimus can overcome the radio-resistance induced by hypoxia. When the fact that mTOR acts upstream of HIF-1α is considered, our data suggest that the restoration of radiation sensitivity by temsirolimus under hypoxia may be associated with the suppression of the HIF-1α pathway. Temsirolimus could therefore be used as a hypoxic cell radio-sensitizer. (author)

  10. Exosome cargo reflects TGF-β1-mediated epithelial-to-mesenchymal transition (EMT) status in A549 human lung adenocarcinoma cells.

    Kim, Jiyeon; Kim, Tae Yeon; Lee, Myung Shin; Mun, Ji Young; Ihm, Chunhwa; Kim, Soon Ae

    2016-09-16

    It has been suggested that tumor cells secrete exosomes to modify the local microenvironment, which then promotes intercellular communication and metastasis. Although exosomes derived from cancer cells may contribute to the epithelial-mesenchymal transition (EMT) in untransformed cells, few studies have defined exosome cargo upon induction of EMT. In this study, we investigated the changes in exosomal cargo from the epithelial to mesenchymal cell phenotype by inducing EMT with transforming growth factor (TGF)-β1 in A549 human lung adenocarcinoma cells. The protein content of the exosomes reflects the change in the cell phenotype. In addition, miR-23a was significantly enriched in the exosomes after mesenchymal transition. Following treatment of exosomes from mesenchymal cells via EMT induction with TGF-β1 to the epithelial cell type, phenotypic changes in protein expression level and cell morphology were observed. Autologous treatment of exosomes enhanced the transcriptional activity and abundance of β-catenin. Our results suggest that the exosomal protein and miRNA content reflects the physiological condition of its source and that exosomes induce phenotypic changes via autocrine signaling. PMID:27492069

  11. THE EFFECT OF IRISQUINONE ON THE GLUTATHIONE SYSTEM AND MRP EXPRESSION OF CISPLATIN-RESISTANT HUMAN LUNG ADENOCARCINOMA CELL LINE (A549DDP)

    LIANG; li

    2001-01-01

    [1] Li DH. A novel radiosensitizer "ANKA" for tumor (Irisquinone) [J]. Chin J Clin Oncol 1999; 26:153.[2]Bordow SB, Haber M, Madafiglio J, et al. Expression of the multidrug resistance-associated protein (MRP) gene correlates with amplification and overexpression of the N-myc oncogene in childhood neuroblastoma [J]. Cancer Res 1994; 54:5036.[3]Cai P, Liu XY, Han FS, et al. Establishment human lung adenocarcinoma cisplatin-resistant cell line A549DDP and the mechanism of its drug resistance [J]. Chin J Clin Oncol 1995; 22:582.[4]Cai P, Liu XY, Wang P. The value of glutathione reductase recycling assay measurement of content of glutathione in human plasma during tumor chemotherapy [J]. Chin J Clin Oncol l994; 21:717.[5]Zhan MC, Liu XY, Cai P, et al. Mechanism of resistance of human cell line A549DDP to cisplatin [J]. Chin J Clin Oncol 1998; 25:726.[6]Wang J, Liu XY, Wu MN, et al. Expression and reversion of drug resistance- and apoptosis- related genes of a DDP-resistant lung adeno-carcinoma cell line A549DDP [J]. Chin J Oncol 1999; 21:422.[7]Ishikawa T. The ATP-dependent glutathione S-conjugate export pump [J]. Treads Biol Sci 1992; 17:463.[8]Goto S, Yoshida K, Morikawa T, et al. Augmen-tation of transport for cisplatin-glutathione adduct in cisplatin-resistant cancer cells [J]. Cancer Res 1995; 55:4297.[9]Fujil R, Mutoh M, Sumizama T, et al. Adenosine triphosphate-dependent transport of leukotriene C4 by membrane vesicles prepared from cis-platinum-resistant human epidermoid carcinoma tumor cells [J]. JNCI 1994; 86:1781.[10]Ishikawa T, Ali-Osman F. Glutathion-associated cis-diamminedichloroplatinum (II) metabolism and ATP-dependent efflux from leukemia cells [J]. J Biol Chem 1993; 268:20116.[11]Ishikawa T, Wrighe CE, Ishizuka H. GS-X pumq is function ally overexpressed in cis-diammine-dichloroplatinum (II)-resistant human leukemia HL-60 cells and downregulated by cell differentiation [J]. J Biol Chem 1994; 269: 29085.

  12. miR-155在肺腺癌 A549细胞侵袭和转移中的作用%Role of miR-155 in invasion and metastasis of lung adenocarcinoma A549 cells

    程田力; 胡成平; 李敏; 顾其华; 安健

    2016-01-01

    Objective To investigate the role and mechanism of miR-155 in invasion and metastasis of lung adenocarcinoma A549 cells.Methods Real-time PCR and fluorescence in situ hybridization were used to detect the miR-155 expression in patients′lung adenocarcinoma and adjacent tissue and lymph nodes.Scratch test and Transwell migration assay were used to assess the effect of miR-155 on the A549 cell migration and invasion capability.Bioinformatics software was used to predict the target genes of miR-155, and using luciferase to assay the target gene.Western blot and real-time PCR were performed to confirm the role of miR-155 expression in the regulation of target gene PTEN.Results The real-time quantitative PCR showed that the miR-155 expression levels in adjacent normal tissue, lung adenocarcinoma and metastatic lymph nodes were 4.1±0.5, 9.6±3.1 and 7.8±2.2, respectively.The in situ hybridization showed that the expression rates of miR-155 in the adjacent normal tissue, lung adenocarcinoma and metastatic lymph nodes were (23.2±15.3)%, (75.4±20.2 )% and (60.4±25.1)%,respectively.The Scratch assay showed that the wound healing rates in the miR-155 mimics group, miR-155 mimics NC group, miR-155 inhibitor group and miR-155 inhibitor NC group at 24 h were (43.2±2.2)%, (21.3±4.2)%, (24.3±5.3)%, and (35.2± 5.1)%, and that at 48 h were (75.2±4.5)%, (52.6±5.2)%, (39.4±4.2)%, and( 51.5±4.3)%, respectively.Dual luciferase reporter gene assay showed that the value of the luciferase in the miR -155 mimics group co-transfected with PTEN 3′UTR-containing wild-type and mutant plasmids were 4.7±0.5 and 7.3±0.7, and the miR-155 mimics luciferase values of the control group co-transfected with PTEN 3′UTR-containing wild-type and mutant plasmids were 7.8±0.9 and 7.5±0.8, respectively.The real-time quantitative fluorescence PCR showed that the relative expression of PTEN protein in the miR-155 mimics group, miR-155 mimics control group, miR-155 mimics inhibitor group

  13. Alteration of membrane lipid biophysical properties and resistance of human lung adenocarcinoma A549 cells to cisplatin

    LIANG; Xingjie; (

    2001-01-01

    [1]Simon, S. M., Schindler, M., Cell biological mechanisms of multidrug resistance in tumors, Proc. Natl. Acad. Sci. USA, 1994, 91: 3497.[2]Ambudkar, S. V., Dey, S., Hrycyna, C. A. et al., Biochemical, cellular, and pharmacological aspects of the multidrug trans-porter, Annu. Rev. Pharmacol. Toxicol., 1999, 39: 361.[3]Dudeja, P. K., Anderson, K. M., Harris, J. S. et al., Reversal of multidrug resistance phenotype by surfactants: Relationship to membrane lipid fluidity, Arch. Biochem. Biophys., 1995, 319 (1): 8309.[4]Collins, J. M., Scott, R. B., Grogan, W. M., Plasma membrane fluidity gradients of human peripheral blood leukocytes, J. Cell Physiol., 1990, 144: 42.[5]Collins, J. M., Dominey, R. N., Grogan, W. M., Shape of the fluidity gradient in the plasma membrane of living Hela cells, J. Lipid Res., 1990, 31: 261.[6]Ashman, R. F., Peckham, D., Alhasan, S. et al., Membrane unpacking and the rapid disposal of apoptotic cells, Immunol. Lett., 1995, 48(3): 159.[7]Sentjurc, M., Zorec, M., Cemazar, M. et al., Effect of vinblastine on cell membrane fluidity in vinblastine-sensitive and -resistant HeLa cells, Cancer Lett., 1998, 130(1-2):183.[8]Regev, R., Assaraf, Y. G., Eytan, G. D. et al., Membrane fluidization by ether, other anesthetics, and certain agents abolish-es P-glycoprotein ATPase activity and modulates efflux from multidrug-resistant cells, Eur. J. Biochem., 1999, 259(1-2): 18.[9]Robert, A. S., Mariamme, S., Katherine, L. S., Altered lipid packing identifies apoptotic thymocytes, Immunol. Lett., 1993, 36: 283.[10] Lagerberg, J. W., Kallen, K. J., Haest, C. W. et al., Factors affecting the amount and the mode of merocyanine 540 binding to the membrane of human erythrocytes, Biochim. Biophys. Acta, 1995, 1235(2): 428.[11] Stillwell, W., Wassall, S. R., Dumaual, A. C. et al., Use of merocyanine (MC540) in quantifying lipid domains and pack-ing in phospholipid vesicles and tumor cells, Biochem. Biophys. Acta, 1993

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

    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.

  15. Expression of P53, P21 in Human Lung Adenocarcinoma A549 Cell Strains under Hypoxia Conditions and the Effect of TSA on Their Expression

    黄宏; 张珍祥; 徐永健; 邵静芳

    2003-01-01

    This paper was designed to investigate the expression of p53, p21of A549 cell strains under hypoxic condition and the effect of trichostatin A (TSA), the inhibitor of histone deacetylasel (HDAC1) on their expression. The authors designed 1 normoxia group (control group) and 6 hypoxia groups (experiemntal group): hypoxia 6 h group (A), TSA+ hypoxia 6 h (B), hypoxia 12 h group (C) ,hypoxia 24 h group (D), TSA+hypoxia 24 h (E), hypoxia 48 h group (F). The expression of HDAC1 in A549 cells was examined by using Western blot and the expression of p53,p21 in A549 cells and the effect of TSA on them were determined by using immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR). The A value expressed by HDAC1 in A549 cell strains was 138±11 in the control group, 78±4, 86±5, 124±3, 120±9 in experimental groups A, C, D, F, respectively. The A value of the expression of the protein and mRNA of p53 in A549 cell strains were 0. 12±0.02, 0. 62±0.02 in the control group, 0. 10±0.03, 0.32±0.03; 0. 11±0.01, 0. 33±0.02; 0. 13±0.03, 0. 58±0.01; 0. 12±0. 02, 0. 56±0.02 in experimental group A, B, D, E, respectively. The A value of the expression of the protein and mRNA of p21 in A549 cell strains were 0. 17±0.03, 0. 62±0. 03 in the control group, 0. 16±0.02, 0. 50±0.02; 0. 14±0.02, 0. 36±0.02; 0. 15±0.03, 0. 49±0.03; 0. 13±0.02, 0. 33 ± 0. 02 in experimental groups A, B, D, E, respectively. These results indicate that the expression of HDAC1 is regulated by hypoxia and the effect of TSA is closely related to the expression of P21 under hypoxia condition.

  16. Construction of Eukaryotic Expression Vector of Human CC10 Gene and Expression of CC10 Protein in Lung Adenocarcinoma A549 Cell Line

    2005-01-01

    A mammalian expression plasmid pcDNA3.1-hCC10 was constructed and identified, then CC10 protein expression in A549 lung cancer cell line was detected. A 273 bp cDNA fragment was amplified from the total RNA of normal lung tissue by using RT-PCR and cloned into expression plasmid cDNA3.1, and the recombinant plasmid was identified by employing double digestion restriction enzymes HindⅢ and BamH Ⅰ and the cDNA sequence was assayed by the Sanger dideoxymediated chain termination method. The segment was then transfected into the A549 lung cancer cell line. The protein expression of CC10 was detected by immunofluorescence and Western blot.Our results showed that the cDNA fragment included the entire coding region (273 bp). The recombinant eukaryotic cell expression vector of pcDNA3.1-hCC10 was successfully constructed, and the sequence of the insert was identical to the published sequence. A549 cells line transfected with the pcDNA3.1-hCC10 expressed high level of CC10 protein. The recombinant plasmid cDNA3. 1hCC10 may serve as an effective tool for the study of tumorogenesis and tumor treatment.

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

    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

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

    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

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

    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.

  20. Inhibition of Oridonin on Human Lung Adenocarcinoma A549 Cells and Its Mechanisms%冬凌草甲素诱导人肺腺癌细胞株A549凋亡及其机制研究

    彭蕾; 顾振纶; 薛仁宇; 周颖; 蒋小岗; 郭次仪

    2010-01-01

    目的:探讨冬凌草甲素(oridnin)对人肺腺癌细胞株A549细胞凋亡的影响.方法:利用MTT法检测oridnin对A549细胞增殖作用的影响;Hoechst 33258染色观察给药后细胞形态改变;透射电镜观察给药后细胞超微结构改变;FITC-AnnexinV/PI双标记检测细胞凋亡率.结果:Hoechst 33258染色和透射电镜观察,oridonin给药后A549细胞出现空泡变性,染色质高度凝集;FTTC-AnnexinV/PI双标记检测oridnin(25,50,100μmol/L)作用细胞48 h后凋亡率分别为1.5%,6.2%,59.7%.结论:Oridonin对A549细胞具有抑制增殖和诱导凋亡作用.

  1. Effect of artemether on the poliferation of human lung adenocarcinoma cell line A549%蒿甲醚对人肺腺癌A549细胞体外生长的影响

    郭燕; 王俊; 章必成; 陈正堂

    2007-01-01

    目的:研究抗疟疾药物蒿甲醚(Artemether)对人肺腺癌A549细胞株体外生长的影响,为蒿甲醚治疗肺癌提供实验依据. 方法:采用单四唑(MTT)比色法检测蒿甲醚对体外培养的人肺腺癌A549细胞的生长抑制作用;用细胞计数法绘制细胞生长曲线,计算对数生长期群体倍增时间;用流式细胞术研究蒿甲醚对细胞周期的影响;采用苏木精-伊红(H-E)染色在光镜下观察凋亡细胞的形态学特征. 结果:蒿甲醚对A549细胞株的半数抑制浓度(IC50)为1.34 mg/L.A549肺腺癌细胞对数生长期群体倍增时间在蒿甲醚作用后(20.7±0.5)h,对照组为(32.2±0.3 )h,两组比较有显著性差异(P< 0.01).A549细胞经蒿甲醚作用后G1期细胞百分比增加(P<0.01),G2或S期细胞减少(P<0.01),凋亡率明显增加(P<0.01). 结论:蒿甲醚对人肺腺癌A549细胞株生长具有显著抑制作用;蒿甲醚的细胞毒作用与其诱导肿瘤细胞凋亡有关.

  2. Inhibitory Effect of Cantharidin on Proliferation of A549 Cells

    WANG Xiao-hua; YIN Yuan-qin; SUI Cheng-guang; MENG Fan-dong; MA Ping; JIANG You-hong

    2007-01-01

    Objective: To study the inhibition of Cantharidin against the proliferation of human lung cancer A549 cells and its mechanism. Methods: MTT assay was employed to determine the inhibition of Cantharidin against proliferation of A549 cells and flow Cytometry was applied to analyze A549 cell cycle and the effect of Cantharidin on cell cycle. Results: Cantharidin showed inhibition against the proliferation of A549 cells, and the inhibition was mediated by blocking A549 cell cycle at G2/M phase significantly. Conclusion: Cantharidin exhibits inhibition against the proliferation of human lung cancer A549 cells.

  3. Cinnamomum verum Component 2-Methoxycinnamaldehyde: A Novel Anticancer Agent with Both Anti-Topoisomerase I and II Activities in Human Lung Adenocarcinoma A549 Cells In Vitro and In Vivo.

    Wong, Ho-Yiu; Tsai, Kuen-daw; Liu, Yi-Heng; Yang, Shu-mei; Chen, Ta-Wei; Cherng, Jonathan; Chou, Kuo-Shen; Chang, Chen-Mei; Yao, Belen T; Cherng, Jaw-Ming

    2016-02-01

    Cinnamomum verum is used to make the spice cinnamon and has been used as a traditional Chinese herbal medicine. We evaluated the anticancer effect of 2-methoxycinnamaldehyde (2-MCA), a constituent of the bark of the plant, and its underlying molecular biomarkers associated with carcinogenesis in human lung adenocarcinoma A549 cells. The results show that 2-MCA suppressed proliferation and induced apoptosis as indicated by an upregulation of pro-apoptotic Bax and Bak genes and downregulation of anti-apoptotic Bcl-2 and Bcl-XL genes, mitochondrial membrane potential loss, cytochrome c release, activation of caspase-3 and -9, and morphological characteristics of apoptosis, including plasma membrane blebbing and long comet tail. In addition, 2-MCA also induced lysosomal vacuolation with increased volume of acidic compartment (VAC) and suppressions of nuclear transcription factors nuclear factor-κB (NF-κB) and both topoisomerase I and II activities. Further study reveals that the growth-inhibitory effect of 2-MCA was also evident in a nude mice model. Taken together, the data suggest that the growth-inhibitory effect of 2-MCA against A549 cells is accompanied by downregulations of NF-κB binding activity and proliferative control involving apoptosis and both topoisomerase I and II activities, together with an upregulation of lysosomal vacuolation and VAC. Our data suggest that 2-MCA could be a potential agent for anticancer therapy. PMID:26676220

  4. 骨髓间充质干细胞参与A549肺腺癌的组织修复★%Bone marrow mesenchymal stem cells are involved in tissue repair of A549 lung adenocarcinoma

    许峰; 张雷; 潘晋坤; 薛利利; 赵晓燕; 李宝平

    2013-01-01

    BACKGROUND:Tumor has been considered as a specific nonhealing trauma. Bone marrow mesenchymal stem cel s participate in tumor mesenchymal reconstitution by tumor tissue homing and differentiation into mesenchyme, resulting in changing tumor microenvironment and affecting tumor growth and transfer. OBJECTIVE:To explore the mechanisms of participation of bone marrow mesenchymal stem cel s in tumor tissue repair in an A549 lung cancer-bearing mouse model. METHODS:Bone marrow mesenchymal stem cel s were isolated in vitro, cultured, and identified using flow cytometry, and then used to establish a mouse model of A549 lung cancer-bearing. In the experimental group, human bone marrow mesenchymal stem cel s were injected into tissue surrounding the tumor. In the control group, an equal volume of PBS was injected. Animal survival condition and tumor size were compared. At 4 weeks, the specimens were harvested. Hematoxylin-eosin staining was used to compare tumor tissue. Masson staining was utilized to compare col agen fiber content. Reverse transcription-PCR was employed to detect the expression ofα-smooth muscle actin. Immunohistochemistry was used to examine the expression of fibroblast specific protein and fibroblast activation protein to reflect the degree of interstitial fibers in tumor tissue in both groups. The expression levels of vascular endothelial growth factor, hepatocyte growth factor, interleukin-6 and tenescin-C were compared between the two groups using immunohistochemistry. RESULTS AND CONCLUSION:Bone marrow mesenchymal stem cel s promoted tumor growth in tumor-bearing mice. The growth rate of tumor tissue in experimental group was faster than the control group (P<0.05). Compared with the control group,α-smooth muscle actin mRNA expression was significantly higher in the experimental group. Immunohistochemistry was used to detect the expression of tumor angiogenesis factors markers (fibroblast specific protein and fibroblast activation protein) in tumor

  5. 艾迪注射液对人肺腺癌A549细胞放疗敏感性的增加作用及其机制%The enhancing radiosensitization effect of Aidi injection on human lung adenocarcinoma A549 cells and its mechanism

    王勇; 刘琴; 朱紫结; 罗辉; 钟小军; 李勇

    2015-01-01

    Objective To observe the radiosensitization effect of Aidi injection on human lung adenocarcinoma A549 cells, and to analyze its possible mechanism.Methods ① A549 cells were treated with different concentrations of Aidi injection (1.875, 3.75, 7.5, 15, 30, 60 mg/mL) for 24 hours, and in the mean time, a blank control group was set up; the effect of Aidi injection on lung adenocarcinoma A549 cells proliferation was detected by methyl thiazolyl tetrazolium (MTT) assay, and the 10% cell growth inhibitor concentration (IC10) was calculated. ② The experiments were divided into blank control, Aidi control, radiation and Aidi pretreatment groups. The Aidi control group was incubated for 24 hours by Aidi injection IC10; the radiotherapy group was given X-ray irradiation of 4 Gy followed by incubation for 24 hours; the Aidi pretreatment group was incubated for 24 hours by Aidi injection IC10 and then given X-ray irradiation of 4 Gy; the blank control group received equal volume of normal saline and was incubated for 24 hours. The survival fraction (SF) value was detected by cell colony formation assay; the protein levels of the serine phosphorylation at 139 locus of histone (γ-H2AX protein), the key protein in homologous recombination repair pathway (Rad51 protein) and the cell autophage characteristic protein (LC3 protein) were detected by Western Blot; the formation of autophagosome was observed by transmission electron microscope.Results Aidi injection possessed the suppression of the growth of human lung adenocarcinoma A549 cells, the proliferation of the cells in various Aidi groups was lower than that in the blank control group, with the increase in drug concentration, the A549 cell growth inhibition ratio (IR) was gradually increased, representing a dose dependent manner, and the IC10 was 3.09 mg/mL. Compared with the blank control group, the SF value in Aidi control group was not significantly different [(94.7±3.85)% vs. (100.0±0.00)%,P > 0.05], the SF value

  6. The effect of ataxia-telangiectasia mutated kinase-dependent hyperphosphorylation of checkpoint kinase-2 on oligodeoxynucleotide 7909 containing CpG motifs-enhanced sensitivity to X-rays in human lung adenocarcinoma A549 cells

    Liu XQ

    2015-06-01

    Full Text Available Xiaoqun Liu,1,* Xiangdong Liu,2,* Tiankui Qiao,1 Wei Chen,1 Sujuan Yuan1 1Department of Oncology, 2Department of Ophthalmology, Affiliated Jinshan Hospital, Fudan University, Shanghai, People’s Republic of China *These authors contributed equally to this work Objective: The aim of the study reported here was to further investigate the potential effect of ataxia-telangiectasia mutated (ATM kinase-dependent hyperphosphorylation of checkpoint kinase-2 (Chk2 on radiosensitivity enhanced by oligodeoxynucleotide 7909 containing CpG motifs (CpG ODN7909 in human lung adenocarcinoma A549 cells. Methods: In vitro A549 cells were randomly separated into control, CpG, X-ray, CpG+X-ray, ATM kinase-small interfering RNA (siRNA+CpG+X-ray (ATM-siRNA, and Chk2-siRNA+CpG+X-ray (Chk2-siRNA groups. siRNAs were adopted to silence the ATM and Chk2 genes. Expression and phosphorylation of ATM kinase and Chk2 were detected by Western blot assay. Cell colonies were observed under inverted phase-contrast microscopy. Cellular survival curves were fitted using a multi-target single-hitting model. Cell cycle and apoptosis were analyzed by flow cytometry. Results: Expression of ATM kinase and Chk2 was similar among the control, CpG, X-ray, and CpG+X-ray groups. Phosphorylated ATM kinase and Chk2 were significantly increased in the CpG+X-ray group compared with in the X-ray group (t=6.00, P<0.01 and t=3.13, P<0.05, respectively, though these were hardly detected in the control and CpG groups. However, expression of ATM kinase and Chk2 was clearly downregulated in the ATM-siRNA and Chk2-siRNA groups, respectively. Similarly, their phosphorylation levels were also significantly decreased in the ATM-siRNA group (t=14.35, P<0.01 and t=8.46, P<0.01, respectively and a significant decrease in phosphorylated Chk2 was observed in the Chk2-siRNA group (t=7.28, P<0.01 when compared with the CpG+X-ray group. Further, the number of A549 cells at Gap 2/mitotic phase and the apoptosis

  7. The biophysical property of A549 cells transferred by VEGF-D.

    Wang, Zhen; Wu, Xiu-Li; Wang, Xu; Tian, Hong-Xia; Chen, Zhi-Hong; Li, Yang-Qiu

    2014-01-01

    Vascular endothelial growth factor-D (VEGF-D) together with VEGF-C is considered to be associated with lymphangiogenesis and angiogenesis and involve in tumorization. This study aims to investigate the influence of exogenous VEGF-D gene on the biophysical property of cell surface of lung adenocarcinoma cell line. A panel of lung adenocarcinoma cell lines were examined the expression of VEGF-D and VEGF-C by real-time PCR. The VEGF-D recombinant plasmid containing enhanced green fluorescence protein (EGFP) was constructed and transfected to the cell line with no expression of VEGF-D and confirmed by real-time PCR and Western blot analysis. Topographic images of cells were obtained by using atomic force microscope (AFM) in contact mode. Unlike VEGF-C, VEGF-D was found to have a very low expression or undetectable expression in lung adenocarcinoma cell lines. The VEGF-D recombinant plasmid had been constructed successfully and was transferred into the human lung adenocarcinoma cell line A549 cells which had no endogenous expression of VEGF-D, and exogenous VEGF-D could be detected in mRNA and protein expression levels in the gene modified cells, while the VEGF-C gene expression had no change after VEGF-D transfection. After transfection, the irregular microspikes or nano clusters could observe on the surface of A549 cells, and VEGF-D transfected A549 cells became more rigid. The exogenous VEGF-D gene might cause the remarkable biophysical architectural changes in the A549 cells, which might as a novel biomarker for evaluation of its biological function. PMID:23526563

  8. Effects of Tumor Suppressor Gene TCF21 on the Proliferation, Migration and Apoptosis of A549 Cells

    Song HU

    2014-04-01

    Full Text Available Background and objective TCF21, a newly discovered gene, exhibits tumor suppressor function in a variety of tumors. This study aims to observe the effects of TCF21 on the proliferation, apoptosis and migration of A549 human lung adenocarcinoma epithelial cells. Methods TCF21 was overexpressed in A549 cells via lentiviral transfection. Fluorescence-based quantitative polymerase chain reaction and Western blot analysis were used to analyze the expression of the target gene. Transwell, proliferation assay, and flow cytometry were applied to detect the effect of TCF21 overexpression on the migration, proliferation, and apoptosis of A549 cells after transfection. Results The proliferation and migration of A549 cells were inhibited, and the apoptotic rate was increased by overexpressing TCF21. Conclusion The tumor suppressor gene, TCF21, significantly inhibits the proliferation and migration, as well as facilitates early apoptosis of A549 cells.

  9. Cytochrome c oxidase is activated by the oncoprotein Ras and is required for A549 lung adenocarcinoma growth

    Telang Sucheta

    2012-08-01

    Full Text Available Abstract Background Constitutive activation of Ras in immortalized bronchial epithelial cells increases electron transport chain activity, oxygen consumption and tricarboxylic acid cycling through unknown mechanisms. We hypothesized that members of the Ras family may stimulate respiration by enhancing the expression of the Vb regulatory subunit of cytochrome c oxidase (COX. Results We found that the introduction of activated H-RasV12 into immortalized human bronchial epithelial cells increased eIF4E-dependent COX Vb protein expression simultaneously with an increase in COX activity and oxygen consumption. In support of the regulation of COX Vb expression by the Ras family, we also found that selective siRNA-mediated inhibition of K-Ras expression in A549 lung adenocarcinoma cells reduced COX Vb protein expression, COX activity, oxygen consumption and the steady-state concentration of ATP. We postulated that COX Vb-mediated activation of COX activity may be required for the anchorage-independent growth of A549 cells as soft agar colonies or as lung xenografts. We transfected the A549 cells with COX Vb small interfering or shRNA and observed a significant reduction of their COX activity, oxygen consumption, ATP and ability to grow in soft agar and as poorly differentiated tumors in athymic mice. Conclusion Taken together, our findings indicate that the activation of Ras increases COX activity and mitochondrial respiration in part via up-regulation of COX Vb and that this regulatory subunit of COX may have utility as a Ras effector target for the development of anti-neoplastic agents.

  10. Role of Hypoxia-inducible factor-1 and its target genes in human lung adenocarcinoma cells after photon- versus carbon ion irradiation; Expression HIF-1-abhaengiger Gene in humanen Lungenadenokarzinom (A549)-Zellen und deren Regulation nach Photonen- und Schwerionenbestrahlung

    Bill, Verena Maria

    2013-11-26

    Exposed to hypoxia tumor cells are notably resistant to photon irradiation. The hypoxiainducible transcription factor 1α (HIF-1α) seems to play a fundamental role in this resistance, while its role after heavy-ion beam remains unknown. The intention of this study was to determine how A549-cells (non-small-cell lung carcinoma) react in different oxygenation states after irradiation with photons or heavy ions, particularly in regards to their expression of HIF-1 target genes. Resistance of hypoxic A549 cells after photon irradiation was documented by cellular and clonogenic survival. In contrast, cellular survival after heavy-ion irradiation in hypoxic cells was not elevated to normoxic cells. Among the oxygen dependent regulation of HIF-1 target genes, gene expression analyses showed an increased expression of GLUT-1, LDH-A, PDK-1 and VEGF after photon irradiation but not after heavy-ion irradiation after 48 hours in normoxic cells. As expected, CDKN1A as inhibitor of cell cycle progression showed higher expression after both radiation forms; interestingly CDKN1A was also in an oxygen dependent manner lightly upregulated. In western blot analyses we demonstrated a significant increase of HIF-1 and GLUT-1 caused by hypoxia, but only a tendency of increased protein level in hypoxia after photon irradiation and no changes after heavy-ion irradiation. Significantly higher protein level of secreted VEGF-A could be measured 72 hours after photon irradiation in normoxic cells by ELISA analyses. Controversially discussed, I could not detect an association between HIF-1 and SCF or Trx-1 in A549-cells in this study. Whereas Trx-1-expression was neither influenced by changed oxygen partial pressure nor irradiation, I could show increased SCF mRNA by quantitative Real Time-PCR and secreted protein level by ELISA after photon irradiation independent of oxygen state. In summary, this study showed that HIF-1 and its target genes (GLUT-1, LDHA; PDK, VEGF) and also SCF was

  11. Radix Tetrastigma hemsleyani flavone inhibits proliferation, migration, and invasion of human lung carcinoma A549 cells

    Zhong LR

    2016-02-01

    Full Text Available Liangrui Zhong,1 Junxian Zheng,2 Qianqian Sun,3 Kemin Wei,2 Yijuan Hu2 1Department of Oncology, Tongde Hospital of Zhejiang Province, Affiliated to Zhejiang Chinese Medical University, 2Department of Chinese Medicine, Zhejiang Academy of Traditional Chinese Medicine, 3Department of Chinese Medicine, Zhejiang Chinese Medical University, Hangzhou, Zhejiang, People’s Republic of China Abstract: Radix Tetrastigma hemsleyani flavone (RTHF is widely used as a traditional herb and has detoxification and anti-inflammatory effects. In this study, we investigated the potential effects of RTHF on the growth and metastasis of human lung adenocarcinoma A549 cells and evaluated its mechanisms. A549 cells were treated with RTHF at various concentrations for different periods. In vitro Cell Counting Kit-8 assay and colony formation methods showed that RTHF had dose- and time-dependent antiproliferation effects on A549 cells. A cell adhesion assay showed that RTHF decreased A549 cell adhesion in a dose-dependent manner. Cell invasion and migration were investigated using the Transwell assay and observed using an inverted microscope; the results showed that cell metastasis was significantly lower in the treatment group than that in the control group (P<0.01. Expression of metastasis-related matrix metalloproteinases (MMPs and tissue inhibitors of metalloproteinases (TIMPs was detected by real-time polymerase chain reaction and Western blotting. The results showed that the expression of MMP-2, MMP-9, and TIMP-1 decreased, while that of TIMP-2 increased significantly in the RTHF group when compared with the results of the control group. These results show that RTHF exhibits antigrowth and antimetastasis activity in lung cancer A549 cells by decreasing the expression of MMP-2/-9 and TIMP-1 and increasing that of TIMP-2. Keywords: flavone, radix Tetrastigma hemsleyani, metastasis, lung cancer

  12. Wogonin has multiple anti-cancer effects by regulating c-Myc/SKP2/Fbw7α and HDAC1/HDAC2 pathways and inducing apoptosis in human lung adenocarcinoma cell line A549.

    Xin-mei Chen

    Full Text Available Wogonin is a plant monoflavonoid which has been reported to inhibit cell growth and/or induce apoptosis in various tumors. The present study examined the apoptosis-inducing activity and underlying mechanism of action of wogonin in A549 cells. The results showed that wogonin was a potent inhibitor of the viability of A549 cells. Apoptotic protein changes detected after exposure to wogonin included decreased XIAP and Mcl-1 expression, increased cleaved-PARP expression and increased release of AIF and cytochrome C. Western blot analysis showed that the activity of c-Myc/Skp2 and HDAC1/HDAC2 pathways, which play important roles in tumor progress, was decreased. Quantitative PCR identified increased levels of c-Myc mRNA and decreased levels of its protein. Protein levels of Fbw7α, GSK3β and Thr58-Myc, which are involved in c-Myc ubiquitin-dependent degradation, were also analyzed. After exposure to wogonin, Fbw7α and GSK3β expression decreased and Thr58-Myc expression increased. However, MG132 was unable to prevent c-Myc degradation. The present results suggest that wogonin has multiple anti-cancer effects associated with degradation of c-Myc, SKP2, HDAC1 and HDAC2. Its ability to induce apoptosis independently of Fbw7α suggests a possible use in drug-resistance cancer related to Fbw7 deficiency. Further studies are needed to determine which pathways are related to c-Myc and Fbw7α reversal and whether Thr58 phosphorylation of c-Myc is dependent on GSK3β.

  13. Growth arrest and apoptosis via caspase activation of dioscoreanone in human non-small-cell lung cancer A549 cells

    Hansakul, Pintusorn; Aree, Kalaya; Tanuchit, Sermkiat; Itharat, Arunporn

    2014-01-01

    Background Dioscoreanone (DN) isolated from Dioscorea membranacea Pierre has been reported to exert potent cytotoxic effects against particular types of cancer. The present study was carried out to investigate the cytotoxicity of DN against a panel of different human lung cancer cell lines. The study further examined the underlying mechanisms of its anticancer activity in the human lung adenocarcinoma cell line A549. Methods Antiproliferative effects of DN were determined by SRB and CFSE assa...

  14. Effect of anti-miR-155 oligonucleotides on proliferation of human lung adenocarcinoma cell line A549%Anti-miR-155反义寡核苷酸对肺腺癌A549细胞增殖的影响

    曹学武; 安江洪; 陈正堂

    2008-01-01

    目的 观察anti-miR-155反义寡核苷酸(AMOs)对肺腺癌A549细胞增殖的影响.方法 A549细胞分为对照组和AMOs处理组,采用AMOs抑制A549细胞内miR-155的活性,液闪计数仪测定[3H]-TdR掺入量,MTT法测定细胞增殖抑制率,流式细胞仪测定细胞周期.结果 与对照组相比,AMOs显著减少A549细胞[3H]-TdR掺入量,随着浓度从10 nmol/L逐渐增加至100 nmol/L,A549细胞[3H]-TdR掺入量亦随之减少.MTT法测定细胞增殖抑制率结果显示,与对照组相比,AMOs显著抑制A549细胞的增殖.流式细胞术检测结果显示AMOs使GO/G1细胞比例显著增加,G2/M期细胞比例显著减少.结论 采用AMOs抑制A549细胞内高水平表达miR-155的活性后,可显著抑制A549细胞的增殖.

