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Sample records for a549 human non-small

  1. Enhancement of radiosensitivity by CpG-oligodeoxyribonucleotide-7909 in human non-small cell lung cancer A549 cells.

    PubMed

    Zha, Lin; Qiao, Tiankui; Yuan, Sujuan; Lei, Linjie

    2010-04-01

    CpG-oligodeoxyribonucleotides (CpG-ODNs), which induce signaling through the toll-like receptor 9, are currently under investigation as immunity stimulators against cancer. It has recently been suggested that CpG-ODNs may also enhance sensitivity to traditional therapies including chemotherapy in certain cancer-cell lines. The purpose of this study was to define the activity of CpG-ODN7909 in increasing radiosensitivity of the human non-small cell lung cancer cell line A549 in vitro. First, a dose- and time-dependent inhibitory effect on cell viability was observed after A549 cells were treated with different concentrations of CpG-ODN7909 (5, 10, 30, and 60 microg/mL). Second, decreased cell clonogenic survival, enhanced cell apoptotic index, accumulated percentage of cells in the G2/M phase, and increased tumor necrosis factor (TNF)-alpha secretion were found after combined treatments with 10 microg/mL of CpG-ODN7909 and radiation compared to either treatment alone (p < 0.05). Furthermore, the toll-like receptor 9 mRNA was found to express in A549. The results suggest that CpG-ODN7909 can increase the radiosensitivity of human non-small cell lung cancer A549 cells, which may be associated with reduced cell clonogenic survival, enhanced apoptosis, prolonged cell-cycle arrest in G2/M, and stimulation of TNF-alpha secretion.

  2. Enhancement of radiosensitivity by roscovitine pretreatment in human non-small cell lung cancer A549 cells.

    PubMed

    Zhang, Feng; Zhang, Tao; Gu, Zhong-Ping; Zhou, Yong-An; Han, Yong; Li, Xiao-Fei; Wang, Xiao-Ping; Cheng, Qing-Shu; Mei, Qi-Bing

    2008-09-01

    Roscovitine has been reported to have anti-proliferative properties and is in process of undergoing clinical trials. In addition to its intrinsic anticancer properties, it has recently been suggested that roscovitine may also enhance the activity of traditional chemo- and radio- therapies in certain cancer cell lines. The purpose of this study was to define the activity of roscovitine in increasing radiosensitivity of human non-small cell lung cancer (NSCLC) cell line A549 cells in vitro. A549 cells were exposed to ionizing radiation (IR) of gamma-ray with or without roscovitine pretreatment. Clonogenic assay was performed and cell cycle and apoptosis were analyzed by flow cytometry. Expression of PARP, Ku70 and Ku80 proteins was detected by Western blot. The active form of caspase-3 positive cells were measured by flow cytometry. Our results showed that roscovitine caused dose-dependent apoptosis in A549 cells. Pretreatment with minimally toxic concentration of roscovitine significantly radiosensitized A549 cells by inhibiting colony formation. We then examined potential mechanisms that may contribute to the enhanced radiation response induced by roscovitine. Our results showed that the combination treatment significantly induced apoptosis in A549 cells compared to roscovitine or IR treatment alone. Meanwhile, in the co-treatment group, the percentage of cells with the active form of caspase-3 was markedly increased, while roscovitine or IR alone had little effect. Roscovitine decreased S phase cells when used alone or in sequential combination with IR. Furthermore, this combination treatment blocked DNA repair process after IR, indicated by down regulation of Ku70 and Ku80 proteins, while the singly used treatment did not. Taken together, these results suggest that roscovitine has the potential to act as a radio-sensitizer in A549 cells by promoting caspase-3 activity and increasing apoptosis, affecting cell cycle distribution and impairing DNA repair process.

  3. Curcumin inhibits interferon-{alpha} induced NF-{kappa}B and COX-2 in human A549 non-small cell lung cancer cells

    SciTech Connect

    Lee, Jeeyun |; Im, Young-Hyuck | E-mail: imyh@smc.samsung.co.kr; Jung, Hae Hyun; Kim, Joo Hyun; Park, Joon Oh |; Kim, Kihyun |; Kim, Won Seog |; Ahn, Jin Seok

    2005-08-26

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

  4. [Inhibitory effect of human mitochondria-targeted MPG recombinant on proliferation of human non-small cell lung cancer multidrug-resistant cell line A549/DDP].

    PubMed

    Yu, Shi-Cang; Qian, Gui-Sheng; Li, Yu-Ying; Lu, Wei-Zhong; Li, Jin; Huang, Gui-Jun

    2006-04-01

    Multidrug resistance is the key obstacle to the improvement of chemotherapy effect of lung cancer. This study was to construct eukaryotic expression vector of human mitochondria-targeted N-methylpurine DNA glycosylase (MPG), and explore its inhibitory effect on proliferation of human non-small cell lung cancer multidrug-resistant cell line A549/DDP. Manganese-superoxide dismutase mitochondria-targeted sequence-MPG fusion gene (mito-MPG) was constructed through splicing by overlap extension (SOE). Recombinant eukaryotic expression vector pCMV-Script/mito-MPG was constructed by molecule-cloning technique, and then transfected into A549/DDP cells. In the stably transfected cells which were screened out by G418, the expression of mito-MPG mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR); its expression in separated and purified mitochondria was detected by Western blot. The proliferation of A549/DDP cells was detected by trypan blue exclusion trial. Cell cycle distribution was analyzed by flow cytometry. The mito-MPG fusion gene was confirmed by DNA sequencing,the recombinant pCMV-Script/mito-MPG was confirmed by restrictive endonuclease digestion and DNA sequencing. mito-MPG mRNA and protein were detected in the cells transfected with pCMV-Script/mito-MPG (MPG group), but not in the cells transfected with pCMV-Script (P group) and untransfected cells (C group). The cell double time were 72.6 h in C group, 73.5 h in P group, and 98.9 h in MPG group. Cell cycle blockage and subdiploid peak were found in MPG group. The proliferation indexes were 51.3% in C group, 54.3% in P group, and 26.1% in MPG group. pCMV-Script/mito-MPG could be constructed and transfected into mitochondria of A549/DDP cells successfully, and inhibit proliferation and induce apoptosis of A549/DDP cells.

  5. Oleifolioside B-mediated autophagy promotes apoptosis in A549 human non-small cell lung cancer cells.

    PubMed

    Jin, Cheng-Yun; Yu, Hai Yang; Park, Cheol; Han, Min Ho; Hong, Su Hyun; Kim, Kyoung-Sook; Lee, Young-Choon; Chang, Young-Chae; Cheong, Jaehun; Moon, Sung-Kwon; Kim, Gi-Young; Moon, Hyung-In; Kim, Wun-Jae; Lee, Jai-Heon; Choi, Yung Hyun

    2013-12-01

    The biochemical mechanisms of cell death by oleifolioside B (OB), a cycloartane-type triterpene glycoside isolated from Dendropanax morbifera Leveille, were investigated in A549 human lung carcinoma cells. Our data indicated that exposure to OB led to caspase activation and typical features of apoptosis; however, apoptotic cell death was not prevented by z-VAD-fmk, a pan-caspase inhibitor, demonstrating that OB-induced apoptosis was independent of caspase activation. Subsequently, we found that OB increased autophagy, as indicated by an increase in monodansylcadaverine fluorescent dye-labeled autophagosome formation and in the levels of the autophagic form of microtubule-associated protein 1 light chain 3 and Atg3, an autophagy-specific gene, which is associated with inhibiting phospho-nuclear factor erythroid 2-related factor 2 (Nrf2) expression. However, pretreatment with bafilomycin A1, an autophagy inhibitor, attenuated OB-induced apoptosis and dephosphorylation of Nrf2. The data suggest that OB-induced autophagy functions as a death mechanism in A549 cells and OB has potential as a novel anticancer agent capable of targeting apoptotic and autophagic cell death and the Nrf2 signaling pathway.

  6. Green tea catechin, epigallocatechin-3-gallate, attenuates the cell viability of human non-small-cell lung cancer A549 cells via reducing Bcl-xL expression.

    PubMed

    Sonoda, Jun-Ichiro; Ikeda, Ryuji; Baba, Yasutaka; Narumi, Keiko; Kawachi, Akio; Tomishige, Erisa; Nishihara, Kazuya; Takeda, Yasuo; Yamada, Katsushi; Sato, Keizo; Motoya, Toshiro

    2014-07-01

    Clinical and epidemiological studies have indicated that the consumption of green tea has a number of beneficial effects on health. Epigallocatechin-3-gallate (EGCg), the major polyphenolic compound present in green tea, has received much attention as an active ingredient. Among the numerous promising profiles of EGCg, the present study focused on the anticancer effects. Apoptosis induced by EGCg and subsequent cell growth suppression have been demonstrated in a number of cell culture studies. However, the underlying mechanism of apoptotic cell death remains unclear. Thus, the aim of the present study was to identify the major molecule that mediates proapoptotic cell death by EGCg. The effect of EGCg on cell proliferation and the induction of mRNA that modulates apoptotic cell death was evaluated in the A549 human non-small-cell lung cancer cell line. In addition, morphological changes were assessed by microscopy in A549 cells that had been treated with 100 μM EGCg for 24 h. The MTT assay revealed that cell proliferation was significantly reduced by EGCg in a dose-dependent manner (3-100 μM). The mRNA expression level of B-cell lymphoma-extra large (Bcl-xL) was decreased in A549 cells following 24 h incubation with 100 μM EGCg. Therefore, the results indicated that the inhibition of cell proliferation by EGCg may be achieved via suppressing the expression of the cell death-inhibiting gene, Bcl-xL.

  7. Green tea catechin, epigallocatechin-3-gallate, attenuates the cell viability of human non-small-cell lung cancer A549 cells via reducing Bcl-xL expression

    PubMed Central

    SONODA, JUN-ICHIRO; IKEDA, RYUJI; BABA, YASUTAKA; NARUMI, KEIKO; KAWACHI, AKIO; TOMISHIGE, ERISA; NISHIHARA, KAZUYA; TAKEDA, YASUO; YAMADA, KATSUSHI; SATO, KEIZO; MOTOYA, TOSHIRO

    2014-01-01

    Clinical and epidemiological studies have indicated that the consumption of green tea has a number of beneficial effects on health. Epigallocatechin-3-gallate (EGCg), the major polyphenolic compound present in green tea, has received much attention as an active ingredient. Among the numerous promising profiles of EGCg, the present study focused on the anticancer effects. Apoptosis induced by EGCg and subsequent cell growth suppression have been demonstrated in a number of cell culture studies. However, the underlying mechanism of apoptotic cell death remains unclear. Thus, the aim of the present study was to identify the major molecule that mediates proapoptotic cell death by EGCg. The effect of EGCg on cell proliferation and the induction of mRNA that modulates apoptotic cell death was evaluated in the A549 human non-small-cell lung cancer cell line. In addition, morphological changes were assessed by microscopy in A549 cells that had been treated with 100 μM EGCg for 24 h. The MTT assay revealed that cell proliferation was significantly reduced by EGCg in a dose-dependent manner (3–100 μM). The mRNA expression level of B-cell lymphoma-extra large (Bcl-xL) was decreased in A549 cells following 24 h incubation with 100 μM EGCg. Therefore, the results indicated that the inhibition of cell proliferation by EGCg may be achieved via suppressing the expression of the cell death-inhibiting gene, Bcl-xL. PMID:24944597

  8. Human Noxin is an anti-apoptotic protein in response to DNA damage of A549 non-small cell lung carcinoma.

    PubMed

    Won, Kyoung-Jae; Im, Joo-Young; Yun, Chae-Ok; Chung, Kyung-Sook; Kim, Young Joo; Lee, Jung-Sun; Jung, Young-Jin; Kim, Bo-Kyung; Song, Kyung Bin; Kim, Young-Ho; Chun, Ho-Kyung; Jung, Kyeong Eun; Kim, Moon-Hee; Won, Misun

    2014-06-01

    Human Noxin (hNoxin, C11Orf82), a homolog of mouse noxin, is highly expressed in colorectal and lung cancer tissues. hNoxin contains a DNA-binding C-domain in RPA1, which mediates DNA metabolic processes, such as DNA replication and DNA repair. Expression of hNoxin is associated with S phase in cancer cells and in normal cells. Expression of hNoxin was induced by ultraviolet (UV) irradiation. Knockdown of hNoxin caused growth inhibition of colorectal and lung cancer cells. The comet assay and western blot analysis revealed that hNoxin knockdown induced apoptosis through activation of p38 mitogen-activated protein kinase (MAPK)/p53 in non-small cell lung carcinoma A549 cells. Furthermore, simultaneous hNoxin knockdown and treatment with DNA-damaging agents, such as camptothecin (CPT) and UV irradiation, enhanced apoptosis, whereas Trichostatin A (TSA) did not. However, transient overexpression of hNoxin rescued cells from DNA damage-induced apoptosis but did not block apoptosis in the absence of DNA damage. These results suggest that hNoxin may be associated with inhibition of apoptosis in response to DNA damage. An adenovirus expressing a short hairpin RNA against hNoxin transcripts significantly suppressed the growth of A549 tumor xenografts, indicating that hNoxin knockdown has in vivo anti-tumor efficacy. Thus, hNoxin is a DNA damage-induced anti-apoptotic protein and potential therapeutic target in cancer. © 2013 UICC.

  9. Combined treatment of curcumin and small molecule inhibitors suppresses proliferation of A549 and H1299 human non-small-cell lung cancer cells.

    PubMed

    Lin, Hui-Ping; Kuo, Li-Kuo; Chuu, Chih-Pin

    2012-01-01

    Curcumin (diferuloylmethane) is a phenolic compound present in turmeric and is ingested daily in many parts of the world. Curcumin has been reported to cause inhibition on proliferation and induction of apoptosis in many human cancer cell lines, including non-small cell lung cancer cells (NSCLC). However, the clinical application of curcumin is restricted by its low bioavailability. In this report, it was observed that combined treatment of a low dosage of curcumin (5-10 µM) with a low concentration (0.1-2.5 µM) of small molecule inhibitors, including AG1478, AG1024, PD173074, LY294002 and caffeic acid phenethyl ester (CAPE) increased the growth inhibition in two human NSCLC cell lines: A549 and H1299 cells. The observation suggested that combined treatment of a low dosage of curcumin with inhibitors against epidermal growth factor receptor (EGFR), insulin-like growth factor 1 (IGF-1R), fibroblast growth factors receptor (FGFR), phosphatidylinositol 3-kinases (PI3K) or NF-κB signaling pathway may be a potential adjuvant therapy beneficial to NSCLC patients.

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

    PubMed

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

    2011-02-01

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

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

    SciTech Connect

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

    2011-02-01

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

  12. Cytotoxicity, DNA damage, and apoptosis induced by titanium dioxide nanoparticles in human non-small cell lung cancer A549 cells.

    PubMed

    Wang, Yurong; Cui, Haiyan; Zhou, Jiaping; Li, Fengjuan; Wang, Jinju; Chen, Mianhua; Liu, Qingdai

    2015-04-01

    Concerns about the risk of titanium dioxide nanoparticles (TiO2 NPs) to human health and environment are gradually increasing due to their wide range of applications. In this study, cytotoxicity, DNA damage, and apoptosis induced by TiO2 NPs (5 nm) in A549 cells were investigated. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays revealed the time- and concentration-dependent cytotoxic effects of TiO2 NPs in a concentration range of 50 to 200 μg/mL. A statistically significant (p < 0.05) induction in DNA damage was observed by the comet assay in cells exposed to 50 to 200 μg/mL TiO2 NPs for 48 h. A significant (p < 0.05) induction in micronucleus formation determined by 4,6-diamino-2-phenylindole (DAPI) staining was also observed at the above concentrations. Typical apoptotic morphological feature and apoptotic bodies in A549 cells induced by TiO2 NPs at the above concentrations were observed by scanning electron micrographs. Flow cytometric analysis demonstrated that the cells treated with TiO2 NPs at concentrations of 100 and 200 μg/mL showed a significant G2/M phase arrest and a significant increased proportion of apoptotic cells. TiO2 NPs also disrupted the mitochondrial membrane potential evaluated by rhodamine 123 staining. Further analysis by quantitative real-time PCR (qRT-PCR) indicated that the expression of caspase-3 and caspase-9 messenger RNA (mRNA) was increased significantly at the concentrations of 100 and 200 μg/mL TiO2 NPs for 48 h. Taken together, these findings suggest that TiO2 NPs can inhibit A549 cell proliferation, cause DNA damage, and induce apoptosis via a mechanism primarily involving the activation of the intrinsic mitochondrial pathway. The assay data provide strong evidence that TiO2 NPs can induce cytotoxicity, significant DNA damage, and apoptosis of A549 cells, suggesting that exposure to TiO2 NPs could cause cell injury and be hazardous to health.

  13. Cephalochromin induces G0/G1 cell cycle arrest and apoptosis in A549 human non-small-cell lung cancer cells by inflicting mitochondrial disruption.

    PubMed

    Hsiao, Che-Jen; Hsiao, George; Chen, Wei-Lin; Wang, Shih-Wei; Chiang, Chun-Ping; Liu, Li-Ya; Guh, Jih-Hwa; Lee, Tzong-Huei; Chung, Chi-Li

    2014-04-25

    The fungus-derived compound cephalochromin, isolated from the fermented broth of Cosmospora vilior YMJ89051501, shows growth-inhibitory and apoptotic activity against human lung cancer A549 cells in a concentration-dependent manner with an IC50 value of 2.8 μM at 48 h. Cephalochromin induced cell cycle arrest at the G0/G1 phase through down-regulation of cyclin D1, cyclin E, Cdk 2, and Cdk 4 expressions. Cephalochromin markedly increased the hypodiploid sub-G1 phase (apoptosis) of the cell cycle at 48 h as measured by flow cytometric analysis. Reactive oxygen species generation and loss of the mitochondrial membrane potential (MMP) were also markedly induced by cephalochromin. Moreover, the immunoblotting assays showed that cephalochromin reduced survivin and Bcl-xL expression and induced the activation of caspase-8, -9, and -3 and the cleavage of poly(ADP-ribose) polymerase, indicating the involvement of a caspase signaling cascade. The caspase inhibitor Z-VAD-fmk significantly suppressed cephalochromin-induced apoptosis. Cephalochromin also triggered LC3 II, autophagic marker, expression. Taken together, this is the first report that cephalochromin induced an antiproliferative effect on human lung cancer cells through mitochondrial disruption and down-regulation of survivin, leading to cell cycle arrest at the G0/G1 phase, loss of MMP, and subsequently apoptotic cell death.

  14. IRE1α-TRAF2-ASK1 pathway is involved in CSTMP-induced apoptosis and ER stress in human non-small cell lung cancer A549 cells.

    PubMed

    Zhang, Jiexia; Liang, Ying; Lin, Yongbin; Liu, Yuanbin; YouYou; Yin, Weiqiang

    2016-08-01

    CSTMP, a Tetramethylpyrazine (TMP) analogue, is designed and synthesized based on the pharmacophores of TMP and resveratrol. Recent studies showed that CSTMP had strong protective effects in endothelial cells apoptosis by its anti-oxidant activity. However, the pharmacological function of CSTMP in cancer have not been elucidated to date. The objective of this study was to investigate the anti-cancer effect of CSTMP against human non-small cell lung cancer (NSCLC) A549 cells and the underlying mechanisms. The cell proliferation and apoptosis were detected by MTT assay and flow cytometry. Caspases activity was determined spectrophotometricaly at 405nm using a microtiter plate reader. Western blot and real-time PCR was used to assess the protein and mRNA expression. Immunoprecipitation was used to examine the protein-protein interactions. CSTMP inhibited the proliferation and induced cell cycle arrest and apoptosis of A549 cells. Caspase3, 8, 9 and PARP-1 activation, and Bax/Bcl-2 ratio analyses demonstrated that the anti-cancer effect of CSTMP in A549 cells was mediated by promoting caspase- and mitochondria-dependent apoptosis. Furthermore, CSTMP induced ER stress in A549 cells as evidenced by elevated levels of GRP78, GRP94, CHOP, IRE1α, TRAF2, p-ASK1 and p-JNK, activation of caspase12 and 4, and enhanced formation of an IRE1α-TRAF2-ASK1 complex. Knockdown of IRE1α by siRNA suppressed activation of IRE1α, TRAF2, p-ASK1 and p-JNK in CSTMP treated A549 cells. In addition, the effects of CSTMP on the formation of an IRE1α-TRAF2-ASK1 complex, caspase- and mitochondria-dependent apoptosis were also reversed by IRE1α siRNA in A549 cells. Collectively, we showed that CSTMP induced apoptosis of A549 cells were through IRE1α-TRAF2-ASK1 complex-mediated ER stress, JNK activation, and mitochondrial dysfunction. These insights on this novel compound CSTMP may provide a novel anti-cancer candidate for the treatment of NSCLC. Copyright © 2016 Elsevier Masson SAS. All

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

    PubMed

    Arafat, Kholoud; Iratni, Rabah; Takahashi, Takashi; Parekh, Khatija; Al Dhaheri, Yusra; Adrian, Thomas E; Attoub, Samir

    2013-01-01

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

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

    SciTech Connect

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

    2013-01-11

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

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

    PubMed

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

    2013-01-11

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

  18. Selective cytotoxicity and combined effects of camptothecin or paclitaxel with sodium-R-alpha lipoate on A549 human non-small cell lung cancer cells.

    PubMed

    Ibrahim, Sherif; Gao, Dayuan; Sinko, Patrick J

    2014-01-01

    Nonsmall cell lung cancer (NSCLC) is the most common type of lung cancer and remains the deadliest form of cancer in the United States and worldwide. New therapies are highly sought after to improve outcome. The effect of sodium-R-alpha lipoate on camptothecin- and paclitaxel-induced cytotoxicity was evaluated on A549 NSCLC and BEAS-2B "normal" lung epithelial cells. Combination indices (CI) and dose reduction indices (DRI) were investigated by studying the cytotoxicity of sodium-R-alpha lipoate (0-16 mM), camptothecin (0-25 nM) and paclitaxel (0-0.06 nM) alone and in combination. 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium-bromide (MTT) was used to assess cytotoxicity. The combinational cytotoxic effects of sodium-R-alpha lipoate with camptothecin or paclitaxel were analyzed using a simulation of dose effects (CompuSyn® 3.01). The effects of sodium-R-alpha lipoate on camptothecin- and paclitaxel-induced cytotoxicity varied based on concentrations and treatment times. It was found that sodium-R-alpha lipoate wasn't cytotoxic toward BEAS-2B cells at any of the concentrations tested. For A549 cells, CIs [(additive (CI = 1); synergistic (CI < 1); antagonistic (CI < 1)] were lower and DRIs were higher for the camptothecin/sodium-R-alpha-lipoate combination (CI = ∼0.17-1.5; DRI = ∼2.2-22.6) than the paclitaxel/sodium-R-alpha-lipoate combination (CI = ∼0.8-9.9; DRI = ∼0.10-5.8) suggesting that the camptothecin regimen was synergistic and that the addition of sodium-R-alpha lipoate was important for reducing the camptothecin dose and potential for adverse effects.

  19. Xanthohumol induces apoptosis and S phase cell cycle arrest in A549 non-small cell lung cancer cells

    PubMed Central

    Yong, Wai Kuan; Ho, Yen Fong; Malek, Sri Nurestri Abd

    2015-01-01

    Background: Xanthohumol, a major prenylated chalcone found in female hop plant, Humulus lupulus, was reported to have various chemopreventive and anti-cancer properties. However, its apoptotic effect on human alveolar adenocarcinoma cell line (A549) of non-small cell lung cancer (NSCLC) was unknown. Objective: This study aimed to investigate the effects of xanthohumol on apoptosis in A549 human NSCLC cells. Materials and Methods: A549 cell proliferation was determined by sulforhodamine B assay. Morphological changes of the cells were studied via phase contrast and fluorescent microscopy. Induction of apoptosis was assessed by Annexin-V fluorescein isothiocyanate/propidium iodide (Annexin V-FITC/PI) staining, DNA fragmentation (TUNEL) assay mitochondrial membrane potential assay, cell cycle analysis, and caspase activity studies. Results: Xanthohumol was found to decrease cell proliferation in A549 cells but had relatively low cytotoxicity on normal human lung fibroblast cell line (MRC-5). Typical cellular and nuclear apoptotic features were also observed in A549 cells treated with xanthohumol. Onset of apoptosis in A549 cells was further confirmed by externalization of phosphatidylserine, changes in mitochondrial membrane potential, and DNA fragmentation in the cells after treatment. Xanthohumol induced accumulation of cells in sub G1 and S phase based on cell cycle analysis and also increased the activities of caspase-3, -8, and -9. Conclusion: This work suggests that xanthohumol as an apoptosis inducer, may be a potent therapeutic compound for NSCLC. PMID:26664015

  20. Proteomic response to 5,6-dimethylxanthenone 4-acetic acid (DMXAA, vadimezan) in human non-small cell lung cancer A549 cells determined by the stable-isotope labeling by amino acids in cell culture (SILAC) approach.

    PubMed

    Pan, Shu-Ting; Zhou, Zhi-Wei; He, Zhi-Xu; Zhang, Xueji; Yang, Tianxin; Yang, Yin-Xue; Wang, Dong; Qiu, Jia-Xuan; Zhou, Shu-Feng

    2015-01-01

    5,6-Dimethylxanthenone 4-acetic acid (DMXAA), also known as ASA404 and vadimezan, is a potent tumor blood vessel-disrupting agent and cytokine inducer used alone or in combination with other cytotoxic agents for the treatment of non-small cell lung cancer (NSCLC) and other cancers. However, the latest Phase III clinical trial has shown frustrating outcomes in the treatment of NSCLC, since the therapeutic targets and underlying mechanism for the anticancer effect of DMXAA are not yet fully understood. This study aimed to examine the proteomic response to DMXAA and unveil the global molecular targets and possible mechanisms for the anticancer effect of DMXAA in NSCLC A549 cells using a stable-isotope labeling by amino acids in cell culture (SILAC) approach. The proteomic data showed that treatment with DMXAA modulated the expression of 588 protein molecules in A549 cells, with 281 protein molecules being up regulated and 306 protein molecules being downregulated. Ingenuity pathway analysis (IPA) identified 256 signaling pathways and 184 cellular functional proteins that were regulated by DMXAA in A549 cells. These targeted molecules and signaling pathways were mostly involved in cell proliferation and survival, redox homeostasis, sugar, amino acid and nucleic acid metabolism, cell migration, and invasion and programed cell death. Subsequently, the effects of DMXAA on cell cycle distribution, apoptosis, autophagy, and reactive oxygen species (ROS) generation were experimentally verified. Flow cytometric analysis showed that DMXAA significantly induced G1 phase arrest in A549 cells. Western blotting assays demonstrated that DMXAA induced apoptosis via a mitochondria-dependent pathway and promoted autophagy, as indicated by the increased level of cytosolic cytochrome c, activation of caspase 3, and enhanced expression of beclin 1 and microtubule-associated protein 1A/1B-light chain 3 (LC3-II) in A549 cells. Moreover, DMXAA significantly promoted intracellular ROS

  1. Intracellular calcium promotes radioresistance of non-small cell lung cancer A549 cells through activating Akt signaling.

    PubMed

    Wang, Yiling; He, Jiantao; Zhang, Shenghui; Yang, Qingbo

    2017-03-01

    Radiotherapy is a major therapeutic approach in non-small cell lung cancer but is restricted by radioresistance. Although Akt signaling promotes radioresistance in non-small cell lung cancer, it is not well understood how Akt signaling is activated. Since intracellular calcium (Ca(2+)) could activate Akt in A549 cells, we investigated the relationship between intracellular calcium (Ca(2+)) and Akt signaling in radioresistant A549 cells by establishing radioresistant non-small cell lung cancer A549 cells. The radioresistant cell line A549 was generated by dose-gradient irradiation of the parental A549 cells. The cell viability, proliferation, and apoptosis were, respectively, assessed using the cell counting kit-8, EdU labeling, and flow cytometry analysis. The phosphorylation of Akt was evaluated by Western blotting, and the intracellular Ca(2+) concentration was assessed by Fluo 4-AM. The radioresistant A549 cells displayed mesenchymal morphology. After additional irradiation, the radioresistant A549 cells showed decreased cell viability and proliferation but increased apoptosis. Moreover, the intracellular Ca(2+) concentration and the phosphorylation level on the Akt473 site in radioresistant A549 cells were higher than those in original cells, whereas the percentage of apoptosis in radioresistant A549 cells was less. All these results could be reversed by verapamil. In conclusion, our study found that intracellular Ca(2+) could promote radioresistance of non-small cell lung cancer cells through phosphorylating of Akt on the 473 site, which contributes to a better understanding on the non-small cell lung cancer radioresistance, and may provide a new target for radioresistance management.

  2. Anti-Proliferative and Apoptosis-Inducing Effect of Theabrownin against Non-small Cell Lung Adenocarcinoma A549 Cells

    PubMed Central

    Wu, Feifei; Zhou, Li; Jin, Wangdong; Yang, Weiji; Wang, Ying; Yan, Bo; Du, Wenlin; Zhang, Qiang; Zhang, Lei; Guo, Yonghua; Zhang, Jin; Shan, Letian; Efferth, Thomas

    2016-01-01

    With the highest cancer incidence rate, lung cancer, especially non-small cell lung cancer (NSCLC), is the leading cause of cancer death in the world. Tea (leaves of Camellia sinensis) has been widely used as a traditional beverage beneficial to human health, including anti-NSCLC activity. Theabrownin (TB) is one major kind of tea pigment responsible for the beneficial effects of tea liquor. However, its effect on NSCLC is unknown. The aim of the present study was to evaluate anti-proliferative and apoptosis-inducing effect of TB on NSCLC (A549) cells, using MTT assay, morphological observation (DAPI staining), in situ terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and annexin-V/PI flow cytometry. Subsequently, the expression of several genes associated with cell proliferation and apoptosis were detected by real time PCR assay to explore its potential underlying mechanism. TB was revealed to inhibit cell proliferation of A549 cells in a concentration-dependent and time-dependent manner. Morphological observation, TUNEL assay and flow cytometric analysis evidenced an apoptosis-inducing effect of TB on A549 cells in a concentration-dependent manner. The real time PCR assay demonstrated that TB down-regulated the expression of TOPO I, TOPO II, and BCL-2, and up-regulated the expression of E2F1, P53, GADD45, BAX, BIM, and CASP 3,7,8,9, which suggests an activation of P53-mediated apoptotic (caspase-dependent) pathway in response to TB treatment. The western blot analysis showed a similar trend for the corresponding protein expression (P53, Bax, Bcl-2, caspase 3,9, and PARP) and further revealed DNA damage as a trigger of the apoptosis (phosphorylation of histone H2A.X). Accordingly, TB can be speculated as a DNA damage inducer and topoisomerase (Topo I and Topo II) inhibitor that can up-regulate P53 expression and subsequently modulate the expression of the downstream genes to induce cell proliferation inhibition and apoptosis of A549 cells

  3. Knockdown of Aurora-B inhibits the growth of non-small cell lung cancer A549 cells.

    PubMed

    Yu, Jing Jing; Zhou, Long Dian; Zhao, Tian Tian; Bai, Wei; Zhou, Jing; Zhang, Wei

    2015-09-01

    Elevated expression of Aurora-B affects cell apoptosis and proliferation in a variety of solid tumors. However, the role of Aurora-B has been poorly evaluated in non-small cell lung cancer (NSCLC). In the present study, it was found that Aurora-B was overexpressed in tissue specimens obtained from 174 patients with lung cancer. It was also demonstrated that knockdown of Aurora-B induces apoptosis and inhibits the growth of lung cancer A549 cells in vitro and in vivo. Furthermore, it was found that silencing Aurora-B decreased the activity of the phosphoinositide 3-kinase (PI3K)/AKT pathway. Therefore, it was concluded that knockdown of Aurora-B induces apoptosis and inhibits growth in NSCLC A549 cells, in addition to inhibiting the activity of the PI3K/AKT signaling pathway. Targeting Aurora-B may provide a novel target for lung cancer therapy.

  4. Venom present in sea anemone (Heteractis magnifica) induces apoptosis in non-small-cell lung cancer A549 cells through activation of mitochondria-mediated pathway.

    PubMed

    Ramezanpour, Mahnaz; da Silva, Karen Burke; Sanderson, Barbara J S

    2014-03-01

    Lung cancer is a major cause of cancer deaths throughout the world and the complexity of apoptosis resistance in lung cancer is apparent. Venom from Heteractis magnifica caused dose-dependent decreases in survival of the human non-small-cell lung cancer cell line, as determined by the MTT and Crystal Violet assays. The H. magnifica venom induced cell cycle arrest and induced apoptosis of A549 cells, as confirmed by annexin V/propidium iodide staining. The venom-induced apoptosis in A549 cells was characterized by cleavage of caspase-3 and a reduction in the mitochondrial membrane potential. Interestingly, crude extracts from H. magnifica had less effect on the survival of non-cancer cell lines. In the non-cancer cells, the mechanism via which cell death occurred was through necrosis not apoptosis. These findings are important for future work using H. magnifica venom for pharmaceutical development to treat human lung cancer.

  5. Tephrosin-induced autophagic cell death in A549 non-small cell lung cancer cells.

    PubMed

    Li, Jing; Wang, Xiao-Lu; Fang, Yu-Chun; Wang, Chang-Yun

    2010-11-01

    Anticancer effect of tephrosin (1) has been documented; however, the molecular mechanisms underlying the cytotoxicity of tephrosin in cancer cells remain unclear. In the present paper, the proliferation inhibition rate of several cancer cells was tested using the MTT assay; cell cycle, reactive oxygen species (ROS), and mitochondrial membrane potential (MMP) were determined by flow cytometry; poly(ADP-ribose) polymerase (PARP) cleavage and heat shock protein 90 (Hsp90) expression were evaluated by Western blotting; autophagy was examined by confocal microscopy and light chain 3 (LC3) conversion assay. The results showed that exposure of the cells to tephrosin induced significant proliferation inhibition in a dose-dependent manner, especially on A549 with G(2)/M being arrested. Tephrosin was not found to induce cell apoptosis as PARP cleavage was not detected after 24 h treatment, but the formation of acidic vesicular organelle of autophagy character was found, and autophagy was further confirmed by the increase in the ratio of LC3-II to LC3-I. It was observed that tephrosin induced ROS generation and Hsp90 expression inhibition. These results indicate that tephrosin induces A549 cancer cell death via the autophagy pathway, and the roles of ROS generation and Hsp90 expression inhibition in this process need further study in the future.

  6. Lysyl oxidase mediates hypoxia-induced radioresistance in non-small cell lung cancer A549 cells

    PubMed Central

    Gong, Chongwen; Gu, Runxia; Jin, Honglin; Sun, Yao; Li, Zhenyu; Wu, Gang

    2016-01-01

    Hypoxia-induced radioresistance has been well known as the main obstacle in cancer radiotherapy. Lysyl oxidase (LOX) was previously demonstrated to play an important role in hypoxia-induced biological behaviors, such as metastasis and angiogenesis, through hypoxia-inducible factor-1α (HIF-1α), which is an important contributing factor to radioresistance in tumor cells. However, how LOX plays a role in hypoxia-induced radioresistance has yet to be determined. Here, we found that LOX expression was in accordance with HIF-1α expression, and LOX expression at the mRNA and protein level, and enzymatic activity were remarkably upregulated in the hypoxic A549 cells, compared with normoxic A549 cells. Inhibition of LOX resulted in the reduction of the ability to repair double-stranded breaks (DSBs), promotion of apoptosis, relief of G2/M cycle arrest, and eventually reduction of hypoxia-induced radioresistance in the hypoxic A549 cells. This suggests that LOX may play an important role in hypoxia-induced radioresistance. Together, our results might suggest a novel potential therapeutic target in the management of non-small cell lung cancer (NSCLC). PMID:26515140

  7. APE1 modulates cellular responses to organophosphate pesticide-induced oxidative damage in non-small cell lung carcinoma A549 cells.

    PubMed

    Thakur, Shweta; Dhiman, Monisha; Mantha, Anil K

    2017-09-08

    Monocrotophos (MCP) and chlorpyrifos (CP) are widely used organophosphate pesticides (OPPs), speculated to be linked with human pathologies including cancer. Owing to the fact that lung cells are most vulnerable to the environmental toxins, the development and progression of lung cancer can be caused by the exposure of OPPs. The present study investigates the oxidative DNA damage response evoked by MCP and CP in human non-small cell lung carcinoma A549 cells. A549 cells were exposed to MCP and CP; cytotoxicity and reactive oxygen species (ROS) generation were measured to select the non-toxic dose. In order to establish whether MCP and CP can initiate the DNA repair and cell survival signalling pathways in A549 cells, qRT-PCR and Western blotting techniques were used to investigate the mRNA and protein expression levels of DNA base excision repair (BER)-pathway enzymes and transcription factors (TFs) involved in cell survival mechanisms. A significant increase in cell viability and ROS generation was observed when exposed to low and moderate doses of MCP and CP at different time points (24, 48 and 72 h) studied. A549 cells displayed a dose-dependent accumulation of apurinic/apyrimidinic (AP) sites after 24 h exposure to MCP advocating for the activation of AP endonuclease-mediated DNA BER-pathway. Cellular responses to MCP- and CP-induced oxidative stress resulted in an imbalance in the mRNA and protein expression of BER-pathway enzymes, viz. PARP1, OGG1, APE1, XRCC1, DNA pol β and DNA ligase III α at different time points. The treatment of OPPs resulted in the upregulation of TFs, viz. Nrf2, c-jun, phospho-c-jun and inducible nitric oxide synthase. Immunofluorescent confocal imaging of A549 cells indicated that MCP and CP induces the translocation of APE1 within the cytoplasm at an early 6 h time point, whereas it promotes nuclear localization after 24 h of treatment, which suggests that APE1 subcellular distribution is dynamically regulated in response to

  8. Identification of curcumin-inhibited extracellular matrix receptors in non-small cell lung cancer A549 cells by RNA sequencing.

    PubMed

    Li, Huiping; Wu, Hongjin; Zhang, Hongfang; Li, Ying; Li, Shuang; Hou, Qiang; Wu, Shixiu; Yang, Shuan-Ying

    2017-06-01

    Curcumin is a potent anti-cancer drug in several types of human cancers. Despite of several preclinical and clinical studies of curcumin, the precise mechanism of curcumin in cancer prevention has remained unclear. In our study, we for the first time investigated whole transcriptome alteration in A549 non-small cell lung cancer (NSCLC) cell lines after treatment with curcumin using RNA sequencing. We found that lots of genes and signaling pathways were significantly altered after curcumin treatment in A549 cells. With bioinformatics approaches (gene ontology, Kyoto Encyclopedia of Genes and Genomes, and STRING), we found that those curcumin altered genes were not only the genes that induce cell death but also those extracellular matrix receptors and mitogen-activated protein kinase signaling pathway genes which regulate cell migration and proliferation. Among those significantly altered genes, eight genes ( COL1A1, COL4A1, COL5A1, LAMA5, ITGA3, ITGA2B, DDIT3, and DUSP1) were further examined by quantitative reverse transcription polymerase chain reaction and western blot analysis in four non-small cell lung cancer cell lines. Both in cell lines and in mouse model, the extracellular matrix receptors including the integrin ( ITGA3 and ITGA2B), collagen ( COL5A1), and laminin ( LAMA5) were significantly inhibited by curcumin at messenger RNA and protein levels. Functional studies confirmed that curcumin not only induced A549 cell death but also repressed cell proliferation and migration by regulating extracellular matrix receptors. Collectively, our study suggests that curcumin may be used as a promising drug candidate for intervening lung cancer in future studies.

  9. miR-107 regulates cisplatin chemosensitivity of A549 non small cell lung cancer cell line by targeting cyclin dependent kinase 8

    PubMed Central

    Zhang, Zhe; Zhang, Lu; Yin, Zhi-Yi; Fan, Xing-Long; Hu, Bo; Wang, Lun-Qing; Zhang, Di

    2014-01-01

    Previous studies demonstrated that the acquired drug resistance of non-small cell lung cancer (NSCLC) was related to deregulation of miRNAs. However, the effects of miR-107 and the mechanism through which miR-107 affects the cisplatin chemoresistance in NSCLC have not been reported. TaqMan RT-PCR or Western blot assay was performed to detect the expression of mature miR-107 and cyclin dependent kinase 8 (CDK8) protein. The viabilities of treated cells were analyzed using MTT assay. We found that the expression level of miR-107 in A549 cells was significantly lower than that in normal human bronchial epithelial cells (0.45 ± 0.26 vs. 1.00 ± 0.29, P = 0.032). The MTT assay showed that the A549 cells transfected with miR-107 mimics were significantly more sensitive to the therapy of cisplatin than control cells. A549 cells transfected with miR-107 mimics showed a decreased CDK8 protein expression. Downregulation of CDK8 expression by siRNAs, A549 cells became more sensitive to the therapy of cisplatin. In addition, the enhanced growth-inhibitory effect by the miR-107 mimic transfection was enhanced after the addition of CDK8 siRNA. In conclusion, the present study provides the first evidence that miR-107 plays a key role in cisplatin resistance by targeting the CDK8 protein in NSCLC cell lines, suggesting that miR-107 can be used to predict a patient’s response to chemotherapy as well as serve as a novel potential maker for NSCLC therapy. PMID:25400821

  10. Novel synthetic chalcones induce apoptosis in the A549 non-small cell lung cancer cells harboring a KRAS mutation.

    PubMed

    Wang, Yiqiang; Hedblom, Andreas; Koerner, Steffi K; Li, Mailin; Jernigan, Finith E; Wegiel, Barbara; Sun, Lijun

    2016-12-01

    A series of novel chalcones were synthesized by the Claisen-Schmidt condensation reaction of tetralones and 5-/6-indolecarboxaldehydes. Treatment of human lung cancer cell line harboring KRAS mutation (A549) with the chalcones induced dose-dependent apoptosis. Cell cycle analyses and Western blotting suggested the critical role of the chalcones in interrupting G2/M transition of cell cycle. SAR study demonstrated that substituent on the indole N atom significantly affects the anticancer activity of the chalcones, with methyl and ethyl providing the more active compounds (EC50: 110-200nM), Compound 1g was found to be >4-fold more active in the A549 cells (EC50: 110nM) than in prostate (PC3) or pancreatic cancer (CLR2119, PAN02) cells. Furthermore, compound 1l selectively induced apoptosis of lung cancer cells A549 (EC50: 0.55μM) but did not show measurable toxicity in the normal lung bronchial epithelial cells (hBEC) at doses as high as 10μM, indicating specificity towards cancer cells. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. Rhizoma Paridis Saponins Induces Cell Cycle Arrest and Apoptosis in Non-Small Cell Lung Carcinoma A549 Cells

    PubMed Central

    Zhang, Jue; Yang, Yixi; Lei, Lei; Tian, Mengliang

    2015-01-01

    Background As a traditional Chinese medicine herb, Chonglou (Paris polyphylla var. chinensis) has been used as anticancer medicine in China in recent decades, as it can induce cell cycle arrest and apoptosis in numerous cancer cells. The saponins extract from the rhizoma of Chonglou [Rhizoma Paridis saponins (RPS)] is known as the main active component for anticancer treatment. However, the molecular mechanism of the anticancer effect of RPS is unknown. Material/Methods The present study evaluated the effect of RPS in non-small-cell lung cancer (NSCLC) A549 cells using the 3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide (MTT) assay and flow cytometry. Subsequently, the expression of several genes associated with cell cycle and apoptosis were detected by reverse transcription-quantitative polymerase chain reaction (qRT-PCR) and Western blotting. Results RPS was revealed to inhibit cell growth, causing a number of cells to accumulate in the G 1 phase of the cell cycle, leading to apoptosis. In addition, the effect was dose-dependent. Moreover, the results of qRT-PCR and Western blotting showed that p53 and cyclin-dependent kinase 2 (CDK2) were significantly downregulated, and that BCL2, BAX, and p21 were upregulated, by RPS treatment. Conclusions We speculated that the RPS could act on a pathway, including p53, p21, BCL2, BAX, and CDK2, and results in G1 cell cycle arrest and apoptosis in NSCLC cells. PMID:26311066

  12. microRNA-99a is downregulated and promotes proliferation, migration and invasion in non-small cell lung cancer A549 and H1299 cells.