  15. Effects of Magnetic Fluid Hyperthermia Induced by An Alternative Magnetic Field on Human Carcinoma A549 Cell in vitro

    Guoqing WANG

    2011-03-01

    Full Text Available Background and objective Magnetic fluid hyperthermia (MFH is a method of heat therapy using nanometer techniques and hyperthermia. It has the advantage of high specificity of targeting. The aim of this study is to detect the effects of MFH induced by an alternating magnetic field on human being carcinoma A549 cells in vitro. Methods A human adenocarcinoma cell line A549 was cultured with various concentrations of ferroferric oxide (Fe3O4 magnetic fluid (1.5-6.0 mg/mL and exposed to an alternative magnetic field (AMF for 30 min. And then the optical density (OD of viable cell, cytotocixity index, growth curve of cells, morphologic changes of cell, cell cycle and aposptosis were measured. Results The proliferation of the A549 cells were remarkably inhibited, the OD value of viable cells decreased and cytotoxity index (CI increased; Apoptosis of the A549 cells were observed to have cell shrinkage, chromatin condensation, margination, unclear fragmentation and intact cell membrane by light and electron microscopy; The cells were inhibited in the stage S. Conclusion MFH induced by AMF could inhibit the proliferation, which promotes apoptosis and arrest at S stage of the A549 cells.

  16. Effects of gene F10 over-expression on the tumorigenicity of A549 cells

    Ya-li SONG

    2012-07-01

    Full Text Available Objective To explore the effects and mechanism of gene F10 over-expression on the tumorigenicity of A549 cells in nude mice. Methods Eighteen SPF nude mice (4-5weeks of age were randomly equally divided into the three groups: A549-WT (vaccination with wild-type strain A549, Mock-A549 (vaccination with controlled cells Mock-A549 transfected by blank vectors and F10+A549 (vaccination with F10+A549 cells which overexpressed F10 gene according to their vaccination and then revaccinated into the subcutaneous tissue of the back with cell suspension (0.1ml with 5×106 cells in each mouse. After vaccination, the mice were observed and weighed once every 3-4 days, then the tumor formation time was recorded, the tumor growth curve drawn and tumor formation rate calculated. The mice were sacrificed and the subcutaneous tumor tissues were paraffin embedded and sectioned, and stained with HE for histopathological examinati on 5 weeks after the vaccination. Meanwhile, the expressions of Caspase-3, Bax and Bcl-2 in the tumor tissue were detected by immunohistochemistry. Results The tumor formation time was significantly different (F=13.523, P=0.000 between the groups of A549-WT (12.0±1.4d, Mock-A549 (11.7±1.0d and F10+A549 (8.5±1.4d, and it was longer in the groups A549-WT and Mock-A549 than that in F10+A549 group (P 0.05. Pathological examination revealed that the tumor tissues in the groups A549-WT and Mock-A549 harbored more necrotic cells, with structure of homogeneously red stained and amorphous material. In F10+A549 group, the number of necrotic cell was less, and cell proliferation was obvious in the edge of tumor tissues, in which, microvessels were found. In groups A549-WT and Mock-A549, the expression of Bcl-2 was rarely observed, while it was obvious in F10+A549 group, with positive cells diffusely distributed. In groups A549-WT and Mock-A549, there was strong the expression of both Bax and Caspase-3, especially in the A549-WT group. But the

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

    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

  18. A novel aminothiazole KY-05009 with potential to inhibit Traf2- and Nck-interacting kinase (TNIK) attenuates TGF-β1-mediated epithelial-to-mesenchymal transition in human lung adenocarcinoma A549 cells.

    Kim, Jiyeon; Moon, Seong-Hee; Kim, Bum Tae; Chae, Chong Hak; Lee, Joo Yun; Kim, Seong Hwan

    2014-01-01

    Transforming growth factor (TGF)-β triggers the epithelial-to-mesenchymal transition (EMT) of cancer cells via well-orchestrated crosstalk between Smad and non-Smad signaling pathways, including Wnt/β-catenin. Since EMT-induced motility and invasion play a critical role in cancer metastasis, EMT-related molecules are emerging as novel targets of anti-cancer therapies. Traf2- and Nck-interacting kinase (TNIK) has recently been considered as a first-in-class anti-cancer target molecule to regulate Wnt signaling pathway, but pharmacologic inhibition of its EMT activity has not yet been studied. Here, using 5-(4-methylbenzamido)-2-(phenylamino)thiazole-4-carboxamide (KY-05009) with TNIK-inhibitory activity, its efficacy to inhibit EMT in cancer cells was validated. The molecular docking/binding study revealed the binding of KY-05009 in the hinge region of TNIK, and the inhibitory activity of KY-05009 against TNIK was confirmed by an ATP competition assay (Ki, 100 nM). In A549 cells, KY-05009 significantly and strongly inhibited the TGF-β-activated EMT through the attenuation of Smad and non-Smad signaling pathways, including the Wnt, NF-κB, FAK-Src-paxillin-related focal adhesion, and MAP kinases (ERK and JNK) signaling pathways. Continuing efforts to identify and validate potential therapeutic targets associated with EMT, such as TNIK, provide new and improved therapies for treating and/or preventing EMT-based disorders, such as cancer metastasis and fibrosis. PMID:25337707

  19. A novel aminothiazole KY-05009 with potential to inhibit Traf2- and Nck-interacting kinase (TNIK attenuates TGF-β1-mediated epithelial-to-mesenchymal transition in human lung adenocarcinoma A549 cells.

    Jiyeon Kim

    Full Text Available Transforming growth factor (TGF-β triggers the epithelial-to-mesenchymal transition (EMT of cancer cells via well-orchestrated crosstalk between Smad and non-Smad signaling pathways, including Wnt/β-catenin. Since EMT-induced motility and invasion play a critical role in cancer metastasis, EMT-related molecules are emerging as novel targets of anti-cancer therapies. Traf2- and Nck-interacting kinase (TNIK has recently been considered as a first-in-class anti-cancer target molecule to regulate Wnt signaling pathway, but pharmacologic inhibition of its EMT activity has not yet been studied. Here, using 5-(4-methylbenzamido-2-(phenylaminothiazole-4-carboxamide (KY-05009 with TNIK-inhibitory activity, its efficacy to inhibit EMT in cancer cells was validated. The molecular docking/binding study revealed the binding of KY-05009 in the hinge region of TNIK, and the inhibitory activity of KY-05009 against TNIK was confirmed by an ATP competition assay (Ki, 100 nM. In A549 cells, KY-05009 significantly and strongly inhibited the TGF-β-activated EMT through the attenuation of Smad and non-Smad signaling pathways, including the Wnt, NF-κB, FAK-Src-paxillin-related focal adhesion, and MAP kinases (ERK and JNK signaling pathways. Continuing efforts to identify and validate potential therapeutic targets associated with EMT, such as TNIK, provide new and improved therapies for treating and/or preventing EMT-based disorders, such as cancer metastasis and fibrosis.

  20. Iron stimulates plasma-activated medium-induced A549 cell injury.

    Adachi, Tetsuo; Nonomura, Saho; Horiba, Minori; Hirayama, Tasuku; Kamiya, Tetsuro; Nagasawa, Hideko; Hara, Hirokazu

    2016-01-01

    Non-thermal atmospheric pressure plasma is applicable to living cells and has emerged as a novel technology for cancer therapy. Plasma has recently been shown to affect cells not only by direct irradiation, but also by indirect treatments with previously prepared plasma-activated medium (PAM). Iron is an indispensable element but is also potentially toxic because it generates the hydroxyl radical (•OH) in the presence of hydrogen peroxide (H2O2) via the Fenton reaction. The aim of the present study was to demonstrate the contribution of iron to PAM-induced A549 adenocarcinoma cell apoptosis. We detected the generation of •OH and elevation of intracellular ferrous ions in PAM-treated cells and found that they were inhibited by iron chelator. The elevations observed in ferrous ions may have been due to their release from the intracellular iron store, ferritin. Hydroxyl radical-induced DNA injury was followed by the activation of poly(ADP-ribose) polymerase-1, depletion of NAD(+) and ATP, and elevations in intracellular Ca(2+). The sensitivities of normal cells such as smooth muscle cells and keratinocytes to PAM were less than that of A549 cells. These results demonstrated that H2O2 in PAM and/or •OH generated in the presence of iron ions disturbed the mitochondrial-nuclear network in cancer cells. PMID:26865334

  1. Exosomes: Decreased Sensitivity of Lung Cancer A549 Cells to Cisplatin

    Xia Xiao; Shaorong Yu; Shuchun Li; Jianzhong Wu; Rong Ma; Haixia Cao; Yanliang Zhu; Jifeng Feng

    2014-01-01

    Exosomes are small extracellular membrane vesicles of endocytic origin released by many cells that could be found in most body fluids. The main functions of exosomes are cellular communication and cellular waste clean-up. This study was conducted to determine the involvement of exosomes in the regulation of sensitivity of the lung cancer cell line A549 to cisplatin (DDP). When DDP was added to A549 cells, exosomes secretion was strengthened. Addition of the secreted exosomes to other A549 cel...

  2. Diallyl trisnlfide induces apoptosis and inhibits proliferation of A549 cells in vitro and in vivo

    Wenjun Li; Bin Hao; Cun Gao; Libo Si; Fei Gao; Hui Tian; Lin Li; Shuhai Li; Weiming Yue; Zhitao Chen; Lei Qi; Wensi Hu; Yingchao Zhu

    2012-01-01

    Lung cancer is the leading cause of cancer-related mortality all over the world.In recent years,pulmonary adenocarcinoma has surpassed squamous cell carcinoma in frequency and is the predominant form of lung cancer in many countries.Epidemiological investigations have shown an inverse relationship between garlic (Allium sativum) consumption and death rate from many cancers.Diallyl trisulfide (DATS) is one of the garlic-derived compounds (also known as:organosulfer compounds,OSC).DATS can induce apoptosis and inhibit the growth of many cancer cell lines.Our study demonstrated that the apoptotic incidents induced by DATS were a mitochondriadependent caspase cascade through a significant decrease of the anti-apoptotic Bcl-2 that resulted in up-regulation of the ratio of Bax/Bcl-2 and the activity of caspase-3,-8,and -9.Eventually,DATS induced the apoptosis and inhibitedthe proliferation in a concentration- and time-dependentmanner.Furthermore,by establishing an animal model of female BALB/c nude mice with A549 xenografts,we found that oral gavage of DATS significantly retarded growth of A549 xenografts in nude mice without causing weight loss or any other side effects compared with the control group.All the evidence both in vitro and in vivo suggested that DATS could be an ideal anti-cancer drug.

  3. Mechanism of Thymosin Beta 10 Inhibiting the Apoptosis 
and Prompting Proliferation in A549 Cells

    Zixuan LI

    2014-11-01

    Full Text Available Background and objective Thymosin beta 10 (Tβ10 is one of β-thymosin family members, has a highly conserved polar 5 kDa peptides. This peptide is now regarded to be a small actin-binding protein and thereby induce depolymerization of the intracellular F-actin networks. Alteration of Tβ10 expression may alter the balance of cell growth, cell death, cell attachment and cell migration. Tβ10 also affects cell metastasis as well as proliferation, apoptosis and vascularization of cancer cells. But function of Tβ10 appear to be rather different between cancer cells, and the molecular mechanisms of β-thymosins to regulate cell apoptosis and proliferation in NSCLC (non-small cell lung cancer cell lines are unclear. In this study, we used lung adenocarcinoma cell line A549, added Tβ10 or down-regulated the expression of Tβ10. We observed the change of apoptosis, proliferation and cell cyclin ability in A549 and the mechanisms underline them were also identified. Methods After A549 was treated with 100 ng/mL recombinant human Tβ10 or siTβ10, apoptosis rate of A549 and cell cycle distribution were detected by flow cytometry (FCM. CCK-8 assay was employed to determine the proliferation of A549. The mRNA level of P53, Caspase-3, Cyclin A and Cyclin E were determined by real-time PCR. The protein level of P53, Caspase-3, Cyclin A and Cyclin E were detected by Western blot. Results Add Tβ10 can inhibit the apoptosis and prompt the proliferation of A549. It can also increase the cell rates of S-phrase and G2/M-phrase, decrease the expression of P53 and Caspase-3, but increase the expression of Cyclin A and Cyclin E. Interferance of Tβ10 can prompt the apoptosis and inhibit the proliferation of A549. It can also increase the cell rates of G0/G1-phrase, increase the expression of P53 and Caspase-3, but decrease the expression of Cyclin A and Cyclin E. Conclusion In lung cancer cell line, Tβ10 can inhibit the apoptosis by increase P53, drive cells into

  4. Ophiopogonin B induces apoptosis, mitotic catastrophe and autophagy in A549 cells.

    Chen, Meijuan; Guo, Yuanyuan; Zhao, Ruolin; Wang, Xiaoxia; Jiang, Miao; Fu, Haian; Zhang, Xu

    2016-07-01

    Ophiopogonin B (OP-B), a saponin compound isolated from Radix Ophiopogon japonicus, was verified to inhibit cell proliferation in numerous non-small cell lung cancer (NSCLC) cells in our previous study. However, the precise mechanisms of action have remained unclear. In the present study, we mainly investigated the effects of OP-B on adenocarcinoma A549 cells to further elaborate the underlying mechanisms of OP-B in different NSCLC cell lines. Detection by high content screening (HCS) and TUNEL assay verified that OP-B induced apoptosis in this cell line, while detection of Caspase-3, Bcl-2 and Bax showed that OP-B induced cell death was caspase and mitochondrial independent. Further experiments showed that OP-B induced cell cycle arrest in the S and G2/M phases by inhibiting the expression of Myt1 and phosphorylation of Histone H3 (Ser10), which resulted in mitotic catastrophe in the cells. Transmission electron microscopy (TEM) observation of cell micro-morphology combined with detection of Atgs by western blot analysis showed that OP-B induced autophagy in this cell line. Autophagy inhibition by the lysosome inhibitor CQ or Beclin1-siRNA knockdown both attenuated cell viability, demonstrated that autophagy also being the vital reason resulted in cell death. More importantly, the xenograft model using A549 cells provided further evidence of the inhibition of OP-B on tumor proliferation. Immunohistochemistry detection of LC3 and Tunel assay both verified that high dose of OP-B (75 mg/kg) induced autophagy and apoptosis in vivo, and western blot detection of p-Histone H3 (Ser10), Survivin and XIAP further indicated the molecular mechanism of OP-B in vivo. As our findings revealed, multiple types of cell death overlapped in OP-B treated A549 cells, it displayed multitarget characteristics of the compounds extracted from the Chinese herbal, which may be used as candidate anticancer medicine in clinic. PMID:27175570

  5. Mimulone-Induced Autophagy through p53-Mediated AMPK/mTOR Pathway Increases Caspase-Mediated Apoptotic Cell Death in A549 Human Lung Cancer Cells

    An, Hyun-Kyu; Kim, Kyoung-Sook; Lee, Ji-Won; Park, Mi-Hyun; Moon, Hyung-In; Park, Shin-Ji; Baik, Ji-Sue; Kim, Cheorl-Ho; Lee, Young-Choon

    2014-01-01

    Anticancer properties and mechanisms of mimulone (MML), C-geranylflavonoid isolated from the Paulownia tomentosa fruits, were firstly elucidated in this study. MML prevented cell proliferation in a dose- and time-dependent way and triggered apoptosis through the extrinsic pathway in A549 human lung adenocarcinoma cells. Furthermore, MML-treated cells displayed autophagic features, such as the formation of autophagic vacuoles, a primary morphological feature of autophagy, and the accumulation ...

  6. Identification and Isolation of Cancer Stem Cells from A549 Cells

    Hui XIA

    2013-08-01

    Full Text Available Background and objective Lung cancer stem cells are the root causes of lung cancer malignant phenotype and potential therapeutic target, the aim of this study is to isolate and characterize the cancer stem cells in the lung adenoearcinomas cell line A549, so as to provide an experimental basis for further stem cell research. Methods The cancer stem cells were isolated from the lung adenoearcinomas cell line A549 using FACS. And the difference of colony formation, cell proliferation and tumorigenicity in vitro were also tested. The expression of CD133 and ABCG2 were evaluated by RT-PCR and Western blot. Results The percentage of SP cells was 5.93% of A549 and 0.32% of A549 after incubation with verapamil. The results showed that there were significantly higher expression of CD133 and ABCG2 on SP cells than that of non-SP cells. And the ability of colony formation, cell proliferation and tumorigenicity in SP cell group were remarkably higher than that in non-SP cell group. Conclusion Our results suggested that the cancer stem cells with higher expression of CD133 and ABCG2 can be isolated from the lung adenoearcinomas cell line A549 using FACS and be used in the further research experiments.

  7. Xylitol induces cell death in lung cancer A549 cells by autophagy.

    Park, Eunjoo; Park, Mi Hee; Na, Hee Sam; Chung, Jin

    2015-05-01

    Xylitol is a widely used anti-caries agent that has anti-inflammatory effects. We have evaluated the potential of xylitol in cancer treatment. It's effects on cell proliferation and cytotoxicity were measured by MTT assay and LDH assay. Cell morphology and autophagy were examined by immunostaining and immunoblotting. Xylitol inhibited cell proliferation in a dose-dependent manner in these cancer cells: A549, Caki, NCI-H23, HCT-15, HL-60, K562, and SK MEL-2. The IC50 of xylitol in human gingival fibroblast cells was higher than in cancer cells, indicating that it is more specific for cancer cells. Moreover, xylitol induced autophagy in A549 cells that was inhibited by 3-methyladenine, an autophagy inhibitor. These results indicate that xylitol has potential in therapy against lung cancer by inhibiting cell proliferation and inducing autophagy of A549 cells. PMID:25650339

  8. β-elemene reverses the drug resistance of lung cancer A549/DDP cells via the mitochondrial apoptosis pathway.

    Yao, Cheng-Cai; Tu, Yuan-Rong; Jiang, Jie; Ye, Sheng-Fang; Du, Hao-Xin; Zhang, Yi

    2014-05-01

    β-elemene (β-ELE) is a new anticancer drug extracted from Curcuma zedoaria Roscoe and has been widely used to treat malignant tumors. Recent studies have demonstrated that β-ELE reverses the drug resistance of tumor cells. To explore the possible mechanisms of action of β-ELE, we investigated its effects on cisplatin-resistant human lung adenocarcinoma A549/DDP cells. The effects of β-ELE on the growth of A549/DDP cells in vitro were determined by MTT assay. Apoptosis was assessed by fluorescence microscopy with Hoechst 33258 staining and flow cytometry with Annexin V-FITC/PI double staining. Mitochondrial membrane potential was assessed using JC-1 fluorescence probe and laser confocal scanning microscopy, and intracellular reactive oxygen species levels were measured by 2',7'-dichlorofluorescein-diacetate staining and flow cytometry. Cytosolic glutathione content was determined using GSH kits. The expression of cytochrome c, caspase-3, procaspase-3 and the Bcl-2 family proteins was assessed by western blotting. The results demonstrated that β-ELE inhibited the proliferation of A549/DDP cells in a time- and dose-dependent manner. Furthermore, β-ELE enhanced the sensitivity of A549/DDP cells to cisplatin and reversed the drug resistance of A549/DDP cells. Consistent with a role in activating apoptosis, β-ELE decreased mitochondrial membrane potential, increased intracellular reactive oxygen species concentration and decreased the cytoplasmic glutathione levels in a time- and dose-dependent manner. The combination of β-ELE and cisplatin enhanced the protein expression of cytochrome c, caspase-3 and Bad, and reduced protein levels of Bcl-2 and procaspase-3 in the A549/DDP lung cancer cells. These results define a pathway of procaspase‑3-β-ELE function that involves decreased mitochondrial membrane potential, leading to apoptosis triggered by the release of cytochrome c into the cytoplasm and the modulation of apoptosis-related genes. The reversal of drug

  9. [Grape seed proanthocyanidins inhibits the invasion and migration of A549 lung cancer cells].

    Zhou, Yehan; Ye, Xiufeng; Shi, Yao; Wang, Ke; Wan, Dan

    2016-02-01

    Objective To explore the effect of grape seed proanthocyanidins (GSPs) on the invasion and migration of A549 lung cancer cells and the underlying mechanism. Methods Trypan blue dye exclusion assay was used to determine the cytotoxic effect of varying doses of GSPs on the BEAS-2B normal human pulmonary epithelial cells. After treated with 0, 10, 20, 40, 80 μg/mL GSP, the proliferation of A549 cells was detected by MTT assay; the invasion and migration of A549 cells were determined by Transwell(TM) assay and scratch wound assay, respectively. The levels of epithelial growth factor receptor (EGFR), E-cadherin, N-cadherin in A549 cells treated with GSPs were detected by Western blotting. Results (0-40) μg/mL GSPs had no significant toxic effect on BEAS-2B cells, while 80 μg/mL GSPs had significant cytotoxicity to BEAS-2B cells. The proliferation of A549 cells was significantly inhibited within limited dosage in a dose-dependent manner, and the abilities of invasion and migration of A549 cells were also inhibited. Western blotting showed that the expression of EGFR and N-cadherin decreased, while E-cadherin increased after GSPs treatment. Conclusion GSPs could inhibit the abilities of proliferation, invasion and migration of A549 cells, which might be related to the dow-regulation of EGFR and N-cadherin and the up-regulation of E-cadherin. PMID:26927375

  10. Influence of suppressor gene p16 on retinoic acid inducing cancer cell A549 differentiation

    2001-01-01

    Objective To investigate the role of suppressor gene p16 in the process of differential regulation of retinoic acid (RA) on the A549 lung cancer cells.Methods Tumor suppressor gene p16 was transferred into A549 cells and the cells were treated with all-trans retinoic acid (ATR) at the dosage of 5×10-6 mol/L for 4 d. After that, the proliferation and differentiation of A549 cells were examined by growth curve and cytometry analysis, the change of lung lineage-specific marker MUC1 was tested by immunohistochemical staining. Meanwhile, Western blot was used to observe the change of p16 protein expression in A549 cells treated with ATRA.Results ATRA could obviously inhibit the growth and induce the differentiation of A549 Cells that were transferred with p16 gene. There were more cells arrested in G1/G0 phase and the expression of MUG1 was markedly down-regulated than in control cells. The expression of p16 protein was up-regulated in A549 cells treated with ATRA.Conclusion Suppressor gene p16 could enhance the effects of RA and proliferated suppression and differential induction of A549 cells.

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

    Won, Joo Yoon; Park, Jong Kuk; Hong, Sung Hee [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)

    2006-07-01

    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.

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

    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

  13. Klotho inhibits growth and promotes apoptosis in human lung cancer cell line A549

    Zhao Weihong

    2010-07-01

    Full Text Available Abstract Background Klotho, as a new anti-aging gene, can shed into circulation and act as a multi-functional humoral factor that influences multiple biological processes. Recently, published studies suggest that klotho can also serve as a potential tumor suppressor. The aim of this study is to investigate the effects and possible mechanisms of action of klotho in human lung cancer cell line A549. Methods In this study, plasmids encoding klotho or klotho specific shRNAs were constructed to overexpress or knockdown klotho in vitro. A549 cells were respectively treated with pCMV6-MYC-KL or klotho specific shRNAs. The MTT assay was used to evaluate the cytotoxic effects of klotho and flow cytometry was utilized to observe and detect the apoptosis of A549 cells induced by klotho. The activation of IGF-1/insulin signal pathways in A549 cells treated by pCMV6-MYC-KL or shRNAs were evaluated by western blotting. The expression levels of bcl-2 and bax transcripts were evaluated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR. Results Overexpression of klotho reduced the proliferation of lung cancer A549 cells, whereas klotho silencing in A549 cells enhanced proliferation. Klotho did not show any effects on HEK-293 cells. Klotho overexpression in A549 cells was associated with reduced IGF-1/insulin-induced phosphorylation of IGF-1R (IGF-1 receptor/IR (insulin receptor (P P P P P Conclusions Klotho can inhibit proliferation and increase apoptosis of A549 cells, this may be partly due to the inhibition of IGF-1/insulin pathways and involving regulating the expression of the apoptosis-related genes bax/bcl-2. Thus, klotho can serve as a potential tumor suppressor in A549 cells.

  14. Epithelial mesenchymal transition of non-small-cell lung cancer cells A549 induced by SPHK1

    Min Ni; Xiao-Lei Shi; Zhi-Gang Qu; Hong Jiang; Zi-Qian Chen; Jun Hu

    2015-01-01

    Objective:To explore the effect and molecular mechanism ofSPHK1 in the invasion and metastasis process of non-small-cell lung cancer cells(A549).Methods:Recombinant retrovirus was used to mediate the production ofA549/vector,A549/SPHK1,A549/scramble, andA549/SPHK1/RNAi that stably expressed or silencedSPHK1.The invasion and migration capacities of A549 cells overexpressing or silencingSPHK1 were determined usingTranswell invasion assay and scratch wound repair experiment.The protein and mRNA expression levels ofE-cadherin, fibronectin, vimentin inA549/vector,A549/SPHK1,A549/scramble,A549/SPHK1/RNAi were detected withWestern blot(WB) and quantitativePCR(QPCR) methods, respectively.Results:Transwell invasion assay and scratch wound repair experiments showed that over-expression of SPHK1 obviously enhanced the invasion and migration capacities ofA549 cells.WB andQPCR detection results showed that, the expression ofE-cadherin(a molecular marker of epithelial cells) and fibronectin, vimentin(molecular markers of mesenchymal cells) inA549 cells was upregulated after overexpression ofSPHK1; whileSPHK1 silencing significantly reduced the invasion and metastasis capacities ofA549cells, upregulated the expression of molecular marker of epithelial cells, and downregulated the expression of molecular marker of mesenchymal cells. Conclusions:SPHK1 promotes epithelial mesenchymal transition of non-small-cell lung cancer cells and affects the invasion and metastasis capacities of these cells.

  15. Radiation-Induced Bystander Effects in A549 Cells Exposed to 6 MV X-rays.

    Yang, Shuning; Xu, Jing; Shao, Weixian; Geng, Chong; Li, Jia; Guo, Feng; Miao, Hui; Shen, Wenbin; Ye, Tao; Liu, Yazhou; Xu, Haiting; Zhang, Xuguang

    2015-07-01

    The aim of the study is to explore the bystander effects in A549 cells that have been exposed to 6MV X-ray. Control group, irradiated group, irradiated conditioned medium (ICM)-received group, and fresh medium group were designed in this study. A549 cells in the logarithmic growth phase were irradiated with 6MV X-ray at 0, 0.5, 1, 1.5, and 2. In ICM-received group, post-irradiation A549 cells were cultured for 3 h and were transferred into non-irradiated A549 cells for further cultivation. Clone forming test was applied to detect the survival fraction of cells. Annexin V-FITC/PI double-staining assay was used to detect the apoptosis of A549 cells 24, 48, 72, and 96 h after 2-Gy 6MV X-ray irradiation, and the curves of apoptosis were drawn. The changes in the cell cycles 4, 48, 72, and 96 h after 2-Gy 6MV X-ray irradiation were detected using PI staining flow cytometry. With the increase of irradiation dose, the survival fraction of A549 cells after the application of 0.5 Gy irradiation was decreasing continuously. In comparison to the control group, the apoptosis rate of the ICM-received group was increased in a time-dependent pattern, with the highest apoptosis rate observed at 72 h (p A549 cell damage, indicating that 6MV X-ray irradiation can induce bystander effect on A549 cells, which reaches a peak at 72 h. PMID:25686868

  16. Effcts of Vitamin C on A549 Cell Proliferation, Apoptosis and Expressions of Caspase, Survivin

    Lanzhen HUANG

    2010-02-01

    Full Text Available Background and objective It was proven that Vitamin C could inhibit the growth of many types of tumors as an antioxidant. The aim of this study is to explore role of Vitamin C in proliferation and apoptosis of lung carcinoma cell line A549 and the underlying mechanism. Methods A549 cells were cultured in vitro and incubated with Vitamin C. The cell viability was measured by growth curve and clonogentic assay. Flow cytometry was used to analyze cell cycle and detect apoptosis. The levels of expression of Caspase-3 mRNA and Survivin mRNA were detected by RT-PCR. Results Vitamin C of 400 μg/mL, 4 mg/mL significantly inhibited the growth of A549 cell lines (P=0.024, P=0.015, respectively. Flow cytometry showed that the cells major stagnation stayed in the G0/G1 and S phase and the apoptotic rate increased with time prolonged. Vitamin C signifiantly up-ragulated the expression of Caspase-3 mRNA, but had no effect on Survivin mRNA. Conclusion Vitamin C can inhibit the proliferation of A549, block A549 cells in G0/G1 and S phase, and induce apoptosis of A549 cells. Apotosis occurred by up-ragulated the expressionof Caspase-3.

  17. Upregulation of Id3 inhibits cell proliferation and induces apoptosis in A549/DDP human lung cancer cells in vitro.

    Chen, Fangfang; Zhao, Qinfei; Wang, Shuxia; Wang, Haiyong; Li, Xiaojun

    2016-07-01

    Inhibitor of DNA binding (Id)3 is a member of the Id multigene family of dominant‑negative helix‑loop-helix transcription factors, which function as oncogenes or tumor suppressors in human cancers. Its upregulation was recently shown to have inhibitory effects on lung cancer, which is the leading cause of cancer‑associated mortality worldwide. As drug resistance represents a major bottleneck of cancer therapy, the present study assessed the ability of Id3 to inhibit cisplatin‑resistant A549 lung adenocarcinoma cells (A549/DDP). A549/DPP cells were transiently transfected with enhanced green fluorescence protein overexpression plasmid (pEGFP) or Id3 overexpression plasmid (Id3/pEGFP), which was confirmed by confocal fluorescence microscopy, PCR and western blot analysis. The effects of Id3 on the viability and apoptosis of A549/DDP were determined using an MTT assay, fluorescence microscopy with Hoechst 33258 staining and flow cytometry following Annexin V/propidium iodide double staining. The results revealed that overexpression of Id3 significantly inhibited the proliferation and viability of A549/DDP cells in a time‑dependent manner. Furthermore, overexpression of Id3 significantly increased the apoptotic rate of A549/DDP cells from 2.73 to 16.92%, confirming the implication of Id3 in the negative control of tumour growth. The results of the present study revealed that overexpression of Id3 may serve as a novel strategy for inhibiting cisplatin‑sensitive lung cancer. Further experiments will be performed to determine whether Id3 overexpression could enhance the sensitivity of lung cancer cells to DDP. PMID:27176047

  18. Runx3 Expression Inhibits Proliferation and Distinctly Alters mRNA Expression of Bax in AGS and A549 Cancer Cells

    Torshabi, Maryam; Faramarzi, Mohammad Ali; Tabatabaei Yazdi, Mojtaba; Ostad, Seyyed Naser; Gharemani, Mohammad Hosein

    2011-01-01

    Runx3, a member of Runt-related transcription factor (Runx) proteins with tumor suppressor effect, is a tissue–restricted and cancer related transcription factor that regulate cell proliferation and growth, as well as differentiation. In the present study, exogenous Run3 was transiently expressed in AGS (human gastric adenocarcinoma), with undetectable Runx3 protein and in A549 (human lung carcinoma) with low levels of endogenous Runx3 protein. The GFP tagged Runx3 was transfected into AGS an...

  19. The effect of β-elemene combined with irradiation on DNA damage and repair in A549 cells

    Objective: To study if β-elemene can increase radiation-induced deoxyribonucleic acid (DNA) damage and decrease the damage repair. Methods: Exponentially growing human lung adenocarcinoma cells (A549) were exposed to 10 or 20 μg/ml β-elemene for 24 h before irradiation.The effect of β-elemene on the in vitro radiosensitivity of A549 cells was evaluated using clonogenic assay. DNA damage and repair were evaluated using comet assay. Results: Exposure to β-elemene before irradiation increased the radiosensitivity of A549 cells. The SERD0 for 10 μg/ml and 20 μg/ml β-elemene was 1.55 and 1.64, respectively. The SERDq for 10 μg/ml and 20 μg/ml β-elemene was 1.43 and 1.75, respectively. Combined treatment, comparing to irradiation or β-elemene treatment alone, induced higher levels of DNA damage and slower rate of damage repair. A549 cells exposed to 20 μg/ml β-elemene followed by irradiation showed a higher levels of tail moment (TM) than those exposed to irradiation or β-elemene alone at 0 h, 2 h, 6 h and 24 h after irradiation. The TM of the three groups at 0 h, 2 h, 6 h and 24 h after irradiation was 7.16±2.61, 0.95±0.65 and 1.81±1.23 (F=231.24, P<0.01), 3.65±2.06, 0.11±0.07 and 1.58±1.40(F=90.22, P<0.01), 2.09±0.83, 0.1±0.05 and 0.45±0.25 (F=238.44, P<0.01), 1.45±1.37, 0.11±0.08 and 0.60±0.40 (F=38.94, P<0.01), respectively. Conclusions: β-elemene can enhance the radiosensitivity of A549 cells through the enhancement of DNA damage and the inhibition of DNA damage repair. (authors)

  20. Wnt/β-catenin signaling regulates cancer stem cells in lung cancer A549 cells

    Wnt/β-catenin signaling plays an important role not only in cancer, but also in cancer stem cells. In this study, we found that β-catenin and OCT-4 was highly expressed in cisplatin (DDP) selected A549 cells. Stimulating A549 cells with lithium chloride (LiCl) resulted in accumulation of β-catenin and up-regulation of a typical Wnt target gene cyclin D1. This stimulation also significantly enhanced proliferation, clone formation, migration and drug resistance abilities in A549 cells. Moreover, the up-regulation of OCT-4, a stem cell marker, was observed through real-time PCR and Western blotting. In a reverse approach, we inhibited Wnt signaling by knocking down the expression of β-catenin using RNA interference technology. This inhibition resulted in down-regulation of the Wnt target gene cyclin D1 as well as the proliferation, clone formation, migration and drug resistance abilities. Meanwhile, the expression of OCT-4 was reduced after the inhibition of Wnt/β-catenin signaling. Taken together, our study provides strong evidence that canonical Wnt signaling plays an important role in lung cancer stem cell properties, and it also regulates OCT-4, a lung cancer stem cell marker.

  1. Effect of Juglone in qinglongyi on cell cycle status and apoptosis in A-549 cells

    ZOU Xiang; KONG Ling-sheng; JI Yu-bin

    2008-01-01

    Objective To explore the inhibition of juglone in Qinglongyi on A-549 cells in vitro. Methods MTT assay was used. Laser confocal scanning microscope was used to observe apoptotic morphology.Changes of cell cycle are studied by flow cytometry analysis. Results MTT assay showed that juglone had a marked growth inhibition in A-549 cells and the IC50 is respectively 3.4×10-5 mol·L-1, 1.8×10-5 mol·L-1 and 2.6×10-6 mol·L-1 after treatment for 24, 48 and 72 h by juglone. Through Laser confocal scanning microscope, we can see that juglone can induce the apoptosis. Cell cycle changes are analyzed by flow cytometry with cells at G1 phase significantly less than those of control and ceils at G2 phase significantly more than those of control. Conclusions It suggests that juglone could apoptosis of A-549 cells with the cell cycle arrest on G2 phase in distinct dose-dependent manner.

  2. MicroRNA-126 inhibits the proliferation of lung cancer cell line A549

    Xun Yang; Bei-Bei Chen; Ming-Hua Zhang; Xin-Rong Wang

    2015-01-01

    Objective:To study the role of microRNA-126 in the development of lung cancer.Methods:The biological function of microRNA-126 was detected using EdU assay and CCK-8 assay;the target gene of microRNA-126 was analyzed using real time RT-PCR and Western blot assay.Results: In A549 cell line, overexpression of microRNA-126 inhibits the proliferation rate; VEGF is the target gene of microRNA-126; microRNA-126 exerts its function via regulating VEGF protein level.Conclusions: microRNA-126 inhibits the proliferation in A549 cell line.

  3. EGCG reverses human neutrophil elastase-induced migration in A549 cells by directly binding to HNE and by regulating α1-AT

    Xiaokaiti, Yilixiati; Wu, Haoming; Chen, Ya; Yang, Haopeng; Duan, Jianhui; Li, Xin; Pan, Yan; Tie, Lu; Zhang, Liangren; Li, Xuejun

    2015-07-01

    Lung carcinogenesis is a complex process that occurs in unregulated inflammatory environment. EGCG has been extensively investigated as a multi-targeting anti-tumor and anti-inflammatory compound. In this study, we demonstrated a novel mechanism by which EGCG reverses the neutrophil elastase-induced migration of A549 cells. We found that neutrophil elastase directly triggered human adenocarcinoma A549 cell migration and that EGCG suppressed the elevation of tumor cell migration induced by neutrophil elastase. We observed that EGCG directly binds to neutrophil elastase and inhibits its enzymatic activity based on the CDOCKER algorithm, MD stimulation by GROMACS, SPR assay and elastase enzymatic activity assay. As the natural inhibitor of neutrophil elastase, α1-antitrypsin is synthesized in tumor cells. We further demonstrated that the expression of α1-antitrypsin was up-regulated after EGCG treatment in neutrophil elastase-treated A549 cells. We preliminarily discovered that the EGCG-mediated induction of α1-antitrypsin expression might be correlated with the regulatory effect of EGCG on the PI3K/Akt pathway. Overall, our results suggest that EGCG ameliorates the neutrophil elastase-induced migration of A549 cells. The mechanism underlying this effect may include two processes: EGCG directly binds to neutrophil elastase and inhibits its enzymatic activity; EGCG enhances the expression of α1-antitrypsin by regulating the PI3K/AKT pathway.

  4. Expressions and Significances of PRL-3 and RhoC in A549 Cell

    Ping ZHANG

    2010-12-01

    Full Text Available Background and objective The expression of phosphatase of regenerating liver-3 (PRL-3 is correlated with Ras homologue C (RhoC in non-small cell lung cancer (NSCLC, suggesting that they have interactions. The aim of this study is to investigate the functions of PRL-3 and RhoC in the migration of A549 cell and the potential mechanism of PRL-3 and RhoC in carcinogenesis and cancer development. Methods PRL-3Ab and RhoCAb were used to block the functions of PRL-3 and RhoC respectively. Wound healing assay was applied to detect the migration of A549 cell and the expression levels of PRL-3 and RhoC were detected by RT-PCR. Results The migration of A549 cell decreased after blockage of PRL-3 and RhoC. The expression of RhoC decreased when PRL-3 was blocked without any changes on the expression of PRL-3. Conclusion PRL-3, RhoC could increase cell migration in A549 cells.

  5. Human lung epithelial cell A549 proteome data after treatment with titanium dioxide and carbon black.

    Vuong, Ngoc Q; Goegan, Patrick; Mohottalage, Susantha; Breznan, Dalibor; Ariganello, Marianne; Williams, Andrew; Elisma, Fred; Karthikeyan, Subramanian; Vincent, Renaud; Kumarathasan, Premkumari

    2016-09-01

    Here, we have described the dataset relevant to the A549 cellular proteome changes after exposure to either titanium dioxide or carbon black particles as compared to the non-exposed controls, "Proteomic changes in human lung epithelial cells (A549) in response to carbon black and titanium dioxide exposures" (Vuong et al., 2016) [1]. Detailed methodologies on the separation of cellular proteins by 2D-GE and the subsequent mass spectrometry analyses using MALDI-TOF-TOF-MS are documented. Particle exposure-specific protein expression changes were measured via 2D-GE spot volume analysis. Protein identification was done by querying mass spectrometry data against SwissProt and RefSeq protein databases using Mascot search engine. Two-way ANOVA analysis data provided information on statistically significant A549 protein expression changes associated with particle exposures. PMID:27508218

  6. Comparison of Cytotoxic Activity of L778123 as a Farnesyltranferase Inhibitor and Doxorubicin against A549 and HT-29 Cell Lines

    Ali Abdollahi

    2013-02-01

    Full Text Available Purpose: Farnesyltransferase (FTase is a zinc-dependent enzyme that adds a farnesyl group to the Ras proteins. L778, 123 is a potent peptidomimetic imidazole-containing FTase inhibitor. Methods: L778123 was synthesized according to known methods and evaluated alone and in combination with doxorubicin against A549 (adenocarcinomic human alveolar basal epithelial cells and HT29 (human colonic adenocarcinoma cell lines by MTT assay. Results: L778123 showed weak cytotoxic activity with IC50 of 100 and 125 for A549 and HT-29 cell lines, respectively. The combination of doxorubicin and L778123 can decrease IC50 of doxorubicin in both cell lines significantly. Conclusion: It can be concluded that L778, 123 can be a good agent for combination therapy.

  7. Experimental Study on A549 Cell Death Mediated by Xenoantigen α-gal 
in Human Serum

    Shengming ZHU

    2012-11-01

    Full Text Available Background and objective The absence of α-gal in humans is caused by the inactivity of α-1,3GT gene. However, humans have pre-existing and abundant anti-gal antibodies. Xenotransplantation procedures have indicated the high potential of introducing α-1,3GT gene to synthesize α-gal for cancer gene therapy by mimicking hyper-acute rejection. The aim of this study is to construct a lung cancer A549 cell line that expressed α-gal, and to observe the antitumor mechanisms mediated by human serum. Methods A549 cells were transfected with pEGFP-N1-GT plasmids constructed in a previous study. A stable transgenic cell line, A549-GT, was then selected and cultivated. The biological characteristics of A549-GT cells, including morphology and proliferation, were examined. α-1,3GT mRNA expression was detected by RT-PCR. Direct immunofluorescence staining and flow cytometry (FCM were used to analyze the synthesis of α-gal in A549-GT. The binding of human serum IgM and C3 with A549-GT were also detected. Results α-1,3GT mRNA was expressed in A549-GT. Direct immunofluorescence staining and FCM indicated a high and stable α-gal expression rate in A549-GT. Compared with parental A549 cells, the biological characteristics of A549-GT were unaltered. α-Gal expression was not detected in the human fetal lung fibroblast cell line MRC-5 even though A549-GT and its culture medium were cultivated with the enzyme. Immunofluorescence staining and FCM also indicated abundant binding between A549-GT treated with human serum and IgM/C3. Conclusion α-Gal expression in tumor cells by gene transduction can induce complement-dependent cytototic antitumor effects.

  8. Treatment of transplanted tumor of lung adenocarcinoma A549 transfected by human somatostatin receptor subtype 2 (hsstr2) gene with 188Re-RC-160

    Background and aim: Radionuclide-labeled somatostatin analogues selectively target somatostatin receptor (SSTR)-expressing tumors as a basis for diagnosis and treatment of these tumors. To those tumors without somatostatin receptor expressed, the hSSTR2 gene was transfected. Express of the hSSTR2 receptor was imaging and the radiotherapeutic effect was evaluated with 188Re-RC-160. Methods: The stable hSSTR2-expressing A549 cells (pcDNA3-hSSTR2 A549) and non-somatostatin receptor expressing A549 cells (pcDNA3 A549) were selected by western blot. Later, a corresponding animal tumor model was established. Expression of the hSSTR2 reporter was imaged using 188Re-RC-160 recognition. Tumors were evaluated for somatostatin receptor expression using immunohistochemistry. The distribution of 188Re-RC-160 in the animal tumor model was measured and the inhibitory effects of 188Re-RC-160 were evaluated by measurement of tumor growth and hematoxylin and eosin and TdT mediated dUTP nick end labeling (TUNEL) staining. Results: In vivo radioimaging revealed specific targeting of 188Re-RC-160 to tumors derived from pcDNA3- hSSTR2 A549 cells, compared to those from pcDNA3 A549 cells. pcDNA3- hSSTR2 A549 tumor growth inhibition was significantly higher in the single 7.4 MBq 188Re-RC-160 treatment group than in the 2x7.4 MBq rhenium-188, RC-160 group, control group, and pcDNA3 A549 tumors (P188Re-RC-160), induced significantly increased tumor-growth inhibition compare with single 7.4 MBq 188Re-RC-160 treatment (P188Re-RC-160 could be effectively used for targeting therapy the A549-derived tumors exogenously expressing hSSTR2, which will offers a potential therapeutic strategy for the treatment of somatostatin receptor-negative cancers.

  9. Dexamethasone-(C21-phosphoramide)-[anti-EGFR]: molecular design, synthetic organic chemistry reactions, and antineoplastic cytotoxic potency against pulmonary adenocarcinoma (A549)

    Coyne, Cody P; Narayanan, Lakshmi

    2016-01-01

    Purpose Corticosteroids are effective in the management of a variety of disease states, such as several forms of neoplasia (leukemia and lymphoma), autoimmune conditions, and severe inflammatory responses. Molecular strategies that selectively “target” delivery of corticosteroids minimize or prevents large amounts of the pharmaceutical moiety from passively diffusing into normal healthy cell populations residing within tissues and organ systems. Materials and methods The covalent immunopharmaceutical, dexamethasone-(C21-phosphoramide)-[anti-EGFR] was synthesized by reacting dexamethasone-21-monophosphate with a carbodiimide reagent to form a dexamethasone phosphate carbodiimide ester that was subsequently reacted with imidazole to create an amine-reactive dexamethasone-(C21-phosphorylimidazolide) intermediate. Monoclonal anti-EGFR immunoglobulin was combined with the amine-reactive dexamethasone-(C21-phosphorylimidazolide) intermediate, resulting in the synthesis of the covalent immunopharmaceutical, dexamethasone-(C21-phosphoramide)-[anti-EGFR]. Following spectrophotometric analysis and validation of retained epidermal growth factor receptor type 1 (EGFR)-binding avidity by cell-ELISA, the selective anti-neoplasic cytotoxic potency of dexamethasone-(C21-phosphoramide)-[anti-EGFR] was established by MTT-based vitality stain methodology using adherent monolayer populations of human pulmonary adenocarcinoma (A549) known to overexpress the tropic membrane receptors EGFR and insulin-like growth factor receptor type 1. Results The dexamethasone:IgG molar-incorporation-index for dexamethasone-(C21-phosphoramide)-[anti-EGFR] was 6.95:1 following exhaustive serial microfiltration. Cytotoxicity analysis: covalent bonding of dexamethasone to monoclonal anti-EGFR immunoglobulin did not significantly modify the ex vivo antineoplastic cytotoxicity of dexamethasone against pulmonary adenocarcinoma at and between the standardized dexamethasone equivalent concentrations of 10

  10. Runx3 Expression Inhibits Proliferation and Distinctly Alters mRNA Expression of Bax in AGS and A549 Cancer Cells

    Torshabi, Maryam; Faramarzi, Mohammad Ali; Tabatabaei Yazdi, Mojtaba; Ostad, Seyyed Naser; Gharemani, Mohammad Hosein

    2011-01-01

    Runx3, a member of Runt-related transcription factor (Runx) proteins with tumor suppressor effect, is a tissue–restricted and cancer related transcription factor that regulate cell proliferation and growth, as well as differentiation. In the present study, exogenous Run3 was transiently expressed in AGS (human gastric adenocarcinoma), with undetectable Runx3 protein and in A549 (human lung carcinoma) with low levels of endogenous Runx3 protein. The GFP tagged Runx3 was transfected into AGS and A549 cells using fugene6 and PolyFect and Runx3 expression was confirmed by fluorescent microscopy and RT-PCR. The effect of Runx3 transfection on cell proliferation was determined by MTT assay and the results were confirmed by the trypan blue dye exclusion method. The effect of Runx3 expression on mRNA expression of BCL2-associated X protein (Bax) was evaluated using RT-PCR. In AGS and A549 cells, Runx3 expression inhibited cell proliferation (p < 0.01). The growth inhibition was less in A549 cells. We show that Runx3 expression increases Bax mRNA expression in AGS cells when compared with control (p < 0.05), but no significant differences in mRNA expression was observed in both examined cells. Runx3 expression has antiproliferative effect in AGS cell perhaps via increase in expression of Bax. The effect of Runx3 on A549 cells’ viability which has endogenous level of Runx3 is not related to Bax. These findings implicate a complex regulation by Runx3 in inhibition of cell proliferation utilizing Bax. PMID:24250365

  11. Aurora A反义寡核苷酸对肺癌细胞A549的作用和对紫杉醇化疗敏感性的影响%The effect of antisense oligodeoxynucleotides targeting Aurora A kinase on cell proliferation and chemosensitivity to paclitaxel in human lung cancer cell line A549

    Rui Meng; Gang Wu; Jing Cheng; Tao Wang

    2007-01-01

    Objective:Aurora A kinase representing a family of evolutionarily conserved mitotic serine/threonine kinases has been found elevated in human lung adenocarcinoma cell line A549.It is suggested that the overexpression of Aurora A contributes to the carcinogenesis,chromosomal instability (CIN),and de-differentiation of lung cancers.To address its possibility as a therapeutic target for lung cancer,we employed the antisense oligodeoxynucleotide (ASODN) technique to inhibi Aurora A expression and investigate its effects on tumor growth and cell cycle of A549.as well as the chemosensitivilty to paclitaxel.Methods:Aurora A ASODN was synthesized and transfected into A549 cells by lipofectAMINE 2000.Aurora A mRNA and protein expression were examined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot respectively.Cell cycle distribution was observed by flow cytometer.MTT assay was used to evaluate cell inhibition ratio before and after transfection.Results:The proliferation of the A549 cell swas inhibited by Aurora AASODN dose and time dependently.It was also observed thal the IC50 of A549 cells after 48 hours'treatmenl of ASODN was about 300 nmol/L and under such circumstances,the Aurora A mRNA and protein expression significantly decreased(P<0.05),along with the induction of accumulation of cells in S phase and the G2-M transition.Furlhermore.cell inhibition ratio of the combination of Aurora AASODN and paclitaxel was higher significantly than paclitaxel(P<0.05)or Aurora AASODN alone (P<0.05).Conclusion:Inhibition of Aurora A expression can result in the suppression of cell growth and chemosensilizina activity to paclitaxel in human lung cancer cell line A549.

  12. Apoptotic Effect of Coix Polysaccharides on A549 Lung Cancer Cells in Vitro

    Luo, Cheng; Liu, Wei; Lu, Xiangyi

    2012-01-01

    Background and objective Coix seeds are commonly used in Traditional Chinese Medicine and ingested through daily diet. The aim of this study is to analyze the apoptotic effect of coix polysaccharides on A549 cells. Methods A fraction of polysaccharides was isolated from coix seeds and extracted by ethanol precipitation. The extract was then purified by dialysis and DEAE-52 ion-exchange chromatography. Cell viability was determined by the MTT assay. Cell morphology was observed by scanning ele...

  13. Effect of evodiamine on the proliferation and apoptosis of A549 human lung cancer cells.

    Lin, Li; Ren, Li; Wen, Liujing; Wang, Yu; Qi, Jin

    2016-09-01

    Evodia rutaecarpa is a plant, which has antitumor activity. Evodiamine is an alkaloid with antitumor activity present in E. rutaecarpa and has potential to be developed into a therapeutic antitumor agent. The present study investigated the effect of evodiamine on the proliferation of A549 human lung cancer cells and the mechanism underlying these effects. The results indicated that evodiamine significantly inhibited proliferation, induced apoptosis and the expression of reactive oxygen species, arrested the cell cycle, regulated the expression of Survivin, Bcl-2 and Cyclin B1, regulated the activity of caspase-3/8 and glutathione in tumor cells, and decreased the activity of AKT/nuclear factor‑κB (NF‑κB) and Sonic hedgehog/GLI family zinc finger 1 (SHH/GLI1) signaling pathways in A549 cells. In conclusion, the evodiamine-induced inhibition of the proliferation of A549 lung cancer cells may be attributable to its ability to promote oxidative injury in the cells, induce apoptosis, arrest the cell cycle and regulate the AKT/NF‑κB and SHH/GLI1 signaling pathways, subsequently controlling the expression of tumor‑associated genes. PMID:27485202

  14. Brusatol Enhances the Radiosensitivity of A549 Cells by Promoting ROS Production and Enhancing DNA Damage

    Xiaohui Sun

    2016-06-01

    Full Text Available NF-E2-related factor 2 (Nrf2 has been identified as a master regulatory factor in the protection of cells from oxidative and electrophilic stress. However, overexpression of Nrf2 in lung cancer may cause chemoresistance, as well as radioresistance. In this study, we examined the relationship between radioresistance and Nrf2 protein levels in H1299, A549, and H460 cells, and finally chose the A549 cell line to continue with due to its strong radioresistance and high Nrf2 protein levels. We found that the Nrf2 inhibitor, brusatol, could prevent the increase and accumulation of Nrf2 after exposure to irradiation. Additionally, following treatment with 80 nM brusatol, A549 cells became sensitive to irradiation, suffering severe DNA damage. Combination treatment with brusatol and ionizing radiation (IR can distinctly increase the level of reactive oxygen species in A549 cells, causing a 1.8-fold increase compared with the control, and a 1.4-fold increase compared with IR alone. In fact, in the treatment with both brusatol and IR, lung cancer cell proliferation is halted, gradually leading to cell death. Because Nrf2 is closely linked to DNA damage repair, inhibiting the function of Nrf2, as in brusatol treatment, may increase the DNA damage caused by radiotherapy or chemotherapy, possibly enhancing the efficacy of chemotherapeutic drugs. Our study is the first to demonstrate brusatol’s ability to enhance the responsiveness of lung cancer cells to irradiation, and its potential application as a natural sensitizer in radiotherapy.

  15. Brusatol Enhances the Radiosensitivity of A549 Cells by Promoting ROS Production and Enhancing DNA Damage.

    Sun, Xiaohui; Wang, Qin; Wang, Yan; Du, Liqing; Xu, Chang; Liu, Qiang

    2016-01-01

    NF-E2-related factor 2 (Nrf2) has been identified as a master regulatory factor in the protection of cells from oxidative and electrophilic stress. However, overexpression of Nrf2 in lung cancer may cause chemoresistance, as well as radioresistance. In this study, we examined the relationship between radioresistance and Nrf2 protein levels in H1299, A549, and H460 cells, and finally chose the A549 cell line to continue with due to its strong radioresistance and high Nrf2 protein levels. We found that the Nrf2 inhibitor, brusatol, could prevent the increase and accumulation of Nrf2 after exposure to irradiation. Additionally, following treatment with 80 nM brusatol, A549 cells became sensitive to irradiation, suffering severe DNA damage. Combination treatment with brusatol and ionizing radiation (IR) can distinctly increase the level of reactive oxygen species in A549 cells, causing a 1.8-fold increase compared with the control, and a 1.4-fold increase compared with IR alone. In fact, in the treatment with both brusatol and IR, lung cancer cell proliferation is halted, gradually leading to cell death. Because Nrf2 is closely linked to DNA damage repair, inhibiting the function of Nrf2, as in brusatol treatment, may increase the DNA damage caused by radiotherapy or chemotherapy, possibly enhancing the efficacy of chemotherapeutic drugs. Our study is the first to demonstrate brusatol's ability to enhance the responsiveness of lung cancer cells to irradiation, and its potential application as a natural sensitizer in radiotherapy. PMID:27347930

  16. Apoptotic Effect of Coix Polysaccharides on A549 Lung Cancer Cells in Vitro

    Cheng LUO

    2012-11-01

    Full Text Available Background and objective Coix seeds are commonly used in Traditional Chinese Medicine and ingested through daily diet. The aim of this study is to analyze the apoptotic effect of coix polysaccharides on A549 cells. Methods A fraction of polysaccharides was isolated from coix seeds and extracted by ethanol precipitation. The extract was then purified by dialysis and DEAE-52 ion-exchange chromatography. Cell viability was determined by the MTT assay. Cell morphology was observed by scanning electronic microscopy (SEM, and cell cycle was detected by flow cytometry (FCM. The relative quantities of caspase-3 and caspase-9 were determined by RT-PCR. Results Coix polysaccharides exerted remarkable inhibitory effects on A549 cell proliferation. Apoptotic bodies were observed by SEM. Apoptotic induction was also verified by DNA accumulation using propidium iodide nucleus staining in the S phase by flow cytometry, as well as by DNA fragmentation using the comet assay. Regarding the molecular mechanism of apoptosis induction, the gene expression of caspase-3 and caspase-9 increased after coix polysaccharide treatment. Conclusion Polysaccharide fraction CP-1 induced A549 cell apoptosis.

  17. Transfection of gene Livin α/β into A549 cells and separate effect on the cell growth

    SUN Jian-guo; LIAO Rong-xia; CHEN Zheng-tang; WANG Zhi-xin; ZHANG Qing; HU Yi-de; WANG Dong-lin

    2005-01-01

    Objective:To express two Livin isoforms (Livin α & β genes) with transfection techniques in A549 cell line respectively in order to observe their effect on growth of cell line. Methods:Two eukaryotic expression vectors of Livin, pcDNA3.1-Livin α & β, were transfected into A549 cell line by electroporation. Then G418-resistant clones were screened. RT-PCR, Northern blot and immunofluorescence cytochemistry were used to detect Livin α & β expression level in the transfected cells. Finally, observation of cell morphology, growth curve assay and colony formation analysis were performed to explore the effect of Livin on growth of the cells. Results:Livin α & β were expressed in transfected A549 cells, and induced a faster cell growth, shorter doubling time and stronger cell colony forming ability, yet had no morphology change.Conclusion:Both isoforms can accelerate the growth of A549 cells, indicating a close relationship between Livin expression and the genesis and development of lung cancer. The expression of Livin α & β in A549 cells provides basis for further study of their different biological functions of anti-apoptosis and of their role in lung cancer cell resistance to radiotherapy and chemotherapy.

  18. T-Type Ca2+ Channel Blocker, KYS05090 Induces Autophagy and Apoptosis in A549 Cells through Inhibiting Glucose Uptake

    Hong-Kun Rim

    2014-07-01

    Full Text Available It has been reported that [3-(1,1'-biphenyl-4-yl-2-(1-methyl-5-dimethylamino-pentylamino-3,4-dihydroquinazolin-4-yl]-N-benzylacetamide 2hydrochloride (KYS05090, a selective T-type Ca2+ channel blocker, reduces tumor volume and weight in the A549 xenograft model, but the molecular mechanism of cell death has not yet been elucidated. In this study, KYS05090 induced autophagy- and apoptosis-mediated cell death in human lung adenocarcinoma A549 cells. Although KYS05090 decreased intracellular Ca2+ levels, it was not directly related with KYS05090-induced cell death. In addition, KYS05090 generated intracellular reactive oxygen species (ROS and reduced glucose uptake, and catalase and methyl pyruvate prevented KYS05090-induced cell death. These results indicate that KYS05090 can lead to autophagy and apoptosis in A549 cells through ROS generation by inhibiting glucose uptake. Our findings suggest that KYS05090 has potential chemotherapeutic value for the treatment of lung cancer.

  19. MicroRNA-1228(*) inhibit apoptosis in A549 cells exposed to fine particulate matter.

    Li, Xiaobo; Ding, Zhen; Zhang, Chengcheng; Zhang, Xin; Meng, Qingtao; Wu, Shenshen; Wang, Shizhi; Yin, Lihong; Pu, Yuepu; Chen, Rui

    2016-05-01

    Studies have reported associations between fine particulate matter (PM2.5) and respiratory disorders; however, the underlying mechanism is not completely clear owing to the complex components of PM2.5. microRNAs (miRNAs) demonstrate tremendous regulation to target genes, which are sensitive to exogenous stimulation, and facilitate the integrative understood of biological responses. Here, significantly modulated miRNA were profiled by miRNA microarray, coupled with bioinformatic analysis; the potential biological function of modulated miRNA were predicted and subsequently validated by cell-based assays. Downregulation of miR-1228-5p (miR-1228(*)) expression in human A549 cells were associated with PM2.5-induced cellular apoptosis through a mitochondria-dependent pathway. Further, overexpression of miR-1228(*) rescued the cellular damages induced by PM2.5. Thus, our results demonstrate that PM2.5-induced A549 apoptosis is initiated by mitochondrial dysfunction and miR-1228(*) could protect A549 cells against apoptosis. The involved pathways and target genes might be used for future mechanistic studies. PMID:26867688

  20. Effect of Long Non-coding RNA SPRY4-IT1 on Invasion and Migration of A549 Cells

    Song CHAI

    2015-08-01

    Full Text Available Background and objective The abnormal expression of human long chain non encoding RNA gene is related to many kinds of tumors. The aim of this study is to investigate the expression of long non-coding RNA maternally expressed gene 3 (SPRY4-IT1 in lung cancer (A549 cells, and to observe the effect of SPRY4-IT1 on the invasion and migration of A549 cells. Methods The levels of SPRY4-IT1 in A549 was detected by Real-time PCR. The effects of SPRY4-IT1 on the invasion and migration of A549 cell were analyzed by MTT and Transwell assay. The expression of matrix metalloproteinase (MMP family proteins was determined by Western blot. Results The invasion and migration of A549 cells were increased after SPRY4-IT1 over-expression. The cell spaces were narrower after SPRY4-IT1 over expression in the wound healing assay. Transwell assays showed that the numbers of transmembrane A549 cells were significantly higher in SPRY4-IT1 over expression group than that in control group (P<0.05. Meanwhile, over expression of SPRY4-IT1 reduced the expression of MMP-2 and MMP-9. Conclusion Over expression of SPRY4-IT1 enhanced the invasion and migration of A549 cells. MMP-2 and MMP-9 might play an important role in this regulation.

  1. Purification and characterization of protease enzyme from actinomycetes and its cytotoxic effect on cancer cell line (A549)

    C Balachandran; V Duraipandiyan; S Ignacimuthu

    2012-01-01

    Objective: To isolate active actinomycetes from soil samples of Northern Himalayas and study their culture characterization, protease production and cytotoxic effects on cancer cell line (A549). Methods: Forty six strains of actinomycetes were isolated from the soil collected from Northern Himalayas, India. Isolation of actinomycetes was performed by serial dilution plate technique. Forty six isolated actinomycetes cultures were grown in ISP 2 medium to study the morphology and biochemical characteristics. Isolated strains were studied for protease enzyme production in skim milk agar medium with solubilising capacity. Seven isolates were studied for melanin pigmentation and different NaCl concentration. Effects of environmental conditions influencing protease enzyme production of seven isolated strains were also studied at different pH, temperature and metal ions (β-mercaptoethanol, dithiothreitol, iodoacetamide, MgSO4, CaCl2 and EDTA). The seven isolates were also studied for lytic enzyme activity using different bacteria and yeast such as Pseudomonas aeruginosa (P. aeruginosa), Enterococcus feacalis (E. feacalis), Escherishia coli (E. coli), Candida albicans (C. albicans), Bacillus subtilis (B. subtilis), Klebsiella pneumonia (K. pneumonia) and Staphylococcus aureus (S. aureus). Results: Isolates ERIA-31 and ERIA-33 produced more protease enzyme activity in modified nutrient agar media compared to other actinomycetes cultures. ERIA-31 and ERIA-33 were tested for cytotoxic effect in human adenocarcinoma cancer cell line (A549). IC50 for ERIA-31 was 57.04 μg/mL and IC50 for ERIA-33 was 55.07 μg/mL. Conclusion: Actinomycete being a protease producing bacteria has the potential for use in industrial purpose, pharmaceuticals, cytotoxic agent and its proteolytic activity. Isolates of ERIA-31 and ERIA-33 produced significant amount of protease enzymes.

  2. Factors influencing recovery from potentially lethal radiation damage in A549 human lung carcinoma cells

    Plateau phase A549 cells exhibit potentially lethal radiation damage recovery (PLDR) that is dependent upon both the pH and the glucose content of the spent medium. At 9-10 days after plating, unfed A549 plateau cultures are acidic (pH 6.5 - 6.7), contain 2 to 4 mM glucose, and exhibit an approximately 40-fold increase in survival when held for 6 hrs in spent medium vs being subcultured immediately after 10 Gy aerobic irradiation. PLDR is maximal 24 hrs. post-irradiation. Adjustment of the pH of the spent medium to 7.5, by NaOH addition, either prior to or immediately post irradiation, nearly completely inhibits PLDR in this cell line. The authors found that medium acidity inhibits glucose utilization, and that alkalinization of spent medium, to pH 7.5, results in stimulation of glucose consumption. Plateau phase cultures depleted of glucose, as a result of medium alkalinization, are not capable of PLDR. In addition to pH effects, they observed that several agents, including nicotinamide, 3-aminobenzamide, caffeine, 2-deoxyglucose and glucosamine, partially inhibit PLDR in A549 plateau phase cultures

  3. Dexamethasone-(C21-phosphoramide-[anti-EGFR]: molecular design, synthetic organic chemistry reactions, and antineoplastic cytotoxic potency against pulmonary adenocarcinoma (A549

    Coyne CP

    2016-08-01

    Full Text Available Cody P Coyne,1 Lakshmi Narayanan2 1Department of Basic Sciences, 2Department of Clinical Sciences, College of Veterinary Medicine, Mississippi State University, Starkville, MS, USA Purpose: Corticosteroids are effective in the management of a variety of disease states, such as several forms of neoplasia (leukemia and lymphoma, autoimmune conditions, and severe inflammatory responses. Molecular strategies that selectively “target” delivery of corticosteroids minimize or prevents large amounts of the pharmaceutical moiety from passively diffusing into normal healthy cell populations residing within tissues and organ systems. Materials and methods: The covalent immunopharmaceutical, dexamethasone-(C21-phosphoramide-[anti-EGFR] was synthesized by reacting dexamethasone-21-monophosphate with a carbodiimide reagent to form a dexamethasone phosphate carbodiimide ester that was subsequently reacted with imidazole to create an amine-reactive dexamethasone-(C21-phosphorylimidazolide intermediate. Monoclonal anti-EGFR immunoglobulin was combined with the amine-reactive dexamethasone-(C21-phosphorylimidazolide intermediate, resulting in the synthesis of the covalent immunopharmaceutical, dexamethasone-(C21-phosphoramide-[anti-EGFR]. Following spectrophotometric analysis and validation of retained epidermal growth factor receptor type 1 (EGFR-binding avidity by cell-ELISA, the selective anti-neoplasic cytotoxic potency of dexamethasone-(C21-phosphoramide-[anti-EGFR] was established by MTT-based vitality stain methodology using adherent monolayer populations of human pulmonary adenocarcinoma (A549 known to overexpress the tropic membrane receptors EGFR and insulin-like growth factor receptor type 1. Results: The dexamethasone:IgG molar-incorporation-index for dexamethasone-(C21-phosphoramide-[anti-EGFR] was 6.95:1 following exhaustive serial microfiltration. Cytotoxicity analysis: covalent bonding of dexamethasone to monoclonal anti-EGFR immunoglobulin

  4. Enhancement of radiation sensitivity by erlotinib and celecoxib in A549 human lung cancer cell line

    Objective: To investigate the role of epidermal growth factor receptor and cyclooxygenase-2 pathways in the erlotinib and celecoxib enhanced radiation sensitivity in A549 human lung cancer cell line. Methods: IC20 of erlotinib and celecoxib on in A549 human lung cancer cells was measured by MTT assay, Clonogenic assays were used to evaluate the antitumor effects of the drugs and X-irradiation. Flow cytometry was used to assess the apoptosis and cell cycle alteration, and Western blot was used for the detection of Akt and phospho-Akt.Results Both erlotinib and celecoxib could inhibit the proliferation of A549 cells in vitro in a dose-dependent manner and their values of IC20 were (5.15 ± 0.14) and (40.32 ± 1.26) μmol/L, respectively. For radiation survival,the values of Dq, D0, SF2 of the combination of two drugs were lower than those of either drug (t=6.62, P<0.05). The SER of celecoxib, erlotinib and their combination were 1.299, 1.503 and 2.217, respectively. Flow cytometry assay showed that both celecoxib and erlotinib could enhance radiation-induced G0/G1 arrest, reduce the cell number in S phase, and enhance radiation-induced apoptosis, especially for the combination of drugs. Western blot assay showed that the expressions of Akt protein were similar in all groups. However, pAkt expression was suppressed by erlotinib and celecoxib, but promoted by radiation. pAkt had the lowest expression in the radiated cells with the treatment of two drugs (t=4.89, P<0.05). Conclusions: The erlotinib and/or celecoxib could enhance radiosensitivity probably by increasing cell apoptosis and reducing the number of S-phase cells with low radiosensitivity. (authors)

  5. Α-MMC and MAP30, two ribosome-inactivating proteins extracted from Momordica charantia, induce cell cycle arrest and apoptosis in A549 human lung carcinoma cells.

    Fan, Xiang; He, Lingli; Meng, Yao; Li, Gangrui; Li, Linli; Meng, Yanfa

    2015-05-01

    α‑Momorcharin (α‑MMC) and momordica anti‑human immunodeficiency virus protein (MAP30), produced by Momordica charantia, are ribosome‑inactivating proteins, which have been reported to exert inhibitory effects on cultured tumor cells. In order to further elucidate the functions of these agents, the present study aimed to investigate the effects of α‑MMC and MAP30 on cell viability, the induction of apoptosis, cell cycle arrest, DNA integrity and superoxide dismutase (SOD) activity. α‑MMC and MAP30 were purified from bitter melon seeds using ammonium sulfate precipitation in combination with sulfopropyl (SP)‑sepharose fast flow, sephacryl S‑100 and macro‑Cap‑SP chromatography. MTT, flow cytometric and DNA fragmentation analyses were then used to determine the effects of α‑MMC and MAP30 on human lung adenocarcinoma epithelial A549 cells. The results revealed that A549 cells were sensitive to α‑MMC and MAP30 cytotoxicity assays in vitro. Cell proliferation was significantly suppressed following α‑MMC and MAP30 treatment in a dose‑ and time‑dependent manner; in addition, the results indicated that MAP30 had a more potent anti‑tumor activity compared with that of α‑MMC. Cell cycle arrest in S phase and a significantly increased apoptotic rate were observed following treatment with α‑MMC and MAP30. Furthermore, DNA integrity analysis revealed that the DNA of A549 cells was degraded following treatment with α‑MMC and MAP30 for 48 h. The pyrogallol autoxidation method and nitrotetrazolium blue chloride staining were used to determine SOD activity, the results of which indicated that α‑MMC and MAP30 did not possess SOD activity. In conclusion, the results of the present study indicated that α‑MMC and MAP30 may have potential as novel therapeutic agents for the prophylaxis and treatment of cancer. PMID:25573293

  6. Inhibitory Effects of Natural Compound Alantolactone on Human Non-small Cell Lung Cancer A549 Cells

    ZONG Min-ru; ZHAO Ying-hao; ZHANG Kun; YANG Long-fei; ZHENG Yong-chen; HE Cheng-yan

    2011-01-01

    Alantolactone is a natural compound identified from the roots of Inula helenium L. that has multiple bio-activities. We examined its inhibitory effects on human non-small cell lung cancer(NSCLC) A549 cells. The antiproliferative effect of alantolactone on A549 cells was investigated via MTT[3'-(4,5dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide]assay and its apoptosis-inducing effect was determined by Hoechst staining and flow cytometry. We found that alantolactone significantly inhibited the proliferation of A549 cells and induced morphological changes typical for apoptosis. Flow cytometry analysis indicates dose-dependent cell cycle retardation at G0/G1 and S stages. The results indicate that alantolactone could be an attractive small-molecular natural compound for further development as a therapeutic drug against NSCLC.

  7. Lentinan exerts synergistic apoptotic effects with paclitaxel in A549 cells via activating ROS-TXNIP-NLRP3 inflammasome

    Liu, Wei; Gu, Jun; Qi, Jun; Zeng, Xiao-Ning; Ji, Juan; Chen, Zheng-Zhen; Sun, Xiu-Lan

    2015-01-01

    Paclitaxel is generally used to treat cancers in clinic as an inhibitor of cell division. However, the acquired resistance in tumours limits its clinical efficacy. Therefore, the aim of this study was to detect whether co-treatment with lentinan enhanced the anti-cancer effects of paclitaxel in A549 cells. We found that the combination of paclitaxel and lentinan resulted in a significantly stronger inhibition on A549 cell proliferation than paclitaxel treatment alone. Co-treatment with paclit...