    PubMed

    Chen, Changjin; Zhao, Ziyi; Liu, Yu; Mu, Dezhi

    2015-03-01

    There is increasing evidence that microRNAs (miRNAs) are able to play a key role in the diagnosis and therapy of cancer. miRNA-99a (miR-99a), which is downregulated in several human malignancies, has been reported as a potential tumor suppressor. However, to the best of our knowledge, the expression and function of miR-99a has not been investigated in human non-small cell lung cancer (NSCLC) at present. The aim of the current study was to evaluate the association between NSCLC and miR-99a. miR-99a expression was analyzed in 15 pairs of NSCLC and non-cancerous tissue samples by reverse transcription-quantitative polymerase chain reaction. In addition, the NSCLC A549 and H1299 cell lines were transfected with miR-99a mimics, and the effect of miR-99a on the cell cycle, cell proliferation, migration and colony formation of A549 and H1299 cells was investigated. It was found that the level of miR-99a expression was significantly downregulated in NSCLC tissues and that ectopic overexpression of miR-99a significantly inhibited the growth of A549 and H1299 cells. Additionally, ectopic overexpression of miR-99a inhibited A549 and H1299 cell migration and invasion by inhibiting epithelial to mesenchymal transition. The downregulation of insulin-like growth factor 1 receptor (IGF-1R) by miR-99a and knockdown of IGF-1R mediated by siRNA were each found to phenocopy the effect of miR-99a overexpression in NSCLC. To the best of our knowledge, the present study demonstrated for the first time that, in NSCLC, miR-99a is downregulated and thus regulates proliferation, colony formation and migration through the IGF-1R pathway, which indicates that miR-99a is a diagnostic biomarker for NSCLC.

  13. Correlation study of biological characteristics of non-small cell lung cancer A549 cells after transfecting plasmid by microbubble ultrasound contrast agent.

    PubMed

    Guo, Xuan-Yan; Lu, Man; Chen, Xue-Qing; He, Fan-Ding; Li, Ang

    2016-06-01

    To explore the role of the abnormal expression of miRNAs in the development process of non-small cell lung cancer and the feasibility of ultrasound microbubble-mediated gene therapy after transfecting antisense miRNA-224 and miRNA-122a plasmids into non-small cell lung cancer A549 cells. Antisense miRNA-224 and miRNA-122a plasmids were transfected into non-small cell lung cancer A549 cells on the optimal ultrasound microbubble-mediated condition. We set up a control group. The cell proliferation activity, apoptosis, invasion ability were detected by MTT assay, Annexin V-PE, Transwell invasion experiment and colony formation assay, respectively. The expression of miRNA-224 decreased and the expression of miRNA-122a rose after the plasmids of target genes were transfected into non-small cell lung cancer A549 cells, and there were significant differences when compared with those of the control group (P < 0.05). After the plasmids of target genes were transfected into A549 cells, the growth of antisense miRNA-224 and miRNA-122a were inhibited, and the differences were significant as compared with the control group (P < 0.05). Besides, the inhibition of miRNA-122a group was the most significant and there was statistically significant difference as compared with miRNA-224 group (t = -4.694, P = 0.009). After the plasmids of target genes were transfected into A549 cells, the proportion of apoptotic cells increased, the invasive cells were decreased and the clone ability reduced, and also there was a significant difference as compared with those of the control group (P < 0.05). What's more, the apoptotic peak appeared in miRNA-122a group. Its invasion ability decreased most obviously (40.25 ± 3.97/visual field), the number of clone ability was 104.93 ± 4.87 and the inhibitory effect was the most obviously. There was statistically significant difference as compared with other groups (P < 0.05). A549 cells transfected by ultrasound microbubble

  14. Investigation of Radiation-induced Transcriptome Profile of Radioresistant Non-small Cell Lung Cancer A549 Cells Using RNA-seq

    PubMed Central

    Yang, Hee Jung; Kim, Namshin; Seong, Ki Moon; Youn, HyeSook; Youn, BuHyun

    2013-01-01

    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. PMID:23533613

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

    PubMed

    Li, Hongmin; Lan, Haitao; Zhang, Ming; An, Ning; Yu, Ruilian; He, Yangke; Gan, Chongzhi

    2016-09-20

    背景与目的 已有的研究表明miR-424可抑制肾癌细胞增殖,抑制宫颈鳞癌细胞的迁移和侵袭能力,而其对非小细胞肺癌(non-small cell lung cancer, NSCLC)细胞的影响目前尚无系统研究。本研究探讨miR-424对NSCLC A549细胞生长和侵袭迁移能力的影响并进一步研讨其分子机制。方法 应用CCK8检测过表达及抑制miR-424的表达对A549细胞增殖的影响。应用Transwell检测过表达及抑制miR-424的表达对A549细胞侵袭能力的影响。应用Western blot检测过表达及抑制miR-424的表达对A549细胞中MMP9和MMP2蛋白水平的影响。构建E2F6 3’UTR区的荧光素酶报告载体,验证miR-424对E2F6的靶向作用。采用Western blot检测过表达及抑制miR-424的表达后,A549细胞中E2F6的表达。结果 过表达miR-424后,A549的生长和侵袭能力显著降低。过表达miR-424后,A549细胞的MMP-2和MMP-9表达下降。荧光素酶活性检测表明miR-424能够抑制E2F6的荧光素酶活性。过表达miR-424后,细胞内E2F6的表达降低。结论 miR-424能够通过调控E2F6而抑制A549的生长和侵袭能力。.

  16. Potent organometallic osmium compounds induce mitochondria-mediated apoptosis and S-phase cell cycle arrest in A549 non-small cell lung cancer cells.

    PubMed

    van Rijt, Sabine H; Romero-Canelón, Isolda; Fu, Ying; Shnyder, Steve D; Sadler, Peter J

    2014-05-01

    The problems of acquired resistance associated with platinum drugs may be addressed by chemotherapeutics based on other transition metals as they offer the possibility of novel mechanisms of action. In this study, the cellular uptake and induction of apoptosis in A549 human non-small cell lung cancer cells of three promising osmium(II) arene complexes containing azopyridine ligands, [Os(η(6)-arene)(p-R-phenylazopyridine)X]PF6, where arene is p-cymene or biphenyl, R is OH or NMe2, and X is Cl or I, were investigated. These complexes showed time-dependent (4–48 h) potent anticancer activity with highest potency after 24 h (IC50 values ranging from 0.1 to 3.6 μM). Cellular uptake of the three compounds as quantified by ICP-MS, was independent of their logP values (hydrophobicity). Furthermore, maximum cell uptake was observed after 24 h, with evident cell efflux of the osmium after 48 and 72 h of exposure, which correlated with the corresponding IC50 values. The most active compound 2, [Os(η(6)-p-cymene)(NMe2-phenylazopyridine)I]PF6, was taken up by lung cancer cells predominately in a temperature-dependent manner indicating that energy-dependent mechanisms are important in the uptake of 2. Cell fractionation studies showed that all three compounds accumulated mainly in cellular membranes. Furthermore, compound 2 induced apoptosis and caused accumulation in the S-phase of the cell cycle. In addition, 2 induced cytochrome c release and alterations in mitochondrial membrane potential even after short exposure times, indicating that mitochondrial apoptotic pathways are involved. This study represents the first steps towards understanding the mode of action of this promising class of new osmium-based chemotherapeutics.

  17. Antiproliferative and antimetastatic action of quercetin on A549 non-small cell lung cancer cells through its effect on the cytoskeleton.

    PubMed

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

    2017-03-01

    To our knowledge, this study is the first to investigate the effect of the dietary flavonoid quercetin on the main cytoskeletal elements, namely microfilaments, microtubules and vimentin intermediate filaments, as well as cytoskeleton-driven processes in A549 non-small cell lung cancer cells. The methyl-thiazol-diphenyl-tetrazolium assay, annexin V/propidium iodide test, electron microscopic examination, cell cycle analysis based on DNA content, real-time PCR assays, in vitro scratch wound-healing assay, fluorescence staining of F-actin, β-tubulin and vimentin were performed to assess the effects of quercetin on A549 cells. Our results showed that quercetin triggered BCL2/BAX-mediated apoptosis, as well as necrosis and mitotic catastrophe, and inhibited the migratory potential of A549 cells. The disassembling effect of quercetin on microfilaments, microtubules and vimentin filaments along with its inhibitory impact on vimentin and N-cadherin expression might account for the decreased migration of A549 cells in response to quercetin treatment. We also suggest that the possible mechanism underlying quercetin-induced mitotic catastrophe involves the perturbation of mitotic microtubules leading to monopolar spindle formation, and, consequently, to the failure of cytokinesis. We further propose that cytokinesis failure could also be a result of the depletion of actin filaments by quercetin. These findings are important to our further understanding of the detailed mechanism of the antitumor activity of quercetin and render this flavonoid a potentially useful candidate for combination therapy with conventional antimicrotubule drugs, nucleic acid-directed agents or novel cytoskeletal-directed agents. Copyright © 2016 Elsevier GmbH. All rights reserved.

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

    PubMed

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

    2016-01-01

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

  19. Sinomenine inhibits A549 human lung cancer cell invasion by mediating the STAT3 signaling pathway

    PubMed Central

    Jiang, Shulong; Gao, Yebo; Hou, Wei; Liu, Rui; Qi, Xin; Xu, Xia; Li, Jie; Bao, Yanju; Zheng, Honggang; Hua, Baojin

    2016-01-01

    Increasing evidence suggests that the failure of lung cancer treatment may occur as a result of tumor invasion and metastasis. Signal transducer and activator of transcription 3 (STAT3), an epithelial-mesenchymal transition-inducing transcription factor, is a key signaling molecule involved in the proliferation, apoptosis, invasion and metastasis of tumor cells. Sinomenine is an alkaloid compound with an antineoplastic potential against a variety of cancer cells. The aim of the present study was to assess the antitumor mechanisms of sinomenine in the A549 human lung cancer cell line. The results demonstrated that sinomenine manifested dose-dependent cytotoxicity and induced apoptosis in A549 cells. The protein expression of Janus kinase 2, STAT3, phosphorylated-STAT3, Snail, N-cadherin and vimentin decreased in sinomenine-treated cells, while E-cadherin protein expression increased. The regulation of STAT3, N-cadherin and E-cadherin by sinomenine was further confirmed by reverse transcription-quantitative polymerase chain reaction and immunofluorescent staining. It was demonstrated that sinomenine exerts inhibitory effects on A549 human lung cancer cell invasion, possibly through the inhibition of STAT3 signaling. These results provide a novel insight into the role of sinomenine in the treatment of non-small cell lung cancer. PMID:27446441

  20. Non-small-cell lung cancer cell lines A549 and NCI-H460 express hypoxanthine guanine phosphoribosyltransferase on the plasma membrane

    PubMed Central

    Townsend, Michelle H; Anderson, Michael D; Weagel, Evita G; Velazquez, Edwin J; Weber, K Scott; Robison, Richard A; O’Neill, Kim L

    2017-01-01

    In both males and females, lung cancer is one of the most lethal cancers worldwide and accounts for >30% of cancer-related deaths. Despite advances in biomarker analysis and tumor characterization, there remains a need to find suitable biomarker antigen targets for treatment in late-stage lung cancer. Previous research on the salvage pathway enzyme TK1 shows a unique relationship with cancer patients as serum levels are raised according to cancer grade. To expand this analysis, the other salvage pathway enzymes were evaluated for possible upregulation within lung cancer. Adenine phosphoribosyltransferase, deoxycytidine kinase, and hypoxanthine guanine phosphoribosyltransferase (HPRT) were assessed for their presentation on two non-small-cell lung cancer cell lines NCI-H460 and A549. In the present study, we show that deoxycytidine kinase and adenine phosphoribosyltransferase have no significant relationship with the membrane of NCI-H460 cells. However, we found significant localization of HPRT to the membrane of NCI-H460 and A549 cells. When treated with anti-HPRT antibodies, the average fluorescence of the cell population increased by 24.3% and 12.9% in NCI-H460 and A549 cells, respectively, in comparison with controls. To ensure that expression was not attributed to cytoplasmic HPRT, confocal microscopy was performed to visualize HPRT binding on the plasma membrane. After staining NCI-H460 cells treated with both fluorescent antibodies and a membrane-specific dye, we observed direct overlap between HPRT and the membrane of the cancer cells. Additionally, gold-conjugated antibodies were used to label and quantify the amount of HPRT on the cell surface using scanning electron microscopy and energy-dispersive analysis X-ray. Further confirming HPRT presence, the gold weight percentage of the sample increased significantly when NCI-H460 cells were exposed to HPRT antibody (P=0.012) in comparison with isotype controls. Our results show that HPRT is localized on the

  1. Non-small-cell lung cancer cell lines A549 and NCI-H460 express hypoxanthine guanine phosphoribosyltransferase on the plasma membrane.

    PubMed

    Townsend, Michelle H; Anderson, Michael D; Weagel, Evita G; Velazquez, Edwin J; Weber, K Scott; Robison, Richard A; O'Neill, Kim L

    2017-01-01

    In both males and females, lung cancer is one of the most lethal cancers worldwide and accounts for >30% of cancer-related deaths. Despite advances in biomarker analysis and tumor characterization, there remains a need to find suitable biomarker antigen targets for treatment in late-stage lung cancer. Previous research on the salvage pathway enzyme TK1 shows a unique relationship with cancer patients as serum levels are raised according to cancer grade. To expand this analysis, the other salvage pathway enzymes were evaluated for possible upregulation within lung cancer. Adenine phosphoribosyltransferase, deoxycytidine kinase, and hypoxanthine guanine phosphoribosyltransferase (HPRT) were assessed for their presentation on two non-small-cell lung cancer cell lines NCI-H460 and A549. In the present study, we show that deoxycytidine kinase and adenine phosphoribosyltransferase have no significant relationship with the membrane of NCI-H460 cells. However, we found significant localization of HPRT to the membrane of NCI-H460 and A549 cells. When treated with anti-HPRT antibodies, the average fluorescence of the cell population increased by 24.3% and 12.9% in NCI-H460 and A549 cells, respectively, in comparison with controls. To ensure that expression was not attributed to cytoplasmic HPRT, confocal microscopy was performed to visualize HPRT binding on the plasma membrane. After staining NCI-H460 cells treated with both fluorescent antibodies and a membrane-specific dye, we observed direct overlap between HPRT and the membrane of the cancer cells. Additionally, gold-conjugated antibodies were used to label and quantify the amount of HPRT on the cell surface using scanning electron microscopy and energy-dispersive analysis X-ray. Further confirming HPRT presence, the gold weight percentage of the sample increased significantly when NCI-H460 cells were exposed to HPRT antibody (P=0.012) in comparison with isotype controls. Our results show that HPRT is localized on the

  2. Suberoylanilide Hydroxamic Acid Treatment Reveals Crosstalks among Proteome, Ubiquitylome and Acetylome in Non-Small Cell Lung Cancer A549 Cell Line

    PubMed Central

    Wu, Quan; Cheng, Zhongyi; Zhu, Jun; Xu, Weiqing; Peng, Xiaojun; Chen, Chuangbin; Li, Wenting; Wang, Fengsong; Cao, Lejie; Yi, Xingling; Wu, Zhiwei; Li, Jing; Fan, Pingsheng

    2015-01-01

    Suberoylanilide hydroxamic acid (SAHA) is a well-known histone deacetylase (HDAC) inhibitor and has been used as practical therapy for breast cancer and non-small cell lung cancer (NSCLC). It is previously demonstrated that SAHA treatment could extensively change the profile of acetylome and proteome in cancer cells. However, little is known about the impact of SAHA on other protein modifications and the crosstalks among different modifications and proteome, hindering the deep understanding of SAHA-mediated cancer therapy. In this work, by using SILAC technique, antibody-based affinity enrichment and high-resolution LC-MS/MS analysis, we investigated quantitative proteome, acetylome and ubiquitylome as well as crosstalks among the three datasets in A549 cells toward SAHA treatment. In total, 2968 proteins, 1099 acetylation sites and 1012 ubiquitination sites were quantified in response to SAHA treatment, respectively. With the aid of intensive bioinformatics, we revealed that the proteome and ubiquitylome were negatively related upon SAHA treatment. Moreover, the impact of SAHA on acetylome resulted in 258 up-regulated and 99 down-regulated acetylation sites at the threshold of 1.5 folds. Finally, we identified 55 common sites with both acetylation and ubiquitination, among which ubiquitination level in 43 sites (78.2%) was positive related to acetylation level. PMID:25825284

  3. Suberoylanilide hydroxamic acid treatment reveals crosstalks among proteome, ubiquitylome and acetylome in non-small cell lung cancer A549 cell line.

    PubMed

    Wu, Quan; Cheng, Zhongyi; Zhu, Jun; Xu, Weiqing; Peng, Xiaojun; Chen, Chuangbin; Li, Wenting; Wang, Fengsong; Cao, Lejie; Yi, Xingling; Wu, Zhiwei; Li, Jing; Fan, Pingsheng

    2015-03-31

    Suberoylanilide hydroxamic acid (SAHA) is a well-known histone deacetylase (HDAC) inhibitor and has been used as practical therapy for breast cancer and non-small cell lung cancer (NSCLC). It is previously demonstrated that SAHA treatment could extensively change the profile of acetylome and proteome in cancer cells. However, little is known about the impact of SAHA on other protein modifications and the crosstalks among different modifications and proteome, hindering the deep understanding of SAHA-mediated cancer therapy. In this work, by using SILAC technique, antibody-based affinity enrichment and high-resolution LC-MS/MS analysis, we investigated quantitative proteome, acetylome and ubiquitylome as well as crosstalks among the three datasets in A549 cells toward SAHA treatment. In total, 2968 proteins, 1099 acetylation sites and 1012 ubiquitination sites were quantified in response to SAHA treatment, respectively. With the aid of intensive bioinformatics, we revealed that the proteome and ubiquitylome were negatively related upon SAHA treatment. Moreover, the impact of SAHA on acetylome resulted in 258 up-regulated and 99 down-regulated acetylation sites at the threshold of 1.5 folds. Finally, we identified 55 common sites with both acetylation and ubiquitination, among which ubiquitination level in 43 sites (78.2%) was positive related to acetylation level.

  4. 5-allyl-7-gen-difluoromethoxychrysin enhances TRAIL-induced apoptosis in human lung carcinoma A549 cells

    PubMed Central

    2011-01-01

    Background 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. Methods 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. Results 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. Conclusions AFMC synergistically enhances TRAIL-mediated apoptosis in NSCLC cells through up-regulating DR5 expression. PMID:21801359

  5. Antitumor efficacy of the cytotoxic RNase, ranpirnase, on A549 human lung cancer xenografts of nude mice.

    PubMed

    Lee, Intae; Kalota, Anna; Gewirtz, Alan M; Shogen, Kuslima

    2007-01-01

    The cytotoxic RNase, ranpirnase (ONCONASE, ONC), may have promising therapeutic implication as an alternative for cisplatin for the treatment of lung cancer, due to inhibition of protein synthesis by t-RNA cleavage. A549 and NCI-H1975 human NSCLC cell lines were cultured in the presence and absence of ONC. Cytotoxicity was monitored using a clonogenic assay. Using an inverted phase and fluorescence microscope, we studied whether apoptosis was induced by ONC in gefitinib-induced apoptosis-resistant A549 tumor cells. The therapeutic effectiveness of ONC was studied via single and multiple administrations on A549 human non-small cell lung cancer (NSCLC), including tumors previously untreatable by cisplatin. ONC-induced changes in ATP levels were also monitored by non-localized phosphorus MR spectroscopy. ONC significantly inhibited the cell growth of A549 tumors. Apoptosis was significantly induced by ONC in a dose-dependent manner. In animal studies, multiple small doses of ONC were more effective than one large single dose for the inhibition of tumor growth with reduced side-effects, probably due to the normalization of leaky tumor vessels. ONC in combination with cisplatin significantly reduced tumor growth of A549 tumors. In large tumors, including those unsuccessfully treated with cisplatin, ONC showed inhibition of tumor growth, while a second treatment of cisplatin did not. During monitoring by non-localized phosphorus MR spectroscopy, ATP levels decreased, likely due to ONC-induced inhibition of oxygen consumption (QO2). ONC significantly inhibited tumor growth of A549 NSCLC cells in both in vitro and in vivo studies. This investigation suggests important potential clinical uses of ONC for the treatment of NSCLC cancer patients.

  6. Coptisine-induced cell cycle arrest at G2/M phase and reactive oxygen species-dependent mitochondria-mediated apoptosis in non-small-cell lung cancer A549 cells.

    PubMed

    Rao, Poorna Chandra; Begum, Sajeli; Sahai, Mahendra; Sriram, D Saketh

    2017-03-01

    This study aimed to explore the effect of coptisine on non-small-cell lung cancer and its mechanism through various in vitro cellular models (A549). Results claimed significant inhibition of proliferation by coptisine against A549, H460, and H2170 cells with IC50 values of 18.09, 29.50, and 21.60 µM, respectively. Also, coptisine exhibited upregulation of pH2AX, cell cycle arrest at G2/M phase, and downregulation of the expression of cyclin B1, cdc2, and cdc25C and upregulation of p21 dose dependently. Furthermore, induction of apoptosis in A549 cells by coptisine was characterized by the activation of caspase 9, caspase 8, and caspase 3, and cleavage of poly adenosine diphosphate ribose polymerase. In addition, coptisine was found to increase reactive oxygen species generation, upregulate Bax/Bcl-2 ratio, disrupt mitochondrial membrane potential, and cause cytochrome c release into the cytosol. Besides, treatment with a reactive oxygen species inhibitor (N-acetyl cysteine) abrogated coptisine-induced growth inhibition, apoptosis, reactive oxygen species generation, and mitochondrial dysfunction. Thus, the mediation of reactive oxygen species in the apoptosis-induced effect of coptisine in A549 cells was corroborated. These findings have offered new insights into the effect and mechanisms of action of coptisine against non-small-cell lung cancer.

  7. Cytotoxicity of withasteroids: withametelin induces cell cycle arrest at G2/M phase and mitochondria-mediated apoptosis in non-small cell lung cancer A549 cells.

    PubMed

    Rao, Poorna Chandra; Begum, Sajeli; Jahromi, Mohammad Ali Farboodniay; Jahromi, Zahra Hosseini; Sriram, Saketh; Sahai, Mahendra

    2016-09-01

    Considerable interest has been gained by withasteroids because of their structural uniqueness and wide spectrum of biological activities. However, limited systematic studies for proving their cytotoxic potential have so far been reported. Hence, an attempt was made to test the cytotoxicity of six withasteroids viz., withametelin (WM), withaphysalin D, withaphysalin E, 12-deoxywithastramonolide, Withaperuvin B, and physalolactone against A549, HT-29, and MDA-MB-231 cancer cell lines. Significant cytotoxic effect of WM against A549 cells (IC50 value of 6.0 μM), MDA-MB-231 cells (IC50 value of 7.6 μM), and HT-29 cells (IC50 value of 8.2 μM) was observed. Withaperuvin B and physalolactone were found to be effective against MDA-MB-231 cells. The significantly active WM arrested the A549 cells at G2/M phase and downregulated the expression of G2/M regulatory proteins such as cdc2, cyclin B1, and cdc25C. Apoptosis induced by WM in A549 cells was associated with the generation of ROS and depletion of MMP. Furthermore, WM treatment resulted in Bax upregulation, Bcl-2 downregulation, translocation of cytochrome c to mitochondria, activation of caspase-9 and -3, and PARP cleavage corroborating the apoptosis induction through intrinsic apoptotic pathway. Thus, WM possessing broader cytotoxic effect is a promising lead molecule which has the potential to be developed as a new therapeutic agent for NSCLC.

  8. Paracrine control of differentiation in the alveolar carcinoma, A549, by human foetal lung fibroblasts.

    PubMed

    Speirs, V; Ray, K P; Freshney, R I

    1991-10-01

    Synthesis of pulmonary surfactant (PS) is necessary for normal functioning of the lungs and its production is indicative of normal differentiated lung. The human alveolar carcinoma, A549, has been found to synthesis and secrete PS in vitro. The purpose of this study was to optimise the culture conditions for PS synthesis by A549 as well as to determine the potential role of foetal lung fibroblasts in the induction of PS by glucocorticoids. A549 cells growing in filter wells produced higher levels of PS in response to steroid, a 5-fold increase on the filter well compared to only a 1.5-fold increase when the cells were cultured on a conventional plastic substrate. A549 cells grown in filter wells responded to coculture with fibroblasts whether in direct contact or separated co-culture. A 20-fold increase in PS over control values was observed in separated steroid-treated co-cultures, suggesting the presence of a diffusible factor. A partially purified factor was isolated from fibroblast conditioned medium which was capable of inducing differentiation and other phenotypic changes in A549, namely induction of PS, reduction of plasminogen activator activity and reduction in the in vivo growth of A549 xenografts in nude mice. These results suggest that, under the correct conditions, A549 cells, although transformed, still retain the capacity to respond to differentiation-inducing signals from normal fibroblasts.

  9. Paracrine control of differentiation in the alveolar carcinoma, A549, by human foetal lung fibroblasts.

    PubMed Central

    Speirs, V.; Ray, K. P.; Freshney, R. I.

    1991-01-01

    Synthesis of pulmonary surfactant (PS) is necessary for normal functioning of the lungs and its production is indicative of normal differentiated lung. The human alveolar carcinoma, A549, has been found to synthesis and secrete PS in vitro. The purpose of this study was to optimise the culture conditions for PS synthesis by A549 as well as to determine the potential role of foetal lung fibroblasts in the induction of PS by glucocorticoids. A549 cells growing in filter wells produced higher levels of PS in response to steroid, a 5-fold increase on the filter well compared to only a 1.5-fold increase when the cells were cultured on a conventional plastic substrate. A549 cells grown in filter wells responded to coculture with fibroblasts whether in direct contact or separated co-culture. A 20-fold increase in PS over control values was observed in separated steroid-treated co-cultures, suggesting the presence of a diffusible factor. A partially purified factor was isolated from fibroblast conditioned medium which was capable of inducing differentiation and other phenotypic changes in A549, namely induction of PS, reduction of plasminogen activator activity and reduction in the in vivo growth of A549 xenografts in nude mice. These results suggest that, under the correct conditions, A549 cells, although transformed, still retain the capacity to respond to differentiation-inducing signals from normal fibroblasts. Images Figure 5 PMID:1654985

  10. Anticancer effect and apoptosis induction by quercetin in the human lung cancer cell line A-549.

    PubMed

    Zheng, Shi-Ying; Li, Yin; Jiang, Dong; Zhao, Jun; Ge, Jin-Feng

    2012-03-01

    The aim of the present study was to investigate the anticancer effect of quercetin (QC) in the human lung cancer cell line A-549 and further study the mechanism of apoptosis induction by QC. Low differentiation potential A-549 human lung cancer cells were treated with QC at different doses and for different times, and the growth inhibitory rates were detected by MTT assay. Apoptosis induced by QC in A-549 cells was observed by Annexin V/PI double staining and flow cytometric assay. The relative tumor growth ratio of the treated/control tumors (T/C) (%) was chosen to represent the tumor growth inhibition of A-549 cell nude mouse xenografts by QC. Apoptosis of the nude mouse xenografts was observed by Annexin V/PI double staining and flow cytometric assay and DNA fragmentation assay. To further determine the molecular mechanism of apoptosis induced by QC, changes in the expression of bcl-2 and bax genes were detected by RT-PCR. Following incubation with QC, the cell growth of the low differentiation potential A-549 human lung cancer cells was dramatically inhibited in a dose-dependent manner. After the cells were exposed to QC for 24, 48 and 72 h, the IC50 value was 1.02 ± 0.05, 1.41 ± 0.20 and 1.14 ± 0.19 µmol/l, respectively. Apoptosis in the A-549 cells induced by QC was noted. The apoptotic subpopulation of A-549 cells was approximately 12.96 and 24.58%, respectively, when cells were incubated with 1.2 µmol/l QC for 48 and 72 h. T/C (%) of A-549 nude mouse xenografts was 44.3, when the nude mice were treated with QC (8 mg/kg). Meanwhile, apoptosis induced by QC was observed in the A-549 nude mouse xenografts. Increased expression of the bax gene and decreased expression of the bc1-2 gene were noted using RT-PCR. Our results provide further evidence of the growth inhibition of the A-549 human lung adenocarcinoma cancer cell line by QC. This effect is associated with the induction of apoptosis in A-549 cells and the molecular mechanism may be related to the

  11. Migration-stimulating factor (MSF) is over-expressed in non-small cell lung cancer and promotes cell migration and invasion in A549 cells over-expressing MSF

    SciTech Connect

    Deng, Xuefeng; Ma, Qunfeng; Zhang, Bo; Jiang, Hong; Zhang, Zhipei; Wang, Yunjie

    2013-10-15

    Migration-stimulating factor (MSF), an oncofetal truncated isoform of fibronectin, is a potent stimulator of cell invasion. However, its distribution and motogenic role in non-small cell lung cancer (NSCLC) have never been identified. In this study, real-time PCR and immunohistochemical staining (IHC) were performed to detect MSF mRNA and protein levels in tumor tissues and matched adjacent tumor-free tissues. Furthermore, to examine the effect of MSF on invasiveness, MSF was upregulated in A549 cells. The invasiveness and viability of A549 cells were then determined using a transwell migration assay and the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) viability assays, respectively. The expression level of MSF in NSCLC tissue was markedly higher than in matched adjacent tumor-free tissue. Additionally, the level of MSF protein expression in stage III and IV NSCLC samples was higher than in stage I and II NSCLC samples. More importantly, we also demonstrated that migration and invasion of A549 cells increased substantially after upregulating MSF, although proliferation remained unchanged. Meanwhile, we found no correlation between increasing motility and invasiveness of MSF-overexpressing cells and expression levels and activities of matrix metalloprotease MMP-2 and MMP-9. Our current study shows that MSF plays a role in migration and invasion of A549 cells and suggests that MSF may be a potential biomarker of NSCLC progression. - Highlights: • MSF expression was upregulated in NSCLC and correlated with TNM stages. • MSF may be a new biomarker for NSCLC progression. • MSF promoted migration and invasion in A549 cells, independent of MMP-2/MMP-9 expression.

  12. Trichomonas vaginalis induces cytopathic effect on human lung alveolar basal carcinoma epithelial cell line A549.

    PubMed

    Salvador-Membreve, Daile Meek C; Jacinto, Sonia D; Rivera, Windell L

    2014-12-01

    Trichomonas vaginalis, the causative agent of trichomoniasis is generally known to inhabit the genitourinary tract. However, several case reports with supporting molecular and immunological identifications have documented its occurrence in the respiratory tract of neonates and adults. In addition, the reports have documented that its occurrence is associated with respiratory failures. The medical significance or consequence of this association is unclear. Thus, to establish the possible outcome from the interaction of T. vaginalis with lung cells, the cytopathic effects of the parasites were evaluated using monolayer cultures of the human lung alveolar basal carcinoma epithelial cell line A549. The possible effect of association of T. vaginalis with A549 epithelial cells was analyzed using phase-contrast, scanning electron microscopy and fluorescence microscopy. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), crystal-violet and TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling) assays were conducted for cytotoxicity testing. The results demonstrate that T. vaginalis: (1) adheres to A549 epithelial cells, suggesting a density-dependent parasite-cell association; (2) adherence on A549 is through flagella, membrane and axostyle; (3) causes cell detachment and cytotoxicity (50-72.4%) to A549 and this effect is a function of parasite density; and (4) induces apoptosis in A549 about 20% after 6 h of incubation. These observations indicate that T. vaginalis causes cytopathic effects on A549 cell. To date, this is the first report showing a possible interaction of T. vaginalis with the lung cells using A549 monolayer cultures. Further studies are recommended to completely elucidate this association.

  13. Irradiated human endothelial progenitor cells induce bystander killing in human non-small cell lung and pancreatic cancer cells.

    PubMed

    Turchan, William T; Shapiro, Ronald H; Sevigny, Garrett V; Chin-Sinex, Helen; Pruden, Benjamin; Mendonca, Marc S

    2016-08-01

    Purpose To investigate whether irradiated human endothelial progenitor cells (hEPC) could induce bystander killing in the A549 non-small cell lung cancer (NSCLC) cells and help explain the improved radiation-induced tumor cures observed in A549 tumor xenografts co-injected with hEPC. Materials and methods We investigated whether co-injection of CBM3 hEPC with A549 NSCLC cells would alter tumor xenograft growth rate or tumor cure after a single dose of 0 or 5 Gy of X-rays. We then utilized dual chamber Transwell dishes, to test whether medium from irradiated CBM3 and CBM4 hEPC would induce bystander cell killing in A549 cells, and as an additional control, in human pancreatic cancer MIA PaCa-2 cells. The CBM3 and CBM4 hEPC were plated into the upper Transwell chamber and the A549 or MIA PaCa-2 cells were plated in the lower Transwell chamber. The top inserts with the CBM3 or CBM4 hEPC cells were subsequently removed, irradiated, and then placed back into the Transwell dish for 3 h to allow for diffusion of any potential bystander factors from the irradiated hEPC in the upper chamber through the permeable membrane to the unirradiated cancer cells in the lower chamber. After the 3 h incubation, the cancer cells were re-plated for clonogenic survival. Results We found that co-injection of CBM3 hEPC with A549 NSCLC cells significantly increased the tumor growth rate compared to A549 cells alone, but paradoxically also increased A549 tumor cure after a single dose of 5 Gy of X-rays (p < 0.05). We hypothesized that irradiated hEPC may be inducing bystander killing in the A549 NSCLC cells in tumor xenografts, thus improving tumor cure. Bystander studies clearly showed that exposure to the medium from irradiated CBM3 and CBM4 hEPC induced significant bystander killing and decreased the surviving fraction of A549 and MIA PaCa-2 cells to 0.46 (46%) ± 0.22 and 0.74 ± 0.07 (74%) respectively (p < 0.005, p < 0.0001). In addition, antibody depletion

  14. Circumvention of drug resistance in human non-small cell lung cancer in vitro by verapamil.

    PubMed

    Merry, S; Courtney, E R; Fetherston, C A; Kaye, S B; Freshney, R I

    1987-10-01

    The sensitivity of 7 human non-small cell lung cancer cell lines to each of 7 cytotoxic drugs was determined. None of the cell lines used in these experiments had been previously exposed to cytotoxic drugs in vitro. A pattern of cross-resistance (P less than 0.05) between the drugs adriamycin (ADR), vincristine (VC) and etoposide (VP16) was noted similar to that seen in other models. The calcium antagonist verapamil (6.6 microM) was shown to increase sensitivity (up to 29-fold) to ADR, VC or VP16 in 5 cell lines. For 2 of the cell lines (A549 and WIL) 2.2 microM verapamil increased VP16 cytotoxicity (up to 4-fold). Drug accumulation studies in 2 cell lines (A549 and SK-MES-1) showed that 6.6 microM verapamil increased intracellular levels of VC up to 4-fold with the greatest increase seen in the cell line (SK-MES-1) for which verapamil produced the greatest increase in cytotoxicity (10-fold). For ADR and VP16 increases in drug accumulation were smaller (up to 1.6-fold). Our data support a potential clinical role for verapamil in overcoming cytotoxic drug resistance in human lung cancer.

  15. Circumvention of drug resistance in human non-small cell lung cancer in vitro by verapamil.

    PubMed Central

    Merry, S.; Courtney, E. R.; Fetherston, C. A.; Kaye, S. B.; Freshney, R. I.

    1987-01-01

    The sensitivity of 7 human non-small cell lung cancer cell lines to each of 7 cytotoxic drugs was determined. None of the cell lines used in these experiments had been previously exposed to cytotoxic drugs in vitro. A pattern of cross-resistance (P less than 0.05) between the drugs adriamycin (ADR), vincristine (VC) and etoposide (VP16) was noted similar to that seen in other models. The calcium antagonist verapamil (6.6 microM) was shown to increase sensitivity (up to 29-fold) to ADR, VC or VP16 in 5 cell lines. For 2 of the cell lines (A549 and WIL) 2.2 microM verapamil increased VP16 cytotoxicity (up to 4-fold). Drug accumulation studies in 2 cell lines (A549 and SK-MES-1) showed that 6.6 microM verapamil increased intracellular levels of VC up to 4-fold with the greatest increase seen in the cell line (SK-MES-1) for which verapamil produced the greatest increase in cytotoxicity (10-fold). For ADR and VP16 increases in drug accumulation were smaller (up to 1.6-fold). Our data support a potential clinical role for verapamil in overcoming cytotoxic drug resistance in human lung cancer. PMID:2825748

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

    PubMed Central

    Zhong, Liangrui; Zheng, Junxian; Sun, Qianqian; Wei, Kemin; Hu, Yijuan

    2016-01-01

    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. PMID:26893573

  17. Integrin α11 regulates IGF2 expression in fibroblasts to enhance tumorigenicity of human non-small-cell lung cancer cells

    PubMed Central

    Zhu, Chang-Qi; Popova, Svetlana N.; Brown, Ewan R. S.; Barsyte-Lovejoy, Dalia; Navab, Roya; Shih, Warren; Li, Ming; Lu, Ming; Jurisica, Igor; Penn, Linda Z.; Gullberg, Donald; Tsao, Ming-Sound

    2007-01-01

    Integrin α11 (ITGA11/α11) is localized to stromal fibroblasts and commonly overexpressed in non-small-cell lung carcinoma (NSCLC). We hypothesized that stromal α11 could be important for the tumorigenicity of NSCLC cells. SV40 immortalized mouse embryonic fibroblasts established from wild-type (WT) and Itga11-deficient [knockout (KO)] mice were tested for their tumorigenicity in immune-deficient mice when implanted alone or coimplanted with the A549 human lung adenocarcinoma cells. A549 coimplanted with the fibroblasts showed a markedly enhanced tumor growth rate compared with A549, WT, or KO, which alone formed only small tumors. Importantly, the growth was significantly greater for A549+WT compared with A549+KO tumors. Reexpression of human α11 cDNA in KO cells rescued a tumor growth rate to that comparable with the A549+WT tumors. These findings were validated in two other NSCLC cell lines, NCI-H460 and NCI-H520. Gene expression profiling indicated that IGF2 mRNA expression level was >200 times lower in A549+KO compared with A549+WT tumors. Stable short-hairpin RNA (shRNA) down-regulation of IGF2 in WT (WTshIGF2) fibroblasts resulted in a decreased growth rate of A549+WTshIGF2, compared with A549+WT tumors. The results indicate that α11 is an important stromal factor in NSCLC and propose a paradigm for carcinoma–stromal interaction indirectly through interaction between the matrix collagen and stromal fibroblasts to stimulate cancer cell growth. PMID:17600088

  18. Aptamer based electrochemical sensor for detection of human lung adenocarcinoma A549 cells

    NASA Astrophysics Data System (ADS)

    Sharma, Rachna; Varun Agrawal, Ved; Sharma, Pradeep; Varshney, R.; Sinha, R. K.; Malhotra, B. D.

    2012-04-01

    We report results of the studies relating to development of an aptamer-based electrochemical biosensor for detection of human lung adenocarcinoma A549 cells. The aminated 85-mer DNA aptamer probe specific for the A549 cells has been covalently immobilized onto silane self assembled monolayer (SAM) onto ITO surface using glutaraldehyde as the crosslinker. The results of cyclic voltammetry and differential pulse voltammetry studies reveal that the aptamer functionalized bioelectrode can specifically detect lung cancer cells in the concentration range of 103 to 107 cells/ml with detection limit of 103 cells/ml within 60 s. The specificity studies of the bioelectrode have been carried out with control KB cells. No significant change in response is observed for control KB cells as compared to that of the A549 target cells.

  19. Functional expression of nicotine influx transporter in A549 human alveolar epithelial cells.

    PubMed

    Tega, Yuma; Yuzurihara, Chihiro; Kubo, Yoshiyuki; Akanuma, Shin-ichi; Ehrhardt, Carsten; Hosoya, Ken-ichi

    2016-02-01

    Nicotine is a potent addictive alkaloid, and is rapidly absorbed through the alveoli of the lung. However, the transport mechanism of nicotine at the human alveolar epithelial barrier has not been investigated in great detail. In the present study, the transport mechanism of nicotine across alveolar epithelium was investigated in vitro using A549 cells, a human adenocarcinoma-derived cell line with an alveolar epithelial cell like phenotype. Nicotine uptake by A549 cells exhibited time-, temperature-, and concentration-dependence with a Km of 50.4 μM. These results suggest that a carrier-mediated transport process is involved in nicotine transport in human alveolar epithelial cells. Nicotine uptake by A549 cells was insensitive to change in extracellular pH. Moreover, nicotine uptake by A549 cells could be inhibited by organic cations such as verapamil and pyrilamine, but not typical substrates of organic cation transporters and β2-agonist. These results suggest that a novel, not yet molecularly identified, organic cation transporter plays a role in nicotine transport which is unlikely to interact with β2-agonist transport. This nicotine influx transporter in human alveolar epithelium might have implications for the rapid absorption of nicotine into the systemic circulation.

  20. Cepharanthine induces apoptosis through reactive oxygen species and mitochondrial dysfunction in human non-small-cell lung cancer cells.

    PubMed

    Hua, Peiyan; Sun, Mei; Zhang, Guangxin; Zhang, Yifan; Tian, Xin; Li, Xin; Cui, Ranji; Zhang, Xingyi

    2015-05-01

    Cepharanthine is a medicinal plant-derived natural compound which possesses potent anti-cancer properties. However, there is little report about its effects on lung cancer cells. In this study, we investigated the effects of cepharanthine on the cell viability and apoptosis in human non-small-cell lung cancer H1299 and A549 cells. It was found that cepharanthine inhibited the growth of H1299 and A549 cells in a dose-dependent manner which was associated with the generation of reactive oxygen species(ROS) and the dissipation of mitochondrial membrane potential (Δψm). These effects were markedly abrogated when cells were pretreated with N-acetylcysteine (NAC), a specific ROS inhibitor, indicating that the apoptosis-inducing effect of cepharanthine in lung cancer cells was mediated by ROS. In addition, cepharanthine triggered apoptosis in non-small lung cancer cells via the upregulation of Bax, downregulation of Bcl-2 and significant activation of caspase-3 and PARP. These results provide the rationale for further research and preclinical investigation of cepharanthine's anti-tumor effect against human non-small-cell lung cancer.

  1. A high-quality secretome of A549 cells aided the discovery of C4b-binding protein as a novel serum biomarker for non-small cell lung cancer.

    PubMed

    Luo, Xiaoyang; Liu, Yansheng; Wang, Rui; Hu, Haichuan; Zeng, Rong; Chen, Haiquan

    2011-04-01

    Cancer secretomes are a promising source for biomarker discovery. The analysis of cancer secretomes still faces some difficulties mainly related to the intracellular contamination, which hinders the qualification and follow-up validations. This study aimed to establish a high-quality secretome of A549 cells by using the cellular proteome as a reference and to test the merits of this refined secretome for biomarker discovery for non-small cell lung cancer (NSCLC). Using one-dimensional gel electrophoresis followed by liquid-chromatography tandem mass spectrometry, we comprehensively investigated the secretome and the concurrent cellular proteome of A549 cells. A high-quality secretome consisting of 382 proteins was refined from 889 initial secretory proteins. More than 85.3% of proteins were annotated as secreted and 76.8% as extracellular or membrane-bound. The discriminative power of the lung-cancer associated secretome was confirmed by gene expression and serum proteomic data. The elevated level of C4b-binding Protein (C4BP) in NSCLC blood was verified by enzyme-linked immunosorbent assays (ELISA, p = 6.07e-6). Moreover, the serum C4BP level in 89 patients showed a strong association with the clinical staging of NSCLC. Our reference-experiment-driven strategy is simple and widely applicable, and may facilitate the identification of novel promising biomarkers of lung cancer.

  2. Matrine induces cell cycle arrest and apoptosis with recovery of the expression of miR-126 in the A549 non-small cell lung cancer cell line

    PubMed Central

    An, Qi; Han, Chao; Zhou, Yubing; Li, Feng; Li, Duolu; Zhang, Xiaojian; Yu, Zujiang; Duan, Zhenfeng; Kan, Quancheng

    2016-01-01

    Non-small cell lung cancer (NSCLC) is the leading cause of cancer-associated mortality in the United States. Chemotherapy prolongs survival rates among patients with advanced disease, however, this is at the cost of clinically significant adverse effects. Matrine is an active component of traditional Chinese medicine and is a promising alternative drug for the treatment of NSCLC. In the present study, the therapeutic effects and the underlying molecular mechanisms of matrine on the A549 NSCLC cell line were investigated. A high concentration of matrine (1.0 mg/ml) significantly (P<0.05) inhibited cell proliferation, by 52.68±3.32%, under which cell shrinkage and disruption were observed. Flow cytometric analysis showed that the proportion of G1/G0 cells was significantly increased, whereas the proportions of S and G2/M cells were significantly decreased (P<0.05) following treatment with matrine for 48 h. These results indicated that cell arrest was induced by matrine. Upregulation of the expression of microRNA (miR)-126, followed by downregulation of the expression of its target gene, vascular endothelial growth factor, were detected following treatment with a low concentration of matrine (0.2 mg/ml) using reverse transcription-quantitative polymerase chain reaction analysis, immunohistochemistry and western blot analysis. In conclusion, matrine induced cell cycle arrest and apoptosis, and recovered the expression of miR-126 in the A549 NSCLC cell line. PMID:27665734

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

    SciTech Connect

    Huang, Chunrong; Zheng, Haichong; He, Wanmei; Lu, Guifang; Li, Xia; Deng, Yubin; Zeng, Mian

    2016-05-20

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

  4. Erlotinib induces the human non-small-cell lung cancer cells apoptosis via activating ROS-dependent JNK pathways.

    PubMed

    Shan, Fenglian; Shao, Zewei; Jiang, Shenghua; Cheng, Zhaozhong

    2016-11-01

    Although erlotinib (ERL) has drawn more and more attention toward its anticancer properties effect, the underlying mechanisms of ERL's anticancer properties effect remain unclear yet. So, the aim of this research was to explore the underlying anticancer mechanisms of ERL and to explore whether the reactive oxygen species (ROS)-dependent c-Jun N-terminal kinase (JNK) pathway contributed to the anticancer properties provided by ERL. In our study, we used MTT assay to detect the anticell growth ability of ERL on human non-small-cell lung cancer cell lines (A549). The extent of cell apoptosis was determined by Hoechst 33342 staining and fluorescence-activated cell sorter (FACS) assay. Then, DCFH-DA and JC-1 staining were used to monitor intracellular reactive oxygen species (ROS) and mitochondrial membrane potential (MMP), respectively. Finally, the effect of ERL on phosphorylation state of JNK protein and downstream apoptosis concerned proteins were detected by western blotting assay. Results showed that ERL significantly suppressed the growth and reproduction of A549 cells with the concentration rising up in vitro. Hoechst 33342 staining and FACS assay also confirmed the proapoptosis effect of ERL on A549 cells with the concentration rising up. Furthermore, exposure of A549 cells to ERL increased the intracellular ROS production. As expected, intracellular ROS activated the proapoptotic JNK signaling pathway and inhibited the activation of EFGR signaling pathway. Our results also revealed that ERL could induce cell-cycle arrest at G0/G1 period. Activation of JNK protein decreased MMP and downregulated content of antiapoptotic protein Bcl-2 concomitant with the upregulated content of proapoptotic protein Bax in A549 cells. In addition, c-Jun and cleaved caspase-3 were also activated by the phosphorylated JNK induced by ERL. All of these proapoptosis effect of ERL was reversed by administration of N-acetylcysteine (NAC), which performed as a ROS scavenger. Our results

  5. Microwave induces apoptosis in A549 human lung carcinoma cell line.

    PubMed

    Song, Xiao-lian; Wang, Chang-hui; Hu, Hai-yang; Yu, Chao; Bai, Chong

    2011-04-01

    Microwaves have other biological effects on cancer as well besides killing tumor cells by coagulation. Some studies showed that microwaves may induce apoptosis in some tumor cells. The apoptotic effect of microwaves may help in clinic to remove residual malignant cells nearby the primary lesion and avoid relapse subsequently. However, there is little evidence on this subject from lung cancer. We studied the effect of microwaves on inducing apoptosis in the human lung carcinoma cell line A549 cells, aiming to identify its effect on apoptosis. A549 cells were radiated by various intensities and durations of microwaves. Apoptosis induction in A549 cells was analyzed by morphological observations, tetrazolium blue color method (MTT) assays, flow cytometry, immunohistochemistry, and image analyses. Morphological changes in A549 cells, including cell shrinking and nuclear pyknosis, were observed after microwave radiation. Microwaves significantly inhibited metabolic activities and induced apoptosis in A549 cells. The results of the MTT assay showed a significant decrease of cell activities in all the radiation groups compared with the normal control (P < 0.01). The low point of cell activities often appeared at 6 - 12 hours after radiation. Apoptosis was also confirmed by flow cytometry. The early stage apoptotic rate reached 6.10% - 17.98% and the advanced stage apoptotic rate + necrosis rate reached 8.04% - 44.06% at 6 hours after microwave irradiation, in contrast to 2.32% and 4.10% in the respective control groups. Down-regulation of Bcl-2 expression and up-regulation of p53 expression were observed by immunohistochemistry after radiation. In most treated groups, the down-regulation of Bcl-2 expression reached its lowest level at 3 - 6 hours after radiation (integrated optical density (IOD)-6 hours: 2.13 ± 0.08 - 5.14 ± 0.13 vs. control: 5.79 ± 0.10, P < 0.01) and the up-regulation of P53 expression peaked at about 3 hours (IOD-3 hours: 2.61 ± 0.13 - 8.07 ± 0

  6. Dichloroacetate alters Warburg metabolism, inhibits cell growth, and increases the X-ray sensitivity of human A549 and H1299 NSC lung cancer cells.

    PubMed

    Allen, Kah Tan; Chin-Sinex, Helen; DeLuca, Thomas; Pomerening, Joseph R; Sherer, Jeremy; Watkins, John B; Foley, John; Jesseph, Jerry M; Mendonca, Marc S

    2015-12-01

    We investigated whether altering Warburg metabolism (aerobic glycolysis) by treatment with the metabolic agent dichloroacetate (DCA) could increase the X-ray-induced cell killing of the radiation-resistant human non-small-cell lung cancer (NSCLC) cell lines A549 and H1299. Treatment with 50mM DCA decreased lactate production and glucose consumption in both A549 and H1299, clear indications of attenuated aerobic glycolysis. In addition, we found that DCA treatment also slowed cell growth, increased population-doubling time, and altered cell cycle distribution. Furthermore, we report that treatment with 50mM DCA significantly increased single and fractionated X-ray-induced cell killing of A549 and H1299 cells. Assay of DNA double-strand break repair by neutral comet assays demonstrated that DCA inhibited both the fast and the slow kinetics of X-ray-induced DSB repair in both A549 and H1299 NSCL cancer cells. Taken together the data suggest a correlation between an attenuated aerobic glycolysis and enhanced cytotoxicity and radiation-induced cell killing in radiation-resistant NSCLC cells.

  7. Fucoidan from Undaria pinnatifida induces apoptosis in A549 human lung carcinoma cells.

    PubMed

    Boo, Hye-Jin; Hyun, Jae-Hee; Kim, Sang-Cheol; Kang, Jung-Il; Kim, Min-Kyoung; Kim, Sun-Yeou; Cho, Heeyeong; Yoo, Eun-Sook; Kang, Hee-Kyoung

    2011-07-01

    Fucoidan, a sulfated polysaccharide, has various biological activities, such as anticancer, antiangiogenic and antiinflammatory effects; however, the mechanisms of action of fucoidan on anticancer activity have not been fully elucidated. The anticancer effects of fucoidan from Undaria pinnatifida on A549 human lung carcinoma cells were examined. Treatment of A549 cells with fucoidan resulted in potent antiproliferative activity. Also, some typical apoptotic characteristics, such as chromatin condensation and an increase in the population of sub-G1 hypodiploid cells, were observed. With respect to the mechanism underlying the induction of apoptosis, fucoidan reduced Bcl-2 expression, but the expression of Bax was increased in a dose-dependent manner compared with the controls. Furthermore, fucoidan induced caspase-9 activation, but decreased the level of procaspase-3. Cleavage of poly-ADP-ribose polymerase (PARP), a vital substrate of effector caspase, was found. The study further investigated the role of the MAPK and PI3K/Akt pathways with respect to the apoptotic effect of fucoidan, and showed that fucoidan activates ERK1/2 in A549 cells. Unlike ERK1/2, however, treatment with fucoidan resulted in the down-regulation of phospho-p38 expression. In addition, fucoidan resulted in the down-regulation of phospho-PI3K/Akt. Together, these results indicate that fucoidan induces apoptosis of A549 human lung cancer cells through down-regulation of p38, PI3K/Akt, and the activation of the ERK1/2 MAPK pathway.

  8. Teroxirone inhibited growth of human non-small cell lung cancer cells by activating p53

    SciTech Connect

    Wang, Jing-Ping; Lin, Kai-Han; Liu, Chun-Yen; Yu, Ya-Chu; Wu, Pei-Tsun; Chiu, Chien-Chih; Su, Chun-Li; Chen, Kwun-Min; Fang, Kang

    2013-11-15

    In this work, we demonstrated that the growth of human non-small-cell-lung-cancer cells H460 and A549 cells can be inhibited by low concentrations of an epoxide derivative, teroxirone, in both in vitro and in vivo models. The cytotoxicity was mediated by apoptotic cell death through DNA damage. The onset of ultimate apoptosis is dependent on the status of p53. Teroxirone caused transient elevation of p53 that activates downstream p21 and procaspase-3 cleavage. The presence of caspase-3 inhibitor reverted apoptotic phenotype. Furthermore, we showed the cytotoxicity of teroxirone in H1299 cells with stable ectopic expression of p53, but not those of mutant p53. A siRNA-mediated knockdown of p53 expression attenuated drug sensitivity. The in vivo experiments demonstrated that teroxirone suppressed growth of xenograft tumors in nude mice. Being a potential therapeutic agent by restraining cell growth through apoptotic death at low concentrations, teroxirone provides a feasible perspective in reversing tumorigenic phenotype of human lung cancer cells. - Highlights: • Teroxirone repressed tumor cell growth in nude mice of human lung cancer cells. • The apoptotic cell death reverted by caspase-3 inhibitor is related to p53 status. • Teroxirone provides a good candidate for lung cancer treatment.

  9. Tomatidine inhibits invasion of human lung adenocarcinoma cell A549 by reducing matrix metalloproteinases expression.

    PubMed

    Yan, Kun-Huang; Lee, Liang-Ming; Yan, Shao-Han; Huang, Hsiang-Ching; Li, Chia-Chen; Lin, Hui-Ting; Chen, Pin-Shern

    2013-05-25

    Tomatidine is an aglycone of glycoalkaloid tomatine in tomato. Tomatidine is found to possess anti-inflammatory properties and may serve as a chemosensitizer in multidrug-resistant tumor cells. However, the effect of tomatidine on cancer cell metastasis remains unclear. This study examines the effect of tomatidine on the migration and invasion of human lung adenocarcinoma A549 cell in vitro. The data demonstrates that tomatidine does not effectively inhibit the viability of A549 cells. When treated with non-toxic doses of tomatidine, cell invasion is markedly suppressed by Boyden chamber invasion assay, while cell migration is not affected. Tomatidine reduces the mRNA level of matrix metalloproteinase-2 (MMP-2), MMP-9 and increases the expression of reversion-inducing cysteine-rich protein with kazal motifs (RECK), as well as tissue inhibitor of metalloproteinase-1 (TIMP-1). The immunoblotting assays indicate that tomatidine is very effective in suppressing the phosphorylation of Akt and extracellular signal regulating kinase (ERK). In addition, tomatidine significantly decreases the nuclear level of nuclear factor kappa B (NF-κB), which suggests that tomatidine inhibits NF-κB activity. Furthermore, the treatment of inhibitors specific for PI3K/Akt (LY294002), ERK (U0126), or NF-κB (pyrrolidine dithiocarbamate) to A549 cells reduced cell invasion and MMP-2/9 expression. The results suggest that tomatidine inhibits the invasion of A549 cells by reducing the expression of MMPs. It also inhibits ERK and Akt signaling pathways and NF-κB activity. These findings demonstrate a new therapeutic potential for tomatidine in anti-metastatic therapy.

  10. Antitumor activity of paederosidic acid in human non-small cell lung cancer cells via inducing mitochondria-mediated apoptosis.

    PubMed

    Yu, Ping; Shi, Lifeng; Song, Meiyan; Meng, Yu

    2017-05-01

    This study was aimed to investigate antitumor activity of paederosidic acid (PA) in human non-small cell lung cancer cells and explore the related mechanisms. The anti-proliferative effects of PA on A549 cells were evaluated by MTT method and the IC50 values were calculated. Furthermore, the PA-induced apoptosis in A549 cells was determined by fluorescence microscope via staining with DAPI and by flow cytometer via staining with FITC conjugated Annexin V/PI. The expression of apoptosis-related or signaling proteins was investigated by Western blotting. Our results demonstrated that PA showed significant anti-tumor activity on lung cancer in vitro; the mechanisms were involved in inducing mitochondria-mediated apoptosis via up-regulation of caspase-3, caspase-8, caspase-9, Bid, Bax, down-regulation of Bcl-2 and stimulating the release of Cyto-C from mitochondria. In addition, JNK phosphorylation levels significantly increased concomitantly with decrease in Akt phosphorylation after treatment with PA in A549 cells. However, JNK siRNA-transfected cells diminished PA-induced caspase-3, 8 and 9, Bid and Bax activaton while enhanced the Bcl-2 activation. Collectively, these results indicated that PA-induced JNK activation played an important functional role in apoptosis. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Dexamethasone suppresses the growth of human non-small cell lung cancer via inducing estrogen sulfotransferase and inactivating estrogen

    PubMed Central

    Wang, Li-jie; Li, Jian; Hao, Fang-ran; Yuan, Yin; Li, Jing-yun; Lu, Wei; Zhou, Tian-yan

    2016-01-01

    Aim: Dexamethasone (DEX) is a widely used synthetic glucocorticoid, which has shown anti-cancer efficacy and anti-estrogenic activity. In this study we explored the possibility that DEX might be used as an endocrine therapeutic agent to treat human non-small cell lung cancer (NSCLC). Methods: The viability and proliferation of human NSCLC cell lines A549 and H1299 were assessed in vitro. Anti-tumor action was also evaluated in A549 xenograft nude mice treated with DEX (2 or 4 mg·kg−1·d−1, ig) or the positive control tamoxifen (50 mg·kg−1·d−1, ig) for 32 d. The expression of estrogen sulfotransferase (EST) in tumor cells and tissues was examined. The intratumoral estrogen levels and uterine estrogen responses were measured. Results: DEX displayed mild cytotoxicity to the NSCLC cells (IC50 >500 μmol/L) compared to tamoxifen (IC50 <50 μmol/L), but it was able to inhibit the cell proliferation at low micromolar ranges. Furthermore, DEX (0.1–10 μmol/L) dose-dependently up-regulated EST expression in the cells, and inhibited the cell migration in vitro. Triclosan, a sulfation inhibitor, was able to diminish DEX-caused inhibition on the cell viability. In A549 xenograft nude mice, DEX or tamoxifen administration remarkably suppressed the tumor growth. Moreover, DEX administration dose-dependently increased EST expression in tumor tissues, and reduced intratumoral estrogen levels as well as the volumes and weights of uterine. Conclusion: DEX suppresses the growth of A549 xenograft tumors via inducing EST and decreasing estradiol levels in tumor tissues, suggesting that DEX may be used as anti-estrogenic agent for the treatment of NSCLC. PMID:27133297

  12. MCM2 is a therapeutic target of lovastatin in human non-small cell lung carcinomas.

    PubMed

    Zhang, Xu; Teng, Yang; Yang, Fang; Wang, Meng; Hong, Xuan; Ye, Lei-Guang; Gao, Yi-Na; Chen, Gong-Yan

    2015-05-01

    Human non-small cell lung carcinoma (NSCLC) is one of the most common cancer worldwide. In previous studies, lovastatin, acting as an inhibitor of 3-hydroxy-3-methylglutaryl Co A (HMG-CoA) reductase, exhibited significant antitumor activity during tumorigenesis. However, whether or not this effect is mediated through changes in minichromosome maintenance (MCM) 2 expression remains unclear. The present study investigated whether lovastatin inhibits proliferation due to MCM2 in NSCLCs. We first assessed the effects of lovastatin on cell anti-proliferation, cell cycle progression and apoptosis in NSCLC cells. We found, by quantitative RT-PCR and western blot analysis, that lovastatin treatment markedly and consistently inhibited the expression of MCM2. Then, to further explore the anticancer mechanism of lovastatin involving MCM2, we silenced MCM2 by siRNA in two cell lines (A549 and GLC-82). Silencing of MCM2 triggered G1/S arrest. Following further examination of cell cycle-related factors, MCM2 knockdown inhibited protein retinoblastoma (Rb), cyclin D1 and CDK4 expression, but increased p21 and p53 expression, suggesting that siMCM2 indeed triggered cell cycle arrest. In addition, siMCM2 induced apoptosis. Finally, lovastatin treatment increased p-JNK, which is involved in the downregulation of MCM2. In conclusion, our data suggest that MCM2 may be a novel therapeutic target of lovastatin treatment in NSCLCs.

  13. Sodium orthovanadate affects growth of some human epithelial cancer cells (A549, HTB44, DU145).

    PubMed

    Klein, Andrzej; Holko, Przemyslaw; Ligeza, Janusz; Kordowiak, Anna M

    2008-01-01

    Within the concentration range of 1-20 microM, orthovanadate (Na3VO4) demonstrated a time and dose-dependent inhibition of autocrine growth of the human carcinoma cell lines A549 (lung), HTB44 (kidney) and DU145 (prostate), as compared to appropriate controls (without Na3VO4). The investigation was conducted by two methods: staining with N-hexa-methylpararosaniline (crystal violet=CV) or bromide3-(4,5-dimethyltio-azo-2)-2,5-diphenyl-tetrazole (MTT). In 5, 10 and 20 microM of Na3VO4 in serum-free medium, the mean values of these two tests for A549 were approximately 40%, 45% or 65% as compared to the appropriate controls. HTB44 had the greatest opportunity (statistically insignificant) at lower vanadium concentrations (up to 10 microM), whereas at 20 microM growth inhibition of these cells was approximately 50% of the controls. DU145 showed approximately 33%, 65% and 98% growth inhibition for 5, 10 and 20 microM of Na3VO4, respectively Additionally, hypothetical curves obtained by a MANOVA test based on the CV results after 72 h incubation with Na3VO4 in serum-free medium, and an example of a time-dependent effect of Na3VO4 on A549 cells, were also presented. Sodium orthovanadate was also examined for its cytotoxic capabilities, especially its ability to induce tumor cell apoptosis; the results were compared with the effect of paclitaxel. The target cells were dyed by differential staining (HOECHST33258 and propidium iodide) after 3 h and 24 h (DU145) or 3 h and 72 h (A549) of incubation with the vanadium compound. Contrary to the two cancer cell lines (viable, apoptotic or necrotic in experimental conditions), the renal HTB44 cells were insensitive up to 15 microM Na3VO4 concentrations. After 3 h incubation with Na3VO4, both lung (A549) and prostate (DU145) cancer cells showed a slight but significant reduction in the percentage of viable cells, and an increased amount of apoptotic cells. In contrast to the lung cells, DU145 prostate cells after 24 h were more

  14. Cimicifuga foetida L. inhibited human respiratory syncytial virus in HEp-2 and A549 cell lines.

    PubMed

    Wang, Kuo Chih; Chang, Jung San; Chiang, Lien Chai; Lin, Chun Ching

    2012-01-01

    Human respiratory syncytial virus (HRSV) causes serious pediatric infection of the lower respiratory tract without effective therapeutic modality. Sheng-Ma-Ge-Gen-Tang (SMGGT; Shoma-kakkon-to) has been proven to be effective at inhibiting HRSV-induced plaque formation, and Cimicifuga foetida is the major constituent of SMGGT. We tested the hypothesis that C. foetida effectively inhibited the cytopathic effects of HRSV by a plaque reduction assay in both human upper (HEp2) and lower (A549) respiratory tract cell lines. Its ability to stimulate anti-viral cytokines was evaluated by an enzyme-linked immunosorbent assay (ELISA). C. foetida dose-dependently inhibited HRSV-induced plaque formation (p < 0.0001) before and after viral inoculation, especially in A549 cells (p < 0.0001). C. foetida dose-dependently inhibited viral attachment (p < 0.0001) and could increase heparins effect on viral attachment. In addition, C. foetida time-dependently and dose-dependently (p < 0.0001) inhibited HRSV internalization. C. foetida could stimulate epithelial cells to secrete IFN-β to counteract viral infection. However, C. foetida did not stimulate TNF-α secretion. Therefore, C. foetida could be useful in managing HRSV infection. This is the first evidence to support that C. foetida possesses antiviral activity.

  15. Previous heat shock treatment inhibits Mayaro virus replication in human lung adenocarcinoma (A549) cells.

    PubMed

    Virgilio, P L; Godinho-Netto, M C; Carvalho Mda, G

    1997-01-01

    Human lung adenocarcinoma cells (A549) were submitted to mild or severe heat shock (42 degrees C or 44 degrees C) for 1 h, while another group of cells was double-heat-shocked (submitted to 42 degrees C for 1 h, returned to 37 degrees C for 3 h, then exposed to 44 degrees C for 1 h). After each heat treatment, the cells were infected with Mayaro virus for 24 h and incubated at 37 degrees C. The results showed that the double-heat-shocked thermotolerant cells exhibited a 10(4)-fold virus titre inhibition, despite the recovery of protein synthesis and original morphology 24 h post-infection. In contrast, cells submitted to mild or severe heat shock exhibited weaker inhibition of Mayaro virus titre (10(2)-fold). The mildly heat-shocked cells also presented a full recovery in protein synthesis, which was not observed in severely heat-shocked cells. These results indicate that exposure of A549 cells to a mild or to a double heat shock treatment before Mayaro virus infection induces an antiviral state.

  16. Role of alpha7-nicotinic acetylcholine receptor in human non-small cell lung cancer proliferation.

    PubMed

    Paleari, L; Catassi, A; Ciarlo, M; Cavalieri, Z; Bruzzo, C; Servent, D; Cesario, A; Chessa, L; Cilli, M; Piccardi, F; Granone, P; Russo, P

    2008-12-01

    Lung cancer is the most common cause of cancer death in the world. Cigarette smoking represents the major risk factor. Nicotine, an active component of cigarettes, can induce cell proliferation, angiogenesis and apoptosis resistance. All these events are mediated through the nicotinic acetylcholine receptor (nAChR) expressed on lung cancer cells. We speculate that new insights into the pathophysiological roles of nAChR may lead to new therapeutic avenues to reduce non-small cell lung cancer (NSCLC) tumour growth. Human samples of NSCLC, cell lines and mouse models were utilized in Western blotting, reverse transcriptase polymerase chain reaction and apoptosis studies. Human NSCLC tissues expressed alpha7-nAChR. This expression was higher in smoking patients with squamous carcinomas than those with adenocarcinomas and in male smoking patients than in females. All the data support the hypothesis that major expression of alpha7-nAChR is related to major activation of the Rb-Raf-1/phospho-ERK/phospho-p90RSK pathway. alpha7-nAChR antagonists, via mitochondria associated apoptosis, inhibited proliferation of human NSCLC primary and established cells. Nicotine stimulates tumour growth in a murine model, A549 cells orthotopically grafted. The effects of nicotine were associated with increases in phospho-ERK in tumours. Proliferation effects of nicotine could be blocked by inhibition of alpha7-nAChR by the high affinity ligand alpha-cobratoxin. These results showed that alpha7-nAChR plays an important role in NSCLC cell growth and tumour progression as well as in cell death.

  17. TUSC3 induces autophagy in human non-small cell lung cancer cells through Wnt/β-catenin signaling

    PubMed Central

    Peng, Yun; Cao, Jun; Yao, Xiao-Yi; Wang, Jian-Xin; Zhong, Mei-Zuo; Gan, Ping-Ping; Li, Jian-Huang

    2017-01-01

    We investigated the effects of tumor suppressor candidate 3 (TUSC3) on autophagy in human non-small cell lung cancer (NSCLC) cells. A total of 118 NSCLC patients (88 males and 30 females) who underwent surgery at our institute were enrolled in the study. Immunohistochemical analysis revealed that TUSC3 protein expression was lower in NSCLC specimens than adjacent normal tissue. Correspondingly, there was greater methylation of TUSC3 in NSCLC than adjacent normal tissue. After transient transfection of A549 NSCLC cells with constructs designed to up-regulate or down-regulate TUSC3 expression, we analyzed the effects of inhibiting the Wnt pathway (XAV939) and autophagy (chloroquine, CQ) on the behavior of NSCLC cells. We also performed TOP/FOP-Flash reporter assays, MTT assays, Annexin V-FITC/propidium iodide staining, and acridine orange staining to evaluate Wnt/β-catenin signaling, cell proliferation, apoptosis, and autophagy, respectively. Expression of Wnt/β-catenin pathway components and autophagy-related proteins was analyzed using qRT-PCR and Western blotting. We found that TUSC3 inhibited cell proliferation and promoted both apoptosis and autophagy in A549 cells. In addition, TUSC3 increased expression of autophagy-related proteins. It also increased expression of Wnt/β-catenin signaling pathway components and promoted nuclear transfer of β-catenin, resulting in activation of Wnt/β-catenin signaling. TUSC3 thus induces autophagy in human NSCLC cells through activation of the Wnt/β-catenin signaling pathway. PMID:28881786

  18. Phenotypic modification of human glioma and non-small cell lung carcinoma by glucocorticoids and other agents.

    PubMed

    McLean, J S; Frame, M C; Freshney, R I; Vaughan, P F; Mackie, A E; Singer, I

    1986-01-01

    Glucocorticoids are cytostatic for human glioma grown at a high cell density in cell culture. The effect is not cytotoxic, appears to involve a modification of the cell surface, and has been detected with methyl prednisolone, dexamethasone, and beta-methasone. Glucocorticoids were also found to reduce malignancy-associated properties (plasminogen activator and endothelial mitogenesis) and enhance differentiation (glutamyl synthetase activity and high affinity GABA uptake). Cytostasis was also seen at high cell densities in non-small cell lung carcinoma with a concomitant reduction in plasminogen activator activity and endothelial mitogenesis. Preliminary data on surfactant production in A549 cells suggests that the repression of malignancy-associated properties is accompanied by an increase in cell differentiation. Treatment of the WIL adenocarcinoma gown as a xenograft in nude mice caused total cessation of growth and massive central necrosis in the tumor.

  19. Ultrafine particles (UFPs) from domestic wood stoves: genotoxicity in human lung carcinoma A549 cells.

    PubMed

    Marabini, Laura; Ozgen, Senem; Turacchi, Silvia; Aminti, Stefania; Arnaboldi, Francesca; Lonati, Giovanni; Fermo, Paola; Corbella, Lorenza; Valli, Gianluigi; Bernardoni, Vera; Dell'Acqua, Manuela; Vecchi, Roberta; Becagli, Silvia; Caruso, Donatella; Corrado, Galli L; Marinovich, Marina

    2017-08-01

    In this paper, results on the potential toxicity of ultrafine particles (UFPs d<100nm) emitted by the combustion of logwood and pellet (hardwood and softwood) are reported. The data were collected during the TOBICUP (TOxicity of BIomass COmbustion generated Ultrafine Particles) project, carried out by a team composed of interdisciplinary research groups. The genotoxic evaluation was performed on A549 cells (human lung carcinomacells) using UFPs whose chemical composition was assessed by a suite of analytical techniques. Comet assay and γ-H2AX evaluation show a significant DNA damage after 24h treatment. The interpretation of the results is based on the correlation among toxicological results, chemical-physical properties of UFPs, and the type and efficiency conditions in residential pellet or logwood stoves. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Genistein inhibits A549 human lung cancer cell proliferation via miR-27a and MET signaling

    PubMed Central

    Yang, Yang; Zang, Aimin; Jia, Youchao; Shang, Yanhong; Zhang, Zhuoqi; Ge, Kun; Zhang, Jinchao; Fan, Wufang; Wang, Bei

    2016-01-01

    Genistein is a soybean isoflavone; in its aglycone it has various biological activities. Animal experiments, clinical studies and epidemiological investigations suggest that genistein has preventative and curative functions for a number of diseases, particularly in cancer. The present study explored the potential anti-cancer effect of genistein by observing its role in inhibiting A549 human lung cancer cell proliferation and investigating the possible mechanism. A549 cells were exposed to various concentrations of genistein (0, 10, 25, 50, 100 and 200 µM; dissolved in physiological saline) for 1, 2 and 3 days. Subsequently, the viability of A549 cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, cell apoptosis was examined using a flow cytometer, caspase 3/9 activity was measured using commercial kits, reverse transcription quantitative polymerase chain reaction was used to analyze the miR-27a expression and western blotting was used to investigate MET protein expression. The results suggested a significant inhibition of A549 cell growth following treatment with genistein in a time- and dose-dependent manner. The current study also indicated that treatment with genistein significantly induces cell apoptosis and promotes caspase-3/9 activation of A549 cells in a dose-dependent manner. Further functional assays revealed that the anti-cancer effect of genistein activated microRNA-27a (miR-27a) expression levels and reduced MET protein expression in A549 cells. In conclusion, the present study demonstrates that genistein inhibits A549 human lung cancer cell proliferation. Furthermore, this study reports, for the first time, a correlation between the anti-cancer effect of genistein and miR-27a-mediated MET signaling. PMID:27602162

  1. Bioluminescent Orthotopic Mouse Models of Human Localized Non-Small Cell Lung Cancer: Feasibility and Identification of Circulating Tumour Cells

    PubMed Central

    Lahon, Benoit; Castier, Yves; Lesèche, Guy; Soria, Jean-Charles; Vozenin, Marie-Catherine; Decraene, Charles; Deutsch, Eric

    2011-01-01

    Background Preclinical models of non-small cell lung cancer (NSCLC) require better clinical relevance to study disease mechanisms and innovative therapeutics. We sought to compare and refine bioluminescent orthotopic mouse models of human localized NSCLC. Methods Athymic nude mice underwent subcutaneous injection (group 1-SC, n = 15, control), percutaneous orthotopic injection (group 2-POI, n = 30), surgical orthotopic implantation of subcutaneously grown tumours (group 3-SOI, n = 25), or transpleural orthotopic injection (group 4-TOI, n = 30) of A549-luciferase cells. Bioluminescent in vivo imaging was then performed weekly. Circulating tumour cells (CTCs) were searched using Cellsearch® system in SC and TOI models. Results Group 2-POI was associated with unexpected direct pleural spreading of the cellular solution in 53% of the cases, forbidding further evaluation of any localized lung tumour. Group 3-SOI was characterized by high perioperative mortality, initially localized lung tumours, and local evolution. Group 4-TOI was associated with low perioperative mortality, initially localized lung tumours, loco regional extension, and distant metastasis. CTCs were detected in 83% of nude mice bearing subcutaneous or orthotopic NSCLC tumours. Conclusions Transpleural orthotopic injection of A549-luc cells in nude mouse lung induces localized tumour, followed by lymphatic extension and specific mortality, and allowed the first time identification of CTCs in a NSCLC mice model. PMID:22022511

  2. 4-Nitroquinoline-1-oxide effects human lung adenocarcinoma A549 cells by regulating the expression of POLD4

    PubMed Central

    HUANG, QIN-MIAO; ZENG, YI-MING; ZHANG, HUA-PING; LV, LIANG-CHAO; YANG, DONG-YONG; LIN, HUI-HUANG

    2016-01-01

    The aim of the present study was to explore the expression of POLD4 in human lung adenocarcinoma A549 cells under 4-nitroquinoline-1-oxide (4NQO) stimulation to investigate the role of POLD4 in smoking-induced lung cancer. The lung cancer A549 cell line was treated with 4NQO, with or without MG132 (an inhibitor of proteasome activity), and subsequently the POLD4 level was determined by western blot analysis. Secondly, the cell sensitivity to 4NQO and Taxol was determined when the POLD4 expression level was downregulated by siRNA. The POLD4 protein levels in the A549 cells decreased following treatment with 4NQO; however, MG132 could reverse this phenotype. Downregulation of the POLD4 expression by siRNA enhanced A549 cell sensitivity to 4NQO, but not to Taxol. In conclusion, 4NQO affects human lung adenocarcinoma A549 cells by regulating the expression of POLD4. PMID:26998273

  3. Jolkinolide A and Jolkinolide B Inhibit Proliferation of A549 Cells and Activity of Human Umbilical Vein Endothelial Cells.

    PubMed

    Shen, Lei; Zhang, Shan-Qiang; Liu, Lei; Sun, Yu; Wu, Yu-Xuan; Xie, Li-Ping; Liu, Ji-Cheng

    2017-01-14

    BACKGROUND Jolkinolide A (JA) and Jolkinolide B (JB) are diterpenoids extracted from the roots of Euphorbia fischeriana Steud and have been shown to have anti-tumor activity. However, their effects on the ability of tumor cells to invade blood vessels and metastasize remain largely unknown. Investigations into the effects of JA and JB on the angiogenesis of tumor tissues may facilitate the identification of new natural drugs with anti-tumor growth and metastasis activities. MATERIAL AND METHODS We used different concentrations of JA and JB (20 μg/ml, 40 μg/ml, 60 μg/ml, 80 μg/ml, and 100 μg/ml) to stimulate A549 cells and then studied the effects on the growth and metastasis of lung cancers. In addition, we used conditional media from A549 cells (A549-CM) stimulated by either JA or JB in different concentrations to culture human umbilical vein endothelial cells (HUVECs). RESULTS We found that both JA and JB significantly inhibited the Akt-STAT3-mTOR signaling pathway and reduced the expression of VEGF in A549 cells, but JB exhibited more significant inhibitory effects than JA. The JB-stimulated A549 cell conditional media had a greater inhibitory effect on the proliferation and migration of HUVECs than did the conditional media of JA-stimulated A549 cells. This effect gradually increased with increasing concentrations of either type of Jolkinolide. CONCLUSIONS Our results suggest that JA and JB inhibited VEGF expression in A549 cells through the inhibition of the Akt-STAT3-mTOR signaling pathway, and directly inhibited the proliferation and migration of HUVECs. These findings are of great significance for the development of new plant-derived chemotherapy agents for the treatment of cancer.

  4. Jolkinolide A and Jolkinolide B Inhibit Proliferation of A549 Cells and Activity of Human Umbilical Vein Endothelial Cells

    PubMed Central

    Shen, Lei; Zhang, Shan-Qiang; Liu, Lei; Sun, Yu; Wu, Yu-Xuan; Xie, Li-Ping; Liu, Ji-Cheng

    2017-01-01

    Background Jolkinolide A (JA) and Jolkinolide B (JB) are diterpenoids extracted from the roots of Euphorbia fischeriana Steud and have been shown to have anti-tumor activity. However, their effects on the ability of tumor cells to invade blood vessels and metastasize remain largely unknown. Investigations into the effects of JA and JB on the angiogenesis of tumor tissues may facilitate the identification of new natural drugs with anti-tumor growth and metastasis activities. Material/Methods We used different concentrations of JA and JB (20 μg/ml, 40 μg/ml, 60 μg/ml, 80 μg/ml, and 100 μg/ml) to stimulate A549 cells and then studied the effects on the growth and metastasis of lung cancers. In addition, we used conditional media from A549 cells (A549-CM) stimulated by either JA or JB in different concentrations to culture human umbilical vein endothelial cells (HUVECs). Results We found that both JA and JB significantly inhibited the Akt-STAT3-mTOR signaling pathway and reduced the expression of VEGF in A549 cells, but JB exhibited more significant inhibitory effects than JA. The JB-stimulated A549 cell conditional media had a greater inhibitory effect on the proliferation and migration of HUVECs than did the conditional media of JA-stimulated A549 cells. This effect gradually increased with increasing concentrations of either type of Jolkinolide. Conclusions Our results suggest that JA and JB inhibited VEGF expression in A549 cells through the inhibition of the Akt-STAT3-mTOR signaling pathway, and directly inhibited the proliferation and migration of HUVECs. These findings are of great significance for the development of new plant-derived chemotherapy agents for the treatment of cancer. PMID:28087861

  5. Schisandrin B inhibits the proliferation of human lung adenocarcinoma A549 cells by inducing cycle arrest and apoptosis

    PubMed Central

    Lv, Xue-Jiao; Zhao, Li-Jing; Hao, Yu-Qiu; Su, Zhen-Zhong; Li, Jun-Yao; Du, Yan-Wei; Zhang, Jie

    2015-01-01

    Lung cancer is the leading cause of cancer death in the world. Schizandrin B (Sch B) is one of the main dibenzocyclooctadiene lignans present in the fruit of Schisandra chinensis (Schisandraceae). Sch B has multiple functions against cancer. The aim of this study was to determine the effect of Sch B on the proliferation, cell cycling, apoptosis and invasion of lung adenocarcinoma A549 cells by MTT, flow cytometry, wound healing and transwell invasion assays. Treatment with Sch B inhibited the proliferation of A549 cells in a dose-dependent manner. Sch B induced cell cycle arrest at G0/G1 phase by down-regulating the expression of cyclin D1, cyclin-dependent kinase (CDK)4, and CDK6, but up-regulating p53 and p21 expression in A549 cells. Furthermore, Sch B triggered A549 cell apoptosis by increasing Bax, cleaved caspase-3, 9, Cyto C, but decreasing Bcl-2 and PCNA expression. In addition, Sch B inhibited the invasion and migration of A549 cells by down-regulating the expressions of HIF-1, VEGF, MMP-9 and MMP-2. Therefore, Sch B has potent anti-tumor activity and may be a promising traditional Chinese medicine for human lung carcinoma. PMID:26221229

  6. Rosemary extract reduces Akt/mTOR/p70S6K activation and inhibits proliferation and survival of A549 human lung cancer cells.

    PubMed

    Moore, Jessy; Megaly, Mark; MacNeil, Adam J; Klentrou, Panagiota; Tsiani, Evangelia

    2016-10-01

    Compounds of plant origin and food components have attracted scientific attention for use as agents for cancer prevention and treatment. Rosemary extract contains polyphenols that were shown to have anti-cancer and other health benefits. The survival pathways of Akt, mammalian target of rapamycin (mTOR) and p70S6K, and the apoptotic protein poly ADP ribose polymerase (PARP) are key modulators of cancer cell growth and survival. In this study, we examined the effects of rosemary extract on proliferation, survival and apoptosis of human non-small cell lung cancer (NSCLC) cells and its influence on signaling events. Human NSCLC adenocarcinoma A549 cells were used. Cell proliferation and clonogenic survival were assessed using specific assays. Immunoblotting was used to examine total and phosphorylated levels of Akt, mTOR and p70S6K, and cleavage of PARP. Rosemary extract dose-dependently inhibited cell proliferation and reduced clonogenic survival of A549 cells, while PARP cleavage, an indicator of apoptosis, was enhanced. Rosemary extract significantly reduced total and phosphorylated/activated Akt, mTOR and p70S6K levels. In conclusion, rosemary extract inhibited proliferation, blocked clonogenic survival, and enhanced apoptosis of A549 lung cancer cells. These effects were associated with inhibition of Akt and downstream mTOR and p70S6K activity. Our data suggest that rosemary extract may have considerable anti-tumor and chemoprevention properties in lung cancer and deserves further systematic investigation in animal models of lung cancer.

  7. Combined toxic effect of airborne heavy metals on human lung cell line A549.

    PubMed

    Choi, Yeowool; Park, Kihong; Kim, Injeong; Kim, Sang D

    2016-11-25

    Many studies have demonstrated that heavy metals existing as a mixture in the atmospheric environment cause adverse effects on human health and are important key factors of cytotoxicity; however, little investigation has been conducted on a toxicological study of a metal mixture from atmospheric fine particulate matter. The objective of this study was to predict the combined effects of heavy metals in aerosol by using in vitro human cells and obtain a suitable mixture toxicity model. Arsenic, nickel, and lead were selected for mixtures exposed to A549 human lung cancer cells. Cell proliferation (WST-1), glutathione (GSH), and interleukin (IL)-8 inhibition were observed and applied to the prediction models of mixture toxicity, concentration addition (CA) and independent action (IA). The total mixture concentrations were set by an IC10-fixed ratio of individual toxicity to be more realistic for mortality and enzyme inhibition tests. The results showed that the IA model was statistically closer to the observed results than the CA model in mortality, indicating dissimilar modes of action. For the GSH inhibition, the results predicted by the IA and CA models were highly overestimated relative to mortality. Meanwhile, the IL-8 results were stable with no significant change in immune reaction related to inflammation. In conclusion, the IA model is a rapid prediction model in heavy metals mixtures; mortality, as a total outcome of cell response, is a good tool for demonstrating the combined toxicity rather than other biochemical responses.

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

    NASA Astrophysics Data System (ADS)

    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.