  8. Adhesion of MRC-5 and A549 cells on poly(dimethylsiloxane) surface modified by proteins.

    Zuchowska, Agnieszka; Kwiatkowski, Piotr; Jastrzebska, Elzbieta; Chudy, Michal; Dybko, Artur; Brzozka, Zbigniew

    2016-02-01

    PDMS is a very popular material used for fabrication of Lab-on-a-Chip systems for biological applications. Although PDMS has numerous advantages, it is a highly hydrophobic material, which inhibits adhesion and proliferation of the cells. PDMS surface modifications are used to enrich growth of the cells. However, due to the fact that each cell type has specific adhesion, it is necessary to optimize the parameters of these modifications. In this paper, we present an investigation of normal (MRC-5) and carcinoma (A549) human lung cell adhesion and proliferation on modified PDMS surfaces. We have chosen these cell types because often they are used as models for basic cancer research. To the best of our knowledge, this is the first presentation of this type of investigation. The combination of a gas-phase processing (oxygen plasma or ultraviolet irradiation) and wet chemical methods based on proteins' adsorption was used in our experiments. Different proteins such as poly-l-lysine, fibronectin, laminin, gelatin, and collagen were incubated with the activated PDMS samples. To compare with other works, here, we also examined how ratio of prepolymer to curing agent (5:1, 10:1, and 20:1) influences PDMS hydrophilicity during further modifications. The highest adhesion of the tested cells was observed for the usage of collagen, regardless of PDMS ratio. However, the MRC-5 cell line demonstrated better adhesion than A549 cells. This is probably due to the difference in their morphology and type (normal/cancer). PMID:26311334

  9. Measuring Attachment and Internalization of Influenza A Virus in A549 Cells by Flow Cytometry.

    Pohl, Marie O; Stertz, Silke

    2015-01-01

    Attachment to target cells followed by internalization are the very first steps of the life cycle of influenza A virus (IAV). We provide here a detailed protocol for measuring relative changes in the amount of viral particles that attach to A549 cells, a human lung epithelial cell line, as well as in the amount of particles that are internalized into the cell. We use biotinylated virus which can be easily detected following staining with Cy3-labeled streptavidin (STV-Cy3). We describe the growth, purification and biotinylation of A/WSN/33, a widely used IAV laboratory strain. Cold-bound biotinylated IAV particles on A549 cells are stained with STV-Cy3 and measured using flow cytometry. To investigate uptake of viral particles, cold-bound virus is allowed to internalize at 37 °C. In order to differentiate between external and internalized viral particles, a blocking step is applied: Free binding spots on the biotin of attached virus on the cell surface are bound by unlabeled streptavidin (STV). Subsequent cell permeabilization and staining with STV-Cy3 then enables detection of internalized viral particles. We present a calculation to determine the relative amount of internalized virus. This assay is suitable to measure effects of drug-treatments or other manipulations on attachment or internalization of IAV. PMID:26575457

  10. Cyclin Y和Cyclin X在肺癌细胞株A549中的细胞定位和功能%The Function Study and Cell Localization of Cyclin Y and Cyclin X in Lung Cancer Cell Line A549

    周世杰; 江姝; 赵晓婷; 岳文涛

    2013-01-01

    [Purpose] To construct pEGFP-N1/CCNY vector and pEGFP-N1/CCNX eukaryotic expression vector,and to explore the location and function of CyclinY/CyclinX in lung caner A549 cell.[Methods] CCNY and CCNX genes were amplified from human lung adenocarcinoma cell line H1299 by PCR.The recombinant plasmids pEGFP-N1/CCNY and pEGFP-N1/CCNX were constructed and transfected into A549 cells.The cellular localization and expression of CyclinY and Cyclin X were detected by fluorescence microscopy and Western Blot.[Results] The recombinant plasmid pEGFP-N1/CCNY and pEGFP-N1/CCNX were constructed successfully.Green fluorescence on the surface of transfected cells was found by fluorescence microscope.Western Blot confirmed Cyclin Y,Cyclin X expression.Cyclin Y and Cyclin X located at cellular membrane and nucleus in recombinant plasmid cell respectively.After transfection,A549-CCNY pEGFPN1 cell viability was 1.36±0.02,A549-CCNX pEGFPN cell viability was 11.45 ±0.05,which was higher than that in A549-pEGFPN1 (1.31±0.03) (P all<0.01).[Conclusion] In A549 cell,Cyclin Y and Cyclin X are differently distributed,Cyclin X plays more important role in promoting proliferation than Cyclin Y.%[目的]构建CCNY和CCNX基因的真核表达载体并观察其在人肺癌细胞株A549中的表达及定位,为进一步探讨Cyclin Y、Cyclin X在肺癌中的细胞定位和功能奠定了基础.[方法]以人肺腺癌细胞株H1299 cDNA为模板扩增CCNY和CCNX基因,并构建CCNY和CCNX过表达真核表达载体.应用荧光显微照相及Western Blot方法鉴定该细胞株中Cyclin Y、Cyclin X的定位及表达.[结果]成功构建pEGFP-N1/CCNY和pEGFP-N1/CCNX真核表达载体.荧光显微照相显示绿色荧光,Western Blot检测证实转染重组质粒细胞表达Cyclin Y、Cyclin X蛋白,Cyclin Y和Cyclin X分别定位于胞膜与胞核.A549-pEGFPN1细胞活性为1.31±0.03,而转染后的A549-CCNY pEGFPN1细胞活性为1.36±0.02,A549-CCNX pEGFPN1细胞活性为1.45±0.05(P<0

  11. High-throughput quantitative proteomic analysis of dengue virus type 2 infected A549 cells.

    Han-Chen Chiu

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

  12. Effect of elemene on radiosensitivity of A549 cells and its possible molecular mechanism

    Objective: To investigate the effect of elemene on the radiosensitivity of A549 cells and its possible molecular mechanism. Methods: The effect of radiosensitivity was detected by colony forming assay. The protein expressions of DNA-PKcs, Bcl-2 and P53 were detected with Western blot. The correlation between the protein expression of DNA-PKcs and Bcl-2, DNA-PKcs and P53 was analyzed. Results: Elemene had radiosensitizing effect on A549 cells, with the SERD0 and SERDq 1.54 ± 0.20 and 1.43±0.15, respectively for 10 μg/ml elemene, and 1.63 ±0.32 and 1.75 ±0.19, respectively for 20 μg/ml elemene. Compared with irradiation group, the expression of DNA-PKcs was reduced significantly in 10, 20 μg/ml elemene combined with radiation group (t=7.52, 8.33, P<0.05), so was for Bcl-2 (t=10.74, 11.33, P<0.05). The expression of P53 protein increased significantly (t=-9.25, 7.66, P<0.05). There was a remarkable negative correlation between the expression of DNA-PKcs and P53 (r=-0.569, P<0.05), and a remarkable positive correlation between DNA-PKcs and Bcl-2 (r=0.755, P<0.05 ). Conclusions: Elemene has radiosensitizing effect on A549 cells, which might be related to down-regulation of DNA-PKcs gene expression, up-regulation of P53 and down-regulation of Bcl-2. (authors)

  13. ANTICANCER ACTIVITY OF OSCILLATORIA TEREBRIFORMIS CYANOBACTERIA IN HUMAN LUNG CANCER CELL LINE A549

    S.Mukund

    2014-04-01

    Full Text Available Purpose: To evaluate the anti-cancer properties of the cyanobacterial extract of Oscillatoria terebriformis Methods: The extract was tested in Human lung cancer cell lines and examined for its effect on cell viability, nuclear morphology and sub-G1 formation. Cell viability was determined by micro culture tetrazolium technique (MTT, nuclear morphology investigated using 4’-6-diamidino-2-phenylindole (DAPI staining technique, and apoptosis assay using DNA fragmentation. Results: The results showed decreasing cell viability in a concentration-dependent manner. Altered cell morphology after treatment with the extract demonstrated that cells experienced apoptosis. Conclusion: The data demonstrate that Oscillatoria Terebriformis extract induced apoptosis in Human lung cancer A549 cells, and therefore, has a potential as an anti-cancer agent.

  14. In vitro and in vivo studies on radiobiological effects of prolonged fraction delivery time in A549 cells

    Jiang, Ling; Xiong, Xiao-Peng; Hu, Chao-su; Ou, Zhou-Luo; Zhu, Guo-Pei; Ying,, Hong-Mei

    2012-01-01

    Intensity-modulated radiation therapy, when used in the clinic, prolongs fraction delivery time. Here we investigated both the in vivoand in vitroradiobiological effects on the A549 cell line, including the effect of different delivery times with the same dose on A549 tumor growth in nude mice. The in vitroeffects were studied with clonogenic assays, using linear-quadratic and incomplete repair models to fit the dose-survival curves. Fractionated irradiation of different doses was given at on...

  15. Anti-Tumor Effect of Heat Shock Protein 70-Peptide Complexes on A-549 Cells

    2007-01-01

    Objective: To investigate the anti-tumor immunity in vitro of heat shock protein 70-peptide complexes (HSP70-PC) from human lung cancer tissue. Methods: HSP70-PC was purified from lung tumor tissues and corresponding non-tumor lung samples with the methods of ADP-affinity chromatography, DEAE ion-exchange chromatography and Western-blot. The activation and proliferation of PBMC induced by different HSP70-PC and tumor cytotoxic reactivity to A549 cells in vitro were measured by the MTT cell proliferation assay. Results: The purified HSP70-PC had a very high purity found by SDS-PAGE and Western-blot. Human lymphocytes were sensitized efficiently by HSP70 preparation purified from lung cancer tissues and a definite cytotoxicity to A-549 cells was observed. There was significant difference with HSP70-PC purified from lung cancer, compared with the control group (P<0.001). Conclusion: High purity of HSP70-PC could be achieved from tumor tissues in this study. HSP70-PC purified from human tumor tissues can induce anti-tumor immunity in vitro mainly implemented by eliciting CTL immunity.

  16. Immune Evasion of Human Lung Carcinoma Cell A549 Suppressed by Human Lymphoma Cell Jurkat via Fas/FasL Pathway

    Hongmei WANG

    2010-07-01

    Full Text Available Background and objective Tumor escape from the host immune system has been a major problem in immunotherapy of human malignancies. FasL/Fas-induced apoptosis plays an important role in various immunological processes. The aim of this study is to investigate the immune evasion in human lung carcinoma cell A549 induced by human lymphoma cell Jurkat via Fas/FasL pathway. Methods Jurkat cells and A549 cells were co-cultured at different proportions. The apoptotic rates of A549 cells were determined by flow cytometry (FCM. Protein levels of Fas, FasL and Caspase-8 in A549 cells were detected by Western blot. Results Survival rates of A549 cells gradually decreased and apoptotic rates of A549 cells were significantly enhanced along with ratio increasing between Jurkat and A549. Meanwhile, the protein levels of Fas and Caspase-8 gradually increased in A549 cells, and the protein levels of FasL had no significant difference in all groups. Conclusion The Jurkat cells might decrease the survival rates of A549 cells and enhanced its apoptosis and immune evasion partly via Fas/FasL pathway.

  17. Blocking the NOTCH pathway can inhibit the growth of CD133-positive A549 cells and sensitize to chemotherapy

    Liu, Juntao; Mao, Zhangfan; Huang, Jie; Xie, Songping; Liu, Tianshu; Mao, Zhifu, E-mail: 48151660@qq.com

    2014-02-21

    Highlights: • Notch signaling pathway members are expressed lower levels in CD133+ cells. • CD133+ cells are not as sensitive as CD133− cells to chemotherapy. • GSI could inhibit the growth of both CD133+ and CD133− cells. • Blockade of Notch signaling pathway enhanced the effect of chemotherapy with CDDP. • DAPT/CDDP co-therapy caused G2/M arrest and elimination in CD133+ cells. - Abstract: Cancer stem cells (CSCs) are believed to play an important role in tumor growth and recurrence. These cells exhibit self-renewal and proliferation properties. CSCs also exhibit significant drug resistance compared with normal tumor cells. Finding new treatments that target CSCs could significantly enhance the effect of chemotherapy and improve patient survival. Notch signaling is known to regulate the development of the lungs by controlling the cell-fate determination of normal stem cells. In this study, we isolated CSCs from the human lung adenocarcinoma cell line A549. CD133 was used as a stem cell marker for fluorescence-activated cell sorting (FACS). We compared the expression of Notch signaling in both CD133+ and CD133− cells and blocked Notch signaling using the γ-secretase inhibitor DAPT (GSI-IX). The effect of combining GSI and cisplatin (CDDP) was also examined in these two types of cells. We observed that both CD133+ and CD133− cells proliferated at similar rates, but the cells exhibited distinctive differences in cell cycle progression. Few CD133+ cells were observed in the G{sub 2}/M phase, and there were half as many cells in S phase compared with the CD133− cells. Furthermore, CD133+ cells exhibited significant resistance to chemotherapy when treated with CDDP. The expression of Notch signaling pathway members, such as Notch1, Notch2 and Hes1, was lower in CD133+ cells. GSI slightly inhibited the proliferation of both cell types and exhibited little effect on the cell cycle. The inhibitory effects of DPP on these two types of cells were

  18. Blocking the NOTCH pathway can inhibit the growth of CD133-positive A549 cells and sensitize to chemotherapy

    Highlights: • Notch signaling pathway members are expressed lower levels in CD133+ cells. • CD133+ cells are not as sensitive as CD133− cells to chemotherapy. • GSI could inhibit the growth of both CD133+ and CD133− cells. • Blockade of Notch signaling pathway enhanced the effect of chemotherapy with CDDP. • DAPT/CDDP co-therapy caused G2/M arrest and elimination in CD133+ cells. - Abstract: Cancer stem cells (CSCs) are believed to play an important role in tumor growth and recurrence. These cells exhibit self-renewal and proliferation properties. CSCs also exhibit significant drug resistance compared with normal tumor cells. Finding new treatments that target CSCs could significantly enhance the effect of chemotherapy and improve patient survival. Notch signaling is known to regulate the development of the lungs by controlling the cell-fate determination of normal stem cells. In this study, we isolated CSCs from the human lung adenocarcinoma cell line A549. CD133 was used as a stem cell marker for fluorescence-activated cell sorting (FACS). We compared the expression of Notch signaling in both CD133+ and CD133− cells and blocked Notch signaling using the γ-secretase inhibitor DAPT (GSI-IX). The effect of combining GSI and cisplatin (CDDP) was also examined in these two types of cells. We observed that both CD133+ and CD133− cells proliferated at similar rates, but the cells exhibited distinctive differences in cell cycle progression. Few CD133+ cells were observed in the G2/M phase, and there were half as many cells in S phase compared with the CD133− cells. Furthermore, CD133+ cells exhibited significant resistance to chemotherapy when treated with CDDP. The expression of Notch signaling pathway members, such as Notch1, Notch2 and Hes1, was lower in CD133+ cells. GSI slightly inhibited the proliferation of both cell types and exhibited little effect on the cell cycle. The inhibitory effects of DPP on these two types of cells were enhanced

  19. Effect of γ-ray irradiation on migration of pulmonary adenocarcinoma cells and its mechanism

    Objective: To investigate the effect of radiation on the migration of the pulmonary adenocarcinoma cell line, A549, and the mechanism involved in this process. Methods: Migration of A549 cells irradiated with 2 and 4 Gy doses of γ-ray was detected using wound healing assay. The level of STAT3 and phosphorylated STAT3 was detected by Western blot. The distribution of p-STAT3 (Y705) in A549 cells was examined by immunofluorescence staining. Finally, the secretion of IL-6 by cultured A549 cells was detected by ELISA. Results: The migration of A549 cells was significantly enhanced by γ-ray radiation at dose levels of either 2 or 4 Gy. Furthermore, radiation was found to activate the phosphorylation of STAT3 and promote the nucleus localization of STAT3. The result of ELISA showed that the secretion of IL-6 increased after 2 or 4 Gy doses of γ-ray. AG490, a special inhibitor of JAK-2, suppressed the radiation-induced phosphorylation of STAT3 and migration of A549 cells. Conclusion: Our results indicated that radiation results in the activation of JAK-2/STAT3 pathway, which triggers the migration of A549 cells. It is possible that the IL-6 is involved in the radiation-induced activation of JAK-2/STAT3 pathway. (authors)

  20. Overexpression of the hydatidiform mole-related gene F10 inhibits apoptosis in A549 cells through downregulation of BCL2-associated X protein and caspase-3.

    Song, Yali; Zhang, Gong; Zhu, Xiulan; Pang, Zhanjun; Xing, Fuqi; Quan, Song

    2012-09-01

    The aim of this study was to investigate how the overexpression of the hydatidiform mole-related gene F10 affects apoptosis in human lung cancer A549 cells. A549 cells were transfected with pEGFP-N1-F10 (A549-F10) or pEGFP-N1 empty vector (A549-empty). Untransfected A549, A549-F10 or A549-empty cells were examined using the MTT cell proliferation assay and the TUNEL-FITC/Hoechst 33258 apoptosis assay. Western blotting was used to examine the expression levels of the pro-apoptotic genes, BCL2-associated X protein (BAX) and caspase-3. F10 was stably expressed in A549 cells. From 12 h, A549-F10 cells proliferated markedly faster than the untransfected and A549-empty cells. F10 overexpression also significantly inhibited apoptosis, as shown by the reduced number of TUNEL and Hoechst 33258 double-positive cells. This inhibition was likely due to an F10-induced reduction in the BAX and caspase-3 levels. The results of this study indicate that F10 overexpression inhibits apoptosis in A549 cells through the downregulation of the pro-apoptotic genes BAX and caspase-3. PMID:23741243

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

    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.

  2. Screening Metastasis-associated Genes from Anoikis Resistant A549 Lung Cancer Cells by Human Genome Array

    Xiaoping WANG

    2010-01-01

    Full Text Available Background and objective As a barrier to metastases, cells normally undergo apoptosis after they lose contact with their extra cellular matrix (ECM. This process has been termed “anoikis”. Tumour cells that acquire malignant potential have developed mechanisms to resist anoikis and thereby survive after detachment from their primary site while traveling through the lymphatic and circulatory systems. This “anoikis resistance” is considered the first step to tumor metastases. The aim of this study was to screen metastasis-associated genes from anoikis resistant and adherent growth A549 lung cancer cell by Human Genome Array. Methods Establish anoikis resistant A549 lung cancer cell lines by using poly-hydroxyethyl methacrylate resin processed petri dishes, which causes cell free from adherent. The different expressed gene between anoikis resistant A549 cell and adherent growth A549 cell was tested using human V2.0 whole-genome oligonucleotide microarray, a product of Capitalbio Corporation, Beijing. Screen metastasis-associated genes. Results 745 different expressed genes were screened, including 63 highly metastasis-associated genes. Conclusion The successfully established anoikis resistant A549 cell lines and screened different expressed genes provide us basis for further research on metastasis of lung cancer.

  3. Panduratin A, a Possible Inhibitor in Metastasized A549 Cells through Inhibition of NF-Kappa B Translocation and Chemoinvasion

    Mohd. Rais Mustafa

    2013-07-01

    Full Text Available In the present study, we investigated the effects of panduratin A (PA, isolated from Boesenbergia rotunda, on apoptosis and chemoinvasion in A549 human non-small cell lung cancer cells. Activation of the executioner procaspase-3 by PA was found to be dose-dependent. Caspase-3 activity was significantly elevated at the 5 µg/mL level of PA treatment and progressed to a maximal level. However, no significant elevated level was detected on procaspase-8. These findings suggest that PA activated caspase-3 but not caspase-8. Numerous nuclei of PA treated A549 cells stained brightly by anti-cleaved PARP antibody through High Content Screening. This result further confirmed that PA induced apoptotic cell death was mediated through activation of caspase-3 and eventually led to PARP cleavage. Treatment of A549 cells with PA resulted in a strong inhibition of NF-κB activation, which was consistent with a decrease in nuclear levels of NF-κB/p65 and NF-κB/p50 and the elevation of p53 and p21. Besides that, we also showed that PA significantly inhibited the invasion of A549 cells in a dose-dependent manner through reducing the secretion of MMP-2 of A549 cells gelatin zymography assay. Our findings not only provide the effects of PA, but may also be important in the design of therapeutic protocols that involve targeting of either p53 or NF-κB.

  4. COPD promotes migration of A549 lung cancer cells: the role of chemokine CCL21

    Kuźnar-Kamińska B

    2016-05-01

    Full Text Available Barbara Kuźnar-Kamińska,1 Justyna Mikuła-Pietrasik,2 Patrycja Sosińska,2 Krzysztof Książek,2 Halina Batura-Gabryel1 1Department of Pulmonology, Allergology and Respiratory Oncology, 2Department of Pathophysiology, Poznań University of Medical Sciences, Poznań, Poland Abstract: Patients with COPD develop lung cancer more frequently than healthy smokers. At the same time, molecular mediators promoting various aspects of cancer cell progression are still elusive. In this report, we examined whether COPD can be coupled with increased migration of non-small-cell lung cancer cells A549 and, if so, whether this effect may be related to altered production and activity of chemokines CCL21, CXCL5, and CXCL12. The study showed that the migration of A549 cells through the polycarbonate membrane and basement membrane extract toward a chemotactic gradient elicited by serum from patients with COPD was markedly higher as compared with serum from healthy donors. The concentration of CCL21 and CXCL12, but not CXCL5, in serum from patients with COPD was also increased. Experiments in which CCL21- and CXCL12-dependent signaling was blocked revealed that increased migration of the cancer cells upon treatment with serum from patients with COPD was mediated exclusively by CCL21. Collectively, our results indicate that COPD may contribute to the progression of lung cancer via CCL21-dependent intensification of cancer cell migration. Keywords: chemokines, COPD, lung cancer, migration

  5. 5-allyl-7-gen-difluoromethoxychrysin enhances TRAIL-induced apoptosis in human lung carcinoma A549 cells

    5-allyl-7-gen-difluoromethoxychrysin (AFMC) is a novel synthetic analogue of chrysin that has been reported to inhibit proliferation in various cancer cell lines. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anti-cancer agent. The cytotoxicity of A549 and WI-38 cells were determined using colorimetry. Apoptosis was detected by flow cytometry (FCM) after propidium iodide (PI) fluorescence staining and agarose gel electrophoresis. Caspase activities were evaluated using enzyme-linked immunosorbent assay (ELISA).The expressions of DR4 and DR5 were analyzed using FCM and western blot. Subtoxic concentrations of AFMC sensitize human non-small cell lung cancer (NSCLC) A549 cells to TRAIL-mediated apoptosis. Combined treatment of A549 cells with AFMC and TRAIL significantly activated caspase-3, -8 and -9. The caspase-3 inhibitor zDEVD-fmk and the caspase-8 inhibitor zIETD-fmk blocked the apoptosis of A549 cells induced by co-treatment with AFMC and TRAIL. In addition, we found that treatment of A549 cells with AFMC significantly induced the expression of death receptor 5 (DR5). AFMC-mediated sensitization of A549 cells to TRAIL was efficiently reduced by administration of a blocking antibody or small interfering RNAs against DR5. AFMC also caused increase of the Sub-G1 cells by TRAIL treatment and increased the expression levels of DR5 in other NSCLC H460 and H157 cell lines. In contrast, AFMC-mediated induction of DR5 expression was not observed in human embryo lung WI-38 cells, and AFMC did not sensitize WI-38 cells to TRAIL-induced apoptosis. AFMC synergistically enhances TRAIL-mediated apoptosis in NSCLC cells through up-regulating DR5 expression

  6. Synthesis, characterization and anticancer activity studies of ruthenium(II) polypyridyl complexes on A549 cells.

    Zeng, Chuan-Chuan; Jiang, Guang-Bin; Lai, Shang-Hai; Zhang, Cheng; Yin, Hui; Tang, Bing; Wan, Dan; Liu, Yun-Jun

    2016-08-01

    Four new ruthenium(II) polypyridyl complexes [Ru(N-N)2(bddp)](ClO4)21-4 (N-N=dmb: 4,4'-dimethyl-2,2'-bipyridine 1, bpy: 2,2'-bipyridine 2, phen: 1,10-phenanthroline 3 and dmp: 2,9-dimethyl-1,10-phenanthroline 4, bddp=benzilo[2,3-b]-1,4-diazabenzo[i]dipyrido[3,2-a:2',3'-c]phenazine) were synthesized and characterized by elemental analysis, ESI-MS and (1)H NMR. The cytotoxicity in vitro of the complexes against BEL-7402, HeLa, MG-63 and A549 cell lines was investigated by MTT method. The complexes show high cytotoxic activity toward the selected cell lines with an IC50 value ranging from 5.3±0.6 to 15.7±3.6μM. The apoptosis was studied with acridine orange (AO)/ethdium bromide (EB) and Hoechst 33258 staining methods. The cellular uptake was investigated with DAPI staining method. The reactive oxygen species (ROS) and mitochondrial membrane potential were performed under fluorescent microscope and flow cytometry. The complexes can induce an increase in the ROS levels and a decrease in the mitochondrial membrane potential. The comet assay was studied with fluorescent microscope. The percentage in apoptotic and necrotic cells and cell cycle arrest were assayed by flow cytometry. The effects of the complexes on the expression of caspases and Bcl-2 family proteins were studied by western blot analysis. The results show that the complexes induce apoptosis in A549 cells through an ROS-mediated mitochondrial dysfunction pathway, which was accompanied by regulating the expression of Bcl-2 family proteins. PMID:27288660

  7. Influence of pEgr1-hsTRAIL plasmid on radiosensitivity and DR4 and DR5 expression levels in lung adencarcinoma A549 cells

    Objective: To measure the changes of the radiosensitivity in human lung adenocarcinoma A549 cells transfected with pEgr1-hsTRAIL plasmid and the effect on death receptor (DR) 4 and DR5 expressions, and to explore the radiosensitizing effect of pEgr1-hsTRAIL plasmid and possible mechanism on inducing apoptosis. Methods: There were normal control, pEgr1-hsTRAIL, 6 Gy X-rays, and pEgr1-hsTRAIL + 6 Gy X-rays groups in the experiment. After the A549 cells were transfected with liposome, and irradiated with X-rays, colony formation assay was used to measure the radiosensitivity, and reverse transcription PCR (RT-PCR) was performed to detect the DR4 and DR5 mRNA expressions, and Western blotting was applied to determine the DR4 and DR5 protein expressions. Results: The D0 values of A549 cells in normal control group and pEgr1-hsTRAIL group were 3.26 and 1.91 Gy, respectively, it indicated that pEgr1-hsTRAIL plasmid could enhance the radiosensitivity in A549 cells. The RT-PCR results showed that as compared with normal control group, the DR4 and DR5 mRNA expression levels in pEgr1-hsTRAIL group had no significant change, but those in 6 Gy X-rays group were increased significantly (P<0.05), and those in pEgr1-hsTRAIL + 6 Gy X-rays group were also increased significantly (P<0.05); the DR5 mRNA expression level in pEgr1-hsTRAIL + 6 Gy X-rays group was higher than that in 6 Gy X-rays group (P<0.05). The Western blotting results showed that the DR4 and DR5 protein expressions in pEgr1-hsTRAIL group did not change obviously compared with normal control group, but those in 6 Gy X-rays and pEgr1-hsTRAIL + 6 Gy X-rays groups were increased, and the DR5 protein expression in pEgr1-hsTRAIL + 6 Gy X-rays group was increased mostly. Conclusion: The recombinant plasmid pEgr1-hsTRAIL can enhance the radiosensitivity of A549 cells, and has the enhancing effect on DR5 expression induced by radiation, but no same effect on DR4 expression. (authors)

  8. High throughput determination of TGFβ1/SMAD3 targets in A549 lung epithelial cells.

    Yingze Zhang

    Full Text Available BACKGROUND: Transforming growth factor beta 1 (TGFβ1 plays a major role in many lung diseases including lung cancer, pulmonary hypertension, and pulmonary fibrosis. TGFβ1 activates a signal transduction cascade that results in the transcriptional regulation of genes in the nucleus, primarily through the DNA-binding transcription factor SMAD3. The objective of this study is to identify genome-wide scale map of SMAD3 binding targets and the molecular pathways and networks affected by the TGFβ1/SMAD3 signaling in lung epithelial cells. METHODOLOGY: We combined chromatin immunoprecipitation with human promoter region microarrays (ChIP-on-chip along with gene expression microarrays to study global transcriptional regulation of the TGFβ1/SMAD3 pathway in human A549 alveolar epithelial cells. The molecular pathways and networks associated with TGFβ1/SMAD3 signaling were identified using computational approaches. Validation of selected target gene expression and direct binding of SMAD3 to promoters were performed by quantitative real time RT-PCR and electrophoretic mobility shift assay on A549 and human primary lung epithelial cells. RESULTS AND CONCLUSIONS: Known TGFβ1 target genes such as SERPINE1, SMAD6, SMAD7, TGFB1 and LTBP3, were found in both ChIP-on-chip and gene expression analyses as well as some previously unrecognized targets such as FOXA2. SMAD3 binding of FOXA2 promoter and changed expression were confirmed. Computational approaches combining ChIP-on-chip and gene expression microarray revealed multiple target molecular pathways affected by the TGFβ1/SMAD3 signaling. Identification of global targets and molecular pathways and networks associated with TGFβ1/SMAD3 signaling allow for a better understanding of the mechanisms that determine epithelial cell phenotypes in fibrogenesis and carcinogenesis as does the discovery of the direct effect of TGFβ1 on FOXA2.

  9. Direct and in vitro observation of growth hormone receptor molecules in A549 human lung epithelial cells by nanodiamond labeling

    Cheng, C.-Y.; Perevedentseva, E.; Tu, J.-S.; Chung, P.-H.; Cheng, C.-L.; Liu, K.-K.; Chao, J.-I.; Chen, P.-H.; Chang, C.-C.

    2007-04-01

    This letter presents direct observation of growth hormone receptor in one single cancer cell using nanodiamond-growth hormone complex as a specific probe. The interaction of surface growth hormone receptor of A549 human lung epithelial cells with growth hormone was observed using nanodiamond's unique spectroscopic signal via confocal Raman mapping. The growth hormone molecules were covalent conjugated to 100nm diameter carboxylated nanodiamonds, which can be recognized specifically by the growth hormone receptors of A549 cell. The Raman spectroscopic signal of diamond provides direct and in vitro observation of growth hormone receptors in physiology condition in a single cell level.

  10. 烹调油烟中细颗粒物PM2.5对A-549细胞存活率和IL-10水平的影响%Effects of PM2.5 in cooking oil fume on cell survival and IL-10 levels in A-549 cells

    冯哲伟; 梁春梅; 操基玉; 郭冬梅; 王勇; 王绩凯

    2011-01-01

    Objective To explore fine particle in cooking oil fume ( FPCOF, PM2. 5 ) on the level of anti-inflammatory factor IL-10 in human lung adenocarcinoma epithelial cell (A-549 cells). Methods A-549 cells were exposed to different concentrations of FPCOF, for 12 h, 24 h and 36 h, respectively, then detecting the cell survival rate with MTT assay and IL-10 level in media supernatant by enzyme linked immunosorbent assay. Results ( 1 ) The survival rate of A-549 cells was reduced time-and dose-dependently, but the difference at 12 h among different doses was not significant, meanwhile the rate in 100 μg/ml PM2.5 + 10 mmol/L NAC group was significantly higher than that in 100 μg/ml PM2.5 group after 24 h or 36 h exposure. (2) There was no obvious effect on IL-10 after 24 h exposure to FPCOF, the rise of IL-10 level was only seen in all the exposed groups after 36 h exposure to FPCOF; additionally, IL-10 level in 100 μg/ml PM2.5 + 10 mmol/L NAC group was significantly lower than that in 100 μg/ml PM2.5 group. Conclusion FPCOF might promote the secretion of anti-inflammatory factor IL-10 in A-549 cells, but its expression was later than the inhibition of cell proliferation caused by FPCOF; and the antioxidant NAC might protect A-549 cells from the adverse effects induced by FPCOF.%探讨烹调油烟中大气细颗粒物PM2.5(FPCOF)对人肺癌上皮细胞(A-549细胞)抗炎因子IL-10分泌的影响.分别用不同浓度的FPCOF对细胞进行染毒,于12 h、24 h和36 h后用MTT法测细胞存活率;用酶联免疫吸附法测染毒24 h和36 h的细胞上清中IL-10的浓度.结果:(1)FPCOF作用于A-549细胞24 h和36 h后,细胞存活率随染毒浓度升高而降低,100μg/ml PM2.5+10 mmol/L NAC组存活率高于100μg/ml PM2.5染毒组.(2)FPCOF对A-549细胞作用24 h后,对抗炎因子IL-10的影响不明显;作用36 h后均可见IL-10水平增高,100 μg/ml PM2.5+10mmol/L NAC组IL-10浓度低于100 μg/ml PM2.5染毒组.提示FPCOF促进A-549

  11. Effect of Inhibiting NGAL Gene Expression on A549 Lung Cancer Cell Migration and Invasion

    Jian TANG

    2015-04-01

    Full Text Available Background and objective To detect the expression of neutrophil gelatinase-assoeiated lipocalin (NGAL in the different differentiations of lung cancer tissues and to study the mechanism of invasion of A549 cells affected by NGAL. Methods The expression of NGAL was detected by immunochemistry in lung cancer tissue and the tissue around edge of the cancer. The effect of NGAL expression on A549 cells was observed by using qRT-PCR and Western blot. The abilities of invasion and metastasis were evaluated by transwell invasion and migration assay, and cell scratch assay in vitro. The protein expression of E-cadherin, Vimentin was measured by immunofluoresence and Western blot. Results The positive expression rate of NGAL was 76.32% (58/76 in the lung cancer, 13.3% (4/30 in adjacent tissue by immunochemistry. NGAL expression levels in the lung cancer tissues were significantly higher than that in adjacent tissues. The rate of migration and invasion in NGAL-siRNA group was 60.4%±6.4% compared to 50.5%±4.4% in the control group, there was a significant difference (P<0.05. Vimentin was suppressed, and E-cadherin was upregulated when NGAL was inhibited. MMP-2 and MMP-9 decreased when NGAL was knocked down. Conclusion The expression level of NGAL is highly expressed in lung cancer. NGAL may be one of important indicators involved in lung cancer infiltrated and transferred. NGAL might be one of potential targets for lung cancer treatment.

  12. Synthesis and cytotoxicity evaluation of 4-amino-4-dehydroxylarctigenin derivatives in glucose-starved A549 tumor cells.

    Lei, Min; Gan, Xianwen; Zhao, Kun; Yu, Qiang; Hu, Lihong

    2015-02-01

    The natural product arctigenin (ATG) demonstrated preferential cytotoxicity to cancer cells under glucose starvation. A series of 4-amino-4-dehydroxylarctigenin derivatives based on lead compound ATG were designed and synthesized by bioisosteric modifications. Their cytotoxicities were evaluated in glucose-starved A549 tumor cells and the results indicated that the 4-amino-4-dehydroxylarctigenin showed more potent cytotoxicity than arctigenin, and the further substituent group on 4-amino would result in the cytotoxicities decreased significantly. 4-Substituted-arctigenin could selectively target on glucose-starved A549 tumor cells which provide an alternative strategy for anticancer drug development with minimal normal tissue toxicity. PMID:25571795

  13. Role of cytoskeleton network in anisosmotic volume changes of intact and permeabilized A549 cells.

    Platonova, Alexandra; Ponomarchuk, Olga; Boudreault, Francis; Kapilevich, Leonid V; Maksimov, Georgy V; Grygorczyk, Ryszard; Orlov, Sergei N

    2015-10-01

    Recently we found that cytoplasm of permeabilized mammalian cells behaves as a hydrogel displaying intrinsic osmosensitivity. This study examined the role of microfilaments and microtubules in the regulation of hydrogel osmosensitivity, volume-sensitive ion transporters, and their contribution to volume modulation of intact cells. We found that intact and digitonin-permeabilized A549 cells displayed similar rate of shrinkage triggered by hyperosmotic medium. It was significantly slowed-down in both cell preparations after disruption of actin microfilaments by cytochalasin B, suggesting that rapid water release by intact cytoplasmic hydrogel contributes to hyperosmotic shrinkage. In hyposmotic swelling experiments, disruption of microtubules by vinblastine attenuated the maximal amplitude of swelling in intact cells and completely abolished it in permeabilized cells. The swelling of intact cells also triggered ~10-fold elevation of furosemide-resistant (86)Rb+ (K+) permeability and the regulatory volume decrease (RVD), both of which were abolished by Ba2+. Interestingly, RVD and K+ permeability remained unaffected in cytocholasin/vinblastine treated cells demonstrating that cytoskeleton disruption has no direct impact on Ba2+-sensitive K+-channels involved in RVD. Our results show, for the first time, that the cytoskeleton network contributes directly to passive cell volume adjustments in anisosmotic media via the modulation of the water retained by the cytoplasmic hydrogel. PMID:26171817

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

    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

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

    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.