  9. Transcriptome Profiles of Human Lung Epithelial Cells A549 Interacting with Aspergillus fumigatus by RNA-Seq

    PubMed Central

    Jia, Xiaodong; Wang, Shuo; Wang, Jing; Chen, Yong; Zhao, Jingya; Tian, Shuguang; Han, Xuelin; Han, Li

    2015-01-01

    Lung epithelial cells constitute the first defense line of host against the inhaled Aspergillus fumigatus; however, the transcriptional response of human alveolar type II epithelial cells was still unclear. Here we used RNA-Seq technology to assess the transcriptome profiles of A549 cells following direct interaction with conidia of A. fumigatus. The total number of identified genes was 19118. Compared with uninfected A549 cells, 459 genes were differentially expressed in cells co-incubated with conidia for 8 h, including 302 up-regulated genes and 157 down-regulated genes. GO and KEGG pathway enrichment analysis showed that most of the up-regulated genes were related to immune response, chemotaxis and inflammatory response and enriched in cytokine-cytokine receptor interaction, JAK-STAT and MAPK signaling pathways. The down-regulated genes were mainly enriched for terms associated with development, hemopoiesis and ion transport. Among them, EGR4 and HIST1H4J gene had the maximum of fold change in up-regulated and down-regulated genes, respectively. Fourteen up-regulated genes and three down-regulated genes were further validated and significant increase on expression of IL-6, IL-8 and TNF-α in A549 cells were confirmed by qRT-PCR during the interaction of A549 cells with A. fumigatus. Besides, western blot showed that expression of two proteins (ARC, EGR1) significantly increased in A549 cells during interaction with A. fumigatus conidia for 8h. Interference of endogenous expression of ARC or EGR1 protein in A549 cells reduced the internalization of A. fumigatus. These results provided important insights into dynamic changes of gene expression in lung epithelial cells, especially its strong immunological response against A. fumigatus infection. PMID:26273834

  10. Different mechanisms for metal-induced adaptation to cadmium in the human lung cell lines A549 and H441.

    PubMed

    Sauvageau, Josée-Anne; Jumarie, Catherine

    2013-06-01

    Sensitivity to Cd and Zn as well as the capacity to develop tolerance were characterized in human lung cells A549 and H441. In the A549 cells, a 2-fold lower LC(50) was obtained for Cd compared to Zn, whereas H441 cells were similarly sensitive to both metals. H441 cells were twice as resistant to Cd as the A549 cells. Higher HSP70, but not metallothionein (MT) or glutathione (GSH) levels, could contribute to this better resistance. A 1.5- and 2-fold increase in the LC(50) for Cd was obtained in the A549 cells pre-exposed to non-cytotoxic concentrations of Cd (20 μM) or Zn (40 μM) for 24 h. On the other hand, only Zn increased H441 cells' resistance to Cd. Maximum Zn- and Cd-induced tolerances were reached as early as 3 and 12 h, respectively. Increases in MT-IIa and HSP70 messenger RNA levels were higher in A549 cells, but cycloheximide eliminated the induction of tolerance only in the H441 cells. Protein synthesis is a prerequisite for metal-induced tolerance to Cd in the H441 cells but not the A549 cells. Results obtained with L-buthionine sulfoximine revealed that GSH synthesis is not responsible for the acquired tolerance in both cell lines. However, GSH plays a critical role against Cd toxicity, and pro-oxidant conditions sensitized cells to Cd with different impacts on the metal-induced mechanisms of acquired tolerance. GSH and catalase both provide antioxidative protection, but only the stress related to low GSH content, not that resulting from catalase inhibition, may be alleviated with Zn.

  11. Pomegranate fruit extract inhibits prosurvival pathways in human A549 lung carcinoma cells and tumor growth in athymic nude mice.

    PubMed

    Khan, Naghma; Hadi, Naghma; Afaq, Farrukh; Syed, Deeba N; Kweon, Mee-Hyang; Mukhtar, Hasan

    2007-01-01

    Developing novel mechanism-based chemopreventive approaches for lung cancer through the use of dietary substances which humans can accept has become an important goal. In the present study, employing normal human bronchial epithelial cells (NHBE) and human lung carcinoma A549 cells, we first compared the growth inhibitory effects of pomegranate fruit extract (PFE). Treatment of PFE (50-150 microg/ml) for 72 h was found to result in a decrease in the viability of A549 cells but had only minimal effects on NHBE cells as assessed by the MTT and Trypan blue assays. PFE treatment of A549 cells also resulted in dose-dependent arrest of cells in G0-G1 phase of the cell cycle (as assessed by DNA cell cycle analysis). We further found that PFE treatment also resulted in (i) induction of WAF1/p21 and KIP1/p27, (ii) decrease in the protein expressions of cyclins D1, D2 and E, and (iii) decrease in cyclin-dependent kinase (cdk) 2, cdk4 and cdk6 expression. The treatment of cells with PFE inhibited (i) phosphorylation of MAPK proteins, (ii) inhibition of PI3K, (iii) phosphorylation of Akt at Thr308, (iv) NF-kappaB and IKKalpha, (v) degradation and phosphorylation of IkappaBalpha, and (vi) Ki-67 and PCNA. We also found that PFE treatment to A549 cells resulted in inhibition of NF-kappaB DNA-binding activity. Oral administration of PFE (0.1 and 0.2%, wt/vol) to athymic nude mice implanted with A549 cells resulted in a significant inhibition in tumor growth. Our results provide a suggestion that PFE can be a useful chemopreventive/chemotherapeutic agent against human lung cancer.

  12. Effect of copper overload on the survival of HepG2 and A-549 human-derived cells.

    PubMed

    Arnal, N; de Alaniz, M J T; Marra, C A

    2013-03-01

    We investigated the effect of copper (Cu) overload (20-160 µM/24 h) in two cell lines of human hepatic (HepG2) and pulmonary (A-549) origin by determining lipid and protein damage and the response of the antioxidant defence system. A-549 cells were more sensitive to Cu overload than HepG2 cells. A marked increase was observed in both the cell lines in the nitrate plus nitrite concentration, protein carbonyls and thiobarbituric acid reactive substances (TBARS). The TBARS increase was consistent with an increment in saturated fatty acids at the expense of polyunsaturated acids in a Cu concentration-dependent fashion. Antioxidant enzymes were stimulated by Cu overload. Superoxide dismutase activity increased significantly in both the cell lines, with greater increases in HepG2 than in A-549 cells. A marked increase in ceruloplasmin and metallothionein content in both the cell types was also observed. Dose-dependent decreases in α-tocopherol and ferric reducing ability were observed. Total glutathione content was lower in A-549 cells and higher in HepG2. Calpain and caspase-3 were differentially activated in a dose-dependent manner under copper-induced reactive oxygen species production. We conclude that Cu exposure of human lung- and liver-derived cells should be considered a reliable experimental system for detailed study of mechanism/mechanisms by which Cu overload exerts its deleterious effects.

  13. Ursolic Acid Reduces Mycobacterium tuberculosis-Induced Nitric Oxide Release in Human Alveolar A549 cells.

    PubMed

    Zerin, Tamanna; Lee, Minjung; Jang, Woong Sik; Nam, Kung-Woo; Song, Ho-Yeon

    2015-07-01

    Alveolar epithelial cells have been functionally implicated in Mycobacterium tuberculosis infection. This study investigated the role of ursolic acid (UA)-a triterpenoid carboxylic acid with potent antioxidant, anti-tumor, anti-inflammatory, and anti-tuberculosis properties in mycobacterial infection of alveolar epithelial A549 cells. We observed that M. tuberculosis successfully entered A549 cells. Cytotoxi-city was mediated by nitric oxide (NO). A549 toxicity peaked along with NO generation 72 h after infection. The NO generated by mycobacterial infection in A549 cells was insufficient to kill mycobacteria, as made evident by the mycobacteria growth indicator tube time to detect (MGIT TTD) and viable cell count assays. Treatment of mycobacteria-infected cells with UA reduced the expression of inducible nitric oxide synthase, NO generation, and eventually improved cell viability. Moreover, UA was found to quench the translocation of the transcription factor, nuclear factor kappa B (NF-κB), from the cytosol to the nucleus in mycobacteria-infected cells. This study is the first to demonstrate the cytotoxic role of NO in the eradication of mycobacteria and the role of UA in reducing this cytotoxicity in A549 cells.

  14. Nedaplatin sensitization of cisplatin-resistant human non-small cell lung cancer cells

    PubMed Central

    WANG, HUAN; ZHU, XIAOLI; HUANG, JING; CHEN, PINGSHENG; HAN, SHUHUA; YAN, XING

    2016-01-01

    Cisplatin (DDP) has been one of the most widely used chemotherapy drugs for advanced non-small cell lung cancer. However, the increase in the number of DDP-resistant cancer cells has become a major impediment in the clinical management of cancer. In the present study, for the first time, the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide assay was used to demonstrate that nedaplatin (NDP) could have a stronger inhibitory effect than DDP alone in DDP-resistant A549 (A549DDP) cells and that it could attenuate the resistance of these cells. Additionally, flow cytometry analysis showed that the apoptosis rate of these resistant cells when exposed to NDP was markedly increased and the number of cells in the G2 stage of the cell cycle was significantly increased. Furthermore, western blot analysis indicated that NDP decreased the protein expression of P-glycoprotein, tumor protein p53 and B-cell lymphoma 2, and increased the expression of Bcl-2-associated X protein, all of which could possibly improve the NDP intracellular drug concentration and promote cell apoptosis. These observations suggested that NDP could have higher efficacy in DDP-resistant lung cancer cells, and further studies applying more detailed analyses are warranted to elucidate the mechanism(s) behind this effect. PMID:27073518

  15. 7-O-geranylquercetin-induced autophagy contributes to apoptosis via ROS generation in human non-small cell lung cancer cells.

    PubMed

    Wang, En-Xia; Zou, Bo-Yang; Shi, Lei; Du, Lin-Ying; Zhu, Yan-Yan; Jiang, Ya-Meng; Ma, Xiao-Dong; Kang, Xiao-Hui; Wang, Chang-Yuan; Zhen, Yu-Hong; Sun, Li-Dan

    2017-07-01

    To investigate the antitumor effects of 7-O-geranylquercetin (GQ), a novel O-alkylated derivative of quercetin, against non-small cell lung cancer (NSCLC) cell lines A549 and NCI-H1975 and the corresponding mechanisms. Cell viability was assessed using MTT assay. The expression of proteins involved in apoptosis and autophagy was measured using western blotting. Besides, apoptosis was determined with DAPI staining, Annexin V-PI staining and transmission electron microscopy (TEM) assay, and autophagy was observed with TEM assay. Cell cycle and reactive oxygen species (ROS) level were detected using flow cytometry. GQ inhibited viability of A549 and NCI-H1975 cells in a dose- and time-dependent manner without apparent cytotoxicity to normal human lung fibroblast cells. GQ down-regulated the expression of apoptosis-related proteins pro-caspase 3 and Bcl-2, and up-regulated the expression of cleaved-PARP and Bax in A549 and NCI-H1975 cells. Meanwhile, GQ-induced cell apoptosis could be attenuated by caspase inhibitor Z-VAD-FMK. Besides, GQ induced autophagosome formation in A549 and NCI-H1975 cells, promoted the expression of autophagy-related proteins LC3-II and Beclin 1, and suppressed the expression of p62. Autophagy inhibition with chloroquine or Beclin 1 siRNA could effectively inhibit GQ-induced apoptosis. Furthermore, GQ treatment increased the generation of ROS, and ROS inhibitor N-acetylcysteine could reverse GQ-induced autophagy and apoptosis. Taken together, GQ could induce apoptosis and autophagy via ROS generation in A549 and NCI-H1975 cells, and GQ-induced autophagy contributed to apoptosis. Our findings highlight that GQ is a promising anticancer agent for the treatment of lung cancer. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Molecular mechanisms underlying mangiferin-induced apoptosis and cell cycle arrest in A549 human lung carcinoma cells

    PubMed Central

    SHI, WEI; DENG, JIAGANG; TONG, RONGSHENG; YANG, YONG; HE, XIA; LV, JIANZHEN; WANG, HAILIAN; DENG, SHAOPING; QI, PING; ZHANG, DINGDING; WANG, YI

    2016-01-01

    Mangiferin, which is a C-glucosylxanthone (1,3,6,7-tetrahydroxyxanthone-C2-β-D-glucoside) purified from plant sources, has recently gained attention due to its various biological activities. The present study aimed to determine the apoptotic effects of mangiferin on A549 human lung adenocarcinoma cells. In vitro studies demonstrated that mangiferin exerted growth-inhibitory and apoptosis-inducing effects against A549 cells. In addition, mangiferin exhibited anti-tumor properties in A549 xenograft mice in vivo. Mangiferin triggered G2/M phase cell cycle arrest via down-regulating the cyclin-dependent kinase 1-cyclin B1 signaling pathway, and induced apoptotic cell death by inhibiting the protein kinase C-nuclear factor-κB pathway. In addition, mangiferin was able to enhance the antiproliferative effects of cisplatin on A549 cells, thus indicating the potential for a combined therapy. Notably, mangiferin exerted anticancer effects in vivo, where it was able to markedly decrease the volume and weight of subcutaneous tumor mass, and expand the lifespan of xenograft mice. The present study clarified the molecular mechanisms underlying mangiferin-induced antitumor activities, and suggested that mangiferin may be considered a potential antineoplastic drug for the future treatment of cancer. PMID:26935347

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

    PubMed

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

    2013-02-04

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

  18. Capsaicin-induced genotoxic stress does not promote apoptosis in A549 human lung and DU145 prostate cancer cells.

    PubMed

    Lewinska, Anna; Jarosz, Paulina; Czech, Joanna; Rzeszutek, Iwona; Bielak-Zmijewska, Anna; Grabowska, Wioleta; Wnuk, Maciej

    2015-02-01

    Capsaicin is the major pungent component of the hot chili peppers of the genus Capsicum, which are consumed worldwide as a food additive. More recently, the selective action of capsaicin against cancer cells has been reported. Capsaicin was found to induce apoptosis and inhibit proliferation of a wide range of cancer cells in vitro, whereas being inactive against normal cells. As data on capsaicin-induced genotoxicity are limited and the effects of capsaicin against human lung A549 and DU145 prostate cancer cells were not explored in detail, we were interested in determining whether capsaicin-associated genotoxicity may also provoke A549 and DU145 cell death. Capsaicin-induced decrease in metabolic activity and cell proliferation, and changes in the cell cycle were limited to high concentrations used (≥ 100 μM), whereas, at lower concentrations, capsaicin stimulated both DNA double strand breaks and micronuclei production. Capsaicin was unable to provoke apoptotic cell death when used up to 250 μM concentrations. Capsaicin induced oxidative stress, but was ineffective in provoking the dissipation of the mitochondrial inner transmembrane potential. A different magnitude of p53 binding protein 1 (53BP1) recruitment contributed to diverse capsaicin-induced genotoxic effects in DU145 and A549 cells. Capsaicin was also found to be a DNA hypermethylating agent in A549 cells. In summary, we have shown that genotoxic effects of capsaicin may contribute to limited susceptibility of DU145 and A549 cancer cells to apoptosis in vitro, which may question the usefulness of capsaicin-based anticancer therapy, at least in a case of lung and prostate cancer.

  19. Involvement of lysosomal dysfunction in silver nanoparticle-induced cellular damage in A549 human lung alveolar epithelial cells.

    PubMed

    Miyayama, Takamitsu; Matsuoka, Masato

    2016-01-01

    While silver nanoparticles (AgNPs) are widely used in consumer and medical products, the mechanism by which AgNPs cause pulmonary cytotoxicity is not clear. AgNP agglomerates are found in endo-lysosomal structures within the cytoplasm of treated cells. In this study, the functional role of lysosomes in AgNP-induced cellular damage was examined in A549 human lung alveolar epithelial cells. We evaluated the intracellular distribution of AgNPs, lysosomal pH, cellular viability, Ag dissolution, and metallothionein (MT) mRNA levels in AgNP-exposed A549 cells that were treated with bafilomycin A1, the lysosomal acidification inhibitor. Exposure of A549 cells to citrate-coated AgNPs (20 nm diameter) for 24 h induced cellular damage and cell death at 100 and 200 μg Ag/ml, respectively. Confocal laser microscopic examination of LysoTracker-stained cells showed that AgNPs colocalized with lysosomes and their agglomeration increased in a dose-dependent manner (50-200 μg Ag/ml). In addition, the fluorescence signals of LysoTracker were reduced following exposure to AgNPs, suggesting the elevation of lysosomal pH. Treatment of A549 cells with 200 nM bafilomycin A1 and AgNPs (50 μg Ag/ml) further reduced the fluorescence signals of LysoTracker. AgNP-induced cell death was also increased by bafilomycin A1 treatment. Finally, treatment with bafilomycin A1 suppressed the dissolution of Ag and decreased the mRNA expression levels of MT-I and MT-II following exposure to AgNPs. The perturbation of lysosomal pH by AgNP exposure may play a role in AgNP agglomeration and subsequent cellular damage in A549 cells.

  20. Physalin A induces G2/M phase cell cycle arrest in human non-small cell lung cancer cells: involvement of the p38 MAPK/ROS pathway.

    PubMed

    Kang, Ning; Jian, Jun-Feng; Cao, Shi-Jie; Zhang, Qiang; Mao, Yi-Wei; Huang, Yi-Yuan; Peng, Yan-Fei; Qiu, Feng; Gao, Xiu-Mei

    2016-04-01

    Physalin A (PA) is an active withanolide isolated from Physalis alkekengi var. franchetii, a traditional Chinese herbal medicine named Jindenglong, which has long been used for the treatment of sore throat, hepatitis, and tumors in China. In the present study, we firstly investigated the effects of PA on proliferation and cell cycle distribution of the human non-small cell lung cancer (NSCLC) A549 cell line, and the potential mechanisms involved. Here, PA inhibited cell growth in dose- and time-dependent manners. Treatment of A549 cells with 28.4 μM PA for 24 h resulted in approximately 50 % cell death. PA increased the amount of intracellular ROS and the proportion of cells in G2/M. G2/M arrest was attenuated by the addition of ROS scavenger NAC. ERK and P38 were triggered by PA through phosphorylation in a time-dependent manner. The phosphorylation of ERK and P38 were not attenuated by the addition of NAC, but the use of the p38 inhibitor could reduce, at least in part, PA-induced ROS and the proportion of cells in G2/M. PA induces G2/M cell cycle arrest in A549 cells involving in the p38 MAPK/ROS pathway. This study suggests that PA might be a promising therapeutic agent against NSCLC.

  1. Albumin nanocapsules containing fenretinide: pre-clinical evaluation of cytotoxic activity in experimental models of human non-small cell lung cancer.

    PubMed

    Pignatta, Sara; Orienti, Isabella; Falconi, Mirella; Teti, Gabriella; Arienti, Chiara; Medri, Laura; Zanoni, Michele; Carloni, Silvia; Zoli, Wainer; Amadori, Dino; Tesei, Anna

    2015-02-01

    The present study deals with the preparation of albumin nanocapsules containing fenretinide and their evaluation in experimental models of human non-small cell lung cancer. These nanocapsules showed enhanced antitumor activity with respect to free fenretinide due to the solubilization effect of albumin on the hydrophobic drug, known to improve bioavailability. The high expression of caveolin-1 on the A549 cell surface further enhanced the antitumor activity of the nanoencapsulated fenretinide. Caveolin-1 favored albumin uptake and improved the efficacy of the fenretinide-loaded albumin nanocapsules, especially in 3-D cultures where the densely packed 3-D structures impaired drug diffusibility and severely reduced the activity of the free drug. The efficacy of the fenretinide albumin nanocapsules was further confirmed in tumor xenograft models of A549 by the significant delay in tumor progression observed with respect to control after intravenous administration of the novel formulation. This study describes the preparation of fenretinide containing albumin nanocapsules and their evaluation in experimental models of non-small cell lung cancer, showing enhanced antitumor activity compared to free fenretinide. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. Bostrycin inhibits proliferation of human lung carcinoma A549 cells via downregulation of the PI3K/Akt pathway

    PubMed Central

    2011-01-01

    Background Bostrycin is a novel compound isolated from marine fungi that inhibits proliferation of many cancer cells. However, the inhibitory effect of bostrycin on lung cancers has not been reported. This study is to investigate the inhibitory effects and mechanism of bostrycin on human lung cancer cells in vitro. Methods We used MTT assay, flow cytometry, microarray, real time PCR, and Western blotting to detect the effect of bostrycin on A549 human pulmonary adenocarcinoma cells. Results We showed a significant inhibition of cell proliferation and induction of apoptosis in bostrycin-treated lung adenocarcinoma cells. Bostrycin treatment caused cell cycle arrest in the G0/G1 phase. We also found the upregulation of microRNA-638 and microRNA-923 in bostrycin-treated cells. further, we found the downregulation of p110α and p-Akt/PKB proteins and increased activity of p27 protein after bostrycin treatment in A549 cells. Conclusions Our study indicated that bostrycin had a significant inhibitory effect on proliferation of A549 cells. It is possible that upregulation of microRNA-638 and microRNA-923 and downregulaton of the PI3K/AKT pathway proteins played a role in induction of cell cycle arrest and apoptosis in bostrycin-treated cells. PMID:21303527

  3. In vitro evaluation of the cellular effect of indium tin oxide nanoparticles using the human lung adenocarcinoma A549 cells.

    PubMed

    Tabei, Yosuke; Sonoda, Akinari; Nakajima, Yoshihiro; Biju, Vasudevanpillai; Makita, Yoji; Yoshida, Yasukazu; Horie, Masanori

    2015-05-01

    Indium tin oxide (ITO) is widely used in liquid crystal displays (LCDs) or plasma and mobile phone displays. Elevated production and usage of ITO in such displays have led to increased concerns over the safety of industrial workers exposed to particulate aerosols produced during cutting, grinding and polishing of these materials. However, the cellular effects of ITO nanoparticles (NPs) are still unclear, although it has been reported that micro-scale ITO particles induce cytotoxicity. The aim of this study was to examine the potential of ITO NPs to induce cytotoxicity, oxidative stress, and DNA damage using human lung adenocarcinoma A549 cells. Here, stable dispersions of a medium containing ITO NPs were obtained using pre-adsorption and centrifugal fractionation methods, and the A549 cells were incubated in this medium. The ITO NPs showed low cytotoxic effects as shown by the WST-1 and LDH assays. Transmission electron microscopy observations showed the cellular uptake of ITO NPs. The ITO NPs increased the intracellular level of reactive oxygen species and the expression of the heme oxygenase 1 gene. Further, the results of alkaline comet assays showed that ITO NPs induced DNA damage. Thus, these results suggest that ITO NPs possess a genotoxic potential on human lung adenocarcinoma A549 cells.

  4. The change of intracellular pH is involved in the cisplatin-resistance of human lung adenocarcinoma A549/DDP cells.

    PubMed

    Huang, Zhenhua; Huang, Youguo

    2005-01-01

    We had reported that the intracellular pH (pHi) of human lung adenocarcinoma A549 cells, which is sensitive to cisplatin, was more acidic than that of cisplatin-resistant A549/DDP cells. The correlation between the change of the pHi and cisplatin-resistance of A549/DDP cells was further studied by altering pHi in consequence of the change of CO2 concentration of the incubator. The pHi alterations of the cells were monitored by using the fluorescence probe of BCECF-AM. The results indicated that the pHi was more alkaline at lower CO2 concentration (2% CO2 in the incubator) and more acidic at higher CO2 concentration (8% CO2 in the incubator) for both A549 and A549/DDP cells compared with those of both A549 and A549/DDP cells cultured at 5% CO2 as the normal condition. Accumulation of bodipy-cisplatin, a fluorescence probe used for drug resistance assays, in A549 cells incubated at 2%, 5%, and 8% CO2 was increased 8.4%, 17.4%, and 23.5% compared to A549/DDP cells, respectively. Intracellular sequestration and distribution of bodipy-cisplatin imaged by laser scanning confocal microscopy indicated that bodipy-cisplatin was more encapsulated in acidic compartments of A549/DDP cells as shown with acridine orange, a dye that specifically labels acidic organelles in the cells. These results can be further confirmed in liposome systems with different pH gradients. It is proposed from the above results that the change of pHi in especially more acidic compartments in A549/DDP cells involves their cisplatin resistance.

  5. Factors involved in depletion of glutathione from A549 human lung carcinoma cells: implications for radiotherapy. [Buthionine sulfoximine

    SciTech Connect

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

    1984-08-01

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

  6. Induction of apoptosis in human lung carcinoma A549 epithelial cells with an ethanol extract of Tremella mesenterica.

    PubMed

    Chen, Nan-Yin; Lai, Hsi-Huai; Hsu, Tai-Hao; Lin, Fang-Yi; Chen, Jian-Zhi; Lo, Hui-Chen

    2008-05-01

    Tremella mesenterica (TM) is a common food and folk medicine widely used in several Asian countries as a tonic for the lungs. In the present study, we compared the effects of extracellular polysaccharides (EPS), intracellular polysaccharides (IPS), and ethanol extract (EE) of Tremella mesenterica on the induction of apoptosis into human lung carcinoma A549 epithelial cells. The EE, but not the EPS or the IPS, almost completely inhibited the growth of A549 cells. The results of Annexin V-FITC/PI staining and flow cytometric analysis indicated that the percentage of Annexin V(+)/PI(-) cells in EE-treated cells increased to 32.8%. The results of further investigation showed a disruption of mitochondrial transmembrane potential (DeltaPsi(m)), the production of reactive oxygen species (ROS), and the activation of caspase-3 protein in EE-treated cells. These findings suggest that EE can decrease cell viability and induce apoptosis in A549 cell lines by activating a mitochondrial pathway.

  7. Vitamin D Analogs Potentiate the Antitumor Effect of Imatinib Mesylate in a Human A549 Lung Tumor Model

    PubMed Central

    Maj, Ewa; Filip-Psurska, Beata; Świtalska, Marta; Kutner, Andrzej; Wietrzyk, Joanna

    2015-01-01

    In previous papers, we presented data on studies on the anticancer activity of the vitamin D3 analogs, named PRI-2191 and PRI-2205, in different cancer models. In this study, we showed the improved antiproliferative activity of a combination of imatinib mesylate (Gleevec, GV) and cytostatic agents in in vitro studies, when used with a third compound, namely PRI-2191, in an A549 human lung cancer model. Furthermore, we analyzed the influence of both PRI-2191, as well as PRI-2205 on the anticancer activity of GV in mice bearing A549 tumors. The route of PRI-2191 analog administration showed a significant impact on the outcome of GV treatment: subcutaneous injection was more efficient and less toxic than oral gavage. Moreover, both vitamin D compounds increased the anticancer activity of GV; however, they might also potentiate some adverse effects. We also evaluated in tumor tissue the expression of VEGF, PDGF-BB, vitamin D receptor, CYP27B1, CYP24, p53 and Bcl-2, as well as PDGF receptors: α and β. We observed the upregulation of p53 expression and the downregulation of Bcl-2, as well as VEGF in A549 tumors as a result of the tested treatment. However, vitamin D analogs did not significantly influence the expression of these proteins. PMID:26580599

  8. In vitro and in vivo therapeutic efficacy of CXCR4 antagonist BKT140 against human non-small cell lung cancer.

    PubMed

    Fahham, Duha; Weiss, Ido D; Abraham, Michal; Beider, Katia; Hanna, Wald; Shlomai, Zippora; Eizenberg, Orly; Zamir, Gideon; Izhar, Uzi; Shapira, Oz M; Peled, Amnon; Wald, Ori

    2012-11-01

    CXCR4/CXCL12 interactions promote non-small cell lung cancer (NSCLC) growth and dissemination. Furthermore, this axis might promote NSCLC resistance to chemotherapy and/or radiotherapy. Therefore, the CXCR4/CXCL12 axis constitutes an attractive therapeutic target for the treatment of NSCLC. We aimed to characterize the therapeutic efficacy of the novel CXCR4 antagonist BKT140 against human NSCLC. We determined the CXCR4 expression in 5 NSCLC cell lines (H358, A549, H460, H1299, and L4). We then tested the colony-forming capacity and proliferation of these cells in the presence of CXCL12 and BKT140. Next, we measured the in vivo growth of A549 and H460 xenografts with or without BKT140 treatment. Finally, we examined, in vitro, the potential antiproliferative effect of BKT140 combined with cisplatin or paclitaxel and after irradiation of NSCLC cells. All tested cell lines expressed CXCR4 and showed increased colony formation in response to CXCL12 stimulation. BKT140 reduced the colony-forming capacity of NSCLC cells. Proliferation assays demonstrated both cytotoxic and cytostatic properties for this peptide. H460 cells were the most sensitive to BKT140 and A549 cells the least. Subcutaneous administration of BKT140 significantly delayed the development of H460 xenografts and showed a similar trend for A549 xenografts. Finally, the antiproliferative effects of BKT140 appears to be additive to those of chemotherapeutic drugs and radiotherapy. Targeting the CXCL12/CXCR4 axis with BKT140 attenuated NSCLC cells tumor growth and augmented the effects of chemotherapy and radiotherapy. Future research will benefit from delineating the downstream mechanism of BKT140 action and defining BKT140 susceptibility markers. Crown Copyright © 2012. Published by Mosby, Inc. All rights reserved.

  9. Therapeutic effects of sorafenib on the A549/DDP human lung adenocarcinoma cell line in vitro.

    PubMed

    Chen, Xiang-Qi; Wang, Yu-Lan; Li, Zhi-Ying; Lin, Ting-Yan

    2014-07-01

    The aim of the present study was to observe the effects of sorafenib on the proliferation, apoptosis and invasion of A549/DDP cisplatin-resistant lung adenocarcinoma cells cultured in vitro. The A549/DDP cisplatin-resistant lung adenocarcinoma cell strain was cultured in vitro, the cell culture group incubated in culture medium only was set as the control group (Group S0) and the four concentration gradients of sorafenib were added to the culture groups as the experimental groups: S1, 2 µmol/l; S2, 4 µmol/l; S3, 8 µmol/l; and S4, 16 µmol/l. The MTT assay was used to determine the growth inhibition rate of the cells, which were respectively subjected to sorafenib treatment for 24, 48 and 72 h. Flow cytometry was used to determine the rate of apoptosis of cells in each group following sorafenib treatment for 72 h. Furthermore, the Transwell invasion experiment was used to determine the effect on A549/DDP cell invasion following sorafenib treatment for 24 h. Based on the MTT assay, it was found that the inhibition rates of A549/DDP cisplatin-resistant lung adenocarcinoma cells in groups S1-4 following sorafenib treatment for 24 h were 4.58±2.82, 14.93±2.62, 37.58±7.13 and 58.39±8.15%, respectively. For 48 h, inhibition rates in S1-4 were 14.98±2.93, 26.28±7.31, 63.00±3.05 and 78.84±3.96%, respectively, and for 72 h, inhibition rates were 18.80±2.82, 32.71±2.55, 75.51±4.73 and 87.50±3.36%, respectively. The difference in the inhibition rates of cells among the experimental groups for the same incubation time showed statistical significance (P<0.05). Flow cytometric analysis indicated that the rate of apoptosis in the control group was 8.88±0.81% following sorafenib treatment for 72 h, and the rates of apoptosis in groups S1-4 were, 12.84±0.24, 17.27±0.78, 21.98±0.75 and 49.67±1.38%, respectively. The rate of apoptosis in each experimental group was higher compared with that in the control group (P<0.05). The difference in the rate of apoptosis

  10. Carbon-Ion Beam Irradiation Effectively Suppresses Migration and Invasion of Human Non-Small-Cell Lung Cancer Cells

    SciTech Connect

    Akino, Yuichi; Teshima, Teruki Kihara, Ayaka; Kodera-Suzumoto, Yuko; Inaoka, Miho; Higashiyama, Shigeki; Furusawa, Yoshiya; Matsuura, Nariaki

    2009-10-01

    Purpose: Control of cancer metastasis is one of the most important issues in cancer treatment. We previously demonstrated that carbon particle irradiation suppresses the metastatic potential of cancer cells, and many studies have reported that photon irradiation promotes it. The purpose of this study was to investigate the effect of carbon beam on non-small-cell lung cancer (NSCLC) cell aggressiveness and gene expression. Methods and Materials: A549 (lung adenocarcinoma) and EBC-1 (lung squamous cell carcinoma) cells were treated with 290 MeV/nucleon carbon ion beam at the Heavy Ion Medical Accelerator in Chiba or with 4-MV X-ray at Osaka University. We tested proliferative, migratory, and invasive activities by cell proliferation assay, Boyden chamber assay, and Matrigel chemoinvasion assay, respectively. cDNA microarray and reverse transcription polymerase chain reaction were also performed to assess mRNA expression alteration. Results: X-irradiation increased cell proliferation of A549 cells at 0.5 Gy, whereas high-dose X-ray reduced migration and invasion of A549 cells. By contrast, carbon beam irradiation did not enhance proliferation, and it reduced the migration and invasion capabilities of both A549 and EBC-1 cells more effectively than did X-irradiation. Carbon beam irradiation induced alteration of various gene expression profiles differently from X-ray irradiation. mRNA expression of ANLN, a homologue of anillin, was suppressed to 60% levels of basal expression in carbon beam-irradiated A549 cells after 12 h. Conclusion: Carbon beam effectively suppresses the metastatic potential of A549 and EBC-1 cells. Carbon beam also has different effects on gene expressions, and downregulation of ANLN was induced only by carbon beam irradiation.

  11. A novel fluorinated thiosemicarbazone derivative- 2-(3,4-difluorobenzylidene) hydrazinecarbothioamide induces apoptosis in human A549 lung cancer cells via ROS-mediated mitochondria-dependent pathway.

    PubMed

    Zhao, Yue; Guo, Chuanlong; Wang, Lijun; Wang, Shuaiyu; Li, Xiangqian; Jiang, Bo; Wu, Ning; Guo, Shuju; Zhang, Renshuai; Liu, Kun; Shi, Dayong

    2017-09-09

    Thiosemicarbazone, a class of compounds with excellent biological activity, especially antitumor activity, have attracted wide attention. In this study, a novel fluorinated thiosemicarbazone derivative, 2-(3,4-difluorobenzylidene) hydrazinecarbothioamide (compound 1) was synthesized and its antitumor activities were further investigated on a non-small cell lung cancer cell line (A549) along with its underlying mechanisms. Compound 1 showed significant anti-proliferative activity on A549 cells, which was further proved by colony formation experiment. Compound 1 also inhibits the invasion of A549 cells in a trans-well culture system. Moreover, compound 1 markedly induced apoptosis on A549 cells, and the ratio of Bcl-2/Bax was decreased while the amount of p53, Cleaved-Caspase 3 and Cleaved-PARP expression were increased significantly. Compound 1 decreased the mitochondrial membrane potential, while the content of reactive oxygen was increased obviously. It is revealed that compound 1 mediated cell cycle arrest in G0/G1 phase by reducing G1 phase dependent proteins, CDK4 and Cyclin D1. As a result, it is indicated that compound 1 induced apoptosis on A549 cells was realized by regulating ROS-mediated mitochondria-dependent signaling pathway. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Cytotoxicity, oxidative stress and genotoxicity induced by glass fibers on human alveolar epithelial cell line A549.

    PubMed

    Rapisarda, Venerando; Loreto, Carla; Ledda, Caterina; Musumeci, Giuseppe; Bracci, Massimo; Santarelli, Lory; Renis, Marcella; Ferrante, Margherita; Cardile, Venera

    2015-04-01

    Man-made vitreous fibers have been widely used as insulation material as asbestos substitutes; however their morphology and composition raises concerns. In 1988 the International Agency for Research on Cancer classified fiberglass, rock wool, slag wool, and ceramic fibers as Group 2B, i.e. possibly carcinogenic to humans. In 2002 it reassigned fiberglass, rock and slag wool, and continuous glass filaments to Group 3, not classifiable as carcinogenic to humans. The aim of this study was to verify the cytotoxic and genotoxic effects and oxidative stress production induced by in vitro exposure of human alveolar epithelial cells A549 to glass fibers with a predominant diameter <3 μm (97%) and length >5 μm (93%). A549 cells were incubated with 5, 50, or 100 μg/ml (2.1, 21, and 42 μg/cm(2), respectively) of glass fibers for 72 h. Cytotoxicity and DNA damage were tested by the MTT and the Comet assay, respectively. Oxidative stress was determined by measuring inducible nitric oxide synthase (iNOS) expression by Western blotting, production of nitric oxide (NO) with Griess reagent, and concentration of reactive oxygen species by fluorescent quantitative analysis with 2',7'-dichlorofluorescein-diacetate (DCFH-DA). The results showed that glass fiber exposure significantly reduced cell viability and increased DNA damage and oxidative stress production in a concentration-dependent manner, demonstrating that glass fibers exert cytotoxic and genotoxic effects related to increased oxidative stress on the human alveolar cell line A549. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. Claudin-18 inhibits cell proliferation and motility mediated by inhibition of phosphorylation of PDK1 and Akt in human lung adenocarcinoma A549 cells.

    PubMed

    Shimobaba, Shun; Taga, Saeko; Akizuki, Risa; Hichino, Asami; Endo, Satoshi; Matsunaga, Toshiyuki; Watanabe, Ryo; Yamaguchi, Masahiko; Yamazaki, Yasuhiro; Sugatani, Junko; Ikari, Akira

    2016-06-01

    Abnormal expression of claudin subtypes has been reported in various cancers. However, the pathological role of each claudin has not been clarified in detail. Claudin-18 was absent in human non-small cell and small cell lung cancers, although it is expressed in normal lung tissues. Here, we examined the effect of claudin-18 expression on the expression of junctional proteins, cell proliferation, and cell motility using human lung adenocarcinoma A549 cells. Real-time PCR and western blotting showed that exogenous expression of claudin-18 had no effect on the expression of junctional proteins including claudin-1, zonula occludens-1 (ZO-1), occludin, and E-cadherin. Claudin-18 was mainly distributed in cell-cell contact areas concomitant with ZO-1. Cell proliferation was significantly decreased at 48 and 72h after seeding of claudin 18-expressing cells. Claudin-18 suppressed cell motility, whereas it increased cell death in anoikis. Claudin-18 decreased phosphorylated (p)-3-phosphoinositide-dependent protein kinase-1 (PDK1) and p-Akt levels without affecting p-epidermal growth factor receptor and p-phosphatidylinositol-3 kinase (PI3K) levels. Furthermore, claudin-18 was bound with PDK1 and suppressed the nuclear localization of PDK1. We suggest that claudin-18 suppresses the abnormal proliferation and motility of lung epithelial cells mediated by inhibition of the PI3K/PDK1/Akt signaling pathway.

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

    SciTech Connect

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

    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.

  15. A Comprehensive Proteomic View of Responses of A549 Type II Alveolar Epithelial Cells to Human Respiratory Syncytial Virus Infection*

    PubMed Central

    Dave, Keyur A.; Norris, Emma L.; Bukreyev, Alexander A.; Headlam, Madeleine J.; Buchholz, Ursula J.; Singh, Toshna; Collins, Peter L.; Gorman, Jeffrey J.

    2014-01-01

    Human respiratory syncytial virus is a major respiratory pathogen for which there are no suitable antivirals or vaccines. A better understanding of the host cell response to this virus may redress this problem. The present report concerns analysis of multiple independent biological replicates of control and 24 h infected lysates of A549 cells by two different proteomic workflows. One workflow involved fractionation of lysates by in-solution protein IEF and individual fractions were digested using trypsin prior to capillary HPLC-LTQ-OrbitrapXL-MS/MS. A second workflow involved digestion of whole cell lysates and analysis by nanoUltraHPLC-LTQ-OrbitrapElite-MS/MS. Both workflows resulted in the quantification of viral proteins exclusively in lysates of infected cells in the relative abundances anticipated from previous studies. Unprecedented numbers (3247 - 5010) of host cell protein groups were also quantified and the infection-specific regulation of a large number (191) of these protein groups was evident based on a stringent false discovery rate cut-off (<1%). Bioinformatic analyses revealed that most of the regulated proteins were potentially regulated by type I, II, and III interferon, TNF-α and noncanonical NF-κB2 mediated antiviral response pathways. Regulation of specific protein groups by infection was validated by quantitative Western blotting and the cytokine-/key regulator-specific nature of their regulation was confirmed by comparable analyses of cytokine treated A549 cells. Overall, it is evident that the workflows described herein have produced the most comprehensive proteomic characterization of host cell responses to human respiratory syncytial virus published to date. These workflows will form the basis for analysis of the impacts of specific genes of human respiratory syncytial virus responses of A549 and other cell lines using a gene-deleted version of the virus. They should also prove valuable for the analysis of the impact of other infectious

  16. A comprehensive proteomic view of responses of A549 type II alveolar epithelial cells to human respiratory syncytial virus infection.

    PubMed

    Dave, Keyur A; Norris, Emma L; Bukreyev, Alexander A; Headlam, Madeleine J; Buchholz, Ursula J; Singh, Toshna; Collins, Peter L; Gorman, Jeffrey J

    2014-12-01

    Human respiratory syncytial virus is a major respiratory pathogen for which there are no suitable antivirals or vaccines. A better understanding of the host cell response to this virus may redress this problem. The present report concerns analysis of multiple independent biological replicates of control and 24 h infected lysates of A549 cells by two different proteomic workflows. One workflow involved fractionation of lysates by in-solution protein IEF and individual fractions were digested using trypsin prior to capillary HPLC-LTQ-OrbitrapXL-MS/MS. A second workflow involved digestion of whole cell lysates and analysis by nanoUltraHPLC-LTQ-OrbitrapElite-MS/MS. Both workflows resulted in the quantification of viral proteins exclusively in lysates of infected cells in the relative abundances anticipated from previous studies. Unprecedented numbers (3247 - 5010) of host cell protein groups were also quantified and the infection-specific regulation of a large number (191) of these protein groups was evident based on a stringent false discovery rate cut-off (<1%). Bioinformatic analyses revealed that most of the regulated proteins were potentially regulated by type I, II, and III interferon, TNF-α and noncanonical NF-κB2 mediated antiviral response pathways. Regulation of specific protein groups by infection was validated by quantitative Western blotting and the cytokine-/key regulator-specific nature of their regulation was confirmed by comparable analyses of cytokine treated A549 cells. Overall, it is evident that the workflows described herein have produced the most comprehensive proteomic characterization of host cell responses to human respiratory syncytial virus published to date. These workflows will form the basis for analysis of the impacts of specific genes of human respiratory syncytial virus responses of A549 and other cell lines using a gene-deleted version of the virus. They should also prove valuable for the analysis of the impact of other infectious

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

    SciTech Connect

    Chen, Tian Jun; Gao, Fei; Yang, Tian; Thakur, Asmitanand; Ren, Hui; Li, Yang; Zhang, Shuo; Wang, Ting; Chen, Ming Wei

    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.

  18. Cardiac glycosides induce autophagy in human non-small cell lung cancer cells through regulation of dual signaling pathways.

    PubMed

    Wang, Yan; Qiu, Qiang; Shen, Jia-Jia; Li, Dian-Dong; Jiang, Xue-Jun; Si, Shu-Yi; Shao, Rong-Guang; Wang, Zhen

    2012-11-01

    Na(+)/K(+)-ATPase targeted cancer therapy has attracted increasing interests of oncologists in lung cancer field. Although multiple anti-cancer mechanisms of cardiac glycosides as Na(+)/K(+)-ATPase inhibitors are revealed, the role of autophagy and related molecular signaling pathway for the class of compounds in human non-small cell lung cancer (NSCLC) cells has not been systematically examined. We herein investigated the anti-cancer effects of two representative cardiac glycosides, digoxin and ouabain, in A549 and H460 cell lines. Both agents caused significant growth inhibition at nanomolar level. The cardiac glycosides were found to induce moderate G(2)/M arrest but not apoptosis at IC(50) level in the NSCLC cell lines. Moreover, autophagy was markedly induced by both agents, as evidenced by the time- and dose-dependent increase of LC3-II, up-regulation of Atg5 and Beclin1, as well as by the observations through acridine orange staining, transmission electron microscopy and quantification of GFP-LC3 fluorescence. Importantly, AMP-activated protein kinase (AMPK) pathway was activated, resulting in mammalian target of rapamycin (mTOR) deactivation during autophagy induction. Moreover, extracellular-signal-regulated kinase 1/2 (ERK1/2) activation was simultaneously found to be involved in the autophagy regulation. Co-treatment with respective inhibitors or siRNAs could either block the autophagic phenotypes and signals, or significantly increase the cellular viability, indicating the drugs-induced autophagy plays tumor-suppressing role. This work provides first evidence showing that the cardiac glycosides induce autophagy in human NSCLC cells through regulation of both mTOR and ERK1/2 signaling pathways. The autophagy may at least partially account for the growth inhibitory effects of the compounds in human NSCLC cells.