  16. Reactive oxygen species involved in apoptosis induction of human respiratory epithelial (A549) cells by Streptococcus agalactiae.

    da Costa, Andréia Ferreira Eduardo; Moraes, João Alfredo; de Oliveira, Jessica Silva Santos; dos Santos, Michelle Hanthequeste Bittencourt; Santos, Gabriela da Silva; Barja-Fidalgo, Christina; Mattos-Guaraldi, Ana Luiza; Nagao, Prescilla Emy

    2016-01-01

    Streptococcus agalactiae (Group B Streptococcus; GBS) is an important pathogen and is associated with pneumonia, sepsis and meningitis in neonates and adults. GBS infections induce cytotoxicity of respiratory epithelial cells (A549) with generation of reactive oxygen species (ROS) and loss of mitochondrial membrane potential (ψm). The apoptosis of A549 cells by GBS was dependent on the activation of caspase-3 and caspase-9 with increased pro-apoptotic Bim and Bax molecules and decreased Bcl-2 pro-survival protein. Treatment of infected A549 cells with ROS inhibitors (diphenyleniodonium chloride or apocynin) prevented intracellular ROS production and apoptosis. Consequently, oxidative stress is included among the cellular events leading to apoptosis during GBS human invasive infections. PMID:26490153

  17. Effect of XPA expression on the chemotherapy sensitivity of A549/DDP cells%着色性干皮病A基因表达对A549/DDP化疗敏感性的影响

    张强; 吴金香; 魏玉平; 郝俊青; 黄山英; 董亮

    2012-01-01

    目的:探讨沉默着色性干皮病A(XPA)基因表达在非小细胞肺癌耐药细胞株顺铂化疗敏感性的影响.方法:采用免疫组化法、实时定量PCR(qPCR)及Western blot方法检测非小细胞肺癌患者肿瘤组织中XPA的表达情况.应用qPCR及Western blot方法检测A549/DDP细胞经XPA-shRNA转染后XPA-mRNA及其蛋白表达.通过MTT法检测沉默XPA基因后A549/DDP细胞凋亡情况及其对顺铂的敏感性.结果:肺癌组织XPA表达水平明显高于癌旁组织;沉默XPA基因能够促进A549/DDP细胞凋亡,并能提高A549/DDP对顺铂的药物敏感性.结论:沉默XPA基因表达能够逆转肺癌A549/DDP细胞对顺铂的耐药性.%AIM; To investigate the influence on platinum-based chemotherapy sensitivity by silencing xeroderma pigmentosum group A (XPA) gene expression in non-small cell lung cancer (NSCLC) drug resistance cell lines (A549/ DDP). METHODS; We detected the expression of XPA in lung normal and tumor tissues by immunohistochemistry, quantitative real-time PCR (qPCR) and Western blotting. We silenced XPA expression in A549/DDP cells by XPA-shRNA transfection, and detected the expression of XPA by qPCR and Western blotting. The cell sensitivity to cisplatin and the apoptosis of A549/DDP cells transfected with XPA-shRNA were determined by MTT assay. RESULTS: The expression of XPA was higher in NSCLC tissues than that in normal lung tissues. Silencing XPA gene increased the apoptosis and sensitivity of A549/DDP cells to cisplatin. CONCLUSION: Silencing XPA gene can partly reverse the cisplatin resistance in human cisplatin-resistant NSCLC cell line A549/DDP.

  18. Inhibiting Effect and Its Mechanism of Ibandronate on the Proliferation of Humanized NSCLC A549 Cells in Vitro

    YAO Qiang; HUA Dong

    2014-01-01

    Objective:To explore the effect of ibandronate on the proliferation and the expression of human telomerase reverse transcriptase (hTERT) of non-small cell lung cancer (NSCLC) A549 cell line in vitro. Methods: Methyl thiazolyl tetrazolium (MTT) assay, microscope, flow cytometry (FCM) and semi-quantitative RT-PCR were employed to detect the cell proliferation, cell cycle as well as the morphological change and the expression of hTERT mRNA of A549 cell line. Results:The data showed that ibandronate could effectively inhibit the proliferation of A549 cell line in time-and concentration-dependent. Under the microscope, the lfoating cells increased gradually as the drug concentration increasing. FCM detection showed that ibandronate could induce the cell cycle stopped in G0/G1 phase and downregulation expression of hTERT. Conclusion:Ibandronate can inhibit the proliferation of A549 cell line in vitro, whose mechanism may be associated with cell cycle arrestted in phase G0/G1 and downregulation expression of hTERT.

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

    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.

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

    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

  1. The mRNA and protein expression of folylpolyglutamate synthetase in methotrexate enantiomer-resistant A549 cell lines%信息动态

    2011-01-01

    Objective To study the expression of folylpolyglutamate synthetase ( FPGS ) in methotrexate ( MTX ) enantiomer-resistant A549 cell lines [ L-( + )-MTX and D-( - )-MTX ]. Methods The expression of FPGS on genetic and protein level was determined by FQ-PCR and Western blot in lung cancer A549 cells, and MTX enantiomer-resistant A549 cells [ L-( + )-MTX and D-( - )-MTX ], with the concentration of drug resistance was 15 μmol/L. Results The genetic expression level of FPGS was ( 0.80 ± 0. 09 ) and ( 2. 04 ± 0. 34 ) folds in L-( + )- MTX/A549 cells and D-( - )-MTX/A549 cells compared with lung cancer A549 cells, there was statistical difference between two groups ( P < 0.05 ). The protein expression level of FPGS was ( 0. 85 ± 0. 12 ) and( 1.62 ± 0. 24 ) folds in L-( + )-MTX/A549 cells and D-( - )-MTX/A549 cells compared with lung cancer A549 cells,there was statistical difference ( P < 0. 05 ). Conclusion The expression level of FPGS on genetic and protein level in drug resistant cells have been changed, and significant difference in two enantiomer-resistant cells are appeared.

  2. Effects of Coptis extract combined with chemotherapeutic agents on ROS production, multidrug resistance, and cell growth in A549 human lung cancer cells

    He Chengwei

    2012-04-01

    Full Text Available Abstract Background Non–small cell lung cancer is associated with high expression of multidrug resistance (MDR proteins and low production of reactive oxygen species (ROS. Coptis extract (COP, a Chinese medicinal herb, and its major constituent, berberine (BER, have anticancer properties. This study aims to investigate the effects of COP and BER combined with chemotherapeutic agents, including fluorouracil (5-FU, camptothecin (CPT, and paclitaxel (TAX, on cell proliferation, ROS production, and MDR in A549 human non-small cell lung cancer cells. Methods A549 cells were treated with different doses of COP and BER, combined with 5-FU, CPT, and TAX. Cell viability was measured by an XTT (2,3-bis-(2-methoxy-4- nitro-5-sulfophenyl-2 H-tetrazolium-5-carboxanilide assay. Intracellular ROS levels were determined by measuring the oxidative conversion of cell permeable 2′,7′-dichlorofluorescein diacetate to fluorescent dichlorofluorescein. MDR of A549 cells was assessed by rhodamine 123 retention assay. Results Both COP and BER significantly inhibited A549 cell growth in a dose-dependent manner. Combinations of COP or BER with chemotherapeutic agents (5-FU, CPT, and TAX exhibited a stronger inhibitory effect on A549 cell growth. In addition, COP and BER increased ROS production and reduced MDR in A549 cells. Conclusion As potential adjuvants to chemotherapy for non–small cell lung cancer, COP and BER increase ROS production, reduce MDR, and enhance the inhibitory effects of chemotherapeutic agents on A549 cell growth.

  3. Impact of CHK2-small interfering RNA on CpG ODN7909-enhanced radiosensitivity in lung cancer A549 cells

    Chen W

    2012-12-01

    Full Text Available Wei Chen,* Xiaoqun Liu,* Tiankui Qiao, Sujuan Yuan Department of Oncology, Jinshan Hospital, Fudan University, Shanghai, People's Republic of China*These authors contributed equally to this workObjective: To investigate the impact of checkpoint kinase 2 (CHK2-small interfering RNA (CHK2-siRNA on the enhancement of radiosensitivity by CpG oligodeoxynucleotide (ODN 7909 in lung cancer A549 cells.Methods: The A549 cells were randomly divided into five groups: control, CpG, X-ray, CpG + X-ray, and CHK2-siRNA + CpG + X-ray. Cell colonization was observed using inverted microscopy. Cell cycle and apoptosis were analyzed by flow cytometry. CHK2 expression was detected by Western blot. CHK2-siRNA was adopted to silence the expression of CHK2.Results: The level of CHK2 phosphorylation was higher in the CpG + X-ray group than in the X-ray group. Increases in G2/mitotic (M phase arrest and apoptosis and a decrease of cell survival rate in the CpG + X-ray group were statistically significant (P < 0.05 when compared with the CHK2-siRNA + CpG + X-ray group in which the expression of CHK2 was obviously inhibited. The combination of CpG ODN7909 and X-ray irradiation was found to enhance the mitotic death of A549 cells. The sensitization enhancement ratio of mean death dose (D0 was 1.42 in the CpG + X-ray group, which was higher than that of the CHK2-siRNA + CpG + X-ray group, in which D0 was 1.05.Conclusion: To a certain extent, the impact of a combination of CpG ODN7909 and X-ray on G2/M phase arrest, apoptosis, and rate of cell survival was attenuated by CHK2-siRNA in human lung adenocarcinoma A549 cells, indicating that increased phosphorylation of CHK2 might be a radiosensitive pathway.Keywords: oligodeoxynucleotide, checkpoint kinase 2, mitotic death, apoptosis, X-ray

  4. In vitro photodynamic effect by phthalocyanine in A549 cell line

    Nevrelova, Pavla; Kolarova, Hana; Bajgar, Robert; Strnad, Miroslav

    2007-03-01

    Photodynamic therapy (PDT) utilizes a combination of sensitizer, visible light and molecular oxygen to generate singlet oxygen and reactive oxygen species (ROS) such as hydrogen peroxide, hydroxyl radical and superoxid anion. Photochemical reactions lead to damage and destruction of cancer cells. The most suitable and effective source of radiation used in PDT is a laser. For this study, a semiconductor laser with output power of 50 mW and 675 nm was selected. In this paper we report a generation of ROS using chloroaluminium disulphonated phthalocyanine (ClAlPcS II) in A549 bronchogenic carcinoma cell line after PDT in vitro. Phthalocyanines, belonging to a new generation of substances for PDT, exhibit effective tissue penetration because of their proper light absorption region, chemical stability and photodynamic stability. The fluorescence measurement with molecular probes, CM-H IIDCFDA and Amplex Red, was performed for detection of ROS generation and hydrogen peroxide release from cells. Our results demonstrated, that irradiation of cells by laser dose of 10 J.cm -2 induces higher rates of fluorescence in cells loaded with phthalocyanine compared to 20 J.cm -2. Furthermore, the production of ROS increases up to sensitizer concentration of 10 μM. The highest ROS generation was observed at laser dose of 10 J.cm -2 and 10 μM ClAlPcS II. The rates of fluorescence for hydrogen peroxid measurements were almost identical with all chosen concentrations at laser doses of 10 and 20 J.cm -2.

  5. Biosynthesis of gold nanoparticles and related cytotoxicity evaluation using A549 cells.

    Sathishkumar, M; Pavagadhi, S; Mahadevan, A; Balasubramanian, R

    2015-04-01

    Biosynthesis of gold nanoparticles (AuNPs) has become an attractive area of research as it is environmentally benign. The toxicity of AuNPs synthesized by chemical routes has been widely studied. However, little is known about the toxicity associated with the biological synthesis of AuNPs. The present study was carried out to synthesize AuNPs using star anise (Illicium verum; a commercially available spice in abundance)and evaluate its toxicity using human epithelial lung cells (A549) in comparison with AuNPs synthesized by the traditional chemical methods (using sodium citrate and sodium borohydride). Apart from cell viability, markers of oxidative stress (reduced glutathione) and cell death (caspases) were also evaluated to understand the mechanisms of toxicity. Cell viability was observed to be 65.7 percent and 72.3 percent in cells exposed to chemically synthesized AuNPs at the highest dose (200nM) as compared to 80.2 percent for biologically synthesized AuNPs. Protective coating/capping of AuNPs by various polyphenolic compounds present in star anise extract appears to be a major contributor to lower toxicity observed in biologically synthesized AuNPs. PMID:24835429

  6. In vitro anticancer activity of fucoidan from Turbinaria conoides against A549 cell lines.

    Marudhupandi, Thangapandi; Ajith Kumar, Thipramalai Thankappan; Lakshmanasenthil, Shanmugaasokan; Suja, Gunasekaran; Vinothkumar, Thirumalairaj

    2015-01-01

    The present study was conducted to evaluate the anticancer activity of fucoidan isolated from brown seaweed Turbinaria conoides. Extracted fucoidan contained 53 ± 0.69% of fucose and 38 ± 0.42% of sulphate, respectively. Functional groups and structural characteristics of the fucoidan were analyzed by FT-IR and NMR. In vitro anticancer effect was studied on A549 cell line. Fucoidan inhibited the growth of cancer cells in a dose-dependent manner and potent anticancer activities were 24.9-73.5% in the concentrations of 31.25-500 μg/ml. The CTC50 value against the cancer cell was found to be 45 μg/ml and the CTC50 value of normal Vero cell line is 325 μg/ml. This study suggests that the fucoidan from T. conoides could be significantly improved if the active component is further purified and tested for further investigation in various cancer cell lines. PMID:25451746

  7. MicroRNA-490-3p inhibits proliferation of A549 lung cancer cells by targeting CCND1

    Highlights: • We examined the level of miR-490-3p in A549 lung cancer cells compared with normal bronchial epithelial cell line. • We are the first to show the function of miR-490-3p in A549 lung cancer cells. • We demonstrate CCND1 may be one of the targets of miR-490-3p. - Abstract: MicroRNAs (miRNAs) are small non-coding RNAs that negatively regulate the translation of messenger RNAs by binding their 3′-untranslated region (3′UTR). In this study, we found that miR-490-3p is significantly down-regulated in A549 lung cancer cells compared with the normal bronchial epithelial cell line. To better characterize the role of miR-490-3p in A549 cells, we performed a gain-of-function analysis by transfecting the A549 cells with chemically synthesized miR-490-3P mimics. Overexpression of miR-490-3P evidently inhibits cell proliferation via G1-phase arrest. We also found that forced expression of miR-490-3P decreased both mRNA and protein levels of CCND1, which plays a key role in G1/S phase transition. In addition, the dual-luciferase reporter assays indicated that miR-490-3P directly targets CCND1 through binding its 3′UTR. These findings indicated miR-490-3P could be a potential suppressor of cellular proliferation

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

    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

  9. 人肺癌A549细胞系肿瘤干细胞的筛选和鉴定%Identiifcation and Isolation of Cancer Stem Cells from A549 Cells

    夏晖; 于长海; 张文; 张宝石; 赵英男; 方芳

    2013-01-01

    Background and objective Lung cancer stem cells are the root causes of lung cancer malignant phe-notype and potential therapeutic target, the aim of this study is to isolate and characterize the cancer stem cells in the lung adenoearcinomas cell line A549, so as to provide an experimental basis for further stem cell research. Methods hTe cancer stem cells were isolated from the lung adenoearcinomas cell line A549 using FACS. And the difference of colony formation, cell proliferation and tumorigenicity in vitro were also tested. hTe expression of CD133 and ABCG2 were evaluated by RT-PCR and Western blot. Results hTe percentage of SP cells was 5.93%of A549 and 0.32%of A549 atfer incubation with verapamil. hTe results showed that there were signiifcantly higher expression of CD133 and ABCG2 on SP cells than that of non-SP cells. And the ability of colony formation, cell proliferation and tumorigenicity in SP cell group were remarkably higher than that in non-SP cell group. Conclusion Our results suggested that the cancer stem cells with higher expression of CD133 and ABCG2 can be isolated from the lung adenoearcinomas cell line A549 using FACS and be used in the further research experiments.%背景与目的肺癌干细胞是肺癌恶性表型的根源和潜在的治疗靶点,从人肺癌A549细胞株中分离肺癌干细胞,观察特异性干细胞标志物分子的表达,为进一步的干细胞研究提供试验基础。方法接种肺癌A549细胞株,经流式细胞术,特异性筛选分离肺癌干细胞,观察克隆形成能力、细胞增殖能力和体外致瘤能力的差别,并分别用RT-PCR和Western blot的方法分析干细胞标志物分子CD133和ABCG2的表达。结果经过流式细胞仪成功分选了人肺腺癌A549细胞系SP细胞亚群,结果表明此SP细胞亚群约占A549细胞总数的5.93%,经维拉帕米处理后Hoechest33342阴性/弱阳性细胞含量下降为0.32%,SP细胞克隆形成能力,细胞增殖能力和

  10. 透明质酸寡糖调节A549/DDP多药耐药作用的研究%Effects of reversing drug resistant of hyaluronate oligomers on A549/DDP cell line of human lung cancer

    张宪真; 王宝中

    2009-01-01

    Objective:To investigate the effects of hyaluronate oligomers on the multiple drug resistance of lung cancer cell lines A549/DDP. Methods: After co-culturing A549/DDP and CD44 monoclonal antibody or hyaluronan oligomers for 48 hours,to detect the following parameters:Hyaluronate contents of the medium by radioimmunoassay,MDR1,MRP,LRP expressions by flow cytometry,survival rate of cells under different concentrations of cisplatin by MTT tests. Results: Hyaluronan oligomers can decrease hyaluronan expression,and MDR1,MRP,LRP expression of A549/DDP.In addition,apoptosis level of cells treated by hyaluronan oligomers increased significantly in higher concentration cisplatin. Conclution: In vitro,hyaluronan oligomers can reverse drug resistance of A549/DDP.%目的:通过研究透明质酸寡糖对人肺腺癌细胞系A549/DDP的P糖蛋白和多药耐药相关蛋白(MRP)、肺耐药蛋白(LRP)表达的影响,探讨透明质酸在引起肿瘤细胞多药耐药中的作用.方法: 将CD44单抗或透明质酸寡糖与A549/DDP细胞共培养48小时,放免法检测培养基中细胞所分泌透明质酸的含量,流式细胞仪检测经上述处理的A549/DDP表面MDR1 、MRP、LRP的表达率,MTT法检测在不同浓度顺铂作用下,各组细胞的存活率.结果: 经透明质酸寡糖处理后A549/DDP,分泌透明质酸较未处理组明显减少(P<0.05);且细胞表面与多药耐药相关的MDR1 、MRP、LRP的表达率均降低(P<0.05).另外,处理后的细胞,在不同浓度顺铂作用时,细胞凋亡率明显增加.结论: 体外条件下,透明质酸寡糖能逆转A549/DDP的耐药.

  11. Cytoskeletal changes as an early event in hydrogen peroxide-induced cell injury: a study in A549 cells.

    Raghu, G.; Striker, L.; Harlan, J; Gown, A.; Striker, G

    1986-01-01

    Hydrogen peroxide (H2O2) and other oxygen metabolites have been implicated in the pathogenesis of cell and tissue injury. The nature of the injury occurring in cells exposed to oxygen metabolites is unknown. A549 cells, derived from human lung carcinoma, were exposed to glucose-glucose oxidase or hydrogen peroxide in vitro. The distribution of actin and cytokeratin filaments, as well as 51chromium (51Cr) release and trypan blue dye exclusion were assessed. Both glucose-glucose oxidase and H2O...

  12. Dexamethasone enhances invasiveness of Aspergillus fumigatus conidia and fibronectin expression in A549 cells

    LI Tao; LI Jing-chao; QI Qian; LI Yu

    2013-01-01

    Background The efficacies of current treatments for invasive aspergillus (IA) are unsatisfactory and new therapeutic targets or regimens to treat IA are urgently needed.Previous studies have indicated that the ability of conidia to invade host cells is critical in IA development and fibronectin has a hand in the conidia adherence process.In the clinical setting,many patients who receive glucocorticoid for extended periods are susceptible to Aspergillus fumigatus (A.fumigatus) infection,for this reason we investigated the effect of glucocorticoid on conidia invasiveness by comparing the invasiveness of A.fumigatus conidia in the type Ⅱ human alveolar cell line (A549) cultured with different concentrations of dexamethasone.We also explored the relationships between dexamethasone and fibronectin expression.Methods Following culture with anti-fibronectin antibodies and/or dexamethasone,type Ⅱ human alveolar A549 cells were infected with conidia of A.fumigatus.After 4 hours,the extracellular free conidia were washed away and the remaining immobilized conidia were released using Triton-X 100 and quantified by counting the colony-forming units.The invasiveness of conidia was measured by calculating the invasion rate (%).The transcription of the fibronectin gene in cells cultured with different concentrations of dexamethasone for 24 hours was tested by fiuorogenic quantitative RT-PCR while the expression of fibronectinin cells cultured for 48 hours was tested by Western blotting and immunocytochemistry.Results A significant reduction in the invasiveness of conidia was seen in the cells cultured with anti-fibronectin antibody ((14.42±1.68)% vs.(19.17±2.53)%,P <0.05),but no significant difference was observed in cells cultured with a combination of anti-fibronectin antibody and dexamethasone (6.37×10-5 mol/L).There was no correlation between the dexamethasone concentration and the invasiveness of conidia after dexamethasone pretreatment of cells for 4 hours

  13. Simulated microgravity alters the metastatic potential of a human lung adenocarcinoma cell line.

    Chang, De; Xu, Huiwen; Guo, Yinghua; Jiang, Xuege; Liu, Yan; Li, Kailong; Pan, Chunxiao; Yuan, Ming; Wang, Junfeng; Li, Tianzhi; Liu, Changting

    2013-03-01

    Simulated microgravity (SM) has been implicated in affecting diverse cellular pathways. Although there is emerging evidence that SM can alter cellular functions, its effect in cancer metastasis has not been addressed. Here, we demonstrate that SM inhibits migration, gelatinolytic activity, and cell proliferation of an A549 human lung adenocarcinoma cell line in vitro. Expression of antigen MKI67 and matrix metalloproteinase-2 (MMP2) was reduced in A549 cells stimulated by clinorotation when compared with the 1×g control condition, while overexpression of each gene improves ability of proliferation and migration, respectively, under SM conditions. These findings suggest that SM reduced the metastatic potential of human lung adenocarcinoma cells by altering the expression of MKI67 and MMP2, thereby inhibiting cell proliferation, migration, and invasion, which may provide some clues to study cancer metastasis in the future. PMID:23404217

  14. CDK-associated Cullin 1 promotes cell proliferation with activation of ERK1/2 in human lung cancer A549 cells

    Highlights: •CDK-associated Cullin 1 (CAC1) expression increases in human lung carcinoma. •CAC1 promotes the proliferation of lung cancer A549 cells. •CAC1 promotes human lung cancer A549 cell proliferation with activation of ERK1/2. -- Abstract: Lung cancer is one of the most common causes of cancer-related death in the world, but the mechanisms remain unknown. In this study, we investigated the expression of CDK-associated Cullin 1 (CAC1) in lung cancer, the effect of CAC1 on the proliferation of human lung cancer A549 cells, and the activation of signaling pathways of mitogen-activated protein kinases (MAPKs). Results showed that CAC1 expression was higher levels in human lung carcinoma than normal lung tissue, and CAC1 siRNA reduced the proliferation of lung cancer A549 cells by decreasing cell activity and cell division in vitro. The proportion of cells treated with CAC1 siRNA increased in the G1 phase and decreased in the S and G2/M phase, indicative of G1 cell cycle arrest. Furthermore, the proportions of early/late apoptosis in lung cancer A549 cells were enhanced with CAC1 siRNA treatment. It was also found that activation of extracellular signal-regulated protein kinase (ERK) and p38 signaling pathways were involved in the proliferation of A549 cells. After CAC1 siRNA treatment, p-ERK1/2 levels decreased, and meanwhile p-p38 level increased, A549 cell proliferation increased when ERK1/2 signaling is activated by PMA. Our findings demonstrated that CAC1 promoted the proliferation of human lung cancer A549 cells with activation of ERK1/2 signaling pathways, suggesting a potential cure target for treatment of human lung cancer

  15. CDK-associated Cullin 1 promotes cell proliferation with activation of ERK1/2 in human lung cancer A549 cells

    Chen, Tian Jun [Respiratory Department, The First Affiliated Hospital, Xi’an Jiaotong University College of Medicine, Xi’an 710061 (China); Gao, Fei [Hua-shan Central Hospital of Xi’an, Xi’an 710043 (China); Yang, Tian; Thakur, Asmitanand; Ren, Hui; Li, Yang; Zhang, Shuo; Wang, Ting [Respiratory Department, The First Affiliated Hospital, Xi’an Jiaotong University College of Medicine, Xi’an 710061 (China); Chen, Ming Wei, E-mail: xjtucmw@163.com [Respiratory Department, The First Affiliated Hospital, Xi’an Jiaotong University College of Medicine, Xi’an 710061 (China)

    2013-07-19

    Highlights: •CDK-associated Cullin 1 (CAC1) expression increases in human lung carcinoma. •CAC1 promotes the proliferation of lung cancer A549 cells. •CAC1 promotes human lung cancer A549 cell proliferation with activation of ERK1/2. -- Abstract: Lung cancer is one of the most common causes of cancer-related death in the world, but the mechanisms remain unknown. In this study, we investigated the expression of CDK-associated Cullin 1 (CAC1) in lung cancer, the effect of CAC1 on the proliferation of human lung cancer A549 cells, and the activation of signaling pathways of mitogen-activated protein kinases (MAPKs). Results showed that CAC1 expression was higher levels in human lung carcinoma than normal lung tissue, and CAC1 siRNA reduced the proliferation of lung cancer A549 cells by decreasing cell activity and cell division in vitro. The proportion of cells treated with CAC1 siRNA increased in the G1 phase and decreased in the S and G2/M phase, indicative of G1 cell cycle arrest. Furthermore, the proportions of early/late apoptosis in lung cancer A549 cells were enhanced with CAC1 siRNA treatment. It was also found that activation of extracellular signal-regulated protein kinase (ERK) and p38 signaling pathways were involved in the proliferation of A549 cells. After CAC1 siRNA treatment, p-ERK1/2 levels decreased, and meanwhile p-p38 level increased, A549 cell proliferation increased when ERK1/2 signaling is activated by PMA. Our findings demonstrated that CAC1 promoted the proliferation of human lung cancer A549 cells with activation of ERK1/2 signaling pathways, suggesting a potential cure target for treatment of human lung cancer.

  16. Characterization of indoor dust from Brazil and evaluation of the cytotoxicity in A549 lung cells.

    Deschamps, E; Weidler, P G; Friedrich, F; Weiss, C; Diabaté, S

    2014-04-01

    Over the past decade, ambient air particulate matter (PM) has been clearly associated with adverse health effects. In Brazil, small and poor communities are exposed to indoor dust derived from both natural sources, identified as blowing soil dust, and anthropogenic particles from mining activities. This study investigates the physicochemical and mineralogical composition of indoor PM10 dust samples collected in Minas Gerais, Brazil, and evaluates its cytotoxicity and inflammatory potential. The mean PM10 mass concentration was 206 μg/m(3). The high dust concentration in the interior of the residences is strongly related to blowing soil dust. The chemical and mineralogical compositions were determined by ICP-OES and XRD, and the most prominent minerals were clays, Fe-oxide, quartz, feldspars, Al(hydr)oxides, zeolites, and anatase, containing the transition metals Fe, Cr, V, Ni, Cu, Zn, Ti, and Mn as well as the metalloid As. The indoor dust samples presented a low water solubility of about 6 %. In vitro experiments were carried out with human lung alveolar carcinoma cells (A549) to study the toxicological effects. The influence of the PM10 dust samples on cell viability, intracellular formation of reactive oxygen species (ROS), and release of the pro-inflammatory cytokine IL-8 was analysed. The indoor dust showed little effects on alamarBlue reduction indicating unaltered mitochondrial activity. However, significant cell membrane damage, ROS production, and IL-8 release were detected in dependence of dose and time. This study will support the implementation of mitigation actions in the investigated area in Brazil. PMID:23990125

  17. NMR studies of the relationship between the changes of membrane lipids and the cisplatin-resistance of A549/DDP cells

    Huang Youguo

    2003-04-01

    Full Text Available Abstract Changes of membrane lipids in cisplatin-sensitive A549 and cisplatin-resistant A549/DDP cells during the apoptotic process induced by a clinical dose of cisplatin (30 μM were detected by 1H and 31P-NMR spectroscopy and by membrane fluidity measurement. The apoptotic phenotypes of the two cell lines were monitored with flow cytometry. The assays of apoptosis showed that significant apoptotic characteristics of the A549 cells were induced when the cells were cultured for 24 hours after treatment with cisplatin, while no apoptotic characteristic could be detected for the resistant A549/DDP cells even after 48 hours. The results of 1H-NMR spectroscopy demonstrated that the CH2/CH3 and Glu/Ct ratios of the membrane of A549 cells increased significantly, but those in A549/DDP cell membranes decreased. In addition, the Chol/CH3 and Eth/Ct ratios decreased for the former but increased for the latter cells under the same conditions. 31P-NMR spectroscopy indicated levels of phosphomonoesters (PME and ATP decreased in A549 but increased in A549/DDP cells after being treated with cisplatin. These results were supported with the data obtained from 1H-NMR measurements. The results clearly indicated that components and properties of membrane phospholipids of the two cell lines were significantly different during the apoptotic process when they were treated with a clinical dose of cisplatin. Plasma membrane fluidity changes during cisplatin treatment as detected with the fluorescence probe TMA-DPH also indicate marked difference between the two cell lines. We provided evidence that there are significant differences in plasma membrane changes during treatment of cisplatin sensitive A549 and resistant A549/DDP cells.

  18. Napsin A transfection interferes the ephethlial-mesenchymal transition of A549 cells%Napsin A基因转染干预A549细胞的上皮-间质转化

    管淑红; 郑金旭; 汤艳; 宋萍; 许清; 刘继柱

    2011-01-01

    目的 探讨Napsin A基因转染至A549细胞对其上皮-间质转化(EMT)的作用和机制.方法 采用慢病毒载体质粒PLJM1构建重组质粒PLJM1-Napsin A,将Napsin A基因转染至A549细胞染色体中并鉴定.用转化生长因子-β1(TGF-β1)刺激A549细胞构建体外EMT模型,倒置显微镜下动态观察细胞形态学的变化,观察Napsin A基因转染对A549细胞在体外EMT模型中细胞EMT和表达黏着斑激酶(FAK)的影响.结果重组质粒PLJM1-Napsin A测序结果与设计序列完全符合,转Napsin A基因A549细胞表达Napsin A蛋白显著高于非转基因细胞组(P<0.01).细胞经TGF-β1刺激后形态上演变为间质细胞,E钙蛋白的mRNA和蛋白表达水平明显下调(P<0.01),相反Ⅰ型胶原则显著上调(P<0.01),提示体外构建EMT模型获得成功.转Napsin A基因A549细胞在TGF-β1干预后,其细胞形态间质改变、E钙蛋白和Ⅰ型胶原的表达量也发生相似变化趋势,但变化幅度显著变小(其中E钙蛋白:P<0.01,Ⅰ型胶原:P<0.05).体外EMT模型中,细胞FAK蛋白表达量增多(P<0.01),但转基因细胞上调趋势明显小于未转基因细胞(P<0.01).结论转染Napsin A基因至A549细胞可以部分阻滞细胞EMT进程,其作用机制可能与抑制整合素信号转导通路有关.%Objective To study the effect and mechanism of Napsin A gene transfection into A549 cells on ephethlial-mesenchymal transition( EMT ) in vitro. Methods A recombinant lentiviral plasmid PLJMI-Napsin A was constructed, then transfected into A549 cell and identified. A549 cells EMT model was estahlished by transforming growth factor beta-1( TGF-β1 )treatment in vitro. The morphology change was observed under inverted microscopy successively. To ohserve the degree of EMT by TGF-β1 intervening A549 cells, the expression of E-cadherin and collagen type Ⅰ was detected by reverse transcription-polymerase chain reaction( RT-PCR )and Western blotting. Finally , in order to investigate

  19. Genomic signature and toxicogenomics comparison of polycationic gene delivery nanosystems in human alveolar epithelial A549 cells

    J Barar

    2009-10-01

    Full Text Available "nBackground and the purpose of the study: Of the gene delivery systems, non-viral polycationic gene delivery nanosystems have been alternatively exploited as a relatively safe delivery reagents compared to viral vectors. However, little is known about the genomic impacts of these delivery systems in target cells/tissues. In this study, the toxicogenomics and genotoxicity potential of some selected polycationic lipid/polymer based nanostructures (i.e., Oligofectamine® (OF, starburst polyamidoamine Polyfect® (PF and diaminobutane (DAB dendrimers were investigated in human alveolar epithelial A549 cells. "nMethods: To study the nature and the ontology of the gene expression changes in A549 cells upon treatment with polycationic nanostructures, MTT assay and microarray gene expression profiling methodology were employed. For microarray analysis, cyanine (Cy3/Cy5 labeled cDNA samples from treated and untreated cells were hybridized on target arrays housing 200 genes. "nResults and major conclusions: The polycationic nanosystems induced significant gene expression changes belonging to different genomic ontologies such as cell defence and apoptosis pathways. These data suggest that polycationic nanosystems can elicit multiple gene expression changes in A549 cells upon their chemical structures and interactions with cellular/subcellular components. Such impacts may interfere with the main goals of the desired genemedicine.

  20. The effects of disodium cromoglycate on enhanced adherence of Haemophilus influenzae to A549 cells infected with respiratory syncytial virus.

    Fukasawa, Chie; Ishiwada, Naruhiko; Ogita, Junko; Hishiki, Haruka; Kohno, Yoichi

    2009-08-01

    Nontypeable Haemophilus influenzae (NTHi) secondary infection often complicates respiratory syncytial virus (RSV) infections. Previous studies have revealed that RSV infections enhance NTHi adherence to airway epithelial cells. In this study, we investigated the effects of disodium cromoglycate (DSCG) and corticosteroids, which are frequently used for the treatment of wheezing often related to RSV infections, on the adherence of NTHi to RSV-infected A549 cells. DSCG inhibited enhanced adherence of NTHi to RSV-infected A549 cells, whereas dexamethasone (Dex) and fluticasone propionate (Fp) did not. DSCG suppressed the expression of ICAM-1, which is one of the NTHi receptors. Furthermore, DSCG exhibited an inhibitory effect on RSV infections. It is suggested that DSCG exerts an anti-RSV effect, and consequently attenuates the expression of NTHi receptors. PMID:19390482

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

    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.