  19. The verapamil transporter expressed in human alveolar epithelial cells (A549) does not interact with β2-receptor agonists.

    PubMed

    Salomon, Johanna J; Ehrhardt, Carsten; Hosoya, Ken-Ichi

    2014-01-01

      Affinity of different organs for verapamil is highly variable and organ-specific. For example, the drug exhibits high levels of accumulation in lung tissues. A transporter recognising verapamil as a substrate has previously been identified in human retinal pigment epithelial (RPE) and in rat retinal capillary endothelial (TR-iBRB2) cells. This transporter is distinct from any of the cloned organic cation transporters. Therefore, we hypothesised that the verapamil transporter is also functionally expressed in the human respiratory mucosa. Moreover, we tested the hypothesis that this transporter interacts with pulmonary administered cationic drugs such as β2-agonists. The uptake of [(3)H]verapamil was studied in A549 human alveolar epithelial cell monolayers at different times and concentrations. The influence of extracellular proton concentration and various organic cations on verapamil uptake was determined. Verapamil uptake into A549 cells was time- and concentration-dependent, sensitive to pH and had a Km value of 39.8 ± 8.2 µM. Verapamil uptake was also sensitive to inhibition by amantadine, quinidine and pyrilamine, but insensitive to other typical modulators of organic cation and choline transporters. Whilst we demonstrated functional activity of the elusive verapamil transporter at the lung epithelium, our data suggest that this transporter does not interact with β2-agonists at therapeutic concentrations.

  20. Coenzyme Q0 from Antrodia cinnamomea in Submerged Cultures Induces Reactive Oxygen Species-Mediated Apoptosis in A549 Human Lung Cancer Cells

    PubMed Central

    Chung, Cheng-Han; Lee, Kung-Ta

    2014-01-01

    We investigated the anticancer effects of Antrodia cinnamomea, a medicinal mushroom from Taiwan, on A549 human lung cancer cells using the ethyl acetate extract from submerged culture filtrates. Our results showed that 2,3-dimethoxy-5-methyl-1,4-benzoquinone (coenzyme Q0; CoQ0) derived from A. cinnamomea submerged culture filtrates has anticancer activity. CoQ0 treatment reduced the viability of A549, HepG2, and SW480 cancer cell lines. Furthermore, CoQ0 induced reactive oxygen species (ROS) generation and apoptosis in A549 cells, which was inhibited by the antioxidant ascorbic acid. To our knowledge, these data demonstrate for the first time that CoQ0 derived from A. cinnamomea submerged culture filtrates exerts its anticancer effect through the induction of ROS-mediated apoptosis in A549 human lung cancer cells. PMID:25431605

  1. Chlorella sorokiniana induces mitochondrial-mediated apoptosis in human non-small cell lung cancer cells and inhibits xenograft tumor growth in vivo.

    PubMed

    Lin, Ping-Yi; Tsai, Ching-Tsan; Chuang, Wan-Ling; Chao, Ya-Hsuan; Pan, I-Horng; Chen, Yu-Kuo; Lin, Chi-Chen; Wang, Bing-Yen

    2017-02-01

    Lung cancer is one of the leading causes of cancer related deaths worldwide. Marine microalgae are a source of biologically active compounds and are widely consumed as a nutritional supplement in East Asian countries. It has been reported that Chlorella or Chlorella extracts have various beneficial pharmacological compounds that modulate immune responses; however, no studies have investigated the anti-cancer effects of Chlorella sorokiniana (CS) on non-small cell lung cancer (NSCLC). In this study, we evaluated the anti-cancer effects of CS in two human NSCLC cell lines (A549 and CL1-5 human lung adenocarcinoma cells), and its effects on tumor growth in a subcutaneous xenograft tumor model. We also investigated the possible molecular mechanisms governing the pharmacological function of CS. Our results showed that exposure of the two cell lines to CS resulted in a concentration-dependent reduction in cell viability. In addition, the percentage of apoptotic cells increased in a dose-dependent manner, suggesting that CS might induce apoptosis in human NSCLC cells. Western blot analysis revealed that exposure to CS resulted in increased protein expression of the cleaved/activated forms of caspase-3, caspase-9, and PARP, except caspase-8. ZDEVD (caspase-3 inhibitor) and Z-LEHD (caspase-9 inhibitor) were sufficient at preventing apoptosis in both A549 and CL1-5 cells, proving that CS induced cell death via the mitochondria-mediated apoptotic pathway. Exposure of A549 and CL1-5 cells to CS for 24 h resulted in decreased expression of Bcl-2 protein and increased expression of Bax protein as well as decreased expression of two IAP family proteins, survivin and XIAP. We demonstrated that CS induces mitochondrial-mediated apoptosis in NSCLC cells via downregulation of Bcl-2, XIAP and survivin. In addition, we also found that the tumors growth of subcutaneous xenograft in vivo was markedly inhibited after oral intake of CS.

  2. Lipoteichoic acids from Staphylococcus aureus stimulate proliferation of human non-small-cell lung cancer cells in vitro.

    PubMed

    Hattar, Katja; Reinert, Christian P; Sibelius, Ulf; Gökyildirim, Mira Y; Subtil, Florentine S B; Wilhelm, Jochen; Eul, Bastian; Dahlem, Gabriele; Grimminger, Friedrich; Seeger, Werner; Grandel, Ulrich

    2017-03-17

    Pulmonary infections are frequent complications in lung cancer and may worsen its outcome and survival. Inflammatory mediators are suspected to promote tumor growth in non-small-cell lung cancer (NSCLC). Hence, bacterial pathogens may affect lung cancer growth by activation of inflammatory signalling. Against this background, we investigated the effect of purified lipoteichoic acids (LTA) of Staphylococcus aureus (S. aureus) on cellular proliferation and liberation of interleukin (IL)-8 in the NSCLC cell lines A549 and H226. A549 as well as H226 cells constitutively expressed TLR-2 mRNA. Even in low concentrations, LTA induced a prominent increase in cellular proliferation of A549 cells as quantified by automatic cell counting. In parallel, metabolic activity of A549 cells was enhanced. The increase in proliferation was accompanied by an increase in IL-8 mRNA expression and a dose- and time-dependent release of IL-8. Cellular proliferation as well as the release of IL-8 was dependent on specific ligation of TLR-2. Interestingly, targeting IL-8 by neutralizing antibodies completely abolished the LTA-induced proliferation of A549 cells. The pro-proliferative effect of LTA could also be reproduced in the squamous NSCLC cell line H226. In summary, LTA of S. aureus induced proliferation of NSCLC cell lines of adeno- and squamous cell carcinoma origin. Ligation of TLR-2 followed by auto- or paracrine signalling by endogenously synthesized IL-8 is centrally involved in LTA-induced tumor cell proliferation. Therefore, pulmonary infections may exert a direct pro-proliferative effect on lung cancer growth.

  3. Cellular uptake and toxic effects of fine and ultrafine metal-sulfate particles in human A549 lung epithelial cells.

    PubMed

    Könczöl, Mathias; Goldenberg, Ella; Ebeling, Sandra; Schäfer, Bianca; Garcia-Käufer, Manuel; Gminski, Richard; Grobéty, Bernard; Rothen-Rutishauser, Barbara; Merfort, Irmgard; Gieré, Reto; Mersch-Sundermann, Volker

    2012-12-17

    Ambient airborne particulate matter is known to cause various adverse health effects in humans. In a recent study on the environmental impacts of coal and tire combustion in a thermal power station, fine crystals of PbSO(4) (anglesite), ZnSO(4)·H(2)O (gunningite), and CaSO(4) (anhydrite) were identified in the stack emissions. Here, we have studied the toxic potential of these sulfate phases as particulates and their uptake in human alveolar epithelial cells (A549). Both PbSO(4) and CaSO(4) yielded no loss of cell viability, as determined by the WST-1 and NR assays. In contrast, a concentration-dependent increase in cytotoxicity was observed for Zn sulfate. For all analyzed sulfates, an increase in the production of reactive oxygen species (ROS), assessed by the DCFH-DA assay and EPR, was observed, although to a varying extent. Again, Zn sulfate was the most active compound. Genotoxicity assays revealed concentration-dependent DNA damage and induction of micronuclei for Zn sulfate and, to a lower extent, for CaSO(4), whereas only slight effects could be found for PbSO(4). Moreover, changes of the cell cycle were observed for Zn sulfate and PbSO(4). It could be shown further that Zn sulfate increased the nuclear factor kappa-B (NF-κB) DNA binding activity and activated JNK. During our TEM investigations, no effect on the appearance of the A549 cells exposed to CaSO(4) compared to the nonexposed cells was observed, and in our experiments, only one CaSO(4) particle was detected in the cytoplasm. In the case of exposure to Zn sulfate, no particles were found in the cytoplasm of A549 cells, but we observed a concentration-dependent increase in the number and size of dark vesicles (presumably zincosomes). After exposure to PbSO(4), the A549 cells contained isolated particles as well as agglomerates both in vesicles and in the cytoplasm. Since these metal-sulfate particles are emitted into the atmosphere via the flue gas of coal-fired power stations, they may be

  4. Extract from Nandina domestica inhibits lipopolysaccharide-induced cyclooxygenase-2 expression in human pulmonary epithelial A549 cells.

    PubMed

    Ueki, Takuro; Akaishi, Tatsuhiro; Okumura, Hidenobu; Abe, Kazuho

    2012-01-01

    Extract from fruits of Nandina domestica THUNBERG (NDE) has been used to improve cough and breathing difficulty in Japan for many years. To explore whether NDE may alleviate respiratory inflammation, we investigated its effect on expression of cyclooxygenase-2 (COX-2) and production of prostaglandin E₂ (PGE₂) in human pulmonary epithelial A549 cells in culture. Treatment with lipopolysaccharide (LPS; 6 µg/mL) resulted in an increase of COX-2 expression and PGE₂ production in A549 cells. Both the LPS-induced COX-2 expression and PGE₂ production were significantly inhibited by NDE (1-10 µg/mL) in a concentration-dependent manner. NDE did not affect COX-1 expression nor COX activity. These results suggest that NDE downregulates LPS-induced COX-2 expression and inhibits PGE₂ production in pulmonary epithelial cells. Furthermore, higenamine and nantenine, two major constituents responsible for tracheal relaxing effect of NDE, did not mimic the inhibitory effect of NDE on LPS-induced COX-2 expression in A549 cells. To identify active constituent(s) of NDE responsible for the anti-inflammatory effect, NDE was introduced in a polyaromatic absorbent resin column and stepwise eluted to yield water fraction, 20% methanol fraction, 40% methanol fraction, 99.8% methanol fraction, and 99.5% acetone fraction. However, none of these five fractions alone inhibited LPS-induced COX-2 expression. On the other hand, exclusion of water fraction from NDE abolished the inhibitory effect of NDE on LPS-induced COX-2 expression. These results suggest that constituent(s) present in water fraction is required but not sufficient for the anti-inflammatory activity of NDE, which may result from interactions among multiple constituents.

  5. Brazilian green propolis induced apoptosis in human lung cancer A549 cells through mitochondrial-mediated pathway.

    PubMed

    Frión-Herrera, Yahima; Díaz-García, Alexis; Ruiz-Fuentes, Jenny; Rodríguez-Sánchez, Hermis; Sforcin, José Maurício

    2015-10-01

    Propolis effect on the growth and apoptosis of human lung adenocarcinoma (A549 cells) was investigated as well as its mechanisms. Cells were incubated with propolis for 72 h, and 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase assays were employed to assess cell viability and the inhibitory concentration (IC). Apoptosis was detected by Acridine Orange/Ethidium Bromide and 4',6-diamidino-2-phenylindole staining after 24 and 48 h of incubation with ¼ IC50 of propolis by testing the mitochondrial membrane potential (ΔΨm) and the expression of apoptosis-related genes (p53, Caspase-3, Bax, Bcl-2, Bcl-XL , Noxa, Puma and p21) by reverse transcription polymerase chain reaction. Propolis displayed antiproliferative and cytotoxic effects on A549 cells in a dose- and time-dependent manner, but it did not suppress the growth of normal Vero cells. An enhanced apoptosis was seen in A549 propolis-treated cells after 48 h compared with the control cells. Propolis decreased mitochondrial membrane potential by overexpression of pro-apoptotic genes (Bax and Noxa) and reduction of the antiapoptotic gene Bcl-XL . The expression level of other genes remained unchanged (p53, Caspse-3 and Bax), whereas p21 expression was increased. Propolis induced caspase-independent apoptosis through a p53-independent mitochondrial pathway, and cell cycle arrest by upregulation of p21. Although propolis induces apoptosis mainly by p53-independent manner, it may be induced by another pathway, and new insights may arise for preventing or treating lung cancer. © 2015 Royal Pharmaceutical Society.

  6. Ultrasensitive cytosensing based on an aptamer modified nanobiosensor with a bioconjugate: Detection of human non-small-cell lung cancer cells.

    PubMed

    Mir, Tanveer A; Yoon, Jang-Hee; Gurudatt, N G; Won, Mi-Sook; Shim, Yoon-Bo

    2015-12-15

    A novel aptamer-based amperometric nanobiosensor was designed for the sensitive and selective detection of A549 human non-small-cell lung cancer (NSCLC) cells. The cytosensing was performed using a MUC1 aptamer probe with a bioconjugate, where the probe was fabricated by the covalent immobilization on a conducting polymer nanocomposite formed through the self-assembly of 4-([2,2':5',2''-terthiophen]-3'-yl) benzoic acid (TTBA) on AuNPs. A bioconjugate composed of hydrazine and aptamer attached on AuNPs was used to reveal the selectively amplified detection signal. The cells were quantitatively analyzed using chronoamperometric measurements, and the results were further compared and confirmed using microscopic and DPV methods based on silver staining cytosensing experiments. The proposed aptasensor showed a high affinity for MUC1 positive lung cancer cells (A549) compared with the other control cancer cells, including human prostate (PC3), MUC1 negative normal lung (MRC-5), and liver tumors (HepG2) cells. An excellent dynamic range of the proposed method was obtained from 15 to 1×10(6) cells/mL with a detection limit of 8 cells/mL.

  7. MicroRNA-1304 suppresses human non-small cell lung cancer cell growth in vitro by targeting heme oxygenase-1

    PubMed Central

    Li, Cheng-gang; Pu, Meng-fan; Li, Chun-zhu; Gao, Man; Liu, Ming-xia; Yu, Cun-zhi; Yan, Hong; Peng, Chun; Zhao, Yang; Li, Yu; Ma, Ze-long; Qi, Xin-ming; Wang, Yi-zheng; Miao, Ling-ling; Ren, Jin

    2017-01-01

    Previous studies have shown that microRNA-1304 (miR-1304) is dysregulated in certain types of cancers, including non-small cell lung cancer (NSCLC), and might be involved in tumor survival and/or growth. In this study we investigated the direct target of miR-1304 and its function in NSCLC in vitro. Human lung adenocarcinoma cell lines (A549 and NCI-H1975) were studied. The cell proliferation and survival were investigated via cell counting, MTT and colony-formation assays. Cell apoptosis and cell cycle were examined using annexin V-PE/7-AAD and PI staining assays, respectively. The dual-luciferase reporter assay was used to verify post-transcriptional regulation of heme oxygenase-1 (HO-1) by miR-1304. CRISPR/Cas9 was used to deplete endogenous miR-1304. Overexpression of MiR-1304 significantly decreased the number and viability of NSCLC cells and colony formation, and induced cell apoptosis and G0/G1 phase cell cycle arrest. HO-1 was demonstrated to be a direct target of miR-1304 in NSCLC cells. Restoration of HO-1 expression by hemin (20 μmol/L) abolished the inhibition of miR-1304 on cell growth and rescued miR-1304-induced apoptosis in A549 cells. Suppression of endogenous miR-1304 with anti-1304 significantly increased HO-1 expression and promoted cell growth and survival in A549 cells. In 17 human NSCLC tissue samples, miR-1304 expression was significantly decreased, while HO-1 expression was significantly increased as compared to normal lung tissues. MicroRNA-1304 is a tumor suppressor and HO-1 is its direct target in NSCLC. The results suggest the potential for miR-1304 as a therapeutic target for NSCLC. PMID:27641735

  8. MicroRNA-1304 suppresses human non-small cell lung cancer cell growth in vitro by targeting heme oxygenase-1.

    PubMed

    Li, Cheng-Gang; Pu, Meng-Fan; Li, Chun-Zhu; Gao, Man; Liu, Ming-Xia; Yu, Cun-Zhi; Yan, Hong; Peng, Chun; Zhao, Yang; Li, Yu; Ma, Ze-Long; Qi, Xin-Ming; Wang, Yi-Zheng; Miao, Ling-Ling; Ren, Jin

    2017-01-01

    Previous studies have shown that microRNA-1304 (miR-1304) is dysregulated in certain types of cancers, including non-small cell lung cancer (NSCLC), and might be involved in tumor survival and/or growth. In this study we investigated the direct target of miR-1304 and its function in NSCLC in vitro. Human lung adenocarcinoma cell lines (A549 and NCI-H1975) were studied. The cell proliferation and survival were investigated via cell counting, MTT and colony-formation assays. Cell apoptosis and cell cycle were examined using annexin V-PE/7-AAD and PI staining assays, respectively. The dual-luciferase reporter assay was used to verify post-transcriptional regulation of heme oxygenase-1 (HO-1) by miR-1304. CRISPR/Cas9 was used to deplete endogenous miR-1304. Overexpression of MiR-1304 significantly decreased the number and viability of NSCLC cells and colony formation, and induced cell apoptosis and G0/G1 phase cell cycle arrest. HO-1 was demonstrated to be a direct target of miR-1304 in NSCLC cells. Restoration of HO-1 expression by hemin (20 μmol/L) abolished the inhibition of miR-1304 on cell growth and rescued miR-1304-induced apoptosis in A549 cells. Suppression of endogenous miR-1304 with anti-1304 significantly increased HO-1 expression and promoted cell growth and survival in A549 cells. In 17 human NSCLC tissue samples, miR-1304 expression was significantly decreased, while HO-1 expression was significantly increased as compared to normal lung tissues. MicroRNA-1304 is a tumor suppressor and HO-1 is its direct target in NSCLC. The results suggest the potential for miR-1304 as a therapeutic target for NSCLC.

  9. Cyclooxygenase-2-dependent expression of angiogenic CXC chemokines ENA-78/CXC Ligand (CXCL) 5 and interleukin-8/CXCL8 in human non-small cell lung cancer.

    PubMed

    Põld, Mehis; Zhu, Li X; Sharma, Sherven; Burdick, Marie D; Lin, Ying; Lee, Peter P N; Põld, Anu; Luo, Jie; Krysan, Kostyantyn; Dohadwala, Mariam; Mao, Jenny T; Batra, Raj K; Strieter, Robert M; Dubinett, Steven M

    2004-03-01

    Elevated tumor cyclooxygenase (COX)-2 activity plays a multifaceted role in non-small cell lung cancer (NSCLC). To elucidate the role of COX-2 in the in vitro and in vivo expression of two known NSCLC angiogenic peptides, CXC ligand (CXCL) 8 and CXCL5, we studied two COX-2 gene-modified NSCLC cell lines, A549 and H157. COX-2 overexpression enhanced the in vitro expression of both CXCL8 and CXCL5. In contrast, specific COX-2 inhibition decreased the production of both peptides as well as nuclear translocation of nuclear factor kappaB. In a severe combined immunodeficient mouse model of human NSCLC, the enhanced tumor growth of COX-2-overexpressing tumors was inhibited by neutralizing anti-CXCL5 and anti-CXCL8 antisera. We conclude that COX-2 contributes to the progression of NSCLC tumorigenesis by enhancing the expression of angiogenic chemokines CXCL8 and CXCL5.

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

    NASA Astrophysics Data System (ADS)

    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.

  11. MicroRNA-221 promotes human non-small cell lung cancer cell H460 growth.

    PubMed

    Xu, Yiming; Zhong, Chongjun; Ding, Shengguang; Huang, Haitao; Shen, Zhenya

    2015-01-01

    MicroRNA (miRNA-221) has been reported to be a regulator of cell proliferation. Here we intended to investigate the role of miRNA-221 in regulating the growth of human non-small cell lung cancer cell line H460. H460 cells were transfected with miRNA-221 mimics/inhibitors or their respective negative controls. Real-time quantitative PCRs (qRT-PCRs) were used to confirm the effects of miRNA-221 mimics and inhibitors in H460 cells while Cell Counting Kit 8 (CCK-8) and 5-Ethynyl-2'-deoxyuridine (EdU) assay were used to access the cell viability and proliferation. P27 and P57, as putative targets of miRNA-221, were determined by qRT-PCRs in H460 cells. We found that overexpression of miRNA-221 led to increased proliferative rate and cell viability in H460 cells while down-regulation of miRNA-221 decreased those effects. P27 but not P57 was identified as a potential target gene of miRNA-221 in H460 as P27 was negatively regulated by miRNA-221 in the protein level. In conclusion, this study suggests that miRNA-221 controls human non-small cell lung cancer cell H460 growth potentially by targeting P57. Inhibition of miRNA-221 represents a novel potential treatment for human non-small cell lung cancer.

  12. In vitro cytotoxicity and morphological assessment of smoke from polymer combustion in human lung derived cells (A549).

    PubMed

    Lestari, F; Hayes, A J; Green, A R; Chattopadhyay, G

    2012-04-01

    The application of polymer and composites in building and modern transport interiors raises concerns of potential health hazards during combustion. Cytotoxicity and morphological assessment of smoke from polymer combustion in human lung derived cells (A549) has been investigated. A laboratory scale vertical tube furnace was used for the generation of combustion products. A range of materials used in the building and transport industry including high density-polyethylene (HDPE), polypropylene (PP), polycarbonate (PC), and polyvinyl chloride (PVC), fiberglass reinforced polymers (FRPs), and melamine faced plywood (MFP) were studied. The exposure of combustion toxicants to human lung cells (A549) at the air/liquid interface was acquired using a Harvard Navicyte Chamber. Cytotoxic effects on human cells were assessed based on cell viability using a selected in vitro cytotoxicity assays, including NRU (neutral red uptake) and ATP (adenosine triphosphate). Morphological assessment on the effects of combustion products in human lung cells from selected materials including PVC, FRP and MFP was assessed using scanning electron microscopy (SEM). The volatile organic compounds from thermal decomposition products were identified using ATD-GCMS (Automatic Thermal Desorption Gas Chromatography Mass Spectrometry). NOAEC (No Observable Adverse Effect Concentration), IC(10) (10% inhibitory concentration), IC(50) (50% inhibitory concentration), and TLC (Total Lethal Concentration) values (mg/l) were generated. The following toxicity ranking was observed from the most toxic material to the least toxic using the NRU assay: PVC>PP>HDPE>PC >FRP-10>MFP>FRP-16; and the ATP assay: PVC>HDPE>PP>FRP-10>FRP-16>MFP>PC. The method described here could potentially be an alternative to current fire toxicity standards.

  13. Water extract of Rheum officinale Baill. induces apoptosis in human lung adenocarcinoma A549 and human breast cancer MCF-7 cell lines.

    PubMed

    Li, Wing-Yan; Chan, Shun-Wan; Guo, De-Jian; Chung, Mei-Kuen; Leung, Tin-Yan; Yu, Peter Hoi-Fu

    2009-07-15

    Rheum officinale Baill. (Da Huang) is one of the herbs commonly used in traditional Chinese medicine formulae against cancer. The traditional decoction is similar to the water extract used in the present study. The water extract of Da Huang was investigated to see if it possesses anticancer effects through apoptotic pathways. Human lung adenocarcinoma A549 and human breast cancer MCF-7 cell lines were treated with different concentrations of Da Huang water extract at different time intervals. Growth inhibition was detected by MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] and colony formation assays; apoptosis was detected by cell morphologic analysis, DNA fragmentation analysis and COMET assay. Da Huang water extract was found to have significant growth inhibitory effects on both A549 and MCF-7 cell lines with IC(50) values 620+/-12.7 and 515+/-10.1 microg/ml, respectively. Growth inhibitory effects were dose- and time-dependent. A significant decrease in cell number, DNA fragmentation and single DNA strand breakages were observed in the Da Huang water extract treated A549 and MCF-7 cells. This suggests that the water extract of Da Huang exerts potential anticancer activity through growth inhibition and apoptosis on MCF-7 and A549 cells lines.

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

    PubMed

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

    2015-01-01

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

  15. Short-Course Treatment With Gefitinib Enhances Curative Potential of Radiation Therapy in a Mouse Model of Human Non-Small Cell Lung Cancer

    SciTech Connect

    Bokobza, Sivan M.; Jiang, Yanyan; Weber, Anika M.; Devery, Aoife M.; Ryan, Anderson J.

    2014-03-15

    Purpose: To evaluate the combination of radiation and an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) in preclinical models of human non-small cell lung cancer. Methods and Materials: Sensitivity to an EGFR TKI (gefitinib) or radiation was assessed using proliferation assays and clonogenic survival assays. Effects on receptor signal transduction pathways (pEGFR, pAKT, pMAPK) and apoptosis (percentage of cleaved PARP Poly (ADP-ribose) polymerase (PARP)) were assessed by Western blotting. Radiation-induced DNA damage was assessed by γH2AX immunofluorescence. Established (≥100 mm{sup 3}) EGFR-mutated (HCC287) or EGFR wild-type (A549) subcutaneous xenografts were treated with radiation (10 Gy, day 1) or gefitinib (50 mg/kg, orally, on days 1-3) or both. Results: In non-small cell lung cancer (NSCLC) cell lines with activating EGFR mutations (PC9 or HCC827), gefitinib treatment markedly reduced pEGFR, pAKT, and pMAPK levels and was associated with an increase in cleaved PARP but not in γH2AX foci. Radiation treatment increased the mean number of γH2AX foci per cell but did not significantly affect EGFR signaling. In contrast, NSCLC cell lines with EGFR T790M (H1975) or wild-type EGFR (A549) were insensitive to gefitinib treatment. The combination of gefitinib and radiation treatment in cell culture produced additive cell killing with no evidence of synergy. In xenograft models, a short course of gefitinib (3 days) did not significantly increase the activity of radiation treatment in wild-type EGFR (A549) tumors (P=.27), whereas this combination markedly increased the activity of radiation (P<.001) or gefitinib alone (P=.002) in EGFR-mutated HCC827 tumors, producing sustained tumor regressions. Conclusions: Gefitinib treatment increases clonogenic cell killing by radiation but only in cell lines sensitive to gefitinib alone. Our data suggest additive rather than synergistic interactions between gefitinib and radiation and that a

  16. Nimesulide, a selective COX-2 inhibitor, acts synergistically with ionizing radiation against A549 human lung cancer cells through the activation of caspase-8 and caspase-3.

    PubMed

    Kim, Byeong Mo; Won, Juyoon; Maeng, Kyung Ah; Han, Young Soo; Yun, Yeon-Sook; Hong, Sung Hee

    2009-05-01

    Several lines of evidence suggest that non-steroidal anti-inflammatory drugs (NSAIDs) have a radiosensitizing effect on cancer cells in vitro and in vivo, but little is known about the underlying cellular mechanism. In this study, we found that the treatment with the NSAID nimesulide significantly increased the sensitivity of A549 human non-small cell lung cancer cells to radiotherapy. The combined nimesulide-radiation treatment increased apoptosis, induced the cleavage of caspase-3, caspase-9, and poly(ADP-ribose) polymerase (PARP), activated caspase-8, and induced cleavage of Bid. A pan-caspase inhibitor, z-VAD-fmk, suppressed this increase in apoptosis and also suppressed the cleavage of caspase-8, caspase-3, and PARP, suggesting a caspase-dependent mechanism. In addition, z-IETD-fmk, a selective caspase-8 inhibitor, suppressed the nimesulide- and radiation-induced cleavage activation of caspase-9, caspase-3, caspase-8, and Bid, and suppressed the concomitant apoptosis, indicating that the nimesulide-induced increase in radiosensitivity was initiated by caspase-8. However, the caspase-3 inhibitor z-DEVD-fmk failed to suppress activation of the caspase-8/Bid pathway, indicating that caspase-3 activation occurred downstream of caspase-8 activation in our experiments. Marked antitumor effects, which were evaluated by measuring protracted tumor regression, were observed when nude mice were treated with a combination of nimesulide at a clinically achievable dose (0.5 mg/kg) and radiation therapy. Our results, demonstrating the radiosensitivity-increasing and tumor growth-inhibiting effects of nimesulide, suggest that nimesulide may be suitable as an adjuvant to enhance the efficacy and selectivity of radiotherapy.

  17. Digoxin downregulates NDRG1 and VEGF through the inhibition of HIF-1α under hypoxic conditions in human lung adenocarcinoma A549 cells.

    PubMed

    Wei, Dong; Peng, Jing-Jing; Gao, Hui; Li, Hua; Li, Dong; Tan, Yong; Zhang, Tao

    2013-04-02

    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.

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

    PubMed

    Chen, Tian Jun; Gao, Fei; Yang, Tian; Thakur, Asmitanand; Ren, Hui; Li, Yang; Zhang, Shuo; Wang, Ting; Chen, Ming Wei

    2013-07-19

    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. Copyright © 2013 Elsevier Inc. All rights reserved.

  19. The histone acetylranseferase hMOF acetylates Nrf2 and regulates anti-drug responses in human non-small cell lung cancer

    PubMed Central

    Chen, Zhiwei; Ye, Xiangyun; Tang, Naiwang; Shen, Shengping; Li, Ziming; Niu, Xiaomin; Lu, Shun; Xu, Ling

    2014-01-01

    BACKGROUND AND PURPOSE The histone acetyltransferase MOF is a member of the MYST family. In mammals, MOF plays critical roles by acetylating histone H4 at K16 and non-histone substrates such as p53. Here we have investigated the role of MOF in human lung cancer and possible new substrates of hMOF. EXPERIMENTAL APPROACH Samples of human non-small cell lung cancer (NSCLC) were used to correlate MOF with clinicopathological parameters and NF–E2-related factor 2 (Nrf2) downstream genes. 293T-cells were used to study interactions between MOF and Nrf2, and acetylation of Nrf2 by MOF. Mouse embryonic fibroblast and A549 cells were utilized to assess involvement of MOF in antioxidative and anti-drug responses. A549 cells were used to analysis the role of MOF in anti-drug response in vitro and in vivo. KEY RESULTS hMOF was overexpressed in human NSCLC tissues and was associated with large tumour size, advanced disease stage and metastasis, and with poor prognosis. hMOF levels were positively correlated with Nrf2-downstream genes. MOF/hMOF physically interacted with and acetylated Nrf2 at Lys588. MOF-mediated acetylation increased nuclear retention of Nrf2 and transcription of its downstream genes. Importantly, MOF/hMOF was essential for anti-oxidative and anti-drug responses in vitro and regulated tumour growth and drug resistance in vivo in an Nrf2-dependent manner. CONCLUSION AND IMPLICATIONS hMOF was overexpressed in human NSCLC and was a predictor of poor survival. hMOF-mediated Nrf2 acetylation and nuclear retention are essential for anti-oxidative and anti-drug responses. hMOF may provide a therapeutic target for the treatment of NSCLC. PMID:24571482

  20. Gene expression modulation in A549 human lung cells in response to combustion-generated nano-sized particles.

    PubMed

    Arenz, Andrea; Hellweg, Christine E; Stojicic, Nevena; Baumstark-Khan, Christa; Grotheer, Horst-Henning

    2006-12-01

    High levels of ambient air pollution are associated in humans with aggravation of asthma and of respiratory and cardiopulmonary morbidity; long-term exposures to particulate matter (PM) have been linked to possible increases in lung cancer risk, chronic respiratory disease, and increased death rates. The Biodiagnostics Group of the DLR Institute of Aerospace Medicine develops cellular test systems capable of monitoring the biological consequences of environmental conditions on humans already on cellular and molecular level. Such bioassays rely on the receptor-reporter principle, where cell lines are transfected with plasmids carrying a reporter gene under control of environment-dependent promoters (receptor), which play a key role in regulating gene expressions in response to extracellular signals. We developed the recombinant human lung epithelial cell line A549-NF-kappaB-EGFP/Neo carrying a genetically encoded fluorescent indicator for monitoring activation of the NF-kappaB signaling pathway in living cells in response to genotoxic and cytotoxic environmental influences. With this cell line we screened several candidate human radiation-responsive genes (GADD45beta, CDKN1A) and NF-kappaB-dependent genes (IL-6, NFkappaBIA, and pNF-kappaB-EGFP) for gene expression changes by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) assay, using cDNA obtained from total RNA isolated at various time points after exposure to combustion generated nano-sized particle samples.

  1. ALKBH3, a human AlkB homologue, contributes to cell survival in human non-small-cell lung cancer

    PubMed Central

    Tasaki, M; Shimada, K; Kimura, H; Tsujikawa, K; Konishi, N

    2011-01-01

    Background: We have demonstrated for the first time that a novel human AlkB homologue, ALKBH3, contributes to prostate cancer development, but its clinical and biological roles in lung cancer remain unclear. Methods: Expression of both mRNA and protein of PCA-1 was examined by RT–PCR and western blotting. We also assessed association with senescence and in vivo ALKBH3 treatment on orthotopic tumour cell inoculation, and analysed it clinicopathologically. Results: We have since found novel biological roles for ALKBH3 in human lung cancers, particularly in adenocarcinoma. Our immunohistochemical analysis of human adenocarcinomas and squamous cell carcinomas of the lung not only showed overexpression of ALKBH3 in these tumours but the percentage of cells positive for ALKBH3 also correlated statistically to recurrence-free survival in adenocarcinoma. Knockdown of ALKBH3 by siRNA transfection induced expression of p21WAF1/Cip1 and p27Kip1 in the human lung adenocarcinoma cell line A549, resulting in cell cycle arrest, senescence and strong suppression of cell growth in vitro. In vivo, peritoneal tumour growth and dissemination was inhibited in nude mice, previously inoculated with the A549 cell line, by intraperitoneal injection of ALKBH3 siRNA + atelocollagen, as demonstrated by the reduction in both number and diameter of tumours developing in the peritoneum. Conclusion: We suggest that ALKBH3 contributes significantly to cancer cell survival and may be a therapeutic target for human adenocarcinoma of the lung. PMID:21285982

  2. p53-independent structure-activity relationships of 3-ring mesogenic compounds' activity as cytotoxic effects against human non-small cell lung cancer lines.

    PubMed

    Fukushi, Saori; Yoshino, Hironori; Yoshizawa, Atsushi; Kashiwakura, Ikuo

    2016-07-25

    We recently demonstrated the cytotoxicity of liquid crystal precursors (hereafter referred to as "mesogenic compounds") in the human non-small cell lung cancer (NSCLC) cell line A549 which carry wild-type p53. p53 mutations are observed in 50 % of NSCLC and contribute to their resistance to chemotherapy. To develop more effective and cancer-specific agents, in this study, we investigated the structure-activity relationships of mesogenic compounds with cytotoxic effects against multiple NSCLC cells. The pharmacological effects of mesogenic compounds were examined in human NSCLC cells (A549, LU99, EBC-1, and H1299) and normal WI-38 human fibroblast. Analyses of the cell cycle, cell-death induction, and capsases expression were performed. The 3-ring compounds possessing terminal alkyl and hydroxyl groups (compounds C1-C5) showed cytotoxicity in NSCLC cells regardless of the p53 status. The compounds C1 and C3, which possess a pyrimidine at the center of the core, induced G2/M arrest, while the compounds without a pyrimidine (C2, C4, and C5) caused G1 arrest; all compounds produced caspase-mediated cell death. These events occurred in a p53-independent manner. Furthermore, it was suggested that compounds induced cell death through p53-independent DNA damage-signaling pathway. Compounds C2, C4, and C5 did not show strong cytotoxicity in WI-38 cells, whereas C1 and C3 did. However, the cytotoxicity of compound C1 against WI-38 cells was improved by modulating the terminal alkyl chain lengths of the compound. We showed the p53-indepdent structure-activity relationships of mesogenic compounds related to the cytotoxic effects. These structure-activity relationships will be helpful in the development of more effective and cancer-specific agents.

  3. Sulforaphene-Carboplatin Combination Synergistically Enhances Apoptosis by Disruption of Mitochondrial Membrane Potential and Cell Cycle Arrest in Human Non-Small Cell Lung Carcinoma.

    PubMed

    Chatterjee, Saswata; Rhee, Yun-Hee; Ahn, Jin-Chul

    2016-09-01

    Worldwide non-small cell lung cancer (NSCLC) causes substantial morbidity and mortality among human populations. Due to the severe side effects and low survival rate of patients with the conventional drugs, implementation of new combination therapies is much needed. The aim of this study was to evaluate the efficacy of a combination therapy with a conventional drug and a natural medicine. We compared the combination of chemotherapy drug carboplatin and the radish-derived isothiocyanate compound sulforaphene, which synergistically induces higher apoptosis and growth inhibition in A549, to the drug alone in human NSCLC cells. We found that this combination group significantly induced higher depolarization of mitochondrial membrane potential (MMP) and intracellular reactive oxygen species generation than the single drug dose, followed by cell cycle arrest at the G0/G1 phase after 24 h of incubation. In addition to that, the Western blot assays showed that combination treatment inhibited the expression of Bcl-2 and successively upregulated the expression of Bax, cytochrome C, apoptosis-inducing factor, caspase-9 and -3, and cleaved poly ADP ribose polymerase. It also modulated the expression of PI3K, p-extracellular signal-regulated kinase (1/2), and p-c-Jun N-terminal kinase indicating the involvement of antiproliferative properties. Further pretreatment with pan-caspase inhibitor Z-VAD-fmk was carried out to confirm the effect of caspases in the combination therapy-induced apoptosis. To summarize, this is the first report that sulforaphene-carboplatin combination treatment synergistically promotes enhanced apoptosis and antiproliferative effect over single drug treatment against A549, human NSCLC cells through caspase activation, MMP disruption, and cell cycle arrest. This study demonstrates that the duel character of this combination therapy may be an effective replacement for conventional therapy alone against NSCLC.

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

    PubMed Central

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

    2014-01-01

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

  5. Raman spectroscopy identifies radiation response in human non-small cell lung cancer xenografts

    NASA Astrophysics Data System (ADS)

    Harder, Samantha J.; Isabelle, Martin; Devorkin, Lindsay; Smazynski, Julian; Beckham, Wayne; Brolo, Alexandre G.; Lum, Julian J.; Jirasek, Andrew

    2016-02-01

    External beam radiation therapy is a standard form of treatment for numerous cancers. Despite this, there are no approved methods to account for patient specific radiation sensitivity. In this report, Raman spectroscopy (RS) was used to identify radiation-induced biochemical changes in human non-small cell lung cancer xenografts. Chemometric analysis revealed unique radiation-related Raman signatures that were specific to nucleic acid, lipid, protein and carbohydrate spectral features. Among these changes was a dramatic shift in the accumulation of glycogen spectral bands for doses of 5 or 15 Gy when compared to unirradiated tumours. When spatial mapping was applied in this analysis there was considerable variability as we found substantial intra- and inter-tumour heterogeneity in the distribution of glycogen and other RS spectral features. Collectively, these data provide unique insight into the biochemical response of tumours, irradiated in vivo, and demonstrate the utility of RS for detecting distinct radiobiological responses in human tumour xenografts.

  6. In vitro and in vivo antitumor activity of scutebarbatine A on human lung carcinoma A549 cell lines.

    PubMed

    Yang, Xiao-Kun; Xu, Ming-Yuan; Xu, Gui-Sen; Zhang, Yu-Lan; Xu, Zhao-Xia

    2014-06-25

    During our systematic study on the anticancer activities of Scutellaria barbata, scutebarbatine A (SBT-A), one of the major alkaloids in S. barbata, was found to have antitumor effects on A549 cells. Thus, we designed the present study to investigate in detail the antitumor effects of SBT-A. The cytotoxic effect of SBT-A on A549 in vitro were determined by an MTT assay and evaluated by IC50 values. Furthermore, results of Hoechst 33258 and Annexin V/PI staining assays demonstrated that SBT-A had significant antitumor effects on A549 cells via apoptosis, in a concentration-dependent manner. What's more, the mechanism was explored by western blotting, and our study revealed that SBT-A can up-regulate the expressions of cytochrome c, caspase-3 and 9, and down-regulate the levels of Bcl-2 in A549 cells. Finally, the antitumor effects of SBT-A were evaluated in vivo by using transplanted tumor nude mice, and the results confirmed that SBT-A has a notable antitumor effect on A549 cancer via mitochondria-mediated apoptosis. Collectively, our results demonstrated that SBT-A showed significant antitumor effects on A549 cells in vivo and in vitro via mitochondria-mediated apoptosis by up-regulating expressions of caspase-3 and 9, and down-regulating Bcl-2.

  7. Flavonoids isolated from Citrus platymamma induced G2/M cell cycle arrest and apoptosis in A549 human lung cancer cells

    PubMed Central

    Nagappan, Arulkumar; Lee, Ho Jeong; Saralamma, Venu Venkatarame Gowda; Park, Hyeon Soo; Hong, Gyeong Eun; Yumnam, Silvia; Raha, Suchismita; Charles, Shobana Nancy; Shin, Sung Chul; Kim, Eun Hee; Lee, Won Sup; Kim, Gon Sup

    2016-01-01

    Citrus platymamma hort. ex Tanaka belongs to the Rutaceae family and is widely used in folk medicines in Korea due to its anti-proliferative, anti-cancer, anti-oxidant, anti-inflammatory and anti-diabetic activities. However, the molecular mechanism of its anti-cancer effect is not well understood. The present study was conducted to elucidate the anti-cancer effect and molecular mechanism of flavonoids from Citrus platymamma (FCP) on A549 cells. FCP displayed concentration-dependent inhibition on A549 cells proliferation. Further, flow cytometry revealed that FCP significantly increased the sub-G1 (apoptotic cell population) and G2/M phase population, and the total number of apoptotic cells, in a dose-dependent manner. Nuclear condensation and fragmentation were also observed upon staining with Hoechst 33342 in FCP-treated A549 cells. Immunoblotting demonstrated a dose-dependent downregulation of cyclin B1, cyclin-dependent kinase 1, cell division cycle 25c, pro-caspases −3, −6, −8 and −9, and poly (adenosine diphosphate-ribose) polymerase (PARP) in FCP-treated A549 cells. In addition, FCP induced caspase-3 activation and subsequent PARP cleavage, and increased the B-cell lymphoma (Bcl)-2-associated X protein/Bcl-extra large ratio in A549 cells. These findings suggest that FCP induced G2/M arrest and apoptosis of A549 cells. The present study provides evidence that FCP may be useful in the treatment of human lung cancer. PMID:27446443

  8. Effects of sodium lactate Ringer's injection on transfection of human protein kinase C-α antisense oligonucleotide in A549 lung cancer cells.

    PubMed

    Wang, Z H; Sun, W W; Han, Y L; Ma, Z

    2016-08-26

    In the present study, we evaluated the effects of four solutions [Dulbecco's modified Eagle's medium (DMEM), sodium lactate Ringer's injection (SLRI), phosphate-buffered saline (PBS), and NaCl] on the transfection of the human protein kinase C-a antisense oligonucleotide (PKC-a ASO) aprinocarsen in human lung carcinoma A549 cells. Specifically, SLRI, DMEM, PBS, or NaCl were used as the growth solutions for A549 cells, and OPTI-MEM was used as the PKC-a ASO diluent for transfection. Additionally, SLRI, DMEM, PBS, or NaCl were used as both the growth solutions and diluents for transfection. The cell viability and transfection efficiency were determined. The results demonstrated that when SLRI was used as either the growth solution or both the growth solution and diluent for aprinocarsen transfection in A549 cells, the effects were close to the best effects observed with DMEM as the growth solution and OPTI-MEM as the diluent, which supported the transfection of aprinocarsen into the cells. Moreover, SLRI resulted in higher transfection efficiency than those of PBS and NaCl. In in vitro experiments, aprinocarsen effectively induced apoptosis in A549 cells. In conclusion, SLRI may replace PBS or NaCl in clinical trials as a transfection solution readily accepted by the human body. To our knowledge, this is the first report demonstrating the use of SLRI as a transfection solution in lung-cancer cell lines.

  9. Selenium-Containing Polysaccharide-Protein Complex in Se-Enriched Ulva fasciata Induces Mitochondria-Mediated Apoptosis in A549 Human Lung Cancer Cells.

    PubMed

    Sun, Xian; Zhong, Yu; Luo, Hongtian; Yang, Yufeng

    2017-07-16

    The role of selenium (Se) and Ulva fasciata as potent cancer chemopreventive and chemotherapeutic agents has been supported by epidemiological, preclinical, and clinical studies. In this study, Se-containing polysaccharide-protein complex (Se-PPC), a novel organoselenium compound, a Se-containing polysaccharide-protein complex in Se-enriched Ulva fasciata, is a potent anti-proliferative agent against human lung cancer A549 cells. Se-PPC markedly inhibited the growth of cancer cells via induction of apoptosis which was accompanied by the formation of apoptotic bodies, an increase in the population of apoptotic sub-G1 phase cells, upregulation of p53, and activation of caspase-3 in A549 cells. Further investigation on intracellular mechanisms indicated that cytochrome C was released from mitochondria into cytosol in A549 cells after Se-PPC treatment. Se-PPC induced depletion of mitochondrial membrane potential (ΔΨm) in A549 cells through regulating the expression of anti-apoptotic (Bcl-2, Bcl-XL) and pro-apoptotic (Bax, Bid) proteins, resulting in disruption of the activation of caspase-9. This is the first report to demonstrate the cytotoxic effect of Se-PPC on human cancer cells and to provide a possible mechanism for this activity. Thus, Se-PPC is a promising novel organoselenium compound with potential to treat human cancers.

  10. Effect of metal ions on HIF-1alpha and Fe homeostasis in human A549 cells.

    PubMed

    Kang, Gi Soo; Li, Qin; Chen, Haobin; Costa, Max

    2006-11-07

    Several metals are carcinogenic but little is known about the mechanisms by which they cause cancer. A pathway that may contribute to metal ion induced carcinogenesis is by hypoxia signaling, which involves a disruption of cellular iron homeostasis by competition with iron transporters or iron-regulated enzymes. To examine the involvement of iron in the hypoxia signaling activity of these metal ions we investigated HIF-1alpha protein stabilization, IRP-1 activity, and ferritin protein levels in human lung carcinoma A459 cells exposed to various agents in serum- and iron-free salt-glucose medium (SGM) or in normal complete medium. We also studied the effects of excess exogenous iron on these responses induced by nickel ion exposure. Our results show the following: (1) SGM enhanced metals-induced HIF-1alpha stabilization and IRP-1 activation (e.g., nickel and cobalt ions). (2) If SGM was reconstituted with a slight excess level (25 microM of FeSO(4)) of iron, this enhancing ability was significantly decreased. (3) The effect of a high level of exogenous iron (500 microM of FeSO(4)) on metal-induced hypoxia and iron metabolism was highly dependent on the order of addition. If treatment with the Fe and metal ions was simultaneous (co-treatment), the effects of nickel ion exposure were overwhelmed, since the added Fe reversed HIF-1alpha stabilization, decreased IRP-1 activity, and increased ferritin level. Pre-treatment with iron was not able to reverse the responses caused by nickel ion exposure. These results imply that it is important to consider the available iron concentration and suitable exposure design when studying metal-induced hypoxia or metal-induced disruption of Fe homeostasis.

  11. Up-Regulation of Pro-Inflammatory Cytokines and Chemokine Production in Avian Influenza H9N2 Virus-Infected Human Lung Epithelial Cell Line (A549).

    PubMed

    Farzin, Hamidreza; Toroghi, Reza; Haghparast, Alireza

    2016-01-01

    Influenza H9N2 virus mostly infects avian species but poses a potential health risk to humans. Little is known about the mammalian host immune responses to H9N2 virus. To obtain insight into the innate immune responses of human lung epithelial cells to the avian H9N2 virus, the expressions of pro-inflammatory cytokines and chemokine in the human airway epithelial cells infected with avian H9N2 virus were examined by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA). H9N2 virus was able to cultivate in the human lung epithelial cell line (A549) and stimulate production of pro-inflammatory cytokines (IL-1β, IL-6) and chemokine (IL-8). Expressions of cytokine genes were up-regulated to a significantly higher level for IL-1β (p < 0.01), IL-6 (p < 0.01 after 12 hours and p < 0.05 after 24 hours) and IL-8 (p < 0.01 after 12 hours and p < 0.001 after 24 hours) in virus-cultured A549 cells as compared with non-virus-cultured cells. The amount of IL-6 and IL-1β proteins secreted into the culture medium was also increased after virus culture infection of A549 cell line compared to non-virus-cultured A549 cells and were significant in both IL-1β (p < 0.05 in 18 hours and p < 0.001 in 24-48 hours harvested supernatant) and IL-6 (p < 0.001). Silencing the p65 component of NF-κB in A549 cells suppressed the stimulatory effects of influenza virus on secretion of pro-inflammatory cytokines and chemokine. The findings in this study will broaden our understanding of host innate immune mechanisms and the pathogenesis of H9N2 influenza viruses in human respiratory epithelium.

  12. Comparative proteomic analysis of paclitaxel sensitive A549 lung adenocarcinoma cell line and its resistant counterpart A549-Taxol.

    PubMed

    Sun, Qiang-Ling; Sha, Hui-Fang; Yang, Xiao-Hua; Bao, Guo-Liang; Lu, Jing; Xie, Yin-Yin

    2011-03-01

    Paclitaxel is used as the first-line chemotherapy for Non-Small Cell Lung Cancer (NSCLC), but acquired resistance becomes a critical problem. Several mechanisms have been proposed in paclitaxel resistance, but they are not sufficient to exhaustively explain this resistance emergence. To better investigate molecular resistance mechanisms, a comparative proteomic approach was carried out to identify differentially expressed proteins between human lung adenocarcinoma A549 cell line (paclitaxel sensitive) and A549-Taxol cell line (acquired resistant). A paclitaxel-resistant subline (A549-Taxol) derived from the parental-sensitive cell line A549 was established by stepwise selection by paclitaxel. Total proteins in the two cell lines were separated by fluorescent differential gel electrophoresis (DIGE). Image analysis was carried out with the DeCyder 2D 6.5 software. Proteins associated with chemoresistance process were identified by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS). Some key molecules were valuated by Western blot. Thirty proteins were identified and grouped into eight main functional classes according to the biological processes in which they are likely to participate, i.e. signal transduction, cytoskeleton, redox reaction, energy and metabolism, and so on. Alterations of these processes might be involved in paclitaxel resistance. Most of the proteins showed mitochondrial and cytoplasm location. The up-regulation of CK8, CK18, ALDH1, CAST and ANX I in A549-Taxol cell line was verified by Western blot, in coincidence with the data obtained from proteomic analysis. For the first time, differentially expressed proteins between paclitaxel-sensitive cell line and paclitaxel-resistant one were explored by comparative proteomic approach in human lung adenocarcinoma. It may be useful for further studying of resistance mechanisms and screening of resistance biomarkers, so as to develop tailored therapeutic

  13. Sensitisation of human lung adenocarcinoma A549 cells to radiotherapy by Nimotuzumab is associated with enhanced apoptosis and cell cycle arrest in the G2/M phase.

    PubMed

    Lin, Shan; Yan, Ying; Liu, Yuan; Gao, Chun-Zi; Shan, Dan; Li, Ying; Han, Bo

    2015-02-01

    The epidermal growth factor receptor (EGFR) plays an important role in tumorigenesis and maintenance of cancers, making it a possible therapeutic target for cancer treatment. Nimotuzumab (h-R3), a humanised monoclonal antibody against EGFR, sensitises human lung adenocarcinoma A549 cells to radiotherapy. We have investigated the underlying molecular mechanism by treating A549 cells with Nimotuzumab (100 μg/mL) alone or in combination with a single dose of 2 Gy irradiation, and analysing apoptosis and cell cycle distribution by flow cytometry. Nimotuzumab significantly enhanced radiation-induced apoptosis of A549 cells as evidenced by increased cell apoptosis (7.15 ± 0.30%) compared with the control group (1.08 ± 0.25%), Nimotuzumab alone group (4.89 ± 0.30%) and irradiation alone group (5.90 ± 0.15%). Combining Nimotuzumab with irradiation significantly arrested cells in the G2/M phase (43. ± 0.36%) compared radiotherapy alone (18.7 ± 0.35%) and single Nimotuzumab treatment (27.2 ± 0.17%). A combination of Nimotuzumab with radiation increased apoptosis and G2/M phase arrest in human lung adenocarcinoma A549 cells, suggesting potential development of combinatorial therapy of Nimotuzumab with radiotherapy for lung cancer. © 2014 International Federation for Cell Biology.

  14. Gliotoxin promotes Aspergillus fumigatus internalization into type II human pneumocyte A549 cells by inducing host phospholipase D activation.

    PubMed

    Jia, Xiaodong; Chen, Fangyan; Pan, Weihua; Yu, Rentao; Tian, Shuguang; Han, Gaige; Fang, Haiqin; Wang, Shuo; Zhao, Jingya; Li, Xianping; Zheng, Dongyu; Tao, Sha; Liao, Wanqing; Han, Xuelin; Han, Li

    2014-06-01

    The internalization of Aspergillus fumigatus into lung epithelial cells is critical for the infection process in the host. Gliotoxin is the most potent toxin produced by A. fumigatus. However, its role in A. fumigatus internalization into the lung epithelial cells is still largely unknown. In the present study, the deletion of the gliP gene regulating the production of gliotoxin in A. fumigatus suppressed the internalization of conidia into the A549 lung epithelial cells, and this suppression could be rescued by the exogenous addition of gliotoxin. At lower concentrations, gliotoxin enhanced the internalization of the conidia of A. fumigatus into A549 cells; in contrast, it inhibited the phagocytosis of J774 macrophages in a dose-dependent manner. Under a concentration of 100 ng/ml, gliotoxin had no effect on A549 cell viability but attenuated ROS production in a dose-dependent manner. Gliotoxin significantly stimulated the phospholipase D activity in the A549 cells at a concentration of 50 ng/ml. This stimulation was blocked by the pretreatment of host cells with PLD1- but not PLD2-specific inhibitor. Morphological cell changes induced by gliotoxin were observed in the A549 cells accompanying with obvious actin cytoskeleton rearrangement and a moderate alteration of phospholipase D distribution. Our data indicated that gliotoxin might be responsible for modulating the A. fumigatus internalization into epithelial cells through phospholipase D1 activation and actin cytoskeleton rearrangement.

  15. Psoralen reverses docetaxel-induced multidrug resistance in A549/D16 human lung cancer cells lines.

    PubMed

    Hsieh, Ming-Ju; Chen, Mu-Kuan; Yu, Ya-Yen; Sheu, Gwo-Tarng; Chiou, Hui-Ling

    2014-06-15

    Chemotherapy is the recommended treatment for advanced-stage cancers. However, the emergence of multidrug resistance (MDR), the ability of cancer cells to become simultaneously resistant to different drugs, limits the efficacy of chemotherapy. Previous studies have shown that herbal medicine or natural food may be feasible for various cancers as potent chemopreventive drug. This study aims to explore the capablility of reversing the multidrug resistance of docetaxel (DOC)-resistant A549 cells (A549/D16) of psoralen and the underlying mechanisms. In this study, results showed that the cell viability of A549/D16 subline is decreased when treated with psoralen plus DOC, while psoralen has no effect on the cell proliferation on A549 and A549/D16 cells. Furthermore, mRNA and proteins levels of ABCB1 were decreased in the presence of psoralen, while decreased ABCB1 activity was also revealed by flow cytometry. Based on these results, we believe that psoralen may be feasible for reversing the multidrug resistance by inhibiting ABCB1 gene and protein expression. Such inhibition will lead to a decrease in ABCB1 activity and anti-cancer drug efflux, which eventually result in drug resistance reversal and therefore, sensitizing drug-resistant cells to death in combination with chemotherapeutic drugs.

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

    PubMed

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

    2016-02-01

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

  17. Bufalin inhibits TGF-β-induced epithelial-to-mesenchymal transition and migration in human lung cancer A549 cells by downregulating TGF-β receptors

    PubMed Central

    ZHAO, LEI; LIU, SHIZHOU; CHE, XIAOFANG; HOU, KEZUO; MA, YANJU; LI, CE; WEN, TI; FAN, YIBO; HU, XUEJUN; LIU, YUNPENG; QU, XIUJUAN

    2015-01-01

    The epithelial-to-mesenchymal transition (EMT) is a well-known prerequisite for cancer cells to acquire the migratory and invasive capacity, and to subsequently metastasize. Bufalin is one of the major active components of the traditional Chinese medicine Chan Su, and accumulating evidence has shown its anticancer effect in multipe types of cancer. However, the role of bufalin in transforming growth factor-β (TGF-β)-induced EMT and migration remains unclear. In the present study, the effect of bufalin on TGF-β-induced EMT and migration was investigated in human lung cancer A549 cells. TGF-β induced EMT in A549 cells and increased their migratory ability, which were markedly suppressed by bufalin. Additionally, TGF-β-induced upregulation of Twist2 and zinc finger E-box binding homeobox 2 (ZEB2), as well as the phosphorylation of Smad2 and Smad3 were also inhibited by bufalin. However, the Smad-independent signaling pathways were not affected. Further analysis showed that the TGF-β receptor I (TβRI) and TGF-β receptor II (TβRII) were downregulated in the presence of bufalin. Pretreatment with SB431542, a potent inhibitor of the phosphorylation of TβRI, significantly attenuated TGF-β-induced EMT, mimicking the effect of bufalin on A549 cells. Taken together, these results suggest that bufalin suppresses TGF-β-induced EMT and migration by downregulating TβRI and TβRII in A549 cells. PMID:26133118

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

    NASA Astrophysics Data System (ADS)

    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.

  19. Alpha-tomatine inactivates PI3K/Akt and ERK signaling pathways in human lung adenocarcinoma A549 cells: effect on metastasis.

    PubMed

    Shih, Yuan-Wei; Shieh, Jiunn-Min; Wu, Pei-Fen; Lee, Yi-Chieh; Chen, Yi-Zhi; Chiang, Tai-An

    2009-08-01

    This study first investigates the anti-metastatic effect of alpha-tomatine in the human lung adenocarcinoma cell line: A549. In this study, we first noted alpha-tomatine inhibited A549 cells invasion and migration by wound-healing assay and Boyden chamber assay. The data also showed alpha-tomatine could inhibit phosphorylation of Akt and extracellular signal-regulated kinase 1 and 2 (ERK1/2), which is involved in the up-regulating matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9) or urokinase-type plasminogen activator (u-PA), whereas it did not affect phosphorylation of c-Jun N-terminal kinase (JNK) and p38. Next, alpha-tomatine significantly decreased the nuclear levels of nuclear factor kappa B (NF-kappaB), c-Fos, and c-Jun. Also, treating A549 cells with alpha-tomatine also leads to a dose-dependent inhibition on the binding abilities of NF-kappaB and activator protein-1 (AP-1). Further, the treatment of inhibitors specific for PI3K (Wortmannin) or ERK (U0126) to A549 cells could cause reduced activities of MMP-2, MMP-9, and u-PA. These results showed alpha-tomatine could inhibit the metastatic ability of A549 cells by reducing MMP-2, MMP-9, and u-PA activities through suppressing phosphoinositide 3-kinase/Akt (PI3K/Akt) or ERK1/2 signaling pathway and inhibition NF-kappaB or AP-1 binding activities. These findings proved alpha-tomatine might be an anti-metastatic agent against human lung adenocarcinoma.

  20. In vitro antiproliferative effect of trastuzumab (Herceptin(®)) combined with cetuximab (Erbitux(®)) in a model of human non-small cell lung cancer expressing EGFR and HER2.

    PubMed

    Privitera, G; Luca, T; Musso, N; Vancheri, C; Crimi, N; Barresi, V; Condorelli, D; Castorina, S

    2016-05-01

    Lung cancer is the leading cause of cancer death. For this reason, new therapies are needed for the treatment of this devastating disease. In this study, we investigated the effects of combining cetuximab and the trastuzumab on the growth of a model of human non-small cell lung carcinoma cell line (A549). The results were compared with those obtained from a human lung squamous carcinoma cell line (NCI-H226). Both cell lines were treated with cetuximab and trastuzumab, alone or in combination, at various concentrations, for 24, 48 and 72 h. Cell proliferation was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. EGFR and HER-2 mRNA expression was detected by reverse transcription polymerase chain reaction, and the gene amplification status of receptors was evaluated by fluorescence in situ hybridisation. The colorimetric proliferation assay showed that trastuzumab combined with cetuximab significantly inhibited A549 cells at a dose of 40 μg/ml after 72 h of treatment (p < 0.05), while no time-dose dependent inhibition was observed in NCI-H226 cells. The combined treatment influenced both levels of EGFR and HER-2 mRNA in A549 cells and only EGFR mRNA levels in NCI-H226 cells. Fluorescence in situ hybridisation showed that both cell lines were aneuploid for the two genes with equally increased EGFR and CEN7 signals, as well as HER-2 and CEN17 signals, indicating a condition of polysomy without amplification. The preliminary results of this study encourage further investigations to elucidate the downstream events involved and to understand how these mechanisms influence non-small cell lung cancers growth.

  1. Winter fine particulate matter from Milan induces morphological and functional alterations in human pulmonary epithelial cells (A549).

    PubMed

    Gualtieri, Maurizio; Mantecca, Paride; Corvaja, Viviana; Longhin, Eleonora; Perrone, Maria Grazia; Bolzacchini, Ezio; Camatini, Marina

    2009-07-10

    Samples of PM(2.5) were gravimetrically collected during the winter 2005/2006 in the urban area of Milan (North Italy). Samples were chemically characterized and the particles were detached from filters to determine their cytotoxic effects on the A549 cell line. Based on the potential toxicological relevance of its components, Milan winter PM(2.5) contained high concentrations of pro-oxidant transition metals and PAHs, while re-suspended particles showed a relatively high frequency of dimensional classes ranging from 40 nm to 300 nm. A549 cells exposed to particle suspensions showed a concentration-dependent decrease in viability, starting from 10 microg/cm(2). Phagocytosis of particles by A549 cells and particle aggregates were morphologically characterized and seemed to depend on both particle concentration and exposure time, with the majority of particles being engulfed in membrane-bound vacuoles after 24h of exposure. The ability of ultrafine particles to penetrate and spread throughout the cells was also verified. Cell membrane lysis and mitochondrial ultrastructural disruption appeared to be the main modifications induced by PM(2.5) on A549 cells. Concomitantly to the adverse effects observed in terms of cell mortality and ultrastructural lesions, a significant intracellular production of reactive oxygen species (ROS) was observed, suggesting that the cytotoxicity, exerted by the winter PM(2.5) in Milan, derived also from its oxidative potential, probably associated with particle-adsorbed metals and PAHs.

  2. Is Human Kallikrein 11 in Non-small Cell Lung Cancer Treated Chemoradiotherapy Associated with Survival?

    PubMed

    Unal, Dilek; Eroglu, Celalettin; Tasdemir, Arzu; Karaman, Hatice; Kurtul, Neslihan; Oguz, Arzu; Goksu, Sema Sezgin; Kaplan, Bunyamin

    2016-01-01

    Involvement of human kallikreins (hKs) in human cancers has been reported and several hKs are promising biomarkers of various cancers. The aim of this study was to evaluate the prognostic significance of hK11 expression in patients with non-metastatic non-small cell lung cancer (NSCLC). The study included 44 patients with NSCLC. hK11 expression was determined by immunohistochemical staining. The estimation of disease-free and overall survival by Kaplan-Meier was 11 months and 17 months, respectively. The estimation of overall survival by Kaplan-Meier was significantly higher in patients with hK11 strongly positive (2+) than in those with hK11 weakly positive (1+) (20 months vs. 11 months, p=0.032). Although not statistically different, the estimation of disease-free survival by Kaplan-Meier was higher in patients with hK11 strongly positive (2+) than in those with hK11 weakly positive (1+) (12 months vs. 9 months, p=0.113). Multivariate Cox regression analysis showed that the overall survival rates were significantly associated with response to chemoradiotherapy and the degree of staining with hK11. The stronger hK11 expression in NSCLC appears to be associated with better survival rates. hK11 may be a prognostic biomarker of NSCLC.

  3. Is Human Kallikrein 11 in Non-small Cell Lung Cancer Treated Chemoradiotherapy Associated with Survival?

    PubMed Central

    Unal, Dilek; Eroglu, Celalettin; Tasdemir, Arzu; Karaman, Hatice; Kurtul, Neslihan; Oguz, Arzu; Goksu, Sema Sezgin; Kaplan, Bunyamin

    2016-01-01

    Purpose Involvement of human kallikreins (hKs) in human cancers has been reported and several hKs are promising biomarkers of various cancers. The aim of this study was to evaluate the prognostic significance of hK11 expression in patients with non-metastatic non-small cell lung cancer (NSCLC). Materials and Methods The study included 44 patients with NSCLC. hK11 expression was determined by immunohistochemical staining. Results The estimation of disease-free and overall survival by Kaplan-Meier was 11 months and 17 months, respectively. The estimation of overall survival by Kaplan-Meier was significantly higher in patients with hK11 strongly positive (2+) than in those with hK11 weakly positive (1+) (20 months vs. 11 months, p=0.032). Although not statistically different, the estimation of disease-free survival by Kaplan-Meier was higher in patients with hK11 strongly positive (2+) than in those with hK11 weakly positive (1+) (12 months vs. 9 months, p=0.113). Multivariate Cox regression analysis showed that the overall survival rates were significantly associated with response to chemoradiotherapy and the degree of staining with hK11. Conclusion The stronger hK11 expression in NSCLC appears to be associated with better survival rates. hK11 may be a prognostic biomarker of NSCLC. PMID:25779361

  4. Glutathione-dependent cell cycle G1 arrest and apoptosis induction in human lung cancer A549 cells caused by methylseleninic acid: comparison with sodium selenite.

    PubMed

    Okuno, Tomofumi; Honda, Eri; Arakawa, Tomohiro; Ogino, Hirofumi; Ueno, Hitoshi

    2014-01-01

    The aim of the present study was to clarify the mechanism underlying the inhibition of cell proliferation in human lung cancer A549 cells by selenium (Se) compounds. Methylseleninic acid (CH3SeO2H, abbreviated as MSA), a synthetic Se compound, is a direct precursor of active methylselenol (CH3SeH) and is considered to be one of beneficial agents for cancer prevention and therapy. Sodium selenite (Na2SeO3), an inorganic Se form, is utilized in clinical Se supplementation. MSA markedly inhibited the growth of A549 cells at a concentration of 2.5×10(-6) mol/L for 1 d. On Day 1, Na2SeO3 also inhibited A549 cell growth at the concentration of 7.5×10(-6) mol/L. These compounds induced cell cycle arrest at the G1 phase and apoptosis under the inhibitory condition. Reduced glutathione (GSH) is critical to MSA or Na2SeO3 metabolism. The depletion of intracellular GSH suppressed Na2SeO3-induced G1 arrest, but promoted Na2SeO3-induced apoptosis. Therefore, Na2SeO3 appears to have directly induced apoptosis. In contrast, the MSA-induced G1 arrest was ameliorated by a marked decrease in GSH content. Additionally, the depletion of GSH slightly suppressed MSA-induced apoptosis. The difference in inhibitory effects between MSA and Na2SeO3 may be due to this variation in GSH-related metabolism. After exposure of A549 cells to MSA, the GSH content was significantly decreased. These results indicate that because MSA-induced G1 arrest and apoptosis induction are enhanced by GSH, the maintenance of GSH is essential for the effective anticancer action of MSA in A549 cells.

  5. Overexpression of polo-like kinase 1 and its clinical significance in human non-small cell lung cancer.

    PubMed

    Wang, Zhao-Xia; Xue, Dong; Liu, Zhi-Li; Lu, Bin-Bin; Bian, Hai-Bo; Pan, Xuan; Yin, Yong-Mei

    2012-01-01

    Polo-like kinase 1 is a serine/threonine kinase which plays an essential role in mitosis and malignant transformation. The aim of this study was to investigate the prognostic significance of polo-like kinase 1 expression and determine its possibility as a therapeutic target in non-small cell lung cancer. Semi-quantitative RT-PCR assay was performed to detect polo-like kinase 1 mRNA expression in non-small cell lung cancer cells or tissues. Immunohistochemistry was performed to detect polo-like kinase 1 protein expression in 100 non-small cell lung cancer tissue samples, and the associations of polo-like kinase 1 expression with clinicopathological factors or prognosis of non-small cell lung cancer patients were evaluated. RNA interference was employed to inhibit endogenous polo-like kinase 1 expression and analyzed the effects of polo-like kinase 1 inhibition on the malignant phenotypes of non-small cell lung cancer cells including growth, apoptosis, radio- or chemoresistance. Also, the possible molecular mechanisms were also investigated. The levels of polo-like kinase 1 mRNA expression in non-small cell lung cancer cell lines or tissues were significantly higher than those in normal human bronchial epithelial cell line or corresponding non-tumor tissues. High polo-like kinase 1 expression was significantly correlated with advanced clinical stage, higher tumor classification and lymph node metastasis of non-small cell lung cancer patients (P=0.001, 0.004 and 0.001, respectively). Meanwhile, high polo-like kinase 1 protein expression was also an independent prognostic molecular marker for non-small cell lung cancer patients (hazard ratio: 2.113; 95% confidence interval: 1.326-3.557; P=0.017). Polo-like kinase 1 inhibition could significantly inhibit in vitro and in vivo proliferation, induce cell arrest of G(2)/M phase and apoptosis enhancement in non-small cell lung cancer cells, which might be activation of the p53 pathway and the Cdc25C/cdc2/cyclin B1 feedback

  6. [Establishment and biological characteristics of a multi-drug resistant cell line A549/Gem.].

    PubMed

    Wang, Weixia; Liu, Xiaoqing; Liu, Guangxian; Lin, Li; Zheng, Xiaoling; Zhu, Yunfeng; Li, Xiaobing

    2008-02-20

    Multi-drug resistance is one of the most important reason why the survival time of non-small cell lung cancer patients is so short. The aim of this study is to establish multi-drug resistant cell line A549/Gem and discuss its biological characters so as to elaborate the possible mechanisms of gemcitabine resistance. Human gemcitabine-resistant non-small cell lung cancer cell line A549/Gem was established by repeated clinical serous peak concentration then low but gradually increasing concentration of gemcitabine from its parental cell human lung adenocarcinoma cell line A549 which is sensitive to gemcitabine. During the course of inducement, monitored its morphology, checked its resistance index and resistant pedigree by MTT method, gathered its growth curve and calculated its doubling time, examined its DNA contents and cell cycles by flow cytometry; at the same time, measured its expression of P53, EGFR, c-erb-B-2, PTEN, PCNA, c-myc, VEGF, MDR-1, Bcl-2, nm23, MMP-9, TIMP-1, CD44v6 Proteins, and RRM1 mRNA. The resistance index of A549/Gem' to gemcitabine was 163.228, and the cell line also exhibited cross-resistance to vinorelbine, taxotere, fluorouraci, etoposide and cisplatin, but kept sensitivity to paclitaxol and oxaliplatin. The doubling time of it was shorter and figures in G0-G1 phase were increased than A549. Compared with A549, A549/Gem' achieved EGFR and c-myc protein expression, nm23 protein expression enhanced, p53, Cerb-B-2 and bcl-2 protein expression reduced, PTEN, PCNA and MDR-1 protein expression vanished, but that of MMP-9, VEGF, CD44v6 and TIMP-1 protein changed trivially. Meanwhile, the expression of RRM1 mRNA was augmented markedly. The resistance index of A549/Gem to gemcitabine was 129.783, and the cell line also held cross-resistance to vinorelbine, taxotere, etoposide, cisplatin and sensitivity to paclitaxol. But the resistance to fluorouracil and sensitivity to oxaliplatin vanished. And the expression of RRM1 mRNA decreased visibly. The

  7. Anti-Inflammatory Effects of Ginsenoside Rg3 via NF-κB Pathway in A549 Cells and Human Asthmatic Lung Tissue

    PubMed Central

    Lee, In-Seung; Uh, InJoon; Kim, Ki-Suk; Kim, Kang-Hoon; Park, Jiyoung; Kim, Yumi; Jung, Ji-Hoon; Jung, Hee-Jae

    2016-01-01

    Objective. There is limited information of the anti-inflammatory effects of Rg3 on inflamed lung cells and tissues. Therefore, we confirmed the anti-inflammatory mechanism of ginsenoside Rg3 in inflamed human airway epithelial cells (A549) and tissues whether Rg3 regulates nuclear factor kappa B (NF-κB) activity. Methods. To induce the inflammation, IL-1β (10 ng/ml) was treated to A549 cells for 4 h. The effects of Rg3 on NF-κB activity and COX-2 expression were evaluated by western blotting analysis in both IL-1β-induced inflamed A549 cell and human asthmatic airway epithelial tissues. Using multiplex cytokines assay, the secretion levels of NF-κB-mediated cytokines/chemokines were measured. Result. Rg3 showed the significant inhibition of NF-κB activity thereby reduced COX-2 expression was determined in both IL-1β-induced inflamed A549 cell and human asthmatic airway epithelial tissues. In addition, among NF-κB-mediated cytokines, the secretion levels of IL-4, TNF-α, and eotaxin were significantly decreased by Rg3 in asthma tissues. Even though there was no significant difference, IL-6, IL-9, and IL-13 secretion showed a lower tendency compared to saline-treated human asthmatic airway epithelial tissues. Conclusion. The results from this study demonstrate the potential of Rg3 as an anti-inflammatory agent through regulating NF-κB activity and reducing the secretion of NF-κB-mediated cytokines/chemokines. PMID:28116321

  8. Human microRNA-24 modulates highly pathogenic avian-origin H5N1 influenza A virus infection in A549 cells by targeting secretory pathway furin.

    PubMed

    Loveday, Emma-Kate; Diederich, Sandra; Pasick, John; Jean, François

    2015-01-01

    A common critical cellular event that many human enveloped viruses share is the requirement for proteolytic cleavage of the viral glycoprotein by furin in the host secretory pathway. For example, the furin-dependent proteolytic activation of highly pathogenic (HP) influenza A (infA) H5 and H7 haemagglutinin precursor (HA0) subtypes is critical for yielding fusion-competent infectious virions. In this study, we hypothesized that viral hijacking of the furin pathway by HP infA viruses to permit cleavage of HA0 could represent a novel molecular mechanism controlling the dynamic production of fusion-competent infectious virus particles during the viral life cycle. We explored the biological role of a newly identified furin-directed human microRNA, miR-24, in this process as a potential post-transcriptional regulator of the furin-mediated activation of HA0 and production of fusion-competent virions in the host secretory pathway. We report that miR-24 and furin are differentially expressed in human A549 cells infected with HP avian-origin infA H5N1. Using miR-24 mimics, we demonstrated a robust decrease in both furin mRNA levels and intracellular furin activity in A549 cells. Importantly, pretreatment of A549 cells with miR-24 mimicked these results: a robust decrease of H5N1 infectious virions and a complete block of H5N1 virus spread that was not observed in A549 cells infected with low-pathogenicity swine-origin infA H1N1 virus. Our results suggest that viral-specific downregulation of furin-directed microRNAs such as miR-24 during the life cycle of HP infA viruses may represent a novel regulatory mechanism that governs furin-mediated proteolytic activation of HA0 glycoproteins and production of infectious virions.

  9. DLC-1 operates as a tumor suppressor gene in human non-small cell lung carcinomas.

    PubMed

    Yuan, Bao-Zhu; Jefferson, Amy M; Baldwin, Kimberly T; Thorgeirsson, Snorri S; Popescu, Nicholas C; Reynolds, Steven H

    2004-02-19

    The deleted in liver cancer (DLC-1) gene at chromosome 8p21-22 is altered mainly by genomic deletion or aberrant promoter methylation in a large number of human cancers such as breast, liver, colon and prostate and is known to have an inhibitory effect on breast and liver tumor cell growth. Given the high frequency of deletion involving region 8p21-22 in human non-small cell lung carcinoma (NSCLC), we examined alterations of DLC-1 in a series of primary tumors and tumor cell lines and tested effects of DLC-1 on tumor cell growth. A significant decrease or absence of the DLC-1 mRNA expression was found in 95% of primary NSCLC (20/21) and 58% of NSCLC cell lines (11/19). Transcriptional silencing of DLC-1 was primarily associated with aberrant DNA methylation, rather than genomic deletion as 5-aza-2'-deoxycytidine induced reactivation of DLC-1 expression in 82% (9/11) NSCLC cell lines showing downregulated DLC-1. It was further evidenced by an aberrant DLC-1 promoter methylation pattern, which was detected by Southern blotting in 73% (8/11) of NSCLC cell lines with downregulation of the gene. The transfer of DLC-1 into three DLC-1 negative cell lines caused a significant inhibition in cell proliferation and/or a decrease in colony formation. Furthermore, stable transfer of DLC-1 abolished tumorigenicity in nude mice of two cell lines, suggesting that DLC-1 plays a role in NSCLC by acting as a bona fide new tumor suppressor gene.

  10. Apoptotic action of peroxisome proliferator-activated receptor-gamma activation in human non small-cell lung cancer is mediated via proline oxidase-induced reactive oxygen species formation.

    PubMed

    Kim, Ki Young; Ahn, Jin Hee; Cheon, Hyae Gyeong

    2007-09-01

    Peroxisome proliferator-activated receptor (PPAR)-gamma ligands have been shown to inhibit human lung cancers by inducing apoptosis and differentiation. In the present study, we elucidated the apoptotic mechanism of PPARgamma activation in human lung cancers by using a novel PPARgamma agonist, 1-(trans-methylimino-N-oxy)-6-(2-morpholinoethoxy)-3-phenyl-(1H-indene-2-carboxylic acid ethyl ester (KR-62980), and rosiglitazone. PPARgamma activation selectively inhibited cell viability of non-small-cell lung cancer with little effect on small-cell lung cancer and normal lung cells. The cell death induced by PPARgamma activation presented apoptotic features of oligonucleosomal DNA fragmentation in A549 human non-small-cell lung cancer cell line. Reactive oxygen species (ROS) production was accompanied by increased expression of proline oxidase (POX), a redox enzyme expressed in mitochondria, upon incubation with the agonists. POX RNA interference treatment blocked PPARgamma-induced ROS formation and cytotoxicity, suggesting that POX plays a functional role in apoptosis through ROS formation. The apoptotic effects by the agonists were antagonized by bisphenol A diglycidyl ether, a PPARgamma antagonist, and by knockdown of PPARgamma expression, indicating the involvement of PPARgamma in these actions. The results of the present study suggest that PPARgamma activation induces apoptotic cell death in non-small-cell lung carcinoma mainly through ROS formation via POX induction.

  11. Effect of silencing SATB1 on proliferation, invasion and apoptosis of A549 human lung adenocarcinoma cells

    PubMed Central

    Huang, Bo; Zhou, Hongli; Wang, Siwang; Lang, Xian Ping; Wang, Xiaodong

    2016-01-01

    The present study aimed to explore the clinical characteristics of special adenine-thymine-rich sequence-binding protein 1 (SATB1) in lung adenocarcinoma and its role in the proliferation, invasion, migration and apoptosis of the lung adenocarcinoma cell line A549. The expression of SATB1 was first studied in tumor tissues of lung adenocarcinoma and adjacent non-tumor tissues. The siRNA green fluorescent protein expression vector of SATB1 was constructed and transfected into the lung adenocarcinoma cell line A549, then a fluorescence microscope was used to study the transfection efficiency. Western blot analysis was adopted to measure the silencing efficiency. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Transwell and scratch assays were used to study cell proliferation, invasion and migration activity, and the apoptosis rate was tested by flow cytometry. SATB1 expression was low in the adjacent non-tumor tissues but high in lung adenocarcinoma tissues, and it was reversely proportional to the differentiation degree. Following transfection with SATB1-siRNA, the expression of SATB1 in A549 cells was blocked (P<0.01). In addition, the proliferation, invasion and migration abilities of cells decreased significantly while the apoptosis rate increased significantly (P<0.01). In conclusion SATB1 is closely associated with the pathogenesis and development of lung adenocarcinoma. PMID:27895736

  12. Monitoring of TGF-β 1-Induced Human Lung Adenocarcinoma A549 Cells Epithelial-Mesenchymal Transformation Process by Measuring Cell Adhesion Force with a Microfluidic Device.

    PubMed

    Li, Yuan; Gao, AnXiu; Yu, Ling

    2016-01-01

    The epithelial-mesenchymal transition (EMT) is a process in which epithelial cells lose their cell polarity and cell-cell adhesion, and gain migratory and invasive properties. It is believed that EMT is associated with initiation and completion of the invasion-metastasis cascade. In this study, an economic approach was developed to fabricate a microfluidic device with less instrumentation requirement for the investigation of EMT by quantifying cell adhesion force. Fluid shear force was precisely controlled by a homemade microfluidic perfusion apparatus and interface. The adhesion capability of the human lung adenocarcinoma cell line A549 on different types of extracellular matrix protein was studied. In addition, effects of transforming growth factor-β (TGF-β) on EMT in A549 cells were investigated by characterizing the adhesion force changes and on-chip fluorescent staining. The results demonstrate that the microfluidic device is a potential tool to characterize the epithelial-mesenchymal transition process by measuring cell adhesion force.

  13. Erucin, a new promising cancer chemopreventive agent from rocket salads, shows anti-proliferative activity on human lung carcinoma A549 cells.

    PubMed

    Melchini, A; Costa, C; Traka, M; Miceli, N; Mithen, R; De Pasquale, R; Trovato, A

    2009-07-01

    Erucin (ER) is a dietary isothiocyanate present in cruciferous vegetables, such as rocket salads (Erucasativa Mill., Diplotaxis sp.), that has been recently considered a promising cancer chemopreventive phytochemical. Biological activity of ER was investigated on human lung adenocarcinoma A549 cells, analyzing its effects on molecular pathways involved in apoptosis and cell cycle arrest, such as PARP-1 cleavage, p53 and p21 protein expression. Our results show that ER affects the A549 cell proliferation, enhancing significantly p53 and p21 protein expression in a dose-dependent manner (p<0.001). PARP-1 cleavage occurs only after exposure to high concentrations of ER (50 microM), in accordance to previous studies showing similar bioactivity of other isothiocyanates (ITCs). Our study reports for the first time that the induction of p53, p21 and PARP-1 cleavage may participate in the anti-proliferative activity of ER in human lung adenocarcinoma A549 cells. Comparison of data with those obtained with the isothiocyanate sulforaphane (SF), structurally related to ER, underlines the strong relationship between structural analogy of ITCs and their biological activity. The ability of dietary compounds to modulate molecular mechanisms that affect cancer cell proliferation is certainly a key point of the cancer prevention potential by functional foods.

  14. Detection and genotype analysis of human papillomavirus in non-small cell lung cancer patients.

    PubMed

    Sarchianaki, Emmanouela; Derdas, Stavros P; Ntaoukakis, Markos; Vakonaki, Elena; Lagoudaki, Eleni D; Lasithiotaki, Ismini; Sarchianaki, Anna; Koutsopoulos, Anastasios; Symvoulakis, Emmanouil K; Spandidos, Demetrios A; Antoniou, Katerina M; Sourvinos, George

    2014-04-01

    Although the role of human papillomavirus (HPV) in the development of uterine cervical cancer is well established, the role of HPV in lung carcinogenesis remains controversial. The detection rates of HPV DNA are subject to a wide variation from 0 to 100%. This is partly influenced by the detection techniques employed. To elucidate the impact of HPV infection on lung parenchyma, we analyzed 100 non-small cell lung cancer (NSCLC) specimens (39 squamous cell carcinomas, 50 adenocarcinomas, 5 samples with characteristics of both squamous cell and adenocarcinoma, 5 undifferentiated and 1 large cell carcinoma) from the region of Crete, Greece. Sixteen non-cancerous samples served as the negative controls. DNA was extracted from 100 paraffin-embedded tissue sections obtained from NSCLC patients. The specimens were examined for the detection of HPV DNA by Real-Time PCR using GP5+/GP6+ primers. Furthermore, the HPV-positive samples were subjected to genotyping. In contrast to the absence of viral genomes in the control samples, HPV DNA was detected in 19 NSCLC specimens (19%). In particular, 4 squamous cell carcinomas, 12 adenocarcinomas, 1 sample with characteristics of both squamous cell and adenocarcinoma, and 2 undifferentiated samples were HPV-positive. The distribution of HPV genotypes was as follows: HPV 16: eight cases (42.1%); HPV 11: three cases (15.8%); HPV 6: one case (5.2%); HPV 59: one case (5.2%); HPV 33: two cases (10.5%); HPV 31: two cases (10.5%) and HPV 18: two cases (10.5%). The presence of HPV in the tumor samples provides evidence of the potential role of HPV in NSCLC and strongly argues for additional research on this issue.