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

    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

  3. Apoptosis signal-regulating kinase 1 mediates denbinobin-induced apoptosis in human lung adenocarcinoma cells

    Pan Shiow-Lin

    2009-05-01

    Full Text Available Abstract In the present study, we explore the role of apoptosis signal-regulating kinase 1 (ASK1 in denbinobin-induced apoptosis in human lung adenocarcinoma (A549 cells. Denbinobin-induced cell apoptosis was attenuated by an ASK1 dominant-negative mutant (ASK1DN, two antioxidants (N-acetyl-L-cysteine (NAC and glutathione (GSH, a c-Jun N-terminal kinase (JNK inhibitor (SP600125, and an activator protein-1 (AP-1 inhibitor (curcumin. Treatment of A549 cells with denbinobin caused increases in ASK1 activity and reactive oxygen species (ROS production, and these effects were inhibited by NAC and GSH. Stimulation of A549 cells with denbinobin caused JNK activation; this effect was markedly inhibited by NAC, GSH, and ASK1DN. Denbinobin induced c-Jun phosphorylation, the formation of an AP-1-specific DNA-protein complex, and Bim expression. Bim knockdown using a bim short interfering RNA strategy also reduced denbinobin-induced A549 cell apoptosis. The denbinobin-mediated increases in c-Jun phosphorylation and Bim expression were inhibited by NAC, GSH, SP600125, ASK1DN, JNK1DN, and JNK2DN. These results suggest that denbinobin might activate ASK1 through ROS production to cause JNK/AP-1 activation, which in turn induces Bim expression, and ultimately results in A549 cell apoptosis.

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

    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

  5. Moringa oleifera Gold Nanoparticles Modulate Oncogenes, Tumor Suppressor Genes, and Caspase-9 Splice Variants in A549 Cells.

    Tiloke, Charlette; Phulukdaree, Alisa; Anand, Krishnan; Gengan, Robert M; Chuturgoon, Anil A

    2016-10-01

    Gold nanoparticles (AuNP's) facilitate cancer cell recognition and can be manufactured by green synthesis using nutrient rich medicinal plants such as Moringa oleifera (MO). Targeting dysregulated oncogenes and tumor suppressor genes is crucial for cancer therapeutics. We investigated the antiproliferative effects of AuNP synthesized from MO aqueous leaf extracts (MLAuNP ) in A549 lung and SNO oesophageal cancer cells. A one-pot green synthesis technique was used to synthesise MLAuNP . A549, SNO cancer cells and normal peripheral blood mononuclear cells (PBMCs) were exposed to MLAuNP and CAuNP to evaluate cytotoxicity (MTT assay); apoptosis was measured by phosphatidylserine (PS) externalization, mitochondrial depolarization (ΔΨm) (flow cytometry), caspase-3/7, -9 activity, and ATP levels (luminometry). The mRNA expression of c-myc, p53, Skp2, Fbw7α, and caspase-9 splice variants was determined using qPCR, while relative protein expression of c-myc, p53, SRp30a, Bax, Bcl-2, Smac/DIABLO, Hsp70, and PARP-1 were determined by Western blotting. MLAuNP and CAuNP were not cytotoxic to PBMCs, whilst its pro-apoptotic properties were confirmed in A549 and SNO cells. MLAuNP significantly increased caspase activity in SNO cells while MLAuNP significantly increased PS externalization, ΔΨm, caspase-9, caspase-3/7 activities, and decreased ATP levels in A549 cells. Also, p53 mRNA and protein levels, SRp30a (P = 0.428), Bax, Smac/DIABLO and PARP-1 24 kDa fragment levels were significantly increased. Conversely, MLAuNP significantly decreased Bcl-2, Hsp70, Skp2, Fbw7α, c-myc mRNA, and protein levels and activated alternate splicing with caspase-9a splice variant being significantly increased. MLAuNP possesses antiproliferative properties and induced apoptosis in A549 cells by activating alternate splicing of caspase-9. J. Cell. Biochem. 117: 2302-2314, 2016. © 2016 Wiley Periodicals, Inc. PMID:26923760

  6. Inflammatory response and genotoxicity of seven wood dusts in the human epithelial cell line A549.

    Bornholdt, Jette; Saber, Anne T; Sharma, Anoop K; Savolainen, Kai; Vogel, Ulla; Wallin, Håkan

    2007-08-15

    Exposure to wood dust is common in many workplaces. Epidemiological studies indicate that occupational exposure to hardwood dusts is more harmful than to softwood dusts. In this study, human epithelial cell line A549 was incubated with well-characterized dusts from six commonly used wood species and from medium density fibreboard (MDF), at concentrations between 10 and 300microg/ml. After 3 and 6h of incubation, genotoxicity was assessed by measurement of DNA damage with the single-cell gel electrophoresis (comet) assay and inflammation was measured by the expression of IL-6 and IL-8 mRNA and by the amount of IL-8 protein. There was a 1.2-1.4-fold increase in DNA strand breaks after incubation with beech, teak, pine and MDF dusts compared with the levels in untreated cells, but after 6h only the increase induced by the MDF dust remained. Increased expression of cellular IL-6 and IL-8 mRNA was induced by all of the wood dusts at both times. Similar to IL-8 mRNA expression, the amounts of secreted IL-8 protein were elevated, except after incubation with oak dust, where a marginal reduction was seen. On the basis of the effects on IL-8 mRNA expression, the wood dusts could be divided into three groups, with teak dust being the most potent, MDF, birch, spruce and pine being intermediate, and beech and oak being the least potent. The induction of DNA strand breaks did not correlate well with the interleukin response. In conclusion, all wood dusts induced cytokine responses, and some dusts induced detectable DNA damage. The inflammatory potency seemed intermediate for dusts from the typical softwoods spruce and pine, whereas the dusts from species linked to cancer, beech and oak, were the least inflammatory. The variation of the effects induced by different wood dusts over time indicates that the DNA damage was not secondary to the cytokine response. Although hardwoods are often considered more harmful than softwoods by regulatory agencies, the current experiments do not

  7. Cytotoxic and genotoxic potencies of single and combined spore extracts of airborne OTA-producing and OTA-non-producing Aspergilli in Human lung A549 cells.

    Šegvić Klarić, Maja; Jakšić Despot, Daniela; Kopjar, Nevenka; Rašić, Dubravka; Kocsubé, Sándor; Varga, János; Peraica, Maja

    2015-10-01

    Aspergillus sclerotiorum (AS) is a well-known producer of ochratoxin A (OTA) while Aspergillus pseudoglaucus (AP) produces a wide range of extrolites with poorly investigated toxicity. These species are frequently co-occur in grain mill aeromycota. The aim of this study was to determine OTA levels in spore extracts using HPLC and immunoaffinity columns, and to examine the cytotoxicity of pure OTA, OTA-positive (AS-OTA(+)) and OTA-negative (AS-OTA(-)) spore extracts, as well as of AP spore extract, on human lung adenocarcinoma cells A549, individually and in combination, using a colorimetric MTT test (540nm). To establish which type of cell death predominated after treatments, a quantitative fluorescent assay with ethidium bromide and acridine orange was used, and the level of primary DNA damage in A549 cells was evaluated using the alkaline comet assay. OTA was detected in spore extracts (0.3-28µg/mL) of 3/6 of the AS strains, while none of the tested AP strains were able to produce OTA. Taking into account the maximum detected concentration of OTA in the spores, the daily intake of OTA by inhalation was calculated to be 1ng/kg body weight (b.w.), which is below the tolerable daily intake for OTA (17ng/kg b.w.). Using the MTT test, the following IC50 values were obtained: single OTA (53μg/mL); AS-OTA(+) (mass concentration 934μg/mL corresponds to 10.5μg/mL of OTA in spore extract); and 2126μg/mL for AP. The highest applied concentration of AS-OTA(-) spore extract (4940μg/mL) decreased cell viability by 30% and IC50 for the extract could not be determined. Single OTA and AS-OTA(+) and combinations (AP+AS-OTA(+) and AP+AS-OTA(-)) in subtoxic concentrations provoked significant primary DNA damage, apoptosis, and to a lesser extent, necrosis in A549 cells. Mixture of AP+AS-OTA(+) and AP+AS-OTA(-) in subtoxic concentrations showed dominant additive interactions. Despite the low calculated daily intake of OTA by inhalation, our results suggest that chronic exposure

  8. Preliminary Proteomic Analysis of A549 Cells Infected with Avian Influenza Virus H7N9 and Influenza A Virus H1N1

    Ding, Xiaoman; Lu, Jiahai; Yu, Ruoxi; Wang, Xin; Wang, Ting; Dong, Fangyuan; Peng, Bo; Wu, Weihua; Liu, Hui; Geng, Yijie; Zhang, Renli; Ma, Hanwu; Cheng, Jinquan; Yu, Muhua; Fang, Shisong

    2016-01-01

    A newly emerged H7N9 influenza virus poses high risk to human beings. However, the pathogenic mechanism of the virus remains unclear. The temporal response of primary human alveolar adenocarcinoma epithelial cells (A549) infected with H7N9 influenza virus and H1N1 influenza A virus (H1N1, pdm09) were evaluated using the proteomics approaches (2D-DIGE combined with MALDI-TOF-MS/MS) at 24, 48 and 72 hours post of the infection (hpi). There were 11, 12 and 33 proteins with significant different expressions (P<0.05) at 24, 48 and 72hpi, especially F-actin-capping protein subunit alpha-1 (CAPZA1), Ornithine aminotransferase (OAT), Poly(rC)-binding protein 1 (PCBP1), Eukaryotic translation initiation factor 5A-1 (EIF5A) and Platelet-activating factor acetylhydrolaseⅠb subunit beta (PAFAH1B2) were validated by western-blot analysis. The functional analysis revealed that the differential proteins in A549 cells involved in regulating cytopathic effect. Among them, the down-regulation of CAPZA1, OAT, PCBP1, EIF5A are related to the death of cells infected by H7N9 influenza virus. This is the first time show that the down-regulation of PAFAH1B2 is related to the later clinical symptoms of patients infected by H7N9 influenza virus. These findings may improve our understanding of pathogenic mechanism of H7N9 influenza virus in proteomics. PMID:27223893

  9. Levels of human equilibrative nucleoside transporter-1 are higher in proliferating regions of A549 tumor cells grown as tumor xenografts in vivo

    3’-Fluoro-3’-deoxythymidine (FLT) has been proposed for positron emission tomography (PET)-based identification of tumor chemosensitivity that is mediated by the human equilibrative nucleoside transporter-1 (ENT1). ENT1 facilitates transport of FLT into cells and elevated levels of FLT are associated with both larger FLT-PET signals and increased response to nucleoside-based chemotherapies. FLT-PET is also used as a measure of tumor proliferation. The present study examined the extent to which ENT1 levels vary in a proliferation-dependent manner in tumor cells in vivo. Methods: The human adenocarcinoma cell line A549 was used to establish tumor xenografts in nude mice. FLT uptake was measured in vivo using PET, and further examined ex vivo using autoradiography. FLT uptake patterns were compared to immunohistochemical (IHC) analysis of ENT1 and the proliferation markers Ki67 and BrdU. Results: Regional differences in FLT uptake matched differences in IHC proliferation markers. All cells stained for ENT1, but the staining intensity was twice as high for Ki67+ cells than for Ki67− cells. Conclusions: Under in vivo conditions, proliferating regions of tumors show increased FLT uptake and higher ENT1 levels than nonproliferating tumor regions.

  10. Mimulone-induced autophagy through p53-mediated AMPK/mTOR pathway increases caspase-mediated apoptotic cell death in A549 human lung cancer cells.

    Hyun-Kyu An

    Full Text Available Anticancer properties and mechanisms of mimulone (MML, C-geranylflavonoid isolated from the Paulownia tomentosa fruits, were firstly elucidated in this study. MML prevented cell proliferation in a dose- and time-dependent way and triggered apoptosis through the extrinsic pathway in A549 human lung adenocarcinoma cells. Furthermore, MML-treated cells displayed autophagic features, such as the formation of autophagic vacuoles, a primary morphological feature of autophagy, and the accumulation of microtubule-associated protein 1 light chain 3 (LC3 puncta, another typical maker of autophagy, as determined by FITC-conjugated immunostaining and monodansylcadaverine (MDC staining, respectively. The expression levels of LC3-I and LC3-II, specific markers of autophagy, were also augmented by MML treatment. Autophagy inhibition by 3-methyladenine (3-MA, pharmacological autophagy inhibitor, and shRNA knockdown of Beclin-1 reduced apoptotic cell death induced by MML. Autophagic flux was not significantly affected by MML treatment and lysosomal inhibitor, chloroquine (CQ suppressed MML-induced autophagy and apoptosis. MML-induced autophagy was promoted by decreases in p53 and p-mTOR levels and increase of p-AMPK. Moreover, inhibition of p53 transactivation by pifithrin-α (PFT-α and knockdown of p53 enhanced induction of autophagy and finally promoted apoptotic cell death. Overall, the results demonstrate that autophagy contributes to the cytotoxicity of MML in cancer cells harboring wild-type p53. This study strongly suggests that MML is a potential candidate for an anticancer agent targeting both autophagy and apoptotic cell death in human lung cancer. Moreover, co-treatment of MML and p53 inhibitor would be more effective in human lung cancer therapy.

  11. Mimulone-induced autophagy through p53-mediated AMPK/mTOR pathway increases caspase-mediated apoptotic cell death in A549 human lung cancer cells.

    An, Hyun-Kyu; Kim, Kyoung-Sook; Lee, Ji-Won; Park, Mi-Hyun; Moon, Hyung-In; Park, Shin-Ji; Baik, Ji-Sue; Kim, Cheorl-Ho; Lee, Young-Choon

    2014-01-01

    Anticancer properties and mechanisms of mimulone (MML), C-geranylflavonoid isolated from the Paulownia tomentosa fruits, were firstly elucidated in this study. MML prevented cell proliferation in a dose- and time-dependent way and triggered apoptosis through the extrinsic pathway in A549 human lung adenocarcinoma cells. Furthermore, MML-treated cells displayed autophagic features, such as the formation of autophagic vacuoles, a primary morphological feature of autophagy, and the accumulation of microtubule-associated protein 1 light chain 3 (LC3) puncta, another typical maker of autophagy, as determined by FITC-conjugated immunostaining and monodansylcadaverine (MDC) staining, respectively. The expression levels of LC3-I and LC3-II, specific markers of autophagy, were also augmented by MML treatment. Autophagy inhibition by 3-methyladenine (3-MA), pharmacological autophagy inhibitor, and shRNA knockdown of Beclin-1 reduced apoptotic cell death induced by MML. Autophagic flux was not significantly affected by MML treatment and lysosomal inhibitor, chloroquine (CQ) suppressed MML-induced autophagy and apoptosis. MML-induced autophagy was promoted by decreases in p53 and p-mTOR levels and increase of p-AMPK. Moreover, inhibition of p53 transactivation by pifithrin-α (PFT-α) and knockdown of p53 enhanced induction of autophagy and finally promoted apoptotic cell death. Overall, the results demonstrate that autophagy contributes to the cytotoxicity of MML in cancer cells harboring wild-type p53. This study strongly suggests that MML is a potential candidate for an anticancer agent targeting both autophagy and apoptotic cell death in human lung cancer. Moreover, co-treatment of MML and p53 inhibitor would be more effective in human lung cancer therapy. PMID:25490748

  12. Role of ATM kinase in the effect of CpG ODN7909 on X-ray-induced G2/M phase arrest and apoptosis in A549 cells

    Objective: To investigate the relationship between the effect of unmethylated cytosine phosphate-guanine oligodeoxynucleotide (CpG ODN) 7909 on X-ray-induced G2/M phase arrest and apoptosis and the phosphorylation of ATM kinase. Methods: Human lung adenocarcinoma A549 cells were randomly classified into five groups, control, CpG group, irradiation group, CpG + irradiation group and ATM siRNA + CpG + irradiation group. Cell survival fraction was evaluated by clonogenic assay. Cell cycle and apoptosis were analyzed by flow cytometry. The expressions of ATM, checkpoint kinase-2 (Chk2) and p53 were detected with Western blot. Results: Compared to the situation under X-ray irradiation alone, decreased cell clonogenic survival, prolonged G2/M arrest, and increased cell apoptosis were observed after the combination treatment with CpG ODN7909 and X-rays (t=13.41, 17.32 and 7.71, P<0.05). Moreover, the phosphorylations on ATM, Chk2, and p53 were increased in the irradiated A549 cells that had been pre-treated with CpG ODN7909. After ATM siRNA interfering, abrogation of G2/M arrest, reduction of apoptosis, and decrease of Chk2 and p53 phosphorylation were found in A549 cells treated with CpG ODN7909 and X-rays (t=26.84, 2.98, 47.24 and 67.47, P<0.05). Conclusions: CpG ODN7909 can enhance the X-ray-induced phosphorylation of ATM kinase in human lung adenocarcinoma cells in vitro, which might be involved in regulating G2/M phase arrest and apoptosis. (authors)

  13. Study on apoptosis of human non-small cell pulmonary carcinoma A549 cells induced by 32P-chromium phosphates in vitro

    Objective: To observe the phasic change and apoptosis of A549 non-small cell lung cancer cells induced by 32P-chromium phosphate in vitro, and establish the dose-response and time-response relationship. Methods: Internal irradiation was conducted by adding 32P-colloid into A549 cell culture system. The initial radioactivities were 0, 93, 180, 278, 370, 463 MBq/L, respectively. Giemsa stain, transmission electron microscopy and TUNEL were used in assessing morphologic, ultra structural pathologic and biochemical characteristics, and the phasic changes and apoptotic rates of cells were studied by flow cytometry. Results: After irradiation of A549 cells, the cell ratio of S + G2-M phase tended to increase within 96 h, then decreased gradually. At 72 h after irradiation the A549 cells showed excited manifestation, and in each irradiation group apoptosis began from 96 h of irradiation, and attained the peak at 120 h. Conclusion: In the lower dosage range, 32P internal irradiation may induce human NSCLC A549 cells to present delayed onset of apoptosis, and the rate of cell apoptosis is positively correlated to the initial radioactivity concentration. (authors)

  14. Platinum(II) phenanthroimidazole G-quadruplex ligand induces selective telomere shortening in A549 cancer cells.

    Mancini, Johanna; Rousseau, Philippe; Castor, Katherine J; Sleiman, Hanadi F; Autexier, Chantal

    2016-02-01

    Telomere maintenance, achieved by the binding of protective shelterin capping proteins to telomeres and by either telomerase or a recombination-based alternative lengthening of telomere (ALT) mechanism, is critical for cell proliferation and survival. Extensive telomere shortening or loss of telomere integrity activates DNA damage checkpoints, leading to cell senescence or death. Although telomerase upregulation is an attractive target for anti-cancer therapy, the lag associated with telomere shortening and the potential activation of ALT pose a challenge. An alternative approach is to modify telomere interactions with binding proteins (telomere uncapping). G-quadruplex ligands stabilize structures generated from single-stranded G-rich 3'-telomere end (G-quadruplex) folding, which in principle, cannot be elongated by telomerase, thus leading to telomere shortening. Ligands can also mediate rapid anti-proliferative effects by telomere uncapping. We previously reported that the G-quadruplex ligand, phenylphenanthroimidazole ethylenediamine platinum(II) (PIP), inhibits telomerase activity in vitro[47]. In the current study, a long-term seeding assay showed that PIP significantly inhibited the seeding capacity of A549 lung cancer cells and to a lesser extent primary MRC5 fibroblast cells. Importantly, treatment with PIP caused a significant dose- and time-dependent decrease in average telomere length of A549 but not MRC5 cells. Moreover, cell cycle analysis revealed a significant increase in G1 arrest upon treatment of A549 cells, but not MRC5 cells. Both apoptosis and cellular senescence may contribute to the anti-proliferative effects of PIP. Our studies validate the development of novel and specific therapeutic ligands targeting telomeric G-quadruplex structures in cancer cells. PMID:26724375

  15. The cytotoxicity of organophosphate flame retardants on HepG2, A549 and Caco-2 cells.

    An, Jing; Hu, Jingwen; Shang, Yu; Zhong, Yufang; Zhang, Xinyu; Yu, Zhiqiang

    2016-09-18

    In order to elucidate the cytotoxicity of organophosphate flame retardants (OPFRs), three human in vitro models, namely the HepG2 hepatoma cells, the A549 lung cancer cells and the Caco-2 colon cancer cells, were chosen to investigate the toxicity of triphenyl phosphate (TPP), tributylphosphate (TBP), tris(2-butoxyexthyl) phosphate (TBEP) and tris (2-chloroisopropyl) phosphate (TCPP). Cytotoxicity was assayed in terms of cell viability, DNA damage status, reactive oxygen species (ROS) level and lactate dehydrogenase (LDH) leakage. The results showed that all these four OPFRs could inhibit cell viability, overproduce ROS level, induce DNA lesions and increase the LDH leakage. In addition, the toxic effects of OPFRs in Caco-2 cells were relatively severer than those in HepG2 and A549 cells, which might result from some possible mechanisms apart from oxidative stress pathway. In conclusion, TBP, TPP, TBEP and TCPP could induce cell toxicity in various cell lines at relatively high concentrations as evidenced by suppression of cell viability, overproduction of ROS, induction of DNA lesions and increase of LDH leakage. Different cell types seemed to have different sensitivities and responses to OPFRs exposure, as well as the underlying potential molecular mechanisms. PMID:27336727

  16. Shikonin Induces Apoptosis, Necrosis, and Premature Senescence of Human A549 Lung Cancer Cells through Upregulation of p53 Expression

    Yueh-Chiao Yeh

    2015-01-01

    Full Text Available Shikonin, a natural naphthoquinone pigment isolated from Lithospermum erythrorhizon, has been reported to suppress growth of various cancer cells. This study was aimed to investigate whether this chemical could also inhibit cell growth of lung cancer cells and, if so, works via what molecular mechanism. To fulfill this, A549 lung cancer cells were treated with shikonin and then subjected to microscopic, biochemical, flow cytometric, and molecular analyses. Compared with the controls, shikonin significantly induced cell apoptosis and reduced proliferation in a dose-dependent manner. Specially, lower concentrations of shikonin (1–2.5 μg/mL cause viability reduction; apoptosis and cellular senescence induction is associated with upregulated expressions of cell cycle- and apoptotic signaling-regulatory proteins, while higher concentrations (5–10 μg/mL precipitate both apoptosis and necrosis. Treatment of cells with pifithrin-α, a specific inhibitor of p53, suppressed shikonin-induced apoptosis and premature senescence, suggesting the role of p53 in mediating the actions of shikonin on regulation of lung cancer cell proliferation. These results indicate the potential and dose-related cytotoxic actions of shikonin on A549 lung cancer cells via p53-mediated cell fate pathways and raise shikonin a promising adjuvant chemotherapeutic agent for treatment of lung cancer in clinical practice.

  17. Predicting the clonogenic survival of A549 cells after modulated x-ray irradiation using the linear quadratic model

    Bromley, Regina; Oliver, Lyn [Northern Sydney Cancer Centre, Radiation Oncology, Royal North Shore Hospital, Sydney, NSW 2065 (Australia); Davey, Ross; Harvie, Rozelle [Department of Medical Oncology, Bill Walsh Cancer Research Laboratories, Royal North Shore Hospital, Sydney, NSW 2065 (Australia); Baldock, Clive [Institute of Medical Physics, School of Physics, Sydney University, NSW 2006 (Australia)

    2009-01-21

    In this study we present two prediction methods, mean dose and summed dose, for predicting the number of A549 cells that will survive after modulated x-ray irradiation. The prediction methods incorporate the dose profile from the modulated x-ray fluence map applied across the cell sample and the linear quadratic (LQ) model. We investigated the clonogenic survival of A549 cells when irradiated using two different modulated x-ray fluence maps. Differences between the measured and predicted surviving fraction were observed for modulated x-ray irradiation. When the x-ray fluence map produced a steep dose gradient across the sample, fewer cells survived in the unirradiated region than expected. When the x-ray fluence map produced a less steep dose gradient across the sample, more cells survived in the unirradiated region than expected. Regardless of the steepness of the dose gradient, more cells survived in the irradiated region than expected for the reference dose range of 1-10 Gy. The change in the cell survival for the unirradiated regions of the two different dose gradients may be an important factor to consider when predicting the number of cells that will survive at the edge of modulated x-ray fields. This investigation provides an improved method of predicting cell survival for modulated x-ray radiation treatment. It highlights the limitations of the LQ model, particularly in its ability to describe the biological response of cells irradiated under these conditions.

  18. Predicting the clonogenic survival of A549 cells after modulated x-ray irradiation using the linear quadratic model

    Bromley, Regina; Oliver, Lyn; Davey, Ross; Harvie, Rozelle; Baldock, Clive

    2009-01-01

    In this study we present two prediction methods, mean dose and summed dose, for predicting the number of A549 cells that will survive after modulated x-ray irradiation. The prediction methods incorporate the dose profile from the modulated x-ray fluence map applied across the cell sample and the linear quadratic (LQ) model. We investigated the clonogenic survival of A549 cells when irradiated using two different modulated x-ray fluence maps. Differences between the measured and predicted surviving fraction were observed for modulated x-ray irradiation. When the x-ray fluence map produced a steep dose gradient across the sample, fewer cells survived in the unirradiated region than expected. When the x-ray fluence map produced a less steep dose gradient across the sample, more cells survived in the unirradiated region than expected. Regardless of the steepness of the dose gradient, more cells survived in the irradiated region than expected for the reference dose range of 1-10 Gy. The change in the cell survival for the unirradiated regions of the two different dose gradients may be an important factor to consider when predicting the number of cells that will survive at the edge of modulated x-ray fields. This investigation provides an improved method of predicting cell survival for modulated x-ray radiation treatment. It highlights the limitations of the LQ model, particularly in its ability to describe the biological response of cells irradiated under these conditions.

  19. Construction of A Eukaryotic Expression Vector Carrying the iNOS Gene and Its Effect on A549 Lung Cancer Cells

    Sujuan YE

    2012-05-01

    Full Text Available Background and objective The iNOS gene is associated with NO-mediated antitumor effects. The aims of this study are to construct a eukaryotic expression plasmid that carries the iNOS gene and to detect the expression levels and antitumor effects of the iNOS gene on A549 lung cancer cells. Methods A DNA fragment of the human iNOS coding sequence was amplified using reverse transcription polymerase chain reaction (RT-PCR. The DNA fragment was subsequently cloned into the multiple cloning sites of the eukaryotic expression vector pVAX. The recombinant plasmid was confirmed using restriction enzyme treatment, PCR, and sequencing and was then transfected into A549 lung cancer cells. The expression of the iNOS gene in the A549 lung cancer cells after transfection was verified by RT-PCR and Western blot analysis. The effects of iNOS on cell apoptosis, proliferation, and migration were identified by staining with Hoechst 3235, an MTT assay, and a scratch assay, respectively. Results The results of the restriction enzyme digestion, PCR, and sequencing verified the successful construction of the eukaryotic expression plasmid pVAX-iNOS. The iNOS gene expression level was increased in the transfected A549 cells. Further experiments also showed increased cell apoptosis among the A549 lung cancer cells transfected with pVAX-iNOS. Meanwhile, the proliferation and migration of A549 cells were significantly inhibited by the enhanced iNOS gene expression. Conclusion The recombinant eukaryotic expression vector pVAX-iNOS was successfully constructed and transfected into A549 cells. The enhanced iNOS gene expression significantly promoted cell apoptosis, whereas the proliferation and migration of A549 cells were inhibited. These findings contribute to the development of novel and effective gene therapies for lung cancer.

  20. Investigation of radiation-induced transcriptome profile of radioresistant non-small cell lung cancer A549 cells using RNA-seq.

    Hee Jung Yang

    Full Text Available Radioresistance is a main impediment to effective radiotherapy for non-small cell lung cancer (NSCLC. Despite several experimental and clinical studies of resistance to radiation, the precise mechanism of radioresistance in NSCLC cells and tissues still remains unclear. This result could be explained by limitation of previous researches such as a partial understanding of the cellular radioresistance mechanism at a single molecule level. In this study, we aimed to investigate extensive radiation responses in radioresistant NSCLC cells and to identify radioresistance-associating factors. For the first time, using RNA-seq, a massive sequencing-based approach, we examined whole-transcriptome alteration in radioresistant NSCLC A549 cells under irradiation, and verified significant radiation-altered genes and their chromosome distribution patterns. Also, bioinformatic approaches (GO analysis and IPA were performed to characterize the radiation responses in radioresistant A549 cells. We found that epithelial-mesenchymal transition (EMT, migration and inflammatory processes could be meaningfully related to regulation of radiation responses in radioresistant A549 cells. Based on the results of bioinformatic analysis for the radiation-induced transcriptome alteration, we selected seven significant radiation-altered genes (SESN2, FN1, TRAF4, CDKN1A, COX-2, DDB2 and FDXR and then compared radiation effects in two types of NSCLC cells with different radiosensitivity (radioresistant A549 cells and radiosensitive NCI-H460 cells. Interestingly, under irradiation, COX-2 showed the most significant difference in mRNA and protein expression between A549 and NCI-H460 cells. IR-induced increase of COX-2 expression was appeared only in radioresistant A549 cells. Collectively, we suggest that COX-2 (also known as prostaglandin-endoperoxide synthase 2 (PTGS2 could have possibility as a putative biomarker for radioresistance in NSCLC cells.

  1. Quercetin metabolites inhibit MMP-2 expression in A549 lung cancer cells by PPAR-γ associated mechanisms.

    Chuang, Cheng-Hung; Yeh, Chiao-Lin; Yeh, Shu-Lan; Lin, En-Shyh; Wang, Li-Yu; Wang, Ying-Hsuna

    2016-07-01

    Our previous study demonstrated that quercetin-metabolite-enriched plasma (QP) but not quercetin itself upregulates peroxisome proliferator-activated receptor gamma (PPAR-γ) expression to induce G2/M arrest in A549 cells. In the present study, we incubated A549 cells with QP as well as quercetin-3-glucuronide (Q3G) and quercetin-3'-sulfate (Q3'S), two major metabolites of quercetin, to investigate the effects of quercetin metabolites on cell invasion and migration, the possible mechanisms and the role of PPAR-γ. We also compared the effects of QP with those of quercetin and troglitazone (TGZ), a PPAR-γ ligand. The results showed that QP significantly suppressed cell invasion and migration, as well as matrix metalloproteinases (MMPs)-2 activity and expression in a dose-dependent manner. The effects of 10% QP on those parameters were similar to those of 10μM quercetin and 20μM TGZ. However, QP and TGZ rather than quercetin itself increased the expressions of nm23-H1 and tissue inhibitor of metalloproteinase (TIMP-2). Furthermore, we demonstrated that Q3G and Q3'S also inhibited the protein expression of MMP-2. GW9662, a PPAR-γ antagonist, significantly diminished such an effect of Q3G and Q3'S. Silencing PPAR-γ expression in A549 cells also significantly diminished the suppression effect of Q3G and Q3'S on MMP-2 expression. Taken together, our study demonstrated that QP inhibited cell invasion and migration through nm23-H1/TIMP-2/MMP-2 associated mechanisms. The upregulation of PPAR-γ by quercetin metabolites such as Q3G and Q3'S could play an important role in the effects of QP. PMID:27260467

  2. 荞麦七提取物对肺癌A549细胞增殖及凋亡的影响%Effects of Fagopyrum cymosum extracts on proliferation and apoptosis of lung cancer cell line A549

    李健; 王晓梅; 杨春娟; 刘帆

    2015-01-01

    Objective To investigate the effects of Fagopyrum cymosum extracts on proliferation and apoptosis of human lung cancer cell line A549. Methods A549 lung cancer cells were processed with aqueous extracts and anthraquinone of Fagopyrum cymosum. Cell viability was detected by trypan blue staining. The inhibition rate of cell proliferation was detected by MTT. The protein expression levels of Csapase 9 and P53 were detected by immunohis-tochemical method. Results The inhibition effects of Fagopyrum cymosum aqueous extracts on lung cancer cell line A549 increased along with higher concentration of the extracts. The inhibition rate at 72 h was significantly higher than the rates at 24 h and 48 h, while there were no significant differences in inhibition rates among the three con-centrations of Fagopyrum cymosum anthraquinone. The induction on Csapase 9 and inhibition on P53 by both extracts were enhanced with the increase of concentration. Conclusion The aqueous extracts and anthraquinone of Fagopy-rum cymosum can inhibit the proliferation of human lung cancer cell line A549 and induce their apoptosis, with the underlying mechanism possibly related to the up-regulation of Caspase 9 and down-regulation of P53.%目的:研究荞麦七提取物对人肺癌A549细胞增殖及凋亡的影响。方法应用荞麦七水提物及荞麦七蒽醌处理肺癌A549细胞,锥虫蓝染色法检测细胞存活率,MTT法检测细胞增殖抑制率,免疫细胞化学法检测Caspase 9和P53蛋白表达水平。结果荞麦七水提取物对肺癌A549细胞增殖的抑制作用随浓度而增强,72 h的抑制率明显较24 h及48 h强,荞麦七蒽醌3种浓度的抑制率之间差异不大。2种提取物对Caspase 9的诱导作用均随着浓度的增大而增强,对P53的抑制作用也随着浓度的增大而增强。结论荞麦七水提物及蒽醌能抑制人肺癌A549细胞的增殖,并诱导其凋亡,其机制可能与上调Caspase 9的表达及下调P53的表达有关。

  3. Polyurethane dispersion containing quaternized ammonium groups: An efficient nanosize gene delivery carrier for A549 cancer cell line transfection.