  15. Curcumin inhibits human non-small cell lung cancer xenografts by targeting STAT3 pathway

    PubMed Central

    Xu, Xiaofang; Zhu, Yuping

    2017-01-01

    Human non-small cell lung cancer (NSCLC) is the most common cause of cancer-related death in men. Signal transducers and activators of transcription 3 (STAT3) is a potential molecular target in angiogenesis-mediated cancer therapy. In this study, we subcutaneously injected athymic nude mice with NCI-H460 cells to induce ectopic xenograft model, and treated the animals with curcumin (100 mg/kg) or vehicle by oral gavage. Tumor size and tumor weight were significantly reduced by curcumin treatment. Besides, curcumin significantly decreased hemoglobin content and mRNA expression of CD31 and CD105 in tumor tissue, suggesting that curcumin could inhibit angiogenesis in NSCLC xenograft. Similarly, we intrathoracally injected athymic nude mice with H1975 cells to induce orthotopic xenograft model, in which curcumin significantly reduced tumor weight as well as improved the survival rate of mice. STAT3 pathway was involved in curcumin-induced tumor inhibition, in which phosphorylation of STAT3 and JAK in ectopic xenograft were both declined after curcumin treatment, and the STAT3-regulated promoter activation of VEGF, Bcl-xL, Cyclin D1 was also significantly reduced after treatment. In in vitro assays, curcumin significantly inhibited cell migration and tube formation of NCI-H460 cells, but transfection with pMXs-Stat3C, a dominant active mutant, could abolish the inhibitory effects of curcumin on the cells, suggesting curcumin inhibited tumor angiogenesis of NCI-H460 cells through the inactivation of STAT3. All data showed that curcumin could be a potential drug targeting STAT3 to treat NSCLC. PMID:28861154

  16. Preferential expansion of pro-inflammatory Tregs in human non-small cell lung cancer

    PubMed Central

    Phillips, Joseph D.; Blatner, Nichole R.; Haghi, Leila; DeCamp, Malcolm M.; Meyerson, Shari L.; Heiferman, Michael J.; Heiferman, Jeffrey R.; Gounari, Fotini; Bentrem, David J.; Khazaie, Khashayarsha

    2016-01-01

    Objectives Lung cancer is the leading cause of cancer-related death in the USA. Regulatory T cells (Tregs) normally function to temper immune responses and decrease inflammation. Previous research has demonstrated different subsets of Tregs with contrasting anti- or pro-inflammatory properties. This study aimed to determine Treg subset distributions and characteristics present in non-small cell lung cancer (NSCLC) patients. Methods Peripheral blood was collected from healthy controls (HC) and NSCLC patients preceding surgical resection, and mononuclear cells were isolated, stained, and analyzed by flow cytometry. Tregs were defined by expression of CD4 and CD25 and classified into CD45RA+Foxp3int (naïve, Fr. I) or CD45RA−Foxp3hi (activated Fr. II). Activated conventional T cells were CD4+CD45RA−Foxp3int (Fr. III). Results Samples from 23 HC and 26 NSCLC patients were collected. Tregs isolated from patients with NSCLC were found to have enhanced suppressive function on naive T cells. Cancer patients had significantly increased frequencies of activated Tregs (fraction II: FrII), 17.5 versus 3.2 % (P < 0.001). FrII Tregs demonstrated increased RORγt and IL17 expression and decreased IL10 expression compared to Tregs from HC, indicating pro-inflammatory characteristics. Conclusions This study demonstrates that a novel subset of Tregs with pro-inflammatory characteristics preferentially expand in NSCLC patients. This Treg subset appears identical to previously reported pro-inflammatory Tregs in human colon cancer patients and in mouse models of polyposis. We expect the pro-inflammatory Tregs in lung cancer to contribute to the immune pathogenesis of disease and propose that targeting this Treg subset may have protective benefits in NSCLC. PMID:26047578

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

    PubMed

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

    2014-01-01

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

  18. Identification of epigallocatechin-3-gallate in green tea polyphenols as a potent inducer of p53-dependent apoptosis in the human lung cancer cell line A549.

    PubMed

    Yamauchi, Rieko; Sasaki, Kaori; Yoshida, Kenichi

    2009-08-01

    The effects of green tea polyphenols on cultured cancer cells have been well characterized, especially the effects of epigallocatechin-3-gallate (EGCg), since EGCg suppresses oncogenic signaling pathways and induces cell cycle arrest or apoptosis by regulating cell cycle-associated proteins. In the present study, we attempted to identify signaling pathways or target molecules regulated by each of or a mixture of green tea polyphenols, including epicatechin (EC), epicatechin-3-gallate (ECg), epigallocatechin (EGC), and EGCg, in the human lung cancer cell line A549. ECg, EGC, and a catechin mixture, in addition to EGCg, significantly decreased cell viability. In contrast, caspase 3/7 activity, an apoptosis indicator, was specifically induced by EGCg. By conducting a series of luciferase-based reporter assays, we revealed that the catechin mixture only up-regulates the p53 reporter. EGCg was a more potent inducer of p53-dependent transcription, and this induction was further supported by the induced level of p53 protein. RNA interference (RNAi)-mediated p53 knockdown completely abolished EGCg-induced apoptosis. Finally, a proteome and western blot analysis using approximately 70 different antibodies failed to detect up-regulated proteins in catechin mixture-treated A549 cells. Taken together, these results indicate that EGCg, among several green tea polyphenols, is a potent apoptosis inducer that functions exclusively through a p53-dependent pathway in A549 cells.

  19. Comparison of oxycodone and morphine on the proliferation, apoptosis and expression of related molecules in the A549 human lung adenocarcinoma cell line

    PubMed Central

    Tian, Mi; Jin, Li; Li, Renqi; Zhu, Sihai; Ji, Muhuo; Li, Weiyan

    2016-01-01

    The present study aimed to compare the effects of oxycodone and morphine hydrochloride on the proliferation, apoptosis and migration of A549 lung cancer cells. A549 human lung cancer cells were cultured in vitro and treated with oxycodone or morphine at various concentrations (10, 20 and 40 µg/ml). Cell migration was determined using a wound healing assay, whereas apoptosis was detected using flow cytometry. Reverse transcription quantitative-polymerase chain reaction was performed in order to assess the apoptosis-related gene expression levels, including p53, B-cell lymphoma (Bcl)-2 and Bcl-2-associated X protein (Bax). The levels of vascular endothelial growth factor (VEGF) and urokinase-type plasminogen activator (uPA) were detected using enzyme-linked immunosorbent assays. The expression levels of intercellular cell adhesion molecule (ICAM)-1 were determined by immunofluorescence. In the present study, oxycodone and morphine induced apoptosis in A549 lung cancer cells with similar potency; however, >20 µg/ml oxycodone was more effective at inhibiting cell proliferation (P<0.05) and migration (P<0.05), as compared with morphine at the same concentration. Oxycodone induced a dose-dependent increase in the expression levels of p53 and Bax apoptosis-related genes, whereas it decreased the gene expression levels of Bcl-2. Furthermore, oxycodone decreased, whereas morphine increased, the expression levels of ICAM-1 in a concentration-dependent manner. In addition, at 40 µg/ml, the expression levels of VEGF and uPA in the morphine group were significantly higher than those demonstrated in the oxycodone group (P<0.05). In conclusion, oxycodone was more effective in inhibiting the proliferation and migration of A549 lung cancer cells, as compared with morphine. PMID:27446244

  20. Effect of Recombinant Human Endostatin on Radiosensitivity in Patients With Non-Small-Cell Lung Cancer

    SciTech Connect

    Jiang Xiaodong; Dai Peng; Wu Jin; Song Daan; Yu Jinming

    2012-07-15

    Purpose: To observe the effects of recombinant human endostatin (RHES) on the radiosensitivity of non-small cell lung cancer (NSCLC). Methods and Materials: First, 10 hypoxia-positive cases of pathology-diagnosed NSCLC selected from 15 patients were used to determine the normalization window, a period during which RHES improves NSCLC hypoxia. Second, 50 hypoxia-positive cases of pathology-diagnosed NSCLC (Stages I-III) were randomly divided into a RHES plus radiotherapy group (25 cases) and a radiotherapy-alone group (25 cases). Intensity = modulated radiotherapy with a total dose of 60 Gy in 30 fractions for 6 weeks was adopted in the two groups. The target area included primary foci and metastatic lymph nodes. In the RHES plus radiotherapy group, RHES (15 mg/day) was intravenously given during the normalization window. Results: After RHES administration, the tumor-to=normal tissue radioactivity ratio and capillary permeability surface were first decreased and then increased, with their lowest points on the fifth day compared with the first day (all p < 0.01). Blood flow was first increased and then decreased, with the highest point on the fifth day, compared with the first and tenth day (all p < 0.01). In the RHES plus radiotherapy group and the radiotherapy-alone group, the total effective rates (complete response plus partial response) were 80% and 44% (p = 0.009), respectively. The median survival times were 21.1 {+-} 0.97 months and 16.5 {+-} 0.95 months (p = 0.004), respectively. The 1-year and 2-year local control rates were 78.9 {+-} 8.4% and 68.1 {+-} 7.8% (p = 0.027) and 63.6 {+-} 7.2% and 43.4 {+-} 5.7% (p = 0.022), respectively. The 1-year and 2-year overall survival rates were 83.3 {+-} 7.2% and 76.6 {+-} 9.3% (p = 0.247) and 46.3 {+-} 2.4% and 37.6 {+-} 9.1% (p = 0.218), respectively. Conclusion: The RHES normalization window is within about 1 week after administration. RHES combined with radiotherapy within the normalization window has better short

  1. Alternative splicing variants of carbonic anhydrase IX in human non-small cell lung cancer.

    PubMed

    Malentacchi, Francesca; Simi, Lisa; Nannelli, Caterina; Andreani, Matteo; Janni, Alberto; Pastorekova, Silvia; Orlando, Claudio

    2009-06-01

    In human cancers, carbonic anhydrase IX (CAIX) contributes to maintain intracellular and extracellular pH under hypoxic conditions, but also influences regulation of cell proliferation and tumor progression. CaIX was previously indicated as an independent prognostic marker in non-small cell lung carcinoma (NSCLC). Very recently a CAIX alternative splicing isoform, generating a transcript lacking of exons 8-9, was detected in cancer cells independently from the levels of hypoxia. This alternative splicing (AS) generates a truncated protein lacking the transmembrane region, the intracellular tail and the C-terminal of the catalytic domain and competes with the full-length (FL) isoform in the regulation of the extracellular pH, mainly in a mild hypoxic status. In the present study we measured the mRNA expression of FL and AS CAIX isoforms in 101 NSCLC and in paired not affected tissues. The two isoforms were coexpressed in all NSCLC and normal tissues but while AS mRNA was prevalent in normal tissues (66+/-3%), the FL isoform was higher in NSCLC (58+/-2%, p=0.001). FL mRNA, but not AS, was statistically increased in NSCLC (p=0.01) and showed a statistical association with lymphnode involvement (p=0.009) and tumor stage (p=0.04). Global survival analysis of cancer/related death showed that high levels of FL mRNA were predictive of unfavorable outcome (p<0.0001) and shorter disease-free survival (p<0.0001). Multivariate analysis indicated that FL is an independent prognostic factor for overall survival and higher levels of mRNA in NSCLC sensibly increase hazard ratio ( approximately sixfold). In conclusion, our results seems to indicate that, at least in NSCLC, FL CAIX is the most accurate surrogate of hypoxic stress and represents the only variant with a prognostic role. These data indicate the importance of a separate measurement of the two isoforms in cancer and the need of an accurate re-evaluation of most studies on the clinical role of CAIX in cancer diagnosis.

  2. Streptococcus pneumoniae ClpL Modulates Adherence to A549 Human Lung Cells through Rap1/Rac1 Activation

    PubMed Central

    Nguyen, Cuong Thach; Le, Nhat-Tu; Tran, Thao Dang-Hien; Kim, Eun-Hye; Park, Sang-Sang; Luong, Truc Thanh; Chung, Kyung-Tae; Pyo, Suhkneung

    2014-01-01

    Caseinolytic protease L (ClpL) is a member of the HSP100/Clp chaperone family, which is found mainly in Gram-positive bacteria. ClpL is highly expressed during infection for refolding of stress-induced denatured proteins, some of which are important for adherence. However, the role of ClpL in modulating pneumococcal virulence is poorly understood. Here, we show that ClpL impairs pneumococcal adherence to A549 lung cells by inducing and activating Rap1 and Rac1, thus increasing phosphorylation of cofilin (inactive form). Moreover, infection with a clpL mutant (ΔclpL) causes a greater degree of filopodium formation than D39 wild-type (WT) infection. Inhibition of Rap1 and Rac1 impairs filopodium formation and pneumococcal adherence. Therefore, ClpL can reduce pneumococcal adherence to A549 cells, likely via modulation of Rap1- and Rac1-mediated filopodium formation. These results demonstrate a potential role for ClpL in pneumococcal resistance to host cell adherence during infection. This study provides insight into further understanding the interactions between hosts and pathogens. PMID:24980975

  3. N-acetylcysteine amide, a thiol antioxidant, prevents bleomycin-induced toxicity in human alveolar basal epithelial cells (A549).

    PubMed

    Tobwala, S; Fan, W; Stoeger, T; Ercal, N

    2013-09-01

    Bleomycin (BLM), a glycopeptide antibiotic from Streptomyces verticillus, is an effective antineoplastic drug. However, its clinical use is restricted due to the wide range of associated toxicities, especially pulmonary toxicity. Oxidative stress has been implicated as an important factor in the development of BLM-induced pulmonary toxicity. Previous studies have indicated disruption of thiol-redox status in lungs (lung epithelial cells) upon BLM treatment. Therefore, this study focused on (1) investigating the oxidative effects of BLM on lung epithelial cells (A549) and (2) elucidating whether a well-known thiol antioxidant, N-acetylcysteine amide (NACA), provides any protection against BLM-induced toxicity. Oxidative stress parameters, such as glutathione (GSH), malondialdehyde (MDA), and antioxidant enzyme activities were altered upon BLM treatment. Loss of mitochondrial membrane potential (ΔΨm), as assessed by fluorescence microscopy, indicated that cytotoxicity is possibly mediated through mitochondrial dysfunction. Pretreatment with NACA reversed the oxidative effects of BLM. NACA decreased the reactive oxygen species (ROS) and MDA levels and restored the intracellular GSH levels. Our data showed that BLM induced A549 cell death by a mechanism involving oxidative stress and mitochondrial dysfunction. NACA had a protective role against BLM-induced toxicity by inhibiting lipid peroxidation, scavenging ROS, and preserving intracellular GSH and ΔΨm. NACA can potentially be developed into a promising adjunctive therapeutic option for patients undergoing chemotherapy with BLM.

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

    PubMed

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

    2017-08-01

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

  5. Inhibitory effect of radiotherapy combined with weekly recombinant human endostatin on the human pulmonary adenocarcinoma A549 xenografts in nude mice.

    PubMed

    Jiang, Xiao-dong; Dai, Peng; Wu, Jin; Song, Da-an; Yu, Jin-ming

    2011-05-01

    The aim of this study was to investigate the inhibitory effect of radiotherapy combined with weekly recombinant human endostatin (RHES) on the human pulmonary adenocarcinoma A549 xenografts in nude mice. The 40 A549 xenograft nude mice models were randomly divided into 4 groups (each group with 10 nude mice). Single radiotherapy group (group 1) was given a single external irradiation (6MV-X ray, 10 Gy) and peritumoral subcutaneous injection of 0.2 ml normal saline every day for 7 days. Single RHES group (group 2) was given peritumoral subcutaneous injection of 0.2 ml RHES (0.75 mg/ml) for 7 days. Combination therapy group (group 3) was given radiotherapy as the same as group 1 and RHES as the same as group 2. Control group was given normal saline as the same as group 1. The tumor volume was smaller in group 3 than in control group from the 8th day after treatment (P<0.05) and tumor regression occurred from the second week after treatment in group 3. On the 15th day after treatment, the inhibitory rates of tumor volume were 69.65%, 92.64% and 116.4% in groups 2, 1 and 3, respectively; MVD number was lower in group 3 than in group 1 (P<0.05); there was no statistical significance in VEGF expression between group 2 and control group as well as between group 3 and group 1 (P>0.05). Apoptosis was marked in group 3. Radiotherapy combined with weekly RHES can significantly inhibit tumor growth and earlier induce tumor regression, which may be related to the improvement of tumor hypoxia and the inhibition of radiation-induced tumor angiogenesis. Short-term application (1 week) of RHES is beneficial to clinical practice.

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

    PubMed

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

    2016-09-16

    MicroRNAs (miRNAs) are potent post-transcriptional regulators of gene expression and play roles in DNA damage response (DDR). PLK1 is identified as a modulator of DNA damage checkpoint. Although down-regulation of PLK1 by certain microRNAs has been reported, little is known about the interplay between PLK1 and miR-509-3-5p in DDR. Here we have demonstrated that miR-509-3-5p repressed PLK1 expression by targeting PLK1 3'-UTR, thereby causing mitotic aberration and growth arrest of human lung cancer A549 cells. Repression of PLK1 by miR-509-3-5p was further evidenced by over-expression of miR-509-3-5p in A549, HepG2 and HCT116p53(-/-) cancer cells, in which PLK1 protein was suppressed. Consistently, miR-509-3-5p was stimulated, while PLK1 protein was down-regulated in A549 cells exposed to CIS and ADR, suggesting that suppression of PLK1 by miR-509-3-5p is a component of CIS/ADR-induced DDR pathway. Flow cytometry and immunofluorescence labeling showed that over-expression of miR-509-3-5p in A549 induced G2/M arrest and aberrant mitosis characterized by abnormal bipolar mitotic spindles, condensed chromosomes, lagging DNA and chromosome bridges. In addition, over-expression of miR-509-3-5p markedly blocked A549 cell proliferation and sensitized the cells to CIS and ADR treatment. Taken together, miR-509-3-5p is a feasible suppressor for cancer by targeting PLK1. Our data may provide aid in potential design of combined chemotherapy and in our better understanding of the roles of microRNAs in response to DNA damage. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. Determination of in vitro free radical scavenging and antiproliferative effect of Pennisetum alopecuroides on cultured A549 human lung cancer cells

    PubMed Central

    Mathew, Githa Elizabeth; Mathew, Bijo; Gokul, S.; Krishna, Rahul; Farisa, M. P.

    2015-01-01

    Context: Pennisetum alopecuroides (Poaceae) is a grass predominantly distributed in tropics and sub tropics. It is used as a cattle feed in many regions. Aim: The objective of the present study was to investigate the in vitro free radical scavenging and antiproliferative activity of ethanol extract of P. alopecuroides (EEPA) on cultured A549 human lung cancer cell lines. Settings and Design: The anti-oxidant activity of ethanol extract was evaluated at dose level 12.5, 25, 50, 100, and 200 μg/ml. The in vitro antiproliferative activity was measured at doses of 10, 50, and 100 μg/ml. Materials and Methods: The free radical scavenging activity of the EEPA was determined by 2,2-Diphenyl-1-picrylhydrazyl (DPPH) method and in vitro antiproliferative activity on A549 human lung cancer cells was conducted by using MTT assay method. Results: The phytochemical screening revealed that the P. alopecuroides contained alkaloids, tannins, saponins, and flavonoids as the major secondary metabolites. The IC50 value of DPPH scavenging activity was found to be 44.41 μg/ml and 31.02 μg/ml  for a mixture of EEPA and standard ascorbic acid, respectively. In vitro MTT assay showed that EEPA had anti-proliferation effects on A549 cells in a dose dependent manner. Conclusions: This is the 1st time a pharmacological exploration of P. alopecuroides grasses has been conducted. We have shown that P. alopecuroides exhibits good free radical scavenging and strong in vitro cytotoxic activities against human lung cancer cell lines. PMID:26120234

  8. Comparative adherence to human A549 cells, plant fibronectin-like protein, and polystyrene surfaces of four Pseudomonas fluorescens strains from different ecological origin.

    PubMed

    Cossard, Elisabeth; Gallet, Olivier; Di Martino, Patrick

    2005-09-01

    The main objective of this study was to compare the adherence properties of four Pseudomonas fluorescens isolates from different ecological niches (human tissue, rhizosphere, drinking water, and cow milk). The substrates used to test P. fluorescens adherence were as follows: cultured human respiratory epithelial cells A549, immobilized plant fibronectin-like protein, and polystyrene. For all the experiments, bacteria were grown at 27 degrees C. The adherence assay to human cells was performed at 37 degrees C, whereas adherence to fibronectin and polystyrene was done at 27 degrees C. The four strains tested adhered to A549 cells but showed different adherence patterns. At 3 h, the milk isolate showed an aggregative adherence phenotype, whereas the three other isolates showed a diffuse adherence pattern. With a longer incubation time of 24 h, the aggregative pattern of the milk isolate disappeared, the adherence of the clinical strain increased, the adherence of the water isolate decreased, and morphological changes in A549 cells were observed with the clinical, water, and soil isolates. The four strains tested formed biofilms on polystyrene dishes. The clinical and milk isolates were the more efficient colonizers of polystyrene surfaces and also the more adherent to immobilized plant fibronectin-like protein. There was no relation between bacterial surface hydrophobicity and P. fluorescens adherence to the substrates tested. The main conclusions of these results are that P. fluorescens is an adherent bacterium, that no clear correlation exists between adherence and ecological habitat, and that P. fluorescens can adhere well to substrates not present in its natural environment.

  9. DUAL INHIBITION OF PI3K/AKT AND mTOR SIGNALING IN HUMAN NON-SMALL CELL LUNG CANCER CELLS BY A DIETARY FLAVONOID FISETIN

    PubMed Central

    Khan, Naghma; Afaq, Farrukh; Khusro, Fatima H.; Adhami, Vaqar Mustafa; Suh, Yewseok; Mukhtar, Hasan

    2011-01-01

    Lung cancer is one of the most commonly occurring malignancies. It has been reported that mTOR is phosphorylated in lung cancer and its activation was more frequent in tumors with over-expression of PI3K/Akt. Therefore, dual inhibitors of PI3K/Akt and mTOR signaling could be valuable agents for treating lung cancer. In the present study, we show that fisetin, a dietary tetrahydroxyflavone inhibits cell-growth with the concomitant suppression of PI3K/Akt and mTOR signaling in human non-small cell lung cancer (NSCLC) cells. Using autodock 4, we found that fisetin physically interacts with the mTOR complex at two sites. Fisetin treatment was also found to reduce the formation of A549 cell colonies in a dose-dependent manner. Treatment of cells with fisetin caused decrease in the protein expression of PI3K (p85 and p110), inhibition of phosphorylation of Akt, mTOR, p70S6K1, eIF-4E and 4E-BP1. Fisetin-treated cells also exhibited dose-dependent inhibition of the constituents of mTOR signaling complex like Rictor, Raptor, GβL and PRAS40. There was increase in the phosphorylation of AMPKα and decrease in the phosphorylation of TSC2 on treatment of cells with fisetin. We also found that treatment of cells with mTOR inhibitor rapamycin and mTOR-siRNA caused decrease in phosphorylation of mTOR and its target proteins which were further downregulated on treatment with fisetin, suggesting that these effects are mediated in part, through mTOR signaling. Our results show that fisetin suppressed PI3K/Akt and mTOR signaling in NSCLC cells and thus, could be developed as a chemotherapeutic agent against human lung cancer. PMID:21618507

  10. Effect of Allium sativum (garlic) diallyl disulfide (DADS) on human non-small cell lung carcinoma H1299 cells.

    PubMed

    Hui, C; Jun, W; Ya, L N; Ming, X

    2008-04-01

    This study was undertaken to elucidate the effect of diallyl disulfide from Allium sativum, an oil-soluble organosulfur compound found in garlic, in suppressing human non-small cell lung carcinoma H1299 cells. A potent increase in apoptotic cells has accompanied 1) a decrease in cell viability, 2) an increase of the fraction of G2/M-phase cells by up to 48.80 %, and 3) a transient increase of the phospho-p42/44 (phosphorylated p42/44 MAPK) in a time- and concentration-dependent manner. These results indicated that diallyl disulfide could induce apoptosis in human non-small cell lung carcinoma H1299 cells via, at least partly, G2/M-phase block of the cell cycle, related to a rise in MAPK phosphorylation.

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

    PubMed

    Kim, Jiyeon; Hwan Kim, Seong

    2013-01-01

    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. 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. 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. 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.

  12. CK2 Inhibitor CX-4945 Blocks TGF-β1-Induced Epithelial-to-Mesenchymal Transition in A549 Human Lung Adenocarcinoma Cells

    PubMed Central

    Kim, Jiyeon; Hwan Kim, Seong

    2013-01-01

    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. PMID:24023938

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

    PubMed

    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.

  14. Licochalcone A Inhibits the Proliferation of Human Lung Cancer Cell Lines A549 and H460 by Inducing G2/M Cell Cycle Arrest and ER Stress.

    PubMed

    Qiu, Chenyu; Zhang, Tingting; Zhang, Wenxin; Zhou, Lina; Yu, Bin; Wang, Wei; Yang, Zhihong; Liu, Zhiguo; Zou, Peng; Liang, Guang

    2017-08-12

    Licochalcone A (LicA), a flavonoid isolated from the famous Chinese medicinal herb Glycyrrhiza uralensis Fisch, has wide spectrum of pharmacological activities. In this study, the anti-cancer effects and potential mechanisms of LicA in non-small cell lung cancer (NSCLC) cells were studied. LicA decreased cell viability and induced apoptosis in a dose-dependent manner in NSCLC cells. LicA inhibited lung cancer cells growth by blocking cell cycle progression at the G2/M transition and inducing apoptosis. LicA treatment decreased the expression of MDM2, Cyclin B1, Cdc2 and Cdc25C in H460 and A549 cancer cell lines. In addition, LicA induced caspase-3 activation and poly-ADP-ribose polymerase (PARP) cleavage, which displayed features of apoptotic signals. Furthermore, LicA increased the expression of endoplasmic reticulum (ER) stress related proteins, such as p-EIF2α and ATF4. These data provide evidence that LicA has the potential to be used in the treatment of lung cancer.

  15. Oxygen Enhancement Ratio in Radiation-Induced Initial DSBs by an Optimized Flow Cytometry-based Gamma-H2AX Analysis in A549 Human Cancer Cells.

    PubMed

    Sunada, Shigeaki; Hirakawa, Hirokazu; Fujimori, Akira; Uesaka, Mitsuru; Okayasu, Ryuichi

    2017-08-22

    High-linear energy transfer (LET) heavy ions cause higher therapeutic effects than low-LET radiation due to lower dependency on oxygen concentration in tumor cell killing. The lethality after irradiation largely depends on DNA double-strand breaks (DSBs), however the detailed LET dependency for DSB induction under oxic and hypoxic conditions has not been reported. Therefore, we evaluated the oxygen enhancement ratio (OER) of heavy ion-induced DSB induction using a highly-optimized flow cytometry-based method of γ-H2AX detection. Non-small cell lung cancer (NSCLC) A549 cells were exposed to X-ray, carbon-ion and iron-ion radiations under oxic or hypoxic condition. As a DSB marker, the γ-H2AX signal was measured 1 h postirradiation and analyzed by flow cytometry. DSB slope values were calculated as DSB induction per Gy. Our method was able to detect high-LET radiation-induced DSBs even from clustered DNA damage sites. We also showed a decrease in OER value in an LET-dependent manner regardless of radiation type. In summary, we demonstrated a simple, quick and highly-optimized flow cytometry-based method of DSB analysis that detects DSBs induced by heavy-ion radiation for hypoxic and nonhypoxic cancer cells. Our study may provide a useful biological basis for heavy-ion radiotherapy.

  16. Profiling ribonucleotide and deoxyribonucleotide pools perturbed by gemcitabine in human non-small cell lung cancer cells

    PubMed Central

    Guo, Jian-Ru; Chen, Qian-Qian; Lam, Christopher Wai Kei; Wang, Cai-Yun; Wong, Vincent Kam Wai; Chang, Zee-Fen; Zhang, Wei

    2016-01-01

    In this study, we investigated the dosage effect of gemcitabine, an inhibitor of ribonucleotide reductase (RR), on cellular levels of ribonucleotides and deoxyribonucleotides using high performance liquid chromatography-electrospray ionization tandem mass spectrometric method. As anticipated, after 4-h incubation of non-small cell lung cancer (A549) cells with gemcitabine at 0.5 and 2 μM, there were consistent reductions in levels of deoxyribonucleoside diphosphates (dNDP) and their corresponding deoxyribonucleoside triphosphates (dNTP). However, after 24-h exposure to 0.5 μM gemcitabine, the amounts of dNTP were increased by about 3 fold, whereas cells after 24-h 2 μM gemcitabine treatment exhibited deoxycytidine diphosphate (dCDP), deoxyadenosine diphosphate (dADP) and deoxyguanosine diphosphate (dGDP) levels less than 50% of control values, with deoxycytidine triphosphate (dCTP) and deoxyguanosine triphosphate (dGTP) returning to the control level. Using cell cycle analysis, we found that 24-h incubation at 0.5 μM gemcitabine resulted in a significant increase in S phase arrest, while 2 μM treatment increased G0/G1 population. Our data demonstrated the correlation between the level of RR and the increased levels of dNTPs in the group of 0.5 μM treatment for 24-h with a markedly reduced level of dFdCTP. Accordingly, we proposed that the dosage of dFdC could determine the arrested phase of cell cycle, in turn affecting the recovery of dNTPs pools. PMID:27845436

  17. Cytotoxicity and genotoxicity of size-fractionated iron oxide (magnetite) in A549 human lung epithelial cells: role of ROS, JNK, and NF-κB.

    PubMed

    Könczöl, Mathias; Ebeling, Sandra; Goldenberg, Ella; Treude, Fabian; Gminski, Richard; Gieré, Reto; Grobéty, Bernard; Rothen-Rutishauser, Barbara; Merfort, Irmgard; Mersch-Sundermann, Volker

    2011-09-19

    Airborne particulate matter (PM) of varying size and composition is known to cause health problems in humans. The iron oxide Fe(3)O(4) (magnetite) may be a major anthropogenic component in ambient PM and is derived mainly from industrial sources. In the present study, we have investigated the effects of four different size fractions of magnetite on signaling pathways, free radical generation, cytotoxicity, and genotoxicity in human alveolar epithelial-like type-II cells (A549). The magnetite particles used in the exposure experiments were characterized by mineralogical and chemical techniques. Four size fractions were investigated: bulk magnetite (0.2-10 μm), respirable fraction (2-3 μm), alveolar fraction (0.5-1.0 μm), and nanoparticles (20-60 nm). After 24 h of exposure, the A549 cells were investigated by transmission electron microscopy (TEM) to study particle uptake. TEM images showed an incorporation of magnetite particles in A549 cells by endocytosis. Particles were found as agglomerates in cytoplasm-bound vesicles, and few particles were detected in the cytoplasm but none in the nucleus. Increased production of reactive oxygen species (ROS), as determined by the 2',7'-dichlorfluorescein-diacetate assay (DCFH-DA), as well as genotoxic effects, as measured by the cytokinesis block-micronucleus test and the Comet assay, were observed for all of the studied fractions after 24 h of exposure. Moreover, activation of c-Jun N-terminal kinases (JNK) without increased nuclear factor kappa-B (NF-κB)-binding activity but delayed IκB-degradation was observed. Interestingly, pretreatment of cells with magnetite and subsequent stimulation with the pro-inflammatory cytokine tumor necrosis factor-alpha (TNFα) led to a reduction of NF-κB DNA binding compared to that in stimulation with TNFα alone. Altogether, these experiments suggest that ROS formation may play an important role in the genotoxicity of magnetite in A549 cells but that activation of JNK seems to be

  18. MicroRNA-7 inhibits cell proliferation, migration and invasion in human non-small cell lung cancer cells by targeting FAK through ERK/MAPK signaling pathway

    PubMed Central

    Shi, Yu-Jia; Chen, Yi; Sun, Yun; Zhang, Qian; Song, Lei; Peng, Li-Ping

    2016-01-01

    Objective To investigate the effects of microRNA-7 (miR-7) on the proliferation, migration and invasion of non-small cell lung cancer NSCLC) cells by targeting FAK through ERK/MAPK signaling pathway. Methods NSCLC tissues and adjacent normal tissues were obtained from 160 NSCLC patients after operation. NSCLC cell lines (A549, H1299 and H1355) and a normal human fetal lung fibroblast cell line (MRC-5) were obtained. NSCLC cells were assigned into miR-7 inhibitors, miR-7 mimics, blank, miR-7 mimics control, miR-7 inhibitors control, FAK siRNA and miR-7 inhibitors + FAK siRNA groups. The expressions of miR-7 and FAK mRNA in tissues and cell lines were detected by qRT-PCR and Western-Blotting. Cell proliferation, migration and invasion were detected by MTT assay, wound scratch assay and Transwell assay. Results Compared with adjacent normal tissues, miR-7 expression was down-regulated, but the mRNA and protein expressions of FAK, ERK and MAPK were up-regulated. Compared with the blank and mimics control groups, miR-7 significantly increased but FAK, ERK and MAPK expressions decreased in miR-7 mimics and FAK siRNA groups. Cell proliferation, migration and invasion were inhibited in the miR-7 mimics and FAK siRNA groups, while opposite regarding miR-7 inhibitors group. Conclusion The miR-7 can inhibit the activation of ERK/MAPK signaling pathway by down-regulating FAK expression, thereby suppressing the proliferation, migration and invasion of NSCLC cells. The miR-7 and its target gene FAK may be novel targets for the diagnosis and treatment of NSCLC. PMID:27764812

  19. Effects of targeted silencing of FOXC1 gene on proliferation and in vitro migration of human non-small-cell lung carcinoma cells

    PubMed Central

    Chen, Sumei; Jiao, Shunchang; Jia, Youchao; Li, Yang

    2016-01-01

    Background: The aim of this study was to evaluate the effects of targeted silencing of forkhead box C1 (FOXC1) gene with small interfering RNA (siRNA) on the proliferation and in vitro migration of human non-small-cell lung carcinoma (NSCLC) A549 and NCIH460 cells, and to explore the molecular mechanism. Methods: These cells were divided into FOXC1 siRNA groups and negative control groups. Results: Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) showed that compared with normal cells and paracancerous tissues, FOXC1 mRNA expressions in NSCLC cells and tissues were significantly higher (P<0.05). qRT-PCR and Western blot showed that FOXC1 siRNA effectively silenced FOXC1 gene expression in NSCLC cells. EdU labeling assay revealed that the proliferative capacity significantly decreased compared with that of normal control group after FOXC1 silencing (P<0.05). Significantly fewer cells in the transfected group migrated than those in negative control group did. After FOXC1 silencing, NSCLC cells were arrested in the G0/G1 phase, which were significantly different from those in negative control group (P<0.05). Compared with negative control group, the expression of cyclin D1 decreased and that of E-cadherin increased. Meanwhile, vimentin and MMP-2 expressions significantly reduced (P<0.05). FOXC1 siRNA effectively silenced FOXC1 gene expressions in NSCLC cells, inhibited their proliferation and invasion, and arrested them in the G0/G1 phase, suggesting that FOXC1 affected proliferation probably by regulating the expression of cell cycle-related protein cyclin D1. Conclusion: Silencing FOXC1 may evidently inhibit the migration of these cells by reversing the EMT process through suppressing cadherin, being associated with the expressions of extracellular MMPs. PMID:27648121

  20. MicroRNA-107 inhibits tumor growth and metastasis by targeting the BDNF-mediated PI3K/AKT pathway in human non-small lung cancer.

    PubMed

    Xia, Huan; Li, Yang; Lv, Xiaohong

    2016-10-01

    Abnormal expression of microRNA-107 (miR-107) was found in non-small cell lung cancer (NSCLC). However, little is known about its role and molecular mechanism in NSCLC progression and metastasis. Therefore, the aims of this study were to clarify the potential role of miR-107 and molecular mechanism in NSCLC progression and metastasis. Quantitative real-time polymerase chain reaction assay showed that miR-107 expression levels were significantly decreased in NSCLC tissue and cell lines. Low miR-107 levels in tumor tissue correlated with advanced TNM stage and lymph node metastasis. Function assays showed that overexpression of miR-107 suppressed cell proliferation, migration and invasion in A549 cells in vitro, and inhibited NSCLC tumor growth in vivo. Further mechanism assays suggested the brain-derived neurotrophic factor (BDNF) was identified as a target gene of miR-107 in NSCLC cells. In addition, BDNF expression was upregulated, and inversely correlated with miR-107 in NSCLC tissues. Enforced overexpression of BDNF effectively reversed the tumor suppressive functions of miR-107 on NSCLC proliferation, migration and invasion. miR-107 overexpression or downregulation of BDNF was able to inhibit activation of PI3K/AKT signaling pathway. Taken together, our findings present the first evidence that miR-107 could suppress NSCLC metastasis by targeting BDNF and indirectly regulating PI3K/AKT signaling pathway, which might lead to a potential therapeutic strategy focusing on miR-107 and BDNF for human NSCLC.

  1. Multidrug Resistant Protein-Three Gene Regulation by the Transcription Factor Nrf2 in Human Bronchial Epithelial and Non-Small Cell Lung Carcinoma

    PubMed Central

    Mahaffey, Christopher M.; Zhang, Hongqiao; Rinna, Alessandra; Holland, William; Mack, Philip C.; Forman, Henry Jay

    2009-01-01

    Multidrug Resistant Proteins (MRP) are members of the ATP-binding cassette superfamily that facilitate detoxification by transporting toxic compounds, including chemotherapeutic drugs, out of cells. Chemotherapy, radiation, and other xenobiotic stresses have been shown to increase levels of select MRPs, although, the underlying mechanism remains largely unknown. Additionally, MRP3 is suspected of playing a role in the drug resistance of non-small cell lung carcinoma (NSCLC). Analysis of the MRP3 promoter revealed the presence of multiple putative electrophile responsive elements (EpRE), sequences that suggested possible regulation of this gene by Nrf2, the key transcription factor that binds to EpRE. The goal of this investigation was to determine whether MRP3 induction was dependent upon the transcription factor Nrf2. Keap1, a key regulator of Nrf2, sequesters Nrf2 in the cytoplasm, preventing entry into the nucleus. The electrophilic lipid peroxidation product, 4-hydroxy-2-nonenal (HNE) has been shown to modify Keap1 allowing Nrf2 to enter the nucleus. We found that HNE up-regulated MRP3 mRNA and protein levels in cell lines with wild type Keap1 (human bronchial epithelial cell line HBE1 and the NSCLC cell line H358), but not in the Keap1 mutant NSCLC cell lines (A549 and H460). Cell lines with mutant Keap1 had constitutively higher MRP3 that was not increased by HNE treatment. In HBE1 cells, silencing of Nrf2 with siRNA inhibited induction of MRP3 and by HNE. Finally, we found that silencing Nrf2 also increased the toxicity of cisplatin in H358 cells. The combined results therefore support the hypothesis that MRP3 induction by HNE involves Nrf2 activation. PMID:19345732

  2. ROS/Autophagy/Nrf2 Pathway Mediated Low-Dose Radiation Induced Radio-Resistance in Human Lung Adenocarcinoma A549 Cell.

    PubMed

    Chen, Ni; Wu, Lijun; Yuan, Hang; Wang, Jun

    2015-01-01

    Low-dose ionizing radiation (LDIR) can induce radio-resistance to following high dose radiation in various mammalian cells. The protective role of LDIR has been thought to be associated with the overall outcomes of cancer radiotherapy. NF-E2 related factor 2 (Nrf2) is a transcription factor that plays pivotal roles in maintaining cellular oxidative equilibrium. Since oxidative stress has been indicated to be a mediator of LDIR induced radio-resistance, the role of Nrf2 in this process was investigated in this research. Our results showed that in human lung adenocarcinoma A549 cell, 5cGy alpha particle induced radio-resistance to following 75cGy alpha particle radiation. The expression level of Nrf2 and its target Heme Oxygenase-1(HO-1) increased after 5cGy radiation. Both the shRNA of Nrf2 and the chemical inhibitor of HO-1 suppressed the induced radio-resistance, indicating the involvement of Nrf2 antioxidant pathway in this process. Further, we found 5cGy radiation stimulated autophagy process in A549. Inhibition of the autophagy process resulted in suppression of the radio-resistance and the induced expression of Nrf2 and HO-1. ROS scavenger N-acetyl-L-cysteine (NAC) blocked the autophagy process induced by 5cGy alpha particle, the upregulation of Nrf2 and HO-1, as well as the induced radio-resistance. In conclusion, ROS elevation caused by LDIR promoted Autophagy/Nrf2-HO-1 and conferred radio-resistance in A549.

  3. ROS/Autophagy/Nrf2 Pathway Mediated Low-Dose Radiation Induced Radio-Resistance in Human Lung Adenocarcinoma A549 Cell

    PubMed Central

    Chen, Ni; Wu, Lijun; Yuan, Hang; Wang, Jun

    2015-01-01

    Low-dose ionizing radiation (LDIR) can induce radio-resistance to following high dose radiation in various mammalian cells. The protective role of LDIR has been thought to be associated with the overall outcomes of cancer radiotherapy. NF-E2 related factor 2 (Nrf2) is a transcription factor that plays pivotal roles in maintaining cellular oxidative equilibrium. Since oxidative stress has been indicated to be a mediator of LDIR induced radio-resistance, the role of Nrf2 in this process was investigated in this research. Our results showed that in human lung adenocarcinoma A549 cell, 5cGy alpha particle induced radio-resistance to following 75cGy alpha particle radiation. The expression level of Nrf2 and its target Heme Oxygenase-1(HO-1) increased after 5cGy radiation. Both the shRNA of Nrf2 and the chemical inhibitor of HO-1 suppressed the induced radio-resistance, indicating the involvement of Nrf2 antioxidant pathway in this process. Further, we found 5cGy radiation stimulated autophagy process in A549. Inhibition of the autophagy process resulted in suppression of the radio-resistance and the induced expression of Nrf2 and HO-1. ROS scavenger N-acetyl-L-cysteine (NAC) blocked the autophagy process induced by 5cGy alpha particle, the upregulation of Nrf2 and HO-1, as well as the induced radio-resistance. In conclusion, ROS elevation caused by LDIR promoted Autophagy/Nrf2-HO-1 and conferred radio-resistance in A549. PMID:26078725

  4. Hyperoside induces both autophagy and apoptosis in non-small cell lung cancer cells in vitro.

    PubMed

    Fu, Ting; Wang, Ling; Jin, Xiang-nan; Sui, Hai-juan; Liu, Zhou; Jin, Ying

    2016-04-01

    Hyperoside (quercetin-3-O-β-D-galactopyranoside) is a flavonol glycoside found in plants of the genera Hypericum and Crataegus, which exhibits anticancer, anti-oxidant, and anti-inflammatory activities. In this study we investigated whether autophagy was involved in the anticancer mechanisms of hyperoside in human non-small cell lung cancer cells in vitro. Human non-small cell lung cancer cell line A549 was tested, and human bronchial epithelial cell line BEAS-2B was used for comparison. The expression of LC3-II, apoptotic and signaling proteins was measured using Western blotting. Autophagosomes were observed with MDC staining, LC3 immunocytochemistry, and GFP-LC3 fusion protein techniques. Cell viability was assessed using MTT assay. Hyperoside (0.5, 1, 2 mmol/L) dose-dependently increased the expression of LC3-II and autophagosome numbers in A549 cells, but had no such effects in BEAS-2B cells. Moreover, hyperoside dose-dependently inhibited the phosphorylation of Akt, mTOR, p70S6K and 4E-BP1, but increased the phosphorylation of ERK1/2 in A549 cells. Insulin (200 nmol/L) markedly enhanced the phosphorylation of Akt and decreased LC3-II expression in A549 cells, which were reversed by pretreatment with hyperoside, whereas the MEK1/2 inhibitor U0126 (20 μmol/L) did not blocked hyperoside-induced LC3-II expression. Finally, hyperoside dose-dependently suppressed the cell viability and induced apoptosis in A549 cells, which were significantly attenuated by pretreatment with the autophagy inhibitor 3-methyladenine (2.5 mmol/L). Hyperoside induces both autophagy and apoptosis in human non-small cell lung cancer cells in vitro. The autophagy is induced through inhibiting the Akt/mTOR/p70S6K signal pathways, which contributes to anticancer actions of hyperoside.