    Yousefpour Marzbali, Mahsa; Yari Khosroushahi, Ahmad; Movassaghpour, AliAkbar; Yeganeh, Hamid

    2016-01-25

    A novel polyurethane containing cationic ammonium groups (QPU) was synthesized and used as vector for gene therapy and cancer gene targeting. The synthesized QPU was characterized by Fourier transform infrared and nuclear magnetic resonance spectroscopy methods. An agarose gel retardation electrophoresis assay was conducted to verify the complete complex formation between QPU and pDNA. The particles size and zeta potential of neat polymers, plasmid DNA, polymers/DNA polyplexes were determined by the dynamic light scattering technique. The polyplexes cytotoxicity was determined using [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and its transfection efficiency was examined qualitatively by fluorescent microscopy and quantitatively by flow cytometery methods. The gel retardation assay, particle size and zeta potential measurements were confirmed that the synthesized cationic polymer could condense DNA efficiently in the physiologic condition. QPU polyplexes showed a significantly lower cytotoxicity compared to Polyfect polyplexes in the examined human cancerous (A549) or normal cells (KDR). Based on our findings, the transfection efficiency by QPU was 2.2 fold higher than Polyfect in the A549 cells whereas in the KDR cells, the cell transfection by Polyfect was 18.1 fold higher than QPU. Due to low cytotoxicity for normal cells and high transfection efficiency in cancer cells, the potential applicability of designed QPU as a non-viral gene carrier for targeting of cancer gene therapy was confirmed. PMID:26658031

  4. In vitro and in vivo studies on radiobiological effects of prolonged fraction delivery time in A549 cells.

    Jiang, Ling; Xiong, Xiao-Peng; Hu, Chao-Su; Ou, Zhou-Luo; Zhu, Guo-Pei; Ying, Hong-Mei

    2013-03-01

    Intensity-modulated radiation therapy, when used in the clinic, prolongs fraction delivery time. Here we investigated both the in vivoand in vitroradiobiological effects on the A549 cell line, including the effect of different delivery times with the same dose on A549 tumor growth in nude mice. The in vitroeffects were studied with clonogenic assays, using linear-quadratic and incomplete repair models to fit the dose-survival curves. Fractionated irradiation of different doses was given at one fraction per day, simulating a clinical dose-time-fractionation pattern. The longer the interval between the exposures, the more cells survived. To investigate the in vivoeffect, we used sixty-four nude mice implanted with A549 cells in the back legs, randomly assigned into eight groups. A 15 Gy radiation dose was divided into different subfractions. The maximum and minimum tumor diameters were recorded to determine tumor growth. Tumor growth was delayed for groups with prolonged delivery time (40 min) compared to the group receiving a single dose of 15 Gy (P< 0.05), and tumors with a 20 min delivery time had delayed growth compared to those with a 40 min delivery time [20' (7.5 Gy × 2 F) vs 40' (7.5 Gy × 2 F), P= 0.035; 20' (3 Gy × 5 F) vs 40' (3 Gy × 5 F); P= 0.054; 20' (1.67 Gy × 9 F) vs 40' (1.67 Gy × 9 F), P= 0.028]. A prolonged delivery time decreased the radiobiological effects, so we strongly recommend keeping the delivery time as short as possible. PMID:23090953

  5. 流感病毒NS1蛋白稳定表达的A549细胞系建立%Establishment of A549 Cell Line Stably Expressing NS1 Protein of Influenza Virus

    李志辉; 曾琳姣; 王慧煜; 梅琳; 刘永飞; 韩雪清

    2013-01-01

    NS1 of influenza A virus is a key multifunctional protein that plays various roles in regulating viral replication mechanisms, disease pathogenesis. In order to establish stable A549 cell line expressing NS1 protein of influenza A Virus, NSl cDNA was obtained by RT-PCR using 2009 A(H1N1) influenza virus total RNA as template. The fragment was cloned in the pMD19-T vector, then the fragment was obtained by BamHI and NdeI digestion, and ligated with pCMV-HA. Linearized pCMV-HA-NS1 and neo were transfect-ed into A549 cells. The stable expressing NSl protein cell line was screened by G418. DNA, RNA, protein levels of NS1 were detected in A549 cells by PCR, RT-PCR and Western blot, the location of the NSl protein in cells was observed by immunofluorescence. The result indicated that NS1 protein was stable expressed in A549 cell line, suggesting that NSl stable expression A549 cell line was successfully constructed, and the NS1 protein is located in nucleus. This stable cell line can be used for further study of biological functions of NS1.%A型流感病毒的NSl(Nonstructurol 1 protein,NSl)蛋白是病毒复制、毒力等的重要调节蛋白.运用RT-PCR方法扩增A/Beijing/501/2009 (H1N1)流感病毒NS1基因,克隆至真核表达载体pCMV-HA,用Lipofectamine 2000将线性化pCMV-HA-NS1与neo基因共同转染A549细胞,通过G418筛选获得阳性重组细胞,并采用PCR、RT-PCR、Western blot技术检测重组细胞中NS1蛋白的表达,通过免疫荧光技术观察NS1蛋白在细胞中的定位.PCR、RT-PCR检测显示NS1基因成功整合进入细胞基因组,并转录为mRNA;Western blot检测显示重组细胞系稳定表达NS1蛋白,免疫荧光显示NS1蛋白定位于细胞核内.表明通过G418筛选,成功构建稳定表达NS1蛋白的重组A549-HA-NS1细胞系,且NS1蛋白定位于细胞核内,为进一步研究NS1蛋白的生物学功能奠定基础.

  6. Expression of epidermal growth factor receptor gene in methotrexate enantiomers-resistant A549 cells and influence on cellular migration ability%氨甲蝶呤对映体耐药A549细胞的表皮生长因子受体基因表达及迁移能力

    张白银; 何晓东; 孙余婕; 张永娟; 嵇金陵; 沈佐君

    2012-01-01

    目的 研究氨甲蝶呤(MTX)对映体耐药人非小细胞肺癌A549细胞的迁移能力以及表皮生长因子受体(EGFR)mRNA的表达.方法 用细胞划痕试验检测L-(+)-MTX/A549细胞和D-(-)-MTX/A549细胞的迁移能力;双层软琼脂克隆试验检测L-(+ )-MTX/A549细胞和D-(-)-MTX/A549细胞的克隆形成率并观察集落的形态;用RT-PCR检测亲本A549细胞、L-(+ )-MTX/A549细胞和D-(-)-MTX/A549细胞中EGFR mRNA的表达.结果 加入MTX 72 h后D-(-)-MTX/A549细胞的迁移能力(1 230.1±40.2)高于L-(+ )-MTX/A549细胞(530.3±25.4);D-(-)-MTX/A549细胞、L-(+ )-MTX/A549细胞和亲本A549细胞的克隆形成率(%)分别为(1.38±0.17)、(1.36±0.13)和(1.37±0.15),差异无统计学意义(P>0.05);亲本A549细胞、L(+)-MTX/A549细胞均有EGFR mRNA表达,其光密度值(IDV)分别为(6 630±64)、(3 697±27),差异有统计学意义(t=103.42,P<0.01).而D-(-)-MTX/A549细胞不表达EGFR.结论 D-(-)-MTX诱导的A549细胞的迁移能力大于L-(+ )-MTX.EGFR基因表达具有手性差异.%Objective To investigate the migration ability of methotrexate (MTX) enantiomers -resistant non-small cell lung cancer ( NSCLC) cell line A549 and the expression of epidermal growth factor receptor (EGFR) in the cells. Methods The migration ability of L-( + )-MTX/A549 and D-( -) -MTX/A549 cells were evaluated by cell scratch assay. The colony formation rates and the morphology of cell cluster of L-( + ) ,MTX/A549 and D-(-) -MTX/A549 were determined by double-layer soft agar colony formation assay. The mRNA expression of EGFR in parental A549 cells, L-( + )-MTX/A549 cells, D-(-)-MTX/A549 cells were detected by RT-PCR. Results The migration ability of D-(-)-MTX/A549 cells (1 230. 1 ±40. 2) was stronger than that of L-( + )-MTX/A549 cells (530.3 ±25.4) at72 h after adding MTT. The rate of colony formation in D-(-)-MTX/A549, L-( + }-MTX/A549 and parental A549 cells was (1.38 ±0.17), (1.36±0.13) and (1.37 ±0. 15) respectively. There was

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

    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 (pbodies' 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. PMID:27243447

  8. Low Dose Hyper-radiosensitivity in Human Lung Cancer Cell Line A549 and Its Possible Mechanisms

    Xiaofang DAI; Dan TAO; Hongge WU; Jing CHENG

    2009-01-01

    The low dose hyper-radiosensitivity (HRS) in human lung cancer cell line A549 was in-vestigated,the changes of ATM kinase,cell cycle and apoptosis of cells at different doses of radiation were observed,and the possible mechanisms were discussed.A549 cells in logarithmic growth phase were irradiated with 60Co γ-rays at doses of 0-2 Gy.Together with flow cytometry for precise cell sorting,cell survival fraction was measured by means of conventional colony-formation assay.The expression of ATM1981Ser-P protein was examined by Western blot 1 h after radiation.Apoptosis was detected by Hoechst 33258 fluorescent staining,and Annexin V-FITC/PI staining flow cytometry 24 h after radiation.Cell cycle distribution was observed by flow cytometly 6,12 and 24 h after ra-diation.The results showed that the expression of ATM1981Ser-P protein was observed at 0.2 Gy,followed by an increase at >0.2 Gy,and reached the peak at 0.5 Gy,with little further increase as the dose exceeded 0.5 Gy.Twenty-four h after radiation,partial cells presented the characteristic mor-phological changes of apoptosis,and the cell apoptosis curve was coincident with the survival curve.As compared with control group,the cell cycle almost had no changes after exposure to 0.1 and 0.2 Gy radiation (P>0.05).After exposure to 0.3,0.4 and 0.5 Cry radiation,G2/M phase arrest occurred 6 and 12 h after radiation (P<0.05),and the ratio of G2/M phase cells was decreased 24 h after radiation (P<0.05).It was concluded that A549 cells displayed the phenomenon of HRS/IRR.The mode of cell death was mainly apoptosis.The activity of ATM and cell cycle change may take an important role in HRS/IRR.

  9. Low dose hyper-radiosensitivity in human lung cancer cell line A549 and its possible mechanisms

    Objective: To study the low dose hyper-radiosensitivity in human lung cancer cell line A549, and its possible mechanisms. Methods: Exponentially growing A549 cells were irradiated with 60Co γ-rays at doses of 0-2 Gy. Together with flow cytometry for precise cell sorting, cell survival fraction was measured by mean of conventional colony-formation assay. ATM1981 Ser-P protein expression was examined by Western blot. Apoptosis was identified by Hoechst 33258 fluorescent staining, and Annexin V-FITC and propidium iodide staining flow cytometry. Cell cycle distribution was observed by flow cytometry. Results: There was an excessive cell killing per unit dose when the doses were below about 0.3 Gy, and the cells exhibited more resistant response at the doses between 0.3 and 0.5 Gy, the cell survival fraction was decreased as the doses over 0.5 Gy. The expression of ATM1981Ser-P protein was first observed at 0.2 Gy, followed by an increase over 0.2 Gy, and reached the peak at 0.5 Gy (compared with 0.2 Gy group, t=7.96, P0.05). 24 hours after irradiation, part cells presented the characteristic morphological change of apoptosis, and the apoptosis curve was coincident with the dose-survival curve. Compared with the control group, the cell cycle had no change post-irradiation to 0.1 and 0.2 Gy. G2/M phase arrest was manifested at 6 and 12 hours post-irradiation to 0.3, 0.4 and 0.5 Gy (t=2.87, 2.88, 4.92 and 3.70, 3.12, 8.11, P2/M phase was decreased at 24 hours post-irradiation (t=3.87, 4.77, 3.01, P<0.05). Conclusions: A549 cells displays the phenomenon of hyper-radiosensitivity (HRS) /induced radioresistance (IRR). The model of cell death induced by low dose irradiation is mainly apoptosis. The activity of ATM and cell cycle change might play an important role in HRS/IRR. (authors)

  10. EXPRESSION AND REVERSION OF DRUG RESISTANCE-AND APOPTOSIS-RELATED GENES OF A DDP-RESISTANT LUNG ADENOCARCINOMA CELL LINE

    2000-01-01

    Objective: To investigate the co-expression of drug resistance- and apoptosis-related genes of cisplatin (CDDP)-selected lung adenocarcinoma cell line A549DDP for compared to the parental cell line A549, and reverse of drug resistance by antisense s-oligodeoxynucleotides (S-ODNs) of differentially expressed genes. Methods: Sense and antisense S-ODN were transferred into A549DDP cells by lipofectin. The expression of drug resistance and apoptosis related genes was examined by RT-PCR, immunocytochemistry and flow cytometry, respectively. Apoptostic cells were identified by DNA electrophoresis and terminal deoxynucleotidyl transferase (TdT)-mediated biotin dUTP nick end-labeling(TUNEL). Drug resistance of tumor cells was detected by a cell viability (MTT) assay. Results: The expression of bc1-2 was positive and that of multidrug resistance-associated protein (MRP) at mRNA and protein level was increased in A549DDP compared to A549 cells. MDR1, c-myc and topoisomeras II (TOPO II) were similarly co-expressed in two cell lines. Both cell lines were negative for c-erbB-2 expression. In A549DDP cells, the expression of bc1-2 and MRP was significantly inhibited by their respective antisense S-ODNs. Antisense S-ODNs could also decrease significantly drug resistance of A549DDP cells to CDDP by promoting cell apoptosis. Conclusion: Both intrinsic and acquired drug resistance were involved in co-expression of multiple MDR-related genes in lung adenocarcinoma. Co- operation of bc1-2 and MRP genes appeared to play an important action to confer the resistance of A549DDP cells to CDDP. Their antisense S-ODNs are responsible for the decrease of drug resistance of this cell line by promoting apoptosis.

  11. Chemosensitization and radiosensitization of a lung cancer cell line A549 induced by a composite polymer micelle.

    Xu, Jing; Zhang, Bi-Cheng; Li, Xiang-Long; Xu, Wen-Hong; Zhou, Juan; Shen, Li; Wei, Qi-Chun

    2016-08-01

    Multidrug resistance (MDR) to Doxorubicin (DOX) remains a major obstacle to successful cancer treatment. The present study sought to overcome the MDR of lung cancer cells and achieve radiosensitization by developing a composite DOX-loaded micelle (M-DOX). M-DOX containing PEG-PCL/Pluronic P105 was prepared by the solvent evaporation method. Lung cancer cell line A549 was adopted in this study. In vitro cytotoxicity, cellular uptake behavior, subcellular distribution, and radiosensitivity were evaluated by the treatment with M-DOX, and free DOX was used as a control. A549 cells treated with M-DOX as opposed to free DOX showed greater cellular uptake as well as greater cytotoxicity. Furthermore, M-DOX reached the mitochondria and lysosome effectively after cellular uptake, and fluorescence used to track M-DOX was found to be surrounding the nucleus. Finally, colony-forming assays demonstrated that M-DOX treatment improved radiosensitization when compared to free DOX. Based on the increased cytotoxicity and radiosensitization, M-DOX could be considered as a promising drug delivery system to overcome MDR in lung cancer therapy. PMID:27585226

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

    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. PMID:27575372

  13. Nonsense and missense mutation of mitochondrial ND6 gene promotes cell migration and invasion in human lung adenocarcinoma

    Previous study showed that mitochondrial ND6 (mitND6) gene missense mutation resulted in NADH dehydrogenase deficiency and was associated with tumor metastasis in several mouse tumor cell lines. In the present study, we investigated the possible role of mitND6 gene nonsense and missense mutations in the metastasis of human lung adenocarcinoma. The presence of mitND6 gene mutations was screened by DNA sequencing of tumor tissues from 87 primary lung adenocarcinoma patients and the correlation of the mutations with the clinical features was analyzed. In addition, we constructed cytoplasmic hybrid cells with denucleared primary lung adenocarcinoma cell as the mitochondria donor and mitochondria depleted lung adenocarcinoma A549 cell as the nuclear donor. Using these cells, we studied the effects of mitND6 gene nonsense and missense mutations on cell migration and invasion through wounding healing and matrigel-coated transwell assay. The effects of mitND6 gene mutations on NADH dehydrogenase activity and ROS production were analyzed by spectrophotometry and flow cytometry. mitND6 gene nonsense and missense mutations were detected in 11 of 87 lung adenocarcinoma specimens and was correlated with the clinical features including age, pathological grade, tumor stage, lymph node metastasis and survival rate. Moreover, A549 cell containing mitND6 gene nonsense and missense mutation exhibited significantly lower activity of NADH dehydrogenase, higher level of ROS, higher capacity of cell migration and invasion, and higher pAKT and pERK1/ERK2 expression level than cells with the wild type mitND6 gene. In addition, NADH dehydrogenase inhibitor rotenone was found to significantly promote the migration and invasion of A549 cells. Our data suggest that mitND6 gene nonsense and missense mutation might promote cell migration and invasion in lung adenocarcinoma, probably by NADH dehydrogenase deficiency induced over-production of ROS

  14. Radiosensitizing Effect of Schinifoline from Zanthoxylum schinifolium Sieb et Zucc on Human Non-Small Cell Lung Cancer A549 Cells: A Preliminary in Vitro Investigation

    Cheng-Fang Wang

    2014-12-01

    Full Text Available Schinifoline (SF, a 4-quinolinone derivative, was found in Zanthoxylum schinifolium for the first time. 4-Quinolinone moieties are thought to have cytotoxic activity and are often used as a tubulin polymerization inhibitors, heterogeneous enzyme inhibitors and antiplatelet agents. However, very little information respect to radiosensitization has focused on SF. This work aimed to investigate the radiosensitizing effect of SF on A549 cells. The cell viability results indicated cytotoxicity of SF on A549 cells, with IC50 values of 33.7 ± 2.4, 21.9 ± 1.9 and 16.8 ± 2.2 μg/mL, respectively, after 6, 12, 24 h treatment with different concentrations, and the 10% or 20% IC50 concentration during 12 h was applied in later experiments. The results of cell proliferative inhibition and clonogenic assay showed that SF enhanced the radiosensitivity of A549 cells when applied before 60Co γ-irradiation and this effect was mainly time and concentration dependent. The flow cytometric data indicated that SF treatment before the irradiation increased the G2/M phase, thus improving the radiosensitivity of A549, leading to cell apoptosis. This paper is the first study that describes the in vitro radiosensitising, cell cycle and apoptotic-inducing effects of schinifoline.

  15. 纳米氧化锌与常规氧化锌对A549细胞的毒性与DNA损伤的比较研究%Comparative study on cytotoxicity and DNA damage in A549 cells caused by nano-and micro-zinc oxide particles

    洪丽玲; 丁树娟; 朱江波; 朱玉平; 张天宝

    2011-01-01

    目的:比较纳米氧化锌(Nano-ZnO)和常规氧化锌(Micro-ZnO)对人肺腺癌A549细胞的毒性及DNA损伤作用.方法:采用不同浓度(25、50、100 μ/ml)的纳米ZnO(30 nm)和常规ZnO(≤1 μm)染毒体外培养的A549细胞,实验同时设对照组(DMEM培养液).分别于染毒后6、12、24、48 h,观察细胞形态及生长情况,采用MTT法和单细胞凝胶电泳技术(彗星实验)检测细胞毒性及对DNA的损伤作用.结果:纳米 ZnO和常规ZnO对A549细胞的半数致死浓度(IC50)分别为53.91和190.15 μg/ml;纳米ZnO在50 μg/ml剂量时对A549细胞就已出现明显的生长抑制作用,并呈明显的剂量和时间依赖效应关系;而常规ZnO在100 μg/ml剂量时才出现细胞生长的抑制.纳米ZnO与常规ZnO的剂量-效应曲线和时间-效应曲线的斜率比较差异均有统计学意义(P<0.05).与对照组相比,纳米ZnO能够导致A549细胞产生DNA损伤;常规ZnO在相同受试剂量下未观察到DNA损伤.结论:纳米ZnO与常规ZnO均对体外A549细胞产生细胞毒性,但纳米ZnO产生毒性的剂量低、出现时间早,并产生了常规ZnO未观察到的遗传毒性.%OBJECTIVE: To compare the cytotoxicity and DNA damage caused by nano- and micro- zinc oxide particles. METHODS: Cultured A549 human lung adenocarcinoma cells were exposed to nano- (30 run) and micro-scale ZnO particles(≤ 1 μm)a the concentrations of 25, 50 and 100 μg/ml for 6, 12, 24 and 48 h. In the mean time, DMEM culture medium was set up as control. Morphological changes were examined after exposure, methyl tetrazolium cytotoricity (MTT) assay and the single cell gel electrophoresis(comet assay) were used In determine cytotoxicity and DNA damage. RESULTS: The IC50 of the nano-ZnO and micro-ZnO on the A549 cells were 53.91 and 190.15 μg/ml, respectively. It was found thai nano-ZnO inhibited A549 cells significantly at 50 μg/ml, showing a dose- and time- dependent manner. But micro-ZnO inhibited A549 cells significantly

  16. Diallyl trisulfide inhibits naphthalene-induced oxidative injury and the production of inflammatory responses in A549 cells and mice.

    Zhang, Fang; Zhang, Yongchun; Wang, Kaiming; Zhu, Xiaosong; Lin, Guimei; Zhao, Zhongxi; Li, Shanzhong; Cai, Jianhua; Cao, Jimin

    2015-12-01

    Diallyl trisulfide (DATS) is a garlic organosulfide that may have a therapeutic potential in the treatment of some diseases. We sought to determine whether DATS could inhibit naphthalene-induced oxidative injury and the production of inflammatory responses in vitro and in vivo. A549 cells were either pre-treated (PreTx, prevention) or concurrently treated (CoTx, treatment) with 20μM naphthalene and either 5 or 10μM DATS. PreTx and CoTx showed the prevention and the treatment potential of DATS to inhibit the generation of naphthalene-induced reactive oxygen species (ROS) in the A549 cells. DATS showed antioxidative activity by elevating the SOD activities in the low dose groups. The mechanistic study showed that the DATS-mediated inhibition of naphthalene-induced oxidative injury and the production of inflammatory responses (i.e., TNF-α, IL-6, and IL-8) were attributed to inhibiting the activity of nuclear factor-kappa B (NF-κB). In addition, DATS inhibited the production of serum nitric oxide NO and myeloperoxidase (MPO) in the lungs of Kunming mice. The histological analysis results indicate that DATS inhibited the naphthalene-induced lung damage, which is consistent with the in vitro study results. The in vivo and in vitro results suggest that DATS may be an effective attenuator of naphthalene-induced lung damage. PMID:26548347

  17. Effect of Integrin α5β1-mediated ERK Signal Pathway on Proliferation 
and Migration of A549 Cells

    Jing BAI

    2011-07-01

    Full Text Available Background and objective Recent studies have shown that integrin α5β1 as a core in the integrin family plays an important role in metastasis, invasion and poor difference of non-small cell lung cancer. In this study, A549 cells were cultured and treated with integrin α5β1 small interfering RNA (siRNA and extracelluar signal-regulated protein kinase (ERK inhibitor PD98059 to investigate the effect of integrin α5β1 on proliferation and migration of A549 cells and explore its signal transduction mechanism. Methods A549 cells were divided into four groups: Untransfection, Lipofectamine, Integrin α5β1 siRNA-transfected group and PD98059 group. The protein expression levels of integrin α5β1 were detected by Western blot analysis and the expression levels of integrin α5β1 mRNA was measured by reverse transcription-polymerase chain reaction (RT-PCR. The protein expression level of ERK1/2, MMP-9 and caspase-3 were measured by Western blot analysis. The proliferation and apoptosis of A549 cells were measured by MTT assay and Annexin-V FITC PI double staining. Results Integrin α5β1 siRNA could inhibit the phosphorylated ratio of ERK by down-regulate the expression of ERK 1/2 proteins. In addition, integrin α5β1 siRNA or PD98059 could inhibit the proliferation of A549 cells, induce the apoptosis of A549 cells, up-regulate the expression of caspase-3 and down-regulate the expression of MMP-9. Conclusion Integrin α5β1 might involves the abnormal proliferation and migration of A549 cells through mediating ERK signal transduction pathway.

  18. Experimental study of targeting MMP-9 deoxyribozyme role of adhesion and migration in human lung adenocarcinoma cancer cell

    Weizhong ZENG

    2008-12-01

    Full Text Available Background and objective Deoxyribozyme has high biocatalytic activity and sequence specificity against the target mRNA and inhibits gene expression at mRNA level. The aim of this study is to investigate the impact of targeting MMP-9 deoxyribozyme to cell adhesion and migration in human lung adenocarcinoma cell line A549. Methods The targeting MMP-9 deoxyribozyme was designed by oligofectamine into human lung adenocarcinoma cell line A549.The expression of MMP-9 in cell was detected by Western blot. The cell adhesion and migration after the intervention of deoxyribozyme was observed. Results After targeting MMP-9 deoxyribozyme intervention, the expression of MMP-9 inthe cells compared with the control group was significantly lower (P <0.01, at the same time, the rate of cell adhesion andmigration were significantly decreased. Conclusion Targeting MMP-9 deoxyribozyme inhibited the expression of MMP-9 in human lung adenocarcinoma cell line A549 and effectively prevent cell adhesion and migration.

  19. Down-regulation of protein kinase Ceta by antisense oligonucleotides sensitises A549 lung cancer cells to vincristine and paclitaxel.

    Sonnemann, Jürgen; Gekeler, Volker; Ahlbrecht, Katrin; Brischwein, Klaus; Liu, Chao; Bader, Peter; Müller, Cornelia; Niethammer, Dietrich; Beck, James F

    2004-06-25

    Previous studies point to protein kinase C (PKC) isozyme eta as a resistance factor in cancer cells. Therefore, we investigated whether down-regulation of PKCeta with second generation antisense oligonucleotides (ODNs) would sensitise A549 human lung carcinoma cells to cytostatics. The effects were compared to the outcome of Bcl-xL down-regulation. Upon treatment with antisense ODNs, PKCeta and Bcl-xL were both significantly reduced on mRNA and protein level. Down-regulation of either PKCeta or Bcl-xL in combination with vincristine or paclitaxel resulted in a significant increase in caspase-3 activity compared to that in the control oligonucleotide treated cells. In addition, PKCeta down-regulation augmented vincristine-induced dissipation of mitochondrial transmembrane potential. In conclusion, these results confirm that PKCeta might represent a considerable resistance factor and an interesting target to improve anticancer chemotherapy. PMID:15159020

  20. Activities of Ten Essential Oils towards Propionibacterium acnes and PC-3, A-549 and MCF-7 Cancer Cells

    Yuangang Zu

    2010-04-01

    Full Text Available Ten essential oils, namely, mint (Mentha spicata L.,Lamiaceae, ginger (Zingiber officinaleRosc.,Zingiberaceae, lemon (Citrus limon Burm.f.,Rutaceae, grapefruit (Citrus paradisi Macf., Rutaceae, jasmine (Jasminum grandiflora L.,Oleaceae, lavender (Mill.,Lamiaceae, chamomile (Matricaria chamomilla L., Compositae, thyme (Thymus vulgaris L., Lamiaceae, rose (Rosa damascena Mill.,Rosaceae and cinnamon (Cinnamomum zeylanicumN. Lauraceae were tested for their antibacterial activities towards Propionibacterium acnes and in vitro toxicology against three human cancer cell lines. Thyme, cinnamon and rose essential oils exhibited the best antibacterial activities towards P. acnes, with inhibition diameters of 40 ± 1.2 mm, 33.5 ± 1.5 mm and 16.5 ± 0.7 mm, and minimal inhibitory concentrations of 0.016% (v/v, 0.016% (v/v and 0.031% (v/v, respectively. Time-kill dynamic procedures showed that thyme, cinnamon, rose, and lavender essential oils exhibited the strongest bactericidal activities at a concentration of 0.25% (v/v, and P. acnes was completely killed after 5 min. The thyme essential oil exhibited the strongest cytotoxicity towards three human cancer cells. Its inhibition concentration 50% (IC50 values on PC-3, A549 and MCF-7 tumor cell lines were 0.010% (v/v, 0.011% (v/v and 0.030% (v/v, respectively. The cytotoxicity of 10 essential oils on human prostate carcinoma cell (PC-3 was significantly stronger than on human lung carcinoma (A549 and human breast cancer (MCF-7 cell lines.

  1. Activities of ten essential oils towards Propionibacterium acnes and PC-3, A-549 and MCF-7 cancer cells.

    Zu, Yuangang; Yu, Huimin; Liang, Lu; Fu, Yujie; Efferth, Thomas; Liu, Xia; Wu, Nan

    2010-05-01

    Ten essential oils, namely, mint (Mentha spicata L., Lamiaceae), ginger (Zingiber officinale Rosc., Zingiberaceae), lemon (Citrus limon Burm.f., Rutaceae), grapefruit (Citrus paradisi Macf., Rutaceae), jasmine (Jasminum grandiflora L., Oleaceae), lavender (Mill., Lamiaceae), chamomile (Matricaria chamomilla L., Compositae), thyme (Thymus vulgaris L., Lamiaceae), rose (Rosa damascena Mill., Rosaceae) and cinnamon (Cinnamomum zeylanicum N. Lauraceae) were tested for their antibacterial activities towards Propionibacterium acnes and in vitro toxicology against three human cancer cell lines. Thyme, cinnamon and rose essential oils exhibited the best antibacterial activities towards P. acnes, with inhibition diameters of 40 +/- 1.2 mm, 33.5 +/- 1.5 mm and 16.5 +/- 0.7 mm, and minimal inhibitory concentrations of 0.016% (v/v), 0.016% (v/v) and 0.031% (v/v), respectively. Time-kill dynamic procedures showed that thyme, cinnamon, rose, and lavender essential oils exhibited the strongest bactericidal activities at a concentration of 0.25% (v/v), and P. acnes was completely killed after 5 min. The thyme essential oil exhibited the strongest cytotoxicity towards three human cancer cells. Its inhibition concentration 50% (IC(50)) values on PC-3, A549 and MCF-7 tumor cell lines were 0.010% (v/v), 0.011% (v/v) and 0.030% (v/v), respectively. The cytotoxicity of 10 essential oils on human prostate carcinoma cell (PC-3) was significantly stronger than on human lung carcinoma (A549) and human breast cancer (MCF-7) cell lines. PMID:20657472

  2. Integrin {beta}1-dependent invasive migration of irradiation-tolerant human lung adenocarcinoma cells in 3D collagen matrix

    Ishihara, Seiichiro [Transdisciplinary Life Science Course, Faculty of Advanced Life Science, Hokkaido University, N10-W8, Kita-ku, Sapporo 060-0810 (Japan); Haga, Hisashi, E-mail: haga@sci.hokudai.ac.jp [Transdisciplinary Life Science Course, Faculty of Advanced Life Science, Hokkaido University, N10-W8, Kita-ku, Sapporo 060-0810 (Japan); Yasuda, Motoaki [Department of Oral Pathobiological Science, Graduate School of Dental Medicine, Hokkaido University, N13-W7, Kita-ku, Sapporo 060-8586 (Japan); Mizutani, Takeomi; Kawabata, Kazushige [Transdisciplinary Life Science Course, Faculty of Advanced Life Science, Hokkaido University, N10-W8, Kita-ku, Sapporo 060-0810 (Japan); Shirato, Hiroki [Department of Radiology, Hokkaido University Graduate School of Medicine, N15-W7, Kita-ku, Sapporo 060-8638 (Japan); Nishioka, Takeshi [Department of Biomedical Sciences and Engineering, Faculty of Health Sciences, Hokkaido University, N12-W5, Kita-ku, Sapporo 060-0812 (Japan)

    2010-06-04

    Radiotherapy is one of the effective therapies used for treating various malignant tumors. However, the emergence of tolerant cells after irradiation remains problematic due to their high metastatic ability, sometimes indicative of poor prognosis. In this study, we showed that subcloned human lung adenocarcinoma cells (A549P-3) that are irradiation-tolerant indicate high invasive activity in vitro, and exhibit an integrin {beta}1 activity-dependent migratory pattern. In collagen gel overlay assay, majority of the A549P-3 cells displayed round morphology and low migration activity, whereas a considerable number of A549P-3IR cells surviving irradiation displayed a spindle morphology and high migration rate. Blocking integrin {beta}1 activity reduced the migration rate of A549P-3IR cells and altered the cell morphology allowing them to assume a round shape. These results suggest that the A549P-3 cells surviving irradiation acquire a highly invasive integrin {beta}1-dependent phenotype, and integrin {beta}1 might be a potentially effective therapeutic target in combination with radiotherapy.

  3. Heat-Modified Citrus Pectin Induces Apoptosis-Like Cell Death and Autophagy in HepG2 and A549 Cancer Cells

    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...

  4. Biological effects of heavy ion and X-ray irradiation on lung cancer cells A549%重离子与X射线照射肺癌细胞A549的生物学效应比较

    杨立娜; 冉俊涛; 张红; 刘圆圆; 孙超; 张秋宁; 王新宇; 王小虎

    2014-01-01

    Objective To compare the effects of carbon heavy ion and X-ray irradiation on survival fraction,cell cycle,cell apoptosis and expression of DNA-PKcs of A549 lung cancer cells.Methods A549 cells were irradiated by carbon heavy ion and X-ray.Cell survival fraction,cell cycle and apoptosis were analyzed by clonogenic formation assay,flow cytometry and Hoechst 33258 staining,respectively.Real time-PCR was performed to detect the expressions of DNA-PKcs and H2AX mRNA.Results Lower cell survival fraction,more G2/M phase arrest and higher apoptosis rate were detected in the A549 cells exposed to carbon heavy ion than X-ray(t =4.77,14.53,14.54,P < 0.05).Expression of DNA-PKcs was up-regulated after irradiation to carbon heavy ion and X-ray(t =10.91,5.05,P < 0.05).Conclusions Both heavy ion and X-ray irradiations enhance the expression of DNA-PKcs,induce apoptosis through regulating cell cycle arrest,and hence reduce survival of A549 cells.Heavy ion irradiation shows more stronger biological effects than X-ray irradiation.%目的 比较碳重离子与X射线对肺癌细胞的生物学效应.方法 对A549细胞分别进行碳重离子和X射线照射,通过克隆形成实验检测照射后细胞存活情况;流式细胞术检测细胞周期分布;通过Hoechst 33258荧光染料对照射后固定的细胞进行染色,计算凋亡率;采用实时荧光定量PCR方法检测照射后48 h细胞内DNA依赖性蛋白激酶催化亚单位(DNA-PKcs)和H2AX的mRNA表达水平.结果 细胞存活曲线显示,碳重离子造成的细胞存活分数远低于X射线,并将细胞周期阻滞于G2/M期(t=4.77、14.53、14.54,P<0.05),导致大部分细胞进入凋亡途径.碳重离子与X射线辐照后DNA-PKcs的表达上调(t=10.91、5.05,P<0.05).结论 碳重离子照射对肺癌细胞造成生物学效应远高于X射线.

  5. Depletion of hepatoma-derived growth factor-related protein-3 induces apoptotic sensitization of radioresistant A549 cells via reactive oxygen species-dependent p53 activation

    Yun, Hong Shik; Hong, Eun-Hee [Division of Radiation Cancer Biology, Korea Institute of Radiological and Medical Sciences, Seoul 139-706 (Korea, Republic of); Department of Chemistry, College of Natural Sciences, Hanyang University, Seoul 133-791 (Korea, Republic of); Lee, Su-Jae [Department of Chemistry, College of Natural Sciences, Hanyang University, Seoul 133-791 (Korea, Republic of); Baek, Jeong-Hwa [Division of Radiation Cancer Biology, Korea Institute of Radiological and Medical Sciences, Seoul 139-706 (Korea, Republic of); Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon 440-746 (Korea, Republic of); Lee, Chang-Woo [Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon 440-746 (Korea, Republic of); Yim, Ji-Hye; Um, Hong-Duck [Division of Radiation Cancer Biology, Korea Institute of Radiological and Medical Sciences, Seoul 139-706 (Korea, Republic of); Hwang, Sang-Gu, E-mail: sgh63@kcch.re.kr [Division of Radiation Cancer Biology, Korea Institute of Radiological and Medical Sciences, Seoul 139-706 (Korea, Republic of)

    2013-09-27

    Highlights: •HRP-3 is a radiation- and anticancer drug-responsive protein in A549 cells. •Depletion of HRP-3 induces apoptosis of radio- and chemoresistant A549 cells. •Depletion of HRP-3 promotes ROS generation via inhibition of the Nrf2/HO-1 pathway. •Depletion of HRP-3 enhances ROS-dependent p53 activation and PUMA expression. -- Abstract: Biomarkers based on functional signaling have the potential to provide greater insight into the pathogenesis of cancer and may offer additional targets for anticancer therapeutics. Here, we identified hepatoma-derived growth factor-related protein-3 (HRP-3) as a radioresistance-related gene and characterized the molecular mechanism by which its encoded protein regulates the radio- and chemoresistant phenotype of lung cancer-derived A549 cells. Knockdown of HRP-3 promoted apoptosis of A549 cells and potentiated the apoptosis-inducing action of radio- and chemotherapy. This increase in apoptosis was associated with a substantial generation of reactive oxygen species (ROS) that was attributable to inhibition of the Nrf2/HO-1 antioxidant pathway and resulted in enhanced ROS-dependent p53 activation and p53-dependent expression of PUMA (p53 upregulated modulator of apoptosis). Therefore, the HRP-3/Nrf2/HO-1/ROS/p53/PUMA cascade is an essential feature of the A549 cell phenotype and a potential radiotherapy target, extending the range of targets in multimodal therapies against lung cancer.

  6. Depletion of hepatoma-derived growth factor-related protein-3 induces apoptotic sensitization of radioresistant A549 cells via reactive oxygen species-dependent p53 activation

    Highlights: •HRP-3 is a radiation- and anticancer drug-responsive protein in A549 cells. •Depletion of HRP-3 induces apoptosis of radio- and chemoresistant A549 cells. •Depletion of HRP-3 promotes ROS generation via inhibition of the Nrf2/HO-1 pathway. •Depletion of HRP-3 enhances ROS-dependent p53 activation and PUMA expression. -- Abstract: Biomarkers based on functional signaling have the potential to provide greater insight into the pathogenesis of cancer and may offer additional targets for anticancer therapeutics. Here, we identified hepatoma-derived growth factor-related protein-3 (HRP-3) as a radioresistance-related gene and characterized the molecular mechanism by which its encoded protein regulates the radio- and chemoresistant phenotype of lung cancer-derived A549 cells. Knockdown of HRP-3 promoted apoptosis of A549 cells and potentiated the apoptosis-inducing action of radio- and chemotherapy. This increase in apoptosis was associated with a substantial generation of reactive oxygen species (ROS) that was attributable to inhibition of the Nrf2/HO-1 antioxidant pathway and resulted in enhanced ROS-dependent p53 activation and p53-dependent expression of PUMA (p53 upregulated modulator of apoptosis). Therefore, the HRP-3/Nrf2/HO-1/ROS/p53/PUMA cascade is an essential feature of the A549 cell phenotype and a potential radiotherapy target, extending the range of targets in multimodal therapies against lung cancer

  7. Psoralidin induces autophagy through ROS generation which inhibits the proliferation of human lung cancer A549 cells

    Wenhui Hao

    2014-09-01

    Full Text Available Psoralidin (PSO, a natural furanocoumarin, is isolated from Psoralea corylifolia L. possessing anti-cancer properties. However, the mechanisms of its effects remain unclear. Herein, we investigated its anti-proliferative effect and potential approaches of action on human lung cancer A549 cells. Cell proliferation and death were measured by MTT and LDH assay respectively. Apoptosis was detected with Hoechst 33342 staining by fluorescence microscopy, Annexin V-FITC by flow cytometry and Western blot analysis for apoptosis-related proteins. The autophagy was evaluated using MDC staining, immunofluorescence assay and Western blot analyses for LC3-I and LC3-II. In addition, the reactive oxygen species (ROS generation was measured by DCFH2-DA with flow cytometry. PSO dramatically decreased the cell viabilities in dose- and time-dependent manner. However, no significant change was observed between the control group and the PSO-treated groups in Hoechst 33342 and Annexin V-FITC staining. The expression of apoptosis-related proteins was not altered significantly either. While the MDC-fluorescence intensity and the expression ratio of LC3-II/LC3-I was remarkably increased after PSO treatment. Autophagy inhibitor 3-MA blocked the production of LC3-II and reduced the cytotoxicity in response to PSO. Furthermore, PSO increased intracellular ROS level which was correlated to the elevation of LC3-II. ROS scavenger N-acetyl cysteine pretreatment not only decreased the ROS level, reduced the expression of LC3-II but also reversed PSO induced cytotoxicity. PSO inhibited the proliferation of A549 cells through autophagy but not apoptosis, which was mediated by inducing ROS production.

  8. Cytotoxicity of carbon nanotube variants: a comparative in vitro exposure study with A549 epithelial and J774 macrophage cells.

    Kumarathasan, Prem; Breznan, Dalibor; Das, Dharani; Salam, Mohamed A; Siddiqui, Yunus; MacKinnon-Roy, Christine; Guan, Jingwen; de Silva, Nimal; Simard, Benoit; Vincent, Renaud

    2015-03-01

    While production of engineered carbon nanotubes (CNTs) has escalated in recent years, knowledge of risk associated with exposure to these materials remains unclear. We report on the cytotoxicity of four CNT variants in human lung epithelial cells (A549) and murine macrophages (J774). Morphology, metal content, aggregation/agglomeration state, pore volume, surface area and modifications were determined for the pristine and oxidized single-walled (SW) and multi-walled (MW) CNTs. Cytotoxicity was evaluated by cellular ATP content, BrdU incorporation, lactate dehydrogenase (LDH) release, and CellTiter-Blue (CTB) reduction assays. All CNTs were more cytotoxic than respirable TiO2 and SiO2 reference particles. Oxidation of CNTs removed most metallic impurities but introduced surface polar functionalities. Although slopes of fold changes for cytotoxicity endpoints were steeper with J774 compared to A549 cells, CNT cytotoxicity ranking in both cell types was assay-dependent. Based on CTB reduction and BrdU incorporation, the cytotoxicity of the polar oxidized CNTs was higher compared to the pristine CNTs. In contrast, pristine CNTs were more cytotoxic than oxidized CNTs when assessed for cellular ATP and LDH. Correlation analyses between CNTs' physico-chemical properties and average relative potency revealed the impact of metal content and surface area on the potency values estimated using ATP and LDH assays, while surface polarity affected the potency values estimated from CTB and BrdU assays. We show that in order to reliably estimate the risk posed by these materials, in vitro toxicity assessment of CNTs should be conducted with well characterized materials, in multiple cellular models using several cytotoxicity assays that report on distinct cellular processes. PMID:24713075

  9. Thrombin stimulation of synthesis and secretion of fibronectin by human A549 epithelial cells and mouse LB fibroblasts

    Thrombin, a serine protease generated at wound sites, takes part in multiple biological functions, including wound healing. The present report elucidates the effect of thrombin on fibronectin (FN) synthesis and secretion in fibroblasts and epithelial cells. Subconfluent cultures of mouse LB fibroblasts and human A549 epithelial cells were exposed to various concentrations of bovine plasma thrombin at 37 degrees C for 16 hr. After exposure, cells were processed for determination of cell-associated and secreted FN by metabolic labeling, immunoprecipitation, immunofluorescence, and peroxidase immunocytochemistry. The correlation of FN production with cell growth was studied by a combined procedure of peroxidase immunocytochemistry and light microscopic autoradiography. The amounts of cell-associated and secreted FN were significantly increased with dose increments of thrombin. The increases were most evident in secreted FN. The increase of cell-associated FN was also evidenced by results from immunofluorescence and immunocytochemical studies. Ultrastructurally, the intracellular FN was localized in rough endoplasmic reticulum, Golgi complexes, and secretory granules, whereas non-released extracellular FN was localized in the plasma membrane of cell-to-cell contacts and in the extracellular fibrils. More intense cytoplasmic FN staining was observed in cells that were not labeled with [3H]-thymidine, indicating that FN production may vary with different phases of cell growth. The results imply that thrombin may play an important role in the early phases of wound healing

  10. Effects of X-ray irradiation on the expression of Pokemon gene in human lung adenocarcinoma cell line

    Objective: To study the dose and time effects of X-ray radiation on the expression of Pokemon gene and protein in human lung adenocarcinoma cell line A549. Methods: A549 cells was exposed to different doses of X-ray (2, 4, 6 and 8 Gy), and the expression of Pokemon mRNA and protein of the cells was detected by using Quantitative real-time PCR and western-blotting at 2, 4, 8, 12, 24, and 48 h after irradiation. 3-( 4, 5-Dimethylthiazole-2-yl )-2, 5-diphenyltetrazolium bromide was used to detect the proliferation of A549 cells at 1, 2, 3, 4, and 5 d after 2 Gy X-ray irradiation. The mock treated A549 cells were used as the control. Results: The expression of Pokemon mRNA trended to decrease after irradiated with 4, 6 and 8 Gy in the earlier period and increased in the later period with statistical difference at the most time points (t =3.40 -154.76, P =0.000 -0.041). The expression of Pokemon protein trended to increase and reached the peak at 8 h after irradiated of 2, 4, 6 and 8 Gy with statistical difference at the most time points (t =4.18 - 89.64, P =0.000 - 0.039). Compared with the control, the proliferation of A549 cells was significantly inhibited during 3 to 5 d after irradiation of 2 Gy (t =2.34 - 18.19, P =0.000 -0.040). Conclusions: X-ray irradiation may increase the expression of Pokemon mRNA and protein in A549 cells, which might be correlated with radiation-resistance of A549 cells. (authors)

  11. Cell division cycle 25 homolog c effects on low-dose hyper-radiosensitivity and induced radioresistance at elevated dosage in A549 cells

    The underlying mechanisms behind both low-dose hyper-radiosensitivity (HRS) and induced radioresistance (IRR), generally occurring at elevated radiation levels, remain unclear; however, elucidation of the relationship between cell cycle division 25 homolog c (Cdc25c) phosphatase and HRS/IRR may provide important insights into this process. Two cell lines with disparate HRS status, A549 and SiHa cells, were selected as cell models for comparison of dose-dependent Cdc25c phosphatase expression subsequent to low-dose irradiation. Knockdown of Cdc25c in A549 cells was mediated by transfection with a pGCsi-RAN-U6neo vector containing hairpin siRNA sequences. S216-phosphorylated Cdc25c protein [p-Cdc25c (Ser216)], cell survival and mitotic ratio were measured by western blot, colony-forming assay and histone H3 phosphorylation analysis. Variant p-Cdc25c (Ser216) expression was observed in the two cell lines after irradiation. The p-Cdc25c (Ser216) expression noted in SiHa cells after administration of 0-1 Gy radiation was similar to the radioresistance model; however, in A549 cells, the dose response for the phosphorylation of the Cdc25c Ser216 residue overlapped the level required to overcome the HRS response. Furthermore, Cdc25c repression prior to low-dose radiation induced more distinct HRS and prevented the development of IRR. The dose required to overcome the HRS response coincided with the effect of early G2-phase checkpoint arrest in A549 cells (approximately 0.3 Gy), and Cdc25c knockdown in A549 cells (approximately 0.5 Gy) corresponded to the phosphorylation of the Cdc25c Ser216 residue. Resultant data confirmed that dose-dependent Cdc25c phosphatase does effectively act as an early G2-phase checkpoint, thus indicating mechanistic importance in the HRS to IRR transition in A549 cells. (author)

  12. β-Escin sodium inhibits inducible nitric oxide synthase expression via downregulation of the JAK/STAT pathway in A549 cells.

    Ji, Deng Bo; Xu, Bo; Liu, Jing Tao; Ran, Fu Xiang; Cui, Jing Rong

    2011-12-01

    β-escin, a triterpene saponin, is one of the major active compounds extracted from horse chestnut (Aesculus hippocastanum) seed. Previous work has found that β-escin sodium has antiinflammatory and antitumor effects. In the present study, we investigated its effect on cell proliferation and inducible nitric-oxide synthase (iNOS) expression in human lung carcinoma A549 cells. β-escin sodium (5-40 µg/mL) inhibited cytokine mixture (CM)-induced nitric oxide (NO) production in A549 cells by reducing the expression of iNOS. β-escin sodium suppressed phosphorylation and nuclear translocation of STAT1 (Tyr701) and STAT3 (Tyr705) induced by CM but did not affect the activation of c-Jun and NF-κB. β-escin sodium inhibited the activation of protein tyrosine kinase JAK2. Pervanadate treatment reversed the β-escin sodium-induced downregulation of STAT3 and STAT1. β-escin sodium treatment enhanced an activating phosphorylation of the phosphatase SHP2. Small interfering RNA-mediated knockdown of SHP2 inhibited β-escin sodium-induced phospho-STAT dephosphorylation. Moreover β-escin sodium reduced the activation of p38 MAPK. Finally, β-escin sodium inhibited the proliferation of A549 cells, did not change the cell membrane's permeability, nuclear morphology and size and the mitochondria's transmembrane potential of A549 cells. Taken together, these results demonstrate that β-escin sodium could downregulate iNOS expression through inhibiting JAK/STAT signaling and p38 MAPK activation in A549 cells. β-escin sodium has a marked antiproliferative effect on A549 cells at least in part by inhibiting the JAK/STAT signaling pathway, but not by a cytotoxic effect. β-escin sodium would be useful as a chemopreventive agent or a therapeutic against inflammatory-associated tumor. © 2011 Wiley Periodicals, Inc. PMID:21400616

  13. Regulation of MAPKs Signaling Contributes to the Growth Inhibition of 1,7-Dihydroxy-3,4-dimethoxyxanthone on Multidrug Resistance A549/Taxol Cells.

    Zuo, Jian; Jiang, Hui; Zhu, Yan-Hong; Wang, Ya-Qin; Zhang, Wen; Luan, Jia-Jie

    2016-01-01

    1,7-Dihydroxy-3,4-dimethoxyxanthone (XAN) is a bioactive compound isolated from Securidaca inappendiculata Hassk. and validated with antiproliferative activities on a panel of cancer cell lines. This study was designed to investigate its growth inhibitory effects on multidrug resistance (MDR) non-small cell lung carcinoma (NSCLC) cell line A549/Taxol and explore the possible linkage between modulation of MAPKs and the bioactivities. Its growth inhibitory potency on the cells was estimated by MTT assay, and flow cytometric analysis was employed to investigate its potential cell cycle arrest and proapoptosis effects. Expressions of hallmark proteins were assessed by Western-Blot method. The results showed A549/Taxol cells were sensitive to XAN. XAN inhibited the proliferation of A549/Taxol cells in the time and concentration dependent manners. It acted as a potent inducer of apoptosis and cell cycle arrest in the cells. Western-Blot investigation validated the proapoptosis and cell cycle arrest activities of XAN and the potential of MDR reversion. Upregulation of p38 by XAN, which accounted for the cell cycle arrest at G2 phase, and the downregulation of ERK associated with the proapoptosis activity were also revealed. Further analysis found p53 may be the central role mediated the bioactivities of MAPKs in A549/Taxol cells. Based on these evidences, a conclusion has been deduced that XAN could be a potential agent for MDR NSCLC therapy targeting specifically MAPKs. PMID:27403196

  14. Regulation of MAPKs Signaling Contributes to the Growth Inhibition of 1,7-Dihydroxy-3,4-dimethoxyxanthone on Multidrug Resistance A549/Taxol Cells

    Jian Zuo

    2016-01-01

    Full Text Available 1,7-Dihydroxy-3,4-dimethoxyxanthone (XAN is a bioactive compound isolated from Securidaca inappendiculata Hassk. and validated with antiproliferative activities on a panel of cancer cell lines. This study was designed to investigate its growth inhibitory effects on multidrug resistance (MDR non-small cell lung carcinoma (NSCLC cell line A549/Taxol and explore the possible linkage between modulation of MAPKs and the bioactivities. Its growth inhibitory potency on the cells was estimated by MTT assay, and flow cytometric analysis was employed to investigate its potential cell cycle arrest and proapoptosis effects. Expressions of hallmark proteins were assessed by Western-Blot method. The results showed A549/Taxol cells were sensitive to XAN. XAN inhibited the proliferation of A549/Taxol cells in the time and concentration dependent manners. It acted as a potent inducer of apoptosis and cell cycle arrest in the cells. Western-Blot investigation validated the proapoptosis and cell cycle arrest activities of XAN and the potential of MDR reversion. Upregulation of p38 by XAN, which accounted for the cell cycle arrest at G2 phase, and the downregulation of ERK associated with the proapoptosis activity were also revealed. Further analysis found p53 may be the central role mediated the bioactivities of MAPKs in A549/Taxol cells. Based on these evidences, a conclusion has been deduced that XAN could be a potential agent for MDR NSCLC therapy targeting specifically MAPKs.

  15. 淫羊藿苷逆转耐甲氨蝶呤肺癌A549细胞转移表型%Icariin reversed metastatic phenotype of methotrexate-resistant lung cancer A549 cells

    吴剑锋; 何晓东; 许卫东; 李道静; 孙利; 沈佐君

    2009-01-01

    目的:研究中药淫羊藿苷(icariin,ICA)作用甲氨蝶呤(methotrexate,MTX)耐药肺癌A549细胞后对细胞转移表型的影响,初步探讨ICA逆转A549/MTX耐药细胞转移表型的作用机制及对肺癌的治疗价值.方法:采用MTT法检测ICA对A549/MTX耐药细胞的半数抑制浓度(half inhibition concentration,IC_(50)).采用双层软琼脂克隆形成实验检测A549/MTX 组和A549/MTX+ICA组细胞的克隆形成率,并观察其集落形态.细胞划痕实验检测A549/MTX组和A549/MTX+ICA组细胞的迁移能力.Transwell小室实验检测细胞侵袭能力的变化.结果:MTT结果显示,无毒剂量的ICA与MTX联合应用后A549/MTX细胞的IC_(50)值为35.50±1.85 μmol/L,比单独应用MTX(同等剂量)后A549/MTX细胞的IC50值(52.17±2.25 μmol/L)有了一定程度的下降.软琼脂实验发现,A549/MTX+ICA组细胞克隆形成率为0.94±0.09,小于A549/MTX组细胞的1.56±1.07(P<0.05).划痕实验显示,72 h后A549/MTX组细胞的迁移能力大于A549/MTX+ICA组细胞(P<0.05).Transwell实验显示,A549/MTX组细胞的穿膜细胞数明显多于A549/MTX+ICA组细胞(P<0.05),说明A549/MTX+ICA组细胞的侵袭浸润能力小于A549/MTX组细胞.结论:中药ICA具有逆转A549/MTX耐药细胞转移表型的作用.

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

    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.

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

    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

  18. Denbinobin induces apoptosis in human lung adenocarcinoma cells via Akt inactivation, Bad activation, and mitochondrial dysfunction.

    Kuo, Chen-Tzu; Hsu, Ming-Jen; Chen, Bing-Chang; Chen, Chien-Chih; Teng, Che-Ming; Pan, Shiow-Lin; Lin, Chien-Huang

    2008-02-28

    Increasing evidence demonstrated that denbinobin, isolated from Ephemerantha lonchophylla, exert cytotoxic effects in cancer cells. The purpose of this study was to investigate whether denbinobin induces apoptosis and the apoptotic mechanism of denbinobin in human lung adenocarcinoma cells (A549). Denbinobin (1-20microM) caused cell death in a concentration-dependent manner. Flow cytometric analysis and annexin V labeling demonstrated that denbinobin increased the percentage of apoptotic cells. A549 cells treated with denbinobin showed typical characteristics of apoptosis including morphological changes and DNA fragmentation. Denbinobin induced caspase 3 activation, and N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk), a broad-spectrum caspase inhibitor, prevented denbinobin-induced cell death. Denbinobin induced the loss of the mitochondrial membrane potential and the release of mitochondrial apoptotic proteins including cytochrome c, second mitochondria derived activator of caspase (Smac), and apoptosis-inducing factor (AIF). In addition, denbinobin-induced Bad activation was accompanied by the dissociation of Bad with 14-3-3 and the association of Bad with Bcl-xL. Furthermore, denbinobin induced Akt inactivation in a time-dependent manner. Transfection of A549 cells with both wild-type and constitutively active Akt significantly suppressed denbinobin-induced Bad activation and cell apoptosis. These results suggest that Akt inactivation, followed by Bad activation, mitochondrial dysfunction, caspase 3 activation, and AIF release, contributes to denbinobin-induced cell apoptosis. PMID:18262737

  19. 11β-HSD2重组真核表达载体构建及对A549细胞IL-6的影响%Construction of 11β-HSD2 recombinant eukaryotic vector and its effect on IL-6 in A549 cells

    陈晓琳; 江红; 王兴友; 王丽娜; 宋何煜; 金帮明

    2013-01-01

    目的 本文旨在构建11β-羟基类固醇脱氢酶2 (11β-HSD2)基因的重组真核表达载体,并检测其转染入哺乳动物细胞后对白介素6(interleukin-6,IL-6)表达水平的影响.方法 用PCR方法扩增11β-HSD2基因的全长cDNA,先亚克隆至pGMT载体,测序正确后再亚克隆至pcDNA3.1 myc-hisC 以构建其重组真核表达载体.将重组的真核表达载体瞬时转染人肺腺癌细胞系A549,用Western blot 方法鉴定表达情况,并用ELISA方法检测IL-6表达水平.结果 测序表明11β-HSD2的重组真核表达载体构建成功,Western blot显示真核表达载体能够在A549细胞中成功表达,并可使IL-6表达水平升高.结论成功构建了11β-HSD2基因的重组真核表达载体,初步验证了该基因的促炎效应.%Objective To establish recombinant eukaryotic vector of 11β-hydroxysteroid dehydrogenase 2 gene in mammalian cell and to detect the expression of interleukin-6 after the vector transfection.Methods The cDNA of 11β-hydroxysteroid dehydrogenase 2 gene was amplified by PCR.After subcloned with pGMT vector and sequenced,the gene was inserted into the eukaryotic vector pcDNA3.1myc hisC.The expression vector was transiently transfected in human lung adenocarcinoma cell line A549 cells,and then the protein was detected using Western blot,interleukin-6 was measured by ELISA.Results Sequencing revealed 11β-hydroxysteroid dehydrogenase 2 recombinant eukaryotic expression vector was constructed successfully,and Western blot indicated that eukaryotic expression vector expressed successfully in A549 cells.Transfection with recombinant eukaryotic vector of 11β hydroxysteroid dehydrogenase 2 gene significantly increased interleukin 6 expression.Conclusions The recombinant eukaryotic vector of 11β-hydroxysteroid dehydrogenase 2 gene is established successfully and could be highly overexpressed in A549 cells.The increase level of interleukin-6 suggests the proinflammatory effect of 11

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

    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.

  1. Quercetin Down-regulates IL-6/STAT-3 Signals to Induce Mitochondrial-mediated Apoptosis in a Non-small-cell Lung-cancer Cell Line, A549

    Avinaba Mukherjee

    2015-03-01

    Full Text Available Objectives: Quercetin, a flavonoid compound, has been reported to induce apoptosis in cancer cells, but its anti-inflammatory effects, which are also closely linked with apoptosis, if any, on non-small-cell lung cancer (NSCLC have not so far been critically examined. In this study, we tried to determine if quercetin had any demonstrable anti-inflammatory potential, which also could significantly contribute to inducing apoptosis in a NSCLC cell line, A549. Methods: In this context, several assays, including cytotoxicity, flow cytometry and fluorimetry, were done. Gene expression was analyzed by using a western blot analysis. Results: Results revealed that quercetin could induce apoptosis in A549 cells through mitochondrial depolarization by causing an imbalance in B-cell lymphoma 2/Bcl2 Antagonist X (Bcl2/Bax ratio and by down-regulating the interleukine-6/signal transducer and activator of transcription 3 (IL-6/STAT3 signaling pathway. An analysis of the data revealed that quercetin could block nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB activity at early hours, which might cause a down-regulation of the IL-6 titer, and the IL-6 expression, in turn, could inhibit p-STAT3 expression. Down-regulation of both the STAT3 and the NF-κB expressions might, therefore, cause down-regulation of Bcl2 activity because both are major upstream effectors of Bcl2. Alteration in Bcl2 responses might result in an imbalance in the Bcl2/Bax ratio, which could ultimately bring about mitochondria mediated apoptosis in A549 cells. Conclusion: Overall, the finding of this study indicates that a quercetin induced anti-inflammatory pathway in A549 cells appeared to make a significant contribution towards induction of apoptosis in NSCLC and, thus, may have a therapeutic use such as a strong apoptosis inducer in cancer cells.

  2. Biological evaluation of new nickel(II) metallates: Synthesis, DNA/protein binding and mitochondrial mediated apoptosis in human lung cancer cells (A549) via ROS hypergeneration and depletion of cellular antioxidant pool.

    Kalaivani, P; Saranya, S; Poornima, P; Prabhakaran, R; Dallemer, F; Vijaya Padma, V; Natarajan, K

    2014-07-23

    A series of novel nickel(II) thiosemicarbazone complexes(1-4) have been prepared and characterized by various spectral, analytical techniques and X-ray crystallography. Further, their efficacy to interact with CT-DNA/BSA has been explored. From the binding studies, it is inferred that complex 4 found to be more active than other complexes. The complexes bound with CT-DNA by intercalation mode. Moreover, static quenching was observed for their interaction with BSA. The new complexes were tested for their in vitro cytotoxicity against human lung adenocarcinoma (A549) cell line. The results showed that the new complexes exhibited significant degree of cytotoxicity at given experimental condition. Further, the results of LDH and NO release supported the cytotoxic nature of the complexes. The observed cytotoxicity of the complexes may be routed through ROS-hypergeneration and lipid-peroxidation with subsequent depletion of cellular antioxidant pool (GSH, SOD, CAT, GPx and GST) resulted in the reduction of mitochondrial-membrane potential, caspase-3 activation and DNA fragmentation. Thus, the data from the present study disclose that the complexes could induce apoptosis in A549 cells through mitochondrial mediated fashion and inhibited the migration of lung cancer cells and by metastasis. PMID:24946146

  3. CpG-ODN 7909 increases radiation sensitivity of radiation-resistant human lung adenocarcinoma cell line by overexpression of Toll-like receptor 9.

    Yan, Li; Xu, Guoxiong; Qiao, Tiankui; Chen, Wei; Yuan, Sujuan; Li, Xuan

    2013-09-01

    Radioresistance is one of the main reasons for the failure of radiotherapy in lung cancer. The aim of this study was to establish a radiation-resistant lung cancer cell line, to evaluate whether CpG oligodeoxyribonucleotide (CpG-ODN) 7909 could increase its radiosensitivity and to explore the relevant mechanisms. The radioresistant cell line, referred to as R-A549, was generated by reduplicative fractionated irradiation from the human lung adenocarcinoma cell line A549. The radioresistance of R-A549 cells were confirmed by the Cell Counting Kit-8 (CCK-8), cell viability assay, and clonogenic assay. Cell growth kinetics, morphological feature, and radiosensitivity were compared between the original A549 cells and R-A549 cells treated with or without CpG-ODN 7909 or radiation. To further explore the potential mechanisms of radiosensitivity, the cell cycle distributions and the expression of Toll-like receptor 9 (TLR-9) were examined by Western blot and flow cytometry. The R-A549 cell line was generated and its radioresistance was further confirmed. CpG-ODN 7909 was found to increase much more radiosensitivity of R-A549 cells under combined treatments with CpG-ODN 7909 and radiation compared with its control group without any treatments. They presented their respective D0 1.33 ± 0.20 Gy versus 1.76 ± 0.25 Gy with N 3.44 ± 1.01 versus 4.96 ± 0.32. Further, there was a larger cell population of R-A549 cells under combined treatment in the G2/M phase compared with the control group after treatment with CpG-ODN7909 or radiation alone at 24 and 48 hour. The expression level of TLR-9 in R-A549 cells was found higher than in A549 cells. These results suggested that CpG-ODN 7909 increased the radiosensitivity of R-A549 cells, which might be mediated via the upregulated TLR-9 and prolonged cell cycle arrest in the G2/M phase compared with A549 cells. PMID:23705865

  4. The mechanism of CpG ODN enhancing the radiosensitivity of lung cancer cell line A549%CpG ODN增强人肺癌细胞株A549放射增敏作用的研究

    颜伟; 孙梯业; 杨春敏; 贾敏; 李静; 史蕊; 唐和兰; 杜斌; 韩全利

    2011-01-01

    Objective To investigate the effect of CpG ODN on the radiosensitivity of lung epithelial cell line A549.Methods The TNF-α,IL-12 and INF-γ secretion by A549 were detected by ELISA;NO level was tested by Griess method ,AP-1 activation within A549 cells was observed using electrophoretic mobility shift assay.Results The inhibitory role was enhanced when CpG ODN 1826(10μg/ml)were combined with β-ray irradiation ,with the increase of TNF-α,IL-2 and INF - γ secretion by cells.CpG ODN1826 combined with β-ray irradiation increased NO leve in A549 cells and inhibited the AP-1 activation within A549 cells.Conclusions CpG ODN1826 can increase the radiosensitivity of lung epithelial cell line A549 and may be tightly related to increasing secretions of IL-12,IFN-γ,TNF-α and NO from cells and the inhibition of AP-1 activation.%目的 初步探讨CpG ODN增强人肺腺上皮细胞株A549放射增敏作用.方法 ELISA法检测细胞TNF-α、IL-12和INF-γ的分泌水平,Griess检测细胞NO的含量并观察CpGODN1826与β射线诱导A549细胞AP-1活化的抑制作用.结果 CpG ODN增加了人肺癌细胞株A549 TNF-α、IL-12、INF-γ和NO的分泌,在联合β射线照射后对A549细胞的杀伤作用更加显著,并显著抑制A549细胞AP-1的活化.结论 CpG ODN对A549有明显的放射增敏作用,其机制可能与CpG ODN增强+4549细胞分泌TNF-α、IL-L2、INF-γ、NO和抑制A549细胞AP-1的活化有关.

  5. Cell cycle inhibitory activity of Piper longum against A549 cell line and its protective effect against metal-induced toxicity in rats.

    Sharma, Amit Kumar; Kumar, Shashank; Chashoo, Gousia; Saxena, Ajit K; Pandey, Abhay K

    2014-10-01

    Anticancer potential of Piper longum fruit against human cancer cell lines (DU-145 prostate, A549 lung, THP-1 leukemia, IGR-OVI-1 ovary and MCF-7 breast) as well as its in vitro and in vivo biochemical efficacy in A1Cl3-induced hepatotoxicity were evaluated in the rats. Dried samples were extracted with several solvents using soxhlet apparatus. Flavonoid content in chloroform, benzene, ethyl alcohol and aqueous extracts of fruit was 19, 14, 12 and 11 μg quercetin equivalent/mg of sample, respectively. Hexane extracts exhibited 90-92% cytotoxicity against most of the test cell lines (A549, THP-1, IGR-OVI-1 and MCF-7), while benzene extract displayed 84-87% cytotoxicity against MCF-7, IGR-OV-1 and THP-1 cell lines. Among extracts, hexane, benzene and acetone extracts demonstrated considerable cytotoxicity (91-95%) against A549 (lung cancer) cell line in Sulforhodamine B dye (SRB) assay. Cell cycle analysis revealed that hexane, benzene and acetone extracts produced 41, 63 and 43% sub-G1 DNA fraction, demonstrating cell cycle inhibitory potential of these extracts against A549 cell line. Chloroform, ethyl alcohol and aqueous extracts displayed 71, 64 and 65% membrane protective activity, respectively in lipid peroxidation inhibition assay. P. longum fruit extracts also ameliorated A1Cl3-induced hepatotoxicity, as indicated by alterations observed in serum enzymes ALP, SGOT and SGPT activity, as well as creatinine and bilirubin contents. In conclusion, study established the cytotoxic and hepatoprotective activity in P. longum extracts. PMID:25630105

  6. Evaluation of the cytotoxicity and the genotoxicity induced by {alpha} radiation in an A549 cell line

    Belchior, Ana, E-mail: anabelchior@itn.pt [Instituto Tecnologico e Nuclear, Estrada Nacional no 10, 2686-953 Sacavem (Portugal) and Universidade de Lisboa, Instituto de Biofisica e Engenharia Biomedica, Campo Grande, 1749-016 Lisboa (Portugal); Gil, Octavia Monteiro [Instituto Tecnologico e Nuclear, Estrada Nacional no 10, 2686-953 Sacavem (Portugal); Almeida, Pedro [Universidade de Lisboa, Instituto de Biofisica e Engenharia Biomedica, Campo Grande, 1749-016 Lisboa (Portugal); Vaz, Pedro [Instituto Tecnologico e Nuclear, Estrada Nacional no 10, 2686-953 Sacavem (Portugal)

    2011-09-15

    Exposure to radon and its progenies represents one of the greatest risks of ionizing radiation from natural sources. Nowadays, these risks are assessed by the extrapolation of biological effects observed from epidemiological data. In the present study, we made a dose response curve, to evaluate the in vitro response of A549 human lung cells to {alpha}-radiation resulting from the decay of a {sup 210}Po source, evaluated by the cytokinesis blocked micronuclei assay. The clonogenic assay was used to measure the survival cell fraction. As expected, the results revealed an increase of cellular damage with increased doses made evident from the increased number of micronuclei (MN) per binucleated cell (BN). Besides this study involving the biological effects induced by direct irradiation, and due to the fact that radiation-induced genomic instability is thought to be an early event in radiation carcinogenesis, we analyzed the genomic instability in early and delayed untargeted effects, by using the medium transfer technique. The obtained results show that unirradiated cells exposed to irradiated medium reveal a higher cellular damage in earlier effects when compared to the delayed effects. The obtained results may provide clues for the biodosimetric determination of radon dose to airway cells at cumulative exposures.

  7. Evaluation of the cytotoxicity and the genotoxicity induced by α radiation in an A549 cell line

    Exposure to radon and its progenies represents one of the greatest risks of ionizing radiation from natural sources. Nowadays, these risks are assessed by the extrapolation of biological effects observed from epidemiological data. In the present study, we made a dose response curve, to evaluate the in vitro response of A549 human lung cells to α-radiation resulting from the decay of a 210Po source, evaluated by the cytokinesis blocked micronuclei assay. The clonogenic assay was used to measure the survival cell fraction. As expected, the results revealed an increase of cellular damage with increased doses made evident from the increased number of micronuclei (MN) per binucleated cell (BN). Besides this study involving the biological effects induced by direct irradiation, and due to the fact that radiation-induced genomic instability is thought to be an early event in radiation carcinogenesis, we analyzed the genomic instability in early and delayed untargeted effects, by using the medium transfer technique. The obtained results show that unirradiated cells exposed to irradiated medium reveal a higher cellular damage in earlier effects when compared to the delayed effects. The obtained results may provide clues for the biodosimetric determination of radon dose to airway cells at cumulative exposures.

  8. Casiopeina IIgly-induced oxidative stress and mitochondrial dysfunction in human lung cancer A549 and H157 cells

    Casiopeinas are a series of mixed chelate copper complexes that are being evaluated as anticancer agents. Their effects in the cell include oxidative damage and mitochondrial dysfunction, yet the molecular mechanisms leading to such effects remain unclear. We tested whether [Cu(4,7-dimethyl-phenanthroline)(glycinate)]NO3 (Casiopeina IIgly or Cas IIgly) could alter cellular glutathione (GSH) levels by redox cycling with GSH to generate ROS and cellular oxidative stress. Cas IIgly induced a dramatic drop in intracellular levels of GSH in human lung cancer H157 and A549 cells, and is able to use GSH as source of electrons to catalyze the Fenton reaction. In both cell lines, the toxicity of Cas IIgly (2.5-5 μM) was potentiated by the GSH synthesis inhibitor L-buthionine sulfoximine (BSO) and diminished by the catalytic antioxidant manganese(III) meso-tetrakis(N,N'-diethylimidazolium-2-yl)porphyrin (MnTDE-1,3-IP5+), thus supporting an important role for oxidative stress. Cas IIgly also caused an over-production of reactive oxygen species (ROS) in the mitochondria and a depolarization of the mitochondrial membrane. Moreover, Cas IIgly produced mitochondrial DNA damage that resulted in an imbalance of the expression of the apoproteins of the mitochondrial respiratory chain, which also can contribute to increased ROS production. These results suggest that Cas IIgly initiates multiple possible sources of ROS over-production leading to mitochondrial dysfunction and cell death.

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

    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.

  10. Histone deacetylase inhibitors stimulate the susceptibility of A549 cells to a plasma-activated medium treatment.

    Adachi, Tetsuo; Kano, Ayame; Nonomura, Saho; Kamiya, Tetsuro; Hara, Hirokazu

    2016-09-15

    The number of potential applications of non-thermal atmospheric pressure plasma (NTAPP) discharges in medicine, particularly in cancer therapy, has increased in recent years. NTAPP has been shown to affect cells not only by direct irradiation, but also by an indirect treatment with previously prepared plasma-activated medium (PAM). Histone deacetylase (HDAC) inhibitors have the potential to enhance susceptibility to anticancer drugs and radiation. The aim of the present study was to demonstrate the advantage of the combined application of PAM and HDAC inhibitors on A549 cancer cell survival and elucidate the underlying mechanisms. Cell death with DNA breaks in the nucleus was greater using combined regimens of PAM and HDAC inhibitors such as trichostatin A (TSA) and valproic acid (VPA) than a single PAM treatment and was accompanied by the activation of poly (ADP-ribose) polymerase-1 (PARP-1), depletion of ATP, and elevations in intracellular calcium levels. Moreover, the expression of Rad 51, a DNA repair factor in homologous recombination pathways, was significantly suppressed by the treatment with HDAC inhibitors. These results demonstrate that HDAC inhibitors may synergistically induce the sensitivity of cancer cells to PAM components. PMID:27470189