  5. Establishment and characterization of human non-small cell lung cancer cell lines.

    PubMed

    Li, Jiangchao; Yang, Hong; Chen, Leilei; Li, Yan; Zhu, Yinghui; Dai, Yongdong; Chen, Kai; Ai, Jiaoyu; Zeng, Tingting; Mao, Xueying; Liu, Lulu; Li, Xiaodong; Guan, Xin-Yuan

    2012-01-01

    Non-small cell lung cancer (NSCLC), a highly malignant tumor, is common in China and is associated with a very poor 5-year survival rate. To better understand the cancer biology of this disease, we report here the establishment of three new NSCLC cell lines, SCC210011, SCC211441 and ACC212102, from the tumor tissue of three NSCLC patients. By histological analysis, we found that all three cell lines displayed the typical features of endothelial cancer cells. The population doubling times of SCC210011, SCC211441 and ACC212102 cells were 42, 38 and 25 h, respectively. Our cytogenetic studies indicated that these cell lines exhibit structural and numerical chromosomal abnormalities. Furthermore, the tumorigenicity in nude mice was confirmed, and H&E staining results revealed that they resembled the primary tissue. These newly established cell lines may serve as useful models for studying the molecular pathogenesis of NSCLC.

  6. miR-129b suppresses cell proliferation in the human lung cancer cell lines A549 and H1299.

    PubMed

    Zheng, L; Qi, Y X; Liu, S; Shi, M L; Yang, W P

    2016-10-17

    Lung cancer is one of the most prevalent malignant tumors, and is one of the primary causes of cancer-associated deaths. In 2002, an estimated 1.18 million lung cancer-associated deaths were recorded, accounting for 18% of cancer-related deaths and 2% of total mortality. Despite the great progress that has been made in lung cancer therapies, the mechanisms underlying lung cancer formation and development remain largely unknown. Meanwhile, the microRNA miR-129 has been shown to be involved in the formation of many types of cancer. Therefore, this study aims to investigate whether miR129b could suppress proliferation of lung cancer cell lines. NSCLC tissue samples were collected from the Department of Respiratory Medicine between April 2013 and December 2015. Ten normal health individuals were recruited as controls. Lung cancer cell lines A549 and H1299 were used to examine the suppressive effects of miR129b. Quantitative real-time PCR was used to detect miR129b expression. The MTT assay was used to analyze cell proliferation. Results indicated that miR-129b is down-regulated in lung cancer cell lines and NSCLC tissues. Furthermore, overexpression of miR-129b inhibited proliferation of lung cancer cells. In conclusion, miR-129b suppresses lung cancer cell proliferation, and can be a potential therapeutic target for treatment of lung cancers.

  7. Chlorogenic acid regulates apoptosis and stem cell marker-related gene expression in A549 human lung cancer cells.

    PubMed

    Yamagata, Kazuo; Izawa, Yuri; Onodera, Daiki; Tagami, Motoki

    2017-09-05

    Previous studies indicated that chlorogenic acid, a compound present in many fruits and vegetables, has anti-cancer activities. We report that chlorogenic acid regulates the expression of apoptosis-related genes and self-renewal-related stem cell markers in cancer cells. The lung cancer cell line A549 was cultured with or without chlorogenic acid. The presence of chlorogenic acid decreased cell proliferation as measured by MTT activity. Polymerase chain reaction (PCR) showed that treatment of cells with chlorogenic acid reduced the expression of BCL2 but increased that of both BAX and CASP3. Chlorogenic acid enhanced annexin V expression as measured using fluorescently labeled annexin V. Chlorogenic acid also induced p38 MAPK and JNK gene expression. Meanwhile, several agents, including SB203580 (p38 MAP kinase inhibitor), N-acetylcysteine (antioxidant inhibitor), dipyridamole (phosphodiesterase inhibitor), and apocynin (NADPH-oxidase inhibitor) blocked chlorogenic acid-induced BAX gene expression. Chlorogenic acid reduced gene expression levels of stem cell-associated markers NANOG, POU5F1, and SOX2. Together these results indicate that chlorogenic acid affects the expression of apoptosis-related genes that are part of oxidative stress and p38 MAP-dependent pathways, as well as genes encoding stem cell markers. In conclusion, chlorogenic acid may contribute to the polyphenolic anti-cancer effect associated with consumption of vegetables and fruits.

  8. Human primary bronchial epithelial cells respond differently to titanium dioxide nanoparticles than the lung epithelial cell lines A549 and BEAS-2B.

    PubMed

    Ekstrand-Hammarström, Barbro; Akfur, Christine M; Andersson, Per Ola; Lejon, Christian; Osterlund, Lars; Bucht, Anders

    2012-09-01

    We have compared the cellular uptake and responses of five preparations of nanocrystalline titanium dioxide (TiO(2)) between normal human bronchial epithelial (NHBE) cells and epithelial cell lines (A549 and BEAS-2B). The P25 nanoparticles, containing both anatase and rutile modifications, induced reactive oxygen species (ROS) and secretion of the neutrophil chemoattractant IL-8 in all three cell types used. Pure anatase and rutile particles provoked differential IL-8 response in A549 and no response in BEAS-2B cells despite similar formation of ROS. The pure TiO(2) modifications also provoked release of the inflammatory mediators: IL-6, G-CSF and VEGF, in NHBE cells but not in the two cell lines. We conclude that the responsiveness of lung epithelial cells is strongly dependent on both the physicochemical properties of TiO(2) nanoparticles and the type of responder cells. The differential pro-inflammatory responsiveness of primary lung epithelial cells compared with immortalized cell lines should be considered in the assessment of adverse reactions to inhaled nanoparticles.

  9. Apigenin induces caspase-dependent apoptosis in human lung cancer A549 cells through Bax- and Bcl-2-triggered mitochondrial pathway.

    PubMed

    Lu, Hsu-Feng; Chie, Yu-Jie; Yang, Ming-Sung; Lee, Ching-Sung; Fu, Jene-John; Yang, Jai-Sing; Tan, Tzu-Wei; Wu, Shin-Hwar; Ma, Yi-Shih; Ip, Siu-Wan; Chung, Jing-Gung

    2010-06-01

    The molecular mechanism and possible signaling pathway of apigenin-induced cytotoxicity and apoptosis in human lung cancer cells has not been reported. We investigated the role of ROS, Ca2+, caspases and Bax proteins and mitochondria membrane potential in apigenin-induced apoptosis in A549 cells. Cells were incubated with different concentrations of apigenin then cell morphological changes, DNA damage, cell viability and apoptosis were determined by Comet assay, and flow cytometric analysis. Sub-G1 phase was also examined. Western blot analysis was used to determined the levels of Bax and Bcl-2 and apoptosis associated proteins, and confocal laser microscope for examining the translocation of associated protein after exposed to apigenin. The results indicated that apigenin induced morphological changes, decreased percentage of viable cells and induced apoptosis dose- and time-dependently. DAPI staining and Comet assay also confirmed that apigenin-induced DNA condensation and damage. The levels of caspase-3, -8 and -9 involved in apigenin-induced apoptosis indicating caspase-dependent pathway was induced by apigenin. Western blotting showed that apigenin promoted cytochrome c levels and also induced dysfunction of mitochondria leading to the release of cytochrome c, AIF and Endo G, causing the activation of caspase-9 and -3, then apoptosis in A549 cells.

  10. Biological impacts of TiO2 on human lung cell lines A549 and H1299: particle size distribution effects

    NASA Astrophysics Data System (ADS)

    Tedja, Roslyn; Marquis, Christopher; Lim, May; Amal, Rose

    2011-09-01

    Increasing use of titanium dioxide (TiO2) nanoparticles in many commercial applications has led to emerging concerns regarding the safety and environmental impact of these materials. In this study, we have investigated the biological impact of nano-TiO2 (with particle primary size of 20 nm Aeroxide P25) on human lung cell lines in vitro and also the effect of particle size distribution on the particle uptake and apparent toxicity. The biological impact of nano-TiO2 is shown to be influenced by the concentration and particle size distribution of the TiO2 and the impact was shown to differ between the two cell lines (A549 and H1299) investigated herein. A549 cell line was shown to be relatively resistant to the total amount of TiO2 particles uptaken, as measured by cell viability and metabolic assays, while H1299 had a much higher capacity to ingest TiO2 particles and aggregates, with consequent evidence of impact at concentrations as low as 30-150 μg/mL TiO2. Evidence gathered from this study suggests that both viability and metabolic assays (measuring metabolic and mitochondrial activities and also cellular ATP level) should be carried out collectively to gain a true assessment of the impact of exposure to TiO2 particles.

  11. Overexpression and activation of hepatocyte growth factor/scatter factor in human non-small-cell lung carcinomas.

    PubMed Central

    Olivero, M.; Rizzo, M.; Madeddu, R.; Casadio, C.; Pennacchietti, S.; Nicotra, M. R.; Prat, M.; Maggi, G.; Arena, N.; Natali, P. G.; Comoglio, P. M.; Di Renzo, M. F.

    1996-01-01

    Hepatocyte growth factor/scatter factor (HGF/SF) stimulates the invasive growth of epithelial cells via the c-MET oncogene-encoded receptor. In normal lung, both the receptor and the ligand are detected, and the latter is known to be a mitogenic and a motogenic factor for both cultured bronchial epithelial cells and non-small-cell carcinoma lines. Here, ligand and receptor expression was examined in 42 samples of primary human non-small-cell lung carcinoma of different histotype. Each carcinoma sample was compared with adjacent normal lung tissue. The Met/HGF receptor was found to be 2 to 10-fold increased in 25% of carcinoma samples (P = 0.0113). The ligand, HGF/SF, was found to be 10 to 100-fold overexpressed in carcinoma samples (P < 0.0001). Notably, while HGF/SF was occasionally detectable and found exclusively as a single-chain inactive precursor in normal tissues, it was constantly in the biologically-active heterodimeric form in carcinomas. Immunohistochemical staining showed homogeneous expression of both the receptor and the ligand in carcinoma samples, whereas staining was barely detectable in their normal counterparts. These data show that HGF/SF is overexpressed and consistently activated in non-small-cell lung carcinomas and may contribute to the invasive growth of lung cancer. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:8980383

  12. Toxic Effects of the Major Components of Diesel Exhaust in Human Alveolar Basal Epithelial Cells (A549)

    PubMed Central

    Rossner, Pavel; Strapacova, Simona; Stolcpartova, Jitka; Schmuczerova, Jana; Milcova, Alena; Neca, Jiri; Vlkova, Veronika; Brzicova, Tana; Machala, Miroslav; Topinka, Jan

    2016-01-01

    We investigated the toxicity of benzo[a]pyrene (B[a]P), 1-nitropyrene (1-NP) and 3-nitrobenzanthrone (3-NBA) in A549 cells. Cells were treated for 4 h and 24 h with: B[a]P (0.1 and 1 μM), 1-NP (1 and 10 μM) and 3-NBA (0.5 and 5 μM). Bulky DNA adducts, lipid peroxidation, DNA and protein oxidation and mRNA expression of CYP1A1, CYP1B1, NQO1, POR, AKR1C2 and COX2 were analyzed. Bulky DNA adducts were induced after both treatment periods; the effect of 1-NP was weak. 3-NBA induced high levels of bulky DNA adducts even after 4-h treatment, suggesting rapid metabolic activation. Oxidative DNA damage was not affected. 1-NP caused protein oxidation and weak induction of lipid peroxidation after 4-h incubation. 3-NBA induced lipid peroxidation after 24-h treatment. Unlike B[a]P, induction of the aryl hydrocarbon receptor, measured as mRNA expression levels of CYP1A1 and CYP1B1, was low after treatment with polycyclic aromatic hydrocarbon (PAH) nitro-derivatives. All test compounds induced mRNA expression of NQO1, POR, and AKR1C2 after 24-h treatment. AKR1C2 expression indicates involvement of processes associated with reactive oxygen species generation. This was supported further by COX2 expression induced by 24-h treatment with 1-NP. In summary, 3-NBA was the most potent genotoxicant, whereas 1-NP exhibited the strongest oxidative properties. PMID:27571070

  13. Toxic Effects of the Major Components of Diesel Exhaust in Human Alveolar Basal Epithelial Cells (A549).

    PubMed

    Rossner, Pavel; Strapacova, Simona; Stolcpartova, Jitka; Schmuczerova, Jana; Milcova, Alena; Neca, Jiri; Vlkova, Veronika; Brzicova, Tana; Machala, Miroslav; Topinka, Jan

    2016-08-26

    We investigated the toxicity of benzo[a]pyrene (B[a]P), 1-nitropyrene (1-NP) and 3-nitrobenzanthrone (3-NBA) in A549 cells. Cells were treated for 4 h and 24 h with: B[a]P (0.1 and 1 μM), 1-NP (1 and 10 μM) and 3-NBA (0.5 and 5 μM). Bulky DNA adducts, lipid peroxidation, DNA and protein oxidation and mRNA expression of CYP1A1, CYP1B1, NQO1, POR, AKR1C2 and COX2 were analyzed. Bulky DNA adducts were induced after both treatment periods; the effect of 1-NP was weak. 3-NBA induced high levels of bulky DNA adducts even after 4-h treatment, suggesting rapid metabolic activation. Oxidative DNA damage was not affected. 1-NP caused protein oxidation and weak induction of lipid peroxidation after 4-h incubation. 3-NBA induced lipid peroxidation after 24-h treatment. Unlike B[a]P, induction of the aryl hydrocarbon receptor, measured as mRNA expression levels of CYP1A1 and CYP1B1, was low after treatment with polycyclic aromatic hydrocarbon (PAH) nitro-derivatives. All test compounds induced mRNA expression of NQO1, POR, and AKR1C2 after 24-h treatment. AKR1C2 expression indicates involvement of processes associated with reactive oxygen species generation. This was supported further by COX2 expression induced by 24-h treatment with 1-NP. In summary, 3-NBA was the most potent genotoxicant, whereas 1-NP exhibited the strongest oxidative properties.

  14. Toxicity of wood smoke particles in human A549 lung epithelial cells: the role of PAHs, soot and zinc.

    PubMed

    Dilger, Marco; Orasche, Jürgen; Zimmermann, Ralf; Paur, Hanns-Rudolf; Diabaté, Silvia; Weiss, Carsten

    2016-12-01

    Indoor air pollution is associated with increased morbidity and mortality. Specifically, the health impact of emissions from domestic burning of biomass and coal is most relevant and is estimated to contribute to over 4 million premature deaths per year worldwide. Wood is the main fuel source for biomass combustion and the shift towards renewable energy sources will further increase emissions from wood combustion even in developed countries. However, little is known about the constituents of wood smoke and biological mechanisms that are responsible for adverse health effects. We exposed A549 lung epithelial cells to collected wood smoke particles and found an increase in cellular reactive oxygen species as well as a response to bioavailable polycyclic aromatic hydrocarbons. In contrast, cell vitality and regulation of the pro-inflammatory cytokine interleukin-8 were not affected. Using a candidate approach, we could recapitulate WSP toxicity by the combined actions of its constituents soot, metals and PAHs. The soot fraction and metals were found to be the most important factors for ROS formation, whereas the PAH response can be mimicked by the model PAH benzo[a]pyrene. Strikingly, PAHs adsorbed to WSPs were even more potent in activating target gene expression than B[a]P individually applied in suspension. As PAHs initiate multiple adverse outcome pathways and are prominent carcinogens, their role as key pollutants in wood smoke and its health effects warrants further investigation. The presented results suggest that each of the investigated constituents soot, metals and PAHs are major contributors to WSP toxicity. Mitigation strategies to prevent adverse health effects of wood combustion should therefore not only aim at reducing the emitted soot and PAHs but also the metal content, through the use of more efficient combustion appliances, and particle precipitation techniques, respectively.

  15. Investigation of the mechanism and apoptotic pathway induced by 4β cinnamido linked podophyllotoxins against human lung cancer cells A549.

    PubMed

    Kamal, Ahmed; Nayak, V Lakshma; Bagul, Chandrakant; Vishnuvardhan, M V P S; Mallareddy, Adla

    2015-11-01

    Apoptosis is essential for normal development and the maintenance of homeostasis. It plays a necessary role to protect against carcinogenesis by eliminating damaged cells. Many studies have demonstrated that the dysregulation of apoptosis results in cancer and this provides an approach to develop therapeutic agents via inducing apoptosis. In our previous studies 4β-cinnamido linked podophyllotoxin conjugates were synthesized and evaluated for their cytotoxic activity in a panel of five human cancer cell lines and the new molecules like 17a and 17f were considered as potential leads. The cytotoxic activity was comparable to etoposide. These observations prompted us to investigate the mechanism underplaying the cytotoxic activity and apoptotic pathway induced by these compounds in human lung cancer cells A459. The results of the present study revealed that these compounds exhibited DNA topoisomerase IIα inhibition and induced mitochondrial mediated apoptosis. It was further confirmed by Mitochondrial membrane potential, Cytochrome c release, cleavage of poly (ADP-ribose) polymerase (PARP), Reactive oxygen species (ROS) generation, regulation of antiapoptotic protein Bcl-2 and pro apoptotic protein Bax studied by Western blot analysis. Annexin V-FITC assay also suggested that these compounds induced cell death by apoptosis. Pretreatment with N-acetyl-L-cysteine (NAC) prevented the generation of ROS. Further, pretreatment with NAC significantly inhibited 17a and 17f induced apoptosis, suggesting that ROS are the key mediators for 17a and 17f induced apoptosis. These data indicate that these compounds might induce apoptosis in A549 cells through a ROS mediated mitochondrial dysfunction pathway. Moreover, these compounds did not significantly inhibit the noncancerous human embryonic kidney cells, HEK-293. Docking studies also elucidate the potential of these molecules to bind to the DNA topoisomerase II. Podophyllotoxin analogs were investigated for their mechanism and

  16. Steroid sulphatase and oestrogen sulphotransferase in human non-small-cell lung carcinoma

    PubMed Central

    Iida, S; Kakinuma, H; Miki, Y; Abe, K; Sakurai, M; Suzuki, S; Niikawa, H; Akahira, J; Suzuki, T; Sasano, H

    2013-01-01

    Background: Steroid sulphatase (STS) is one of the steroid-metabolising enzymes involved in desulphating inactive steroid sulphates and oestrogen sulphotransferase (EST) sulphates active oestrogen. The roles of both STS and EST have not been examined in oestrogen-dependent non-small-cell lung cancer (NSCLC). Methods: We evaluated the immunoreactivity of STS and EST in NSCLC cases using immunohistochemistry. The function of STS and EST was further demonstrated using NSCLC cell lines. Results: The immunoreactivity of STS and EST was detected in 49.5% and 27.8% of NSCLC cases, respectively. The immunoreactivity of STS was significantly higher in female adenocarcinoma cases. The STS-positive NSCLCs were also significantly correlated in an inversed manner with tumour size and cell proliferation and tended to be associated with better clinical outcome. However, the immunoreactivity of EST was significantly correlated with intracellular oestradiol concentration. Results of in vitro analysis demonstrated that oestrone sulphate (E1-S) induced and pregnenolone sulphate (Preg-S) inhibited the proliferation in STS-expressing cell lines. The inhibition by Preg-S was reversed by a specific progesterone receptor blocker. Simultaneous addition of E1-S and Preg-S significantly suppressed the proliferation. Conclusion: In NSCLC patients, STS is considered a good prognostic factor. Results of our present study also indicated the benefits of potential progesterone therapy for NSCLC patients. PMID:23531699

  17. Deoxypodophyllotoxin triggers necroptosis in human non-small cell lung cancer NCI-H460 cells.

    PubMed

    Wu, Meijuan; Jiang, Zhenzhou; Duan, Huaqin; Sun, Lixin; Zhang, Shuang; Chen, Mi; Wang, Yun; Gao, Qin; Song, Yuming; Zhu, Xiong; Zhang, Luyong

    2013-10-01

    Deoxypodophyllotoxin (DPT), a naturally occurring microtubule destabilizer, inhibits tubulin polymerization and causes cell cycle arrest at G2/M phase in tumor cells. However, the anti-tumor effect and specific mechanism of DPT in non-small cell lung cancer (NSCLC) are still poorly understood. In this study, we determined the anti-tumor effect and potential mechanism of DPT in the NSCLC cell line, NCI-H460 (H460). First, we demonstrated that DPT significantly inhibits the proliferation of H460 cells in vitro and the growth of H460 xenografts in vivo. In further studies, DPT triggered necroptosis in H460 cells with the following characteristics: (I) necrotic cell death morphology; (II) autophagy; (III) loss of plasma membrane integrity; (IV) loss of mitochondria membrane potential; (V) elevation of reactive oxygen species levels; and (VI) specific inhibition of necroptosis via a small molecule, necrostatin-1. This study also revealed that DPT has a similar effect towards the drug-sensitive cancer cell line, H460, and the drug-resistant cell line, H460/Bcl-xL. To our knowledge, this is the first report to document the induction of necroptosis by a microtubule-targeting agent to circumvent cancer drug resistance, thereby providing a new potential choice for clinical cancer therapy, especially drug-resistant cancer therapy.

  18. Three-dimensional quantitative structure-activity relationship study on anti-cancer activity of 3,4-dihydroquinazoline derivatives against human lung cancer A549 cells

    NASA Astrophysics Data System (ADS)

    Cho, Sehyeon; Choi, Min Ji; Kim, Minju; Lee, Sunhoe; Lee, Jinsung; Lee, Seok Joon; Cho, Haelim; Lee, Kyung-Tae; Lee, Jae Yeol

    2015-03-01

    A series of 3,4-dihydroquinazoline derivatives with anti-cancer activities against human lung cancer A549 cells were subjected to three-dimensional quantitative structure-activity relationship (3D-QSAR) studies using the comparative molecular similarity indices analysis (CoMSIA) approaches. The most potent compound, 1 was used to align the molecules. As a result, the best prediction was obtained with CoMSIA combined the steric, electrostatic, hydrophobic, hydrogen bond donor, and hydrogen bond acceptor fields (q2 = 0.720, r2 = 0.897). This model was validated by an external test set of 6 compounds giving satisfactory predictive r2 value of 0.923 as well as the scrambling stability test. This model would guide the design of potent 3,4-dihydroquinazoline derivatives as anti-cancer agent for the treatment of human lung cancer.

  19. Role of {alpha}{sub v}{beta}{sub 5} integrin receptor in endocytosis of crocidolite and its effect on intracellular glutathione levels in human lung epithelial (A549) cells

    SciTech Connect

    Pande, Priyadarshini; Mosleh, Tariq A.; Aust, Ann E. . E-mail: aaust@cc.usu.edu

    2006-01-15

    Crocidolite, containing 27% iron by weight, is the most carcinogenic form of asbestos. Crocidolite fibers are endocytized by {alpha}{sub v}{beta}{sub 5} integrin receptors in rabbit pleural mesothelial cells. We show here that crocidolite fibers are endocytized in human lung epithelial (A549) cells and in primary small airway epithelial (SAEC) cells. Presence of the integrin {alpha}{sub v}{beta}{sub 5} blocking antibody, P1F6, significantly reduced the uptake of crocidolite fibers in A549 cells. Thus, the integrin {alpha}{sub v}{beta}{sub 5} receptor is involved in endocytosis of crocidolite fibers in A549 cells as well. Previously, it has been observed that asbestos fibers lead to changes in the intracellular redox environment, i.e. a marked decrease in intracellular glutathione concentrations and an increase in the extracellular glutathione in A549 cells. In addition, the decrease in intracellular glutathione was found to be largely independent of iron present on the surface of the fiber. A549 cells were treated with crocidolite in the presence of endocytosis inhibitor cytochalasin D. Our data indicate that, upon preventing endocytosis, we were able to reverse the decrease in total intracellular glutathione. The decrease in total intracellular glutathione could also be prevented in the presence of the monoclonal antibody P1F6. Thus, we observed that endocytosis of crocidolite fibers via integrin {alpha}{sub v}{beta}{sub 5} receptor is linked to the marked decrease in total intracellular glutathione in A549 cells.

  20. Factors associated with human small aggressive non small cell lung cancer.

    PubMed

    Tammemagi, C Martin; Freedman, Matthew T; Church, Timothy R; Oken, Martin M; Hocking, William G; Kvale, Paul A; Hu, Ping; Riley, Thomas L; Ragard, Lawrence R; Prorok, Philip C; Berg, Christine D

    2007-10-01

    Some non-small cell lung cancers (NSCLC) progress to distant lymph nodes or metastasize while relatively small. Such small aggressive NSCLCs (SA-NSCLC) are no longer resectable with curative intent, carry a grave prognosis, and may involve unique biological pathways. This is a study of factors associated with SA-NSCLC. A nested case-case study was embedded in the National Cancer Institute's Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial. SA-NSCLC cases had stage T1, N3, and/or M1 NSCLC (n = 48) and non-SA-NSCLC cases had T2 to T3, N0 to N2, and M0 NSCLC (n = 329). Associations were assessed by multiple logistic regression. SA-NSCLCs were associated with younger age at diagnosis [odds ratio (OR)(>or=65 versus <65), 0.44; 95% confidence interval (95% CI), 0.22-0.88], female gender, family history of lung cancer, and the interaction gender*family history of lung cancer and were inversely associated with ibuprofen use (OR(yes versus no), 0.29; 95% CI, 0.11-0.76). The ORs for associating gender (women versus men) with SA-NSCLC in those with and without a family history of lung cancer were 11.76 (95% CI, 2.00-69.22) and 1.86 (95% CI, 0.88-3.96), respectively. These associations held adjusted for histology and time from screening to diagnosis and when alternative controls were assessed. SA-NSCLC was associated with female gender, especially in those with a family history of lung cancer. If these exploratory findings, which are subject to bias, are validated as causal, elucidation of the genetic and female factors involved may improve understanding of cancer progression and lead to preventions and therapies. Ibuprofen may inhibit lung cancer progression.

  1. Reversine Induced Multinucleated Cells, Cell Apoptosis and Autophagy in Human Non-Small Cell Lung Cancer Cells

    PubMed Central

    Lin, Ching-Yen; Chen, Yih-Yuan; Chen, Ping-Tzu; Tseng, Ya-Shih

    2016-01-01

    Reversine, an A3 adenosine receptor antagonist, has been shown to induce differentiated myogenic-lineage committed cells to become multipotent mesenchymal progenitor cells. We and others have reported that reversine has an effect on human tumor suppression. This study revealed anti-tumor effects of reversine on proliferation, apoptosis and autophagy induction in human non-small cell lung cancer cells. Treatment of these cells with reversine suppressed cell growth in a time- and dosage-dependent manner. Moreover, polyploidy occurred after reversine treatment. In addition, caspase-dependent apoptosis and activation of autophagy by reversine in a dosage-dependent manner were also observed. We demonstrated in this study that reversine contributes to growth inhibition, apoptosis and autophagy induction in human lung cancer cells. Therefore, reversine used as a potential therapeutic agent for human lung cancer is worthy of further investigation. PMID:27385117

  2. Dose-biomarker-response modeling of the anticancer effect of ethaselen in a human non-small cell lung cancer xenograft mouse model

    PubMed Central

    Ye, Suo-fu; Li, Jian; Ji, Shuang-min; Zeng, Hui-hui; Lu, Wei

    2017-01-01

    Thioredoxin reductase (TrxR) is a component of several redox-sensitive signaling cascades that mediate important biological processes such as cell survival, maturation, growth, migration and inhibition of apoptosis. The expression levels of TrxR1 in some human carcinoma cell lines are nearly 10 times higher than those in normal cells. Ethaselen is a novel antitumor candidate that exerts potent inhibition on non-small cell lung cancer (NSCLC) by targeting TrxR. In this study we explored the relationship between the ethaselen dose and TrxR activity level and the relationship between TrxR degradation and tumor apoptosis in a human lung carcinoma A549 xenograft model. BALB/c nude mice implanted with human NSCLC cell line A54 were administered ethaselen (36, 72, 108 mg·kg−1·d−1, ig) or vehicle for 10 d. The tumor size and TrxR activity levels in tumor tissues were daily recorded and detected. Based on the experimental data, NONMEM 7.2 was used to develop an integrated dose-biomarker-response model for describing the quantitative relationship between ethaselen dose and tumor eradication effects. The time course of TrxR activity levels was modeled using an indirect response model (IDR model), in which the influence of the tumor growth rates on Kin with the linear correction factor γ1 (0.021 d/mm). The drug binding-inhibition effects on Kout was described using a sigmoidal Emax model with Smax (5.95), SC50 (136 mg/kg) and Hill's coefficient γ2 (2.29). The influence of TrxR activity inhibition on tumor eradication was characterized by an Emax model with an Emax (130 mm3/d) and EC50 (0.0676). This model was further validated using a visual predictive check (VPC) and was used to predict the efficacy of different doses. In conclusion, the properties and characteristics of ethaselen acting on TrxR degradation and subsequently resulting in tumor apoptosis are characterized by the IDR model and integrated dose-biomarker-response model with high goodness-of-fit and great

  3. Fyn mediates transforming growth factor-beta1-induced down-regulation of E-cadherin in human A549 lung cancer cells.

    PubMed

    Kim, An Na; Jeon, Woo-Kwang; Lim, Kyu-Hyoung; Lee, Hui-Young; Kim, Woo Jin; Kim, Byung-Chul

    2011-04-01

    Transforming growth factor-beta (TGF-β) signaling positively contributes to the regulation of tumor metastasis. However, the underlying molecular mechanisms are less well defined. We here show that Fyn, a member of Src family tyrosine kinases, plays a critical role in mediating TGF-β1-induced down-regulation of E-cadherin in human A549 lung cancer cells. Blockade of Fyn with siRNA knockdown or ligand-binding defective mutant significantly lowered the ability of TGF-β1 to repress E-cadherin expression. Furthermore, our results demonstrated that Fyn facilitates TGF-β1-mediated suppression of E-cadherin through p38 kinase-dependent induction of Snail. Collectively, our findings identify a Fyn-p38-Snail cascade as a new signaling pathway mediating oncogenic TGF-β function.

  4. Separation of an aqueous extract Inonotus obliquus (Chaga). A novel look at the efficiency of its influence on proliferation of A549 human lung carcinoma cells.

    PubMed

    Mazurkiewicz, Witold; Rydel, Katarzyna; Pogocki, Dariusz; Lemieszek, Marta Kinga; Langner, Ewa; Rzeski, Wojciech

    2010-01-01

    Aqueous extract of Inonotus obliquus was hydrolyzed in dilute hydrochloric acid. The products were extracted applying organic solvents, and separated chromatographically on a silica gel-packed column. Eluted fractions were analyzed by means of GC-MS. The presence of hydrocarbons, alcohols, phenols and various carbonyl compounds in analyzed fractions has been detected and quantified. Preliminarily experiments on the influence of certain separated samples on the proliferation of A549 human lung carcinoma cells were performed. Therefore, we hypothesize that the major antiproliferative effects are related to the presence of benzaldehyde, which is a benzyl alcohol metabolite formed in situ in the cells culture with the yield moderated by the presence of trace amounts of "high molecular mass compounds".

  5. Cytotoxicity and genotoxicity in human lung epithelial A549 cells caused by airborne volatile organic compounds emitted from pine wood and oriented strand boards.

    PubMed

    Gminski, Richard; Tang, Tao; Mersch-Sundermann, Volker

    2010-06-16

    Due to the massive reduction of air-change rates in modern, energy-saving houses and dwellings, the contribution of volatile organic compound (VOCs) emissions from wood-based materials to indoor air quality has become increasingly important. To evaluate toxicity of VOC mixtures typically emitted from pine wood and oriented strand boards (OSB) and their main constituents (selected terpenes and aldehydes), cytotoxicity and genotoxicity were investigated in human A549 lung cells. To facilitate exposure directly via gas phase, a 250 L emission chamber was combined with a Vitrocell exposure system. VOC exposure concentrations were measured by GC/MSD. Biological effects were determined after an exposure time of 1h by measuring cytotoxicity (erythrosine B staining) and genotoxicity (comet assay). Neither cytotoxic nor genotoxic effects were observed for VOC mixtures emitted from pine wood or OSB at loading factors of approximately 13 m(2)/m(3) (worst case conditions) of the panels (with maximum VOC levels of about 80 mg/m(3)) in comparison to clean air. While alpha-pinene and Delta(3)-carene did not induce toxic effects even at exposure concentrations of up to 1800 mg/m(3) and 600 mg/m(3), respectively, hexanal showed a cytotoxic effect at 2000 mg/m(3). The alpha,beta-unsaturated aldehydes 2-heptenal and 2-octenal caused genotoxic effects in concentrations exceeding 100mg/m(3) and 40 mg/m(3), respectively. In conclusion, high concentrations of VOCs and VOC mixtures emitted from pine wood and OSB did not lead to adverse effects in A549 human lung cells even at concentrations 10(2) to 10(5)-fold higher than those found in normal indoor air. Attention must be paid to mutagenic and possibly carcinogenic alpha,beta-unsaturated aldehydes.

  6. Multidimensional effects of biologically synthesized silver nanoparticles in Helicobacter pylori, Helicobacter felis, and human lung (L132) and lung carcinoma A549 cells

    NASA Astrophysics Data System (ADS)

    Gurunathan, Sangiliyandi; Jeong, Jae-Kyo; Han, Jae Woong; Zhang, Xi-Feng; Park, Jung Hyun; Kim, Jin-Hoi

    2015-02-01

    Silver nanoparticles (AgNPs) are prominent group of nanomaterials and are recognized for their diverse applications in various health sectors. This study aimed to synthesize the AgNPs using the leaf extract of Artemisia princeps as a bio-reductant. Furthermore, we evaluated the multidimensional effect of the biologically synthesized AgNPs in Helicobacter pylori, Helicobacter felis, and human lung (L132) and lung carcinoma (A549) cells. UV-visible (UV-vis) spectroscopy confirmed the synthesis of AgNPs. X-ray diffraction (XRD) indicated that the AgNPs are specifically indexed to a crystal structure. The results from Fourier transform infrared spectroscopy (FTIR) indicate that biomolecules are involved in the synthesis and stabilization of AgNPs. Dynamic light scattering (DLS) studies showed the average size distribution of the particle between 10 and 40 nm, and transmission electron microscopy (TEM) confirmed that the AgNPs were significantly well separated and spherical with an average size of 20 nm. AgNPs caused dose-dependent decrease in cell viability and biofilm formation and increase in reactive oxygen species (ROS) generation and DNA fragmentation in H. pylori and H. felis. Furthermore, AgNPs induced mitochondrial-mediated apoptosis in A549 cells; conversely, AgNPs had no significant effects on L132 cells. The results from this study suggest that AgNPs could cause cell-specific apoptosis in mammalian cells. Our findings demonstrate that this environmentally friendly method for the synthesis of AgNPs and that the prepared AgNPs have multidimensional effects such as anti-bacterial and anti-biofilm activity against H. pylori and H. felis and also cytotoxic effects against human cancer cells. This report describes comprehensively the effects of AgNPs on bacteria and mammalian cells. We believe that biologically synthesized AgNPs will open a new avenue towards various biotechnological and biomedical applications in the near future.

  7. The lncRNA XIST exhibits oncogenic properties via regulation of miR-449a and Bcl-2 in human non-small cell lung cancer This article has been corrected since Advanced Online Publication, and an erratum is also printed in this issue.

    PubMed Central

    Zhang, Ya-long; Li, Xue-bing; Hou, Yan-xu; Fang, Nian-zhen; You, Jia-cong; Zhou, Qing-hua

    2017-01-01

    Long non-coding RNAs (lncRNAs) are associated with the occurrence, development and prognoses of non-small cell lung cancer (NSCLC). In the present study, we investigated the functional mechanisms of the lncRNA XIST in two human NSCLC cell lines, A549 and NCI-H1299. In all the 5 NSCLC cell lines (NL9980, NCI-H1299, NCI-H460, SPC-A-1 and A549) tested, the expression levels of XIST were significantly elevated, as compared with those in normal human bronchial epithelial cell line BEAS-2B. In A549 and NCI-H1299 cells, knockdown of XIST by siRNA significantly inhibited the cell proliferation, migration and invasion, and promoted cell apoptosis. Furthermore, XIST knockdown elevated the expression of E-cadherin, and suppressed the expression of Bcl-2. Moreover, knockdown of XIST significantly suppressed the tumor growth in NSCLC A549 xenograft mouse model. Bioinformatic analysis and luciferase reporter assays revealed that XIST was negatively regulated by miR-449a. We further identified reciprocal repression between XIST and miR-449a, which eventually influenced the expression of Bcl-2: XIST functioned as a miRNA sponge of miR-449a, which was a negative regulator of Bcl-2. These data show that expression of the lncRNA XIST is associated with an increased growth rate and metastatic potential in NSCLC A549 and NCI-H1299 cells partially through miR-449a, and suggest that XIST may be a potential prognostic factor and therapeutic target for patients with NSCLC. PMID:28248928

  8. In vitro toxicoproteomic analysis of A549 human lung epithelial cells exposed to urban air particulate matter and its water-soluble and insoluble fractions.

    PubMed

    Vuong, Ngoc Q; Breznan, Dalibor; Goegan, Patrick; O'Brien, Julie S; Williams, Andrew; Karthikeyan, Subramanian; Kumarathasan, Premkumari; Vincent, Renaud

    2017-10-02

    Toxicity of airborne particulate matter (PM) is difficult to assess because PM composition is complex and variable due to source contribution and atmospheric transformation. In this study, we used an in vitro toxicoproteomic approach to identify the toxicity mechanisms associated with different subfractions of Ottawa urban dust (EHC-93). A549 human lung epithelial cells were exposed to 0, 60, 140 and 200 μg/cm(2) doses of EHC-93 (total), its insoluble and soluble fractions for 24 h. Multiple cytotoxicity assays and proteomic analyses were used to assess particle toxicity in the exposed cells. The cytotoxicity data based on cellular ATP, BrdU incorporation and LDH leakage indicated that the insoluble, but not the soluble, fraction is responsible for the toxicity of EHC-93 in A549 cells. Two-dimensional gel electrophoresis results revealed that the expressions of 206 protein spots were significantly altered after particle exposures, where 154 were identified by MALDI-TOF-TOF-MS/MS. The results from cytotoxicity assays and proteomic analyses converged to a similar finding that the effects of the total and insoluble fraction may be alike, but their effects were distinguishable, and their effects were significantly different from the soluble fraction. Furthermore, the toxic potency of EHC-93 total is not equal to the sum of its insoluble and soluble fractions, implying inter-component interactions between insoluble and soluble materials resulting in synergistic or antagonistic cytotoxic effects. Pathway analysis based on the low toxicity dose (60 μg/cm(2)) indicated that the two subfractions can alter the expression of those proteins involved in pathways including cell death, cell proliferation and inflammatory response in a distinguishable manner. For example, the insoluble and soluble fractions differentially affected the secretion of pro-inflammatory cytokines such as MCP-1 and IL-8 and distinctly altered the expression of those proteins (e.g., TREM1, PDIA3 and

  9. Therapeutic effects of gold nanoparticles synthesized using Musa paradisiaca peel extract against multiple antibiotic resistant Enterococcus faecalis biofilms and human lung cancer cells (A549).

    PubMed

    Vijayakumar, S; Vaseeharan, B; Malaikozhundan, B; Gopi, N; Ekambaram, P; Pachaiappan, R; Velusamy, P; Murugan, K; Benelli, G; Suresh Kumar, R; Suriyanarayanamoorthy, M

    2017-01-01

    Botanical-mediated synthesis of nanomaterials is currently emerging as a cheap and eco-friendly nanotechnology, since it does not involve the use of toxic chemicals. In the present study, we focused on the synthesis of gold nanoparticles using the aqueous peel extract of Musa paradisiaca (MPPE-AuNPs) following a facile and cheap fabrication process. The green synthesized MPPE-AuNPs were bio-physically characterized by UV-Vis spectroscopy, FTIR, XRD, TEM, Zeta potential analysis and EDX. MPPE-AuNPs were crystalline in nature, spherical to triangular in shape, with particle size ranging within 50 nm. The biofilm inhibition activity of MPPE-AuNPs was higher against multiple antibiotic resistant (MARS) Gram-positive Enterococcus faecalis. Light and confocal laser scanning microscopic observations evidenced that the MPPE-AuNPs effectively inhibited the biofilm of E. faecalis when tested at 100 μg mL(-1). Cytotoxicity studies demonstrated that MPPE-AuNPs were effective in inhibiting the viability of human A549 lung cancer cells at higher concentrations of 100 μg mL(-1). The morphological changes in the MPPE-AuNPs treated A549 lung cancer cells were visualized under phase-contrast microscopy. Furthermore, the ecotoxicity of MPPE-AuNPs on the freshwater micro crustacean Ceriodaphnia cornuta were evaluated. Notably, no mortality was recorded in MPPE-AuNPs treated C. cornuta at 250 μg mL(-1). This study concludes that MPPE-AuNPs are non-toxic, eco-friendly and act as a multipurpose potential biomaterial for biomedical applications. Copyright © 2016 Elsevier Ltd. All rights reserved.

  10. Avastin® in combination with gemcitabine and cisplatin significantly inhibits tumor angiogenesis and increases the survival rate of human A549 tumor-bearing mice

    PubMed Central

    LIU, YING; XIA, XIZHENG; ZHOU, MINGKAI; LIU, XIAOJUN

    2015-01-01

    The aim of this study was to investigate the effect of Avastin® in combination with gemcitabine and cisplatin (GP) on the tumor growth of A549 tumor-bearing mice and the potential anti-tumor mechanism. A total of 30 human A549 tumor-bearing nude mice were randomly divided into the Avastin, chemotherapy and combined treatment groups for treatment with an intraperitoneal injection of Avastin (5 mg/kg) (Avastin group); an intraperitoneal injection of gemcitabine (4 mg/kg) and cisplatin (4 mg/kg) (chemotherapy group); or intraperitoneal injections of Avastin and GP (combined treatment group). The mice were observed for 30 days and the tumor growth, survival and body weight of the mice in the three groups were analyzed. The protein level of vascular endothelial growth factor (VEGF) in the tumor tissues was analyzed by ELISA. The vascular density and structural changes of the tumor were analyzed using immunohistochemistry. Compared with the Avastin and chemotherapy groups, the tumor growth of mice in the combined treatment group was significantly inhibited, and the survival rate of the mice was increased significantly. No difference in body weight was observed among the three groups of mice (P>0.05). The levels of VEGF in the combined treatment group tumor tissues were significantly reduced compared with those in the chemotherapy group tumor tissues (P<0.05). Furthermore, the vessel density of the tumor tissue in the combined treatment group was significantly reduced compared with that in the chemotherapy group (P<0.05), and the number of normal vessels in the combined treatment group tumors was significantly higher than that in the chemotherapy group tumors after 7 days of treatment (P<0.05). In conclusion, Avastin can significantly decrease the level of VEGF in tumor tissue, inhibit tumor angiogenesis and promote the normalization of tumor vascular structure, which may explain the enhanced efficacy of Avastin in combination with chemotherapy. PMID:26136956

  11. Comparative physicochemical and biological characterization of NIST Interim Reference Material PM2.5 and SRM 1648 in human A549 and mouse RAW264.7 cells.

    PubMed

    Mitkus, Robert J; Powell, Jan L; Zeisler, Rolf; Squibb, Katherine S

    2013-12-01

    The epidemiological association between exposure to fine particulate matter (PM2.5) and adverse health effects is well-known. Here we report the size distribution, metals content, endotoxin content, and biological activity of National Institute of Standards and Technology (NIST) Interim Reference Material (RM) PM2.5. Biological activity was measured in vitro by effects on cell viability and the release of four inflammatory immune mediators, from human A549 alveolar epithelial cells or murine RAW264.7 monocytes. A dose range covering three orders of magnitude (1-1000μg/mL) was tested, and biological activity was compared to an existing Standard Reference Material (SRM) for urban PM (NIST SRM 1648). Robust release of IL-8 and MCP-1 from A549 cells was observed in response to IRM PM2.5 exposures. Significant TNF-α, but not IL-6, secretion from RAW264.7 cells was observed in response to both IRM PM2.5 and SRM 1648 particle types. Cytokine or chemokine release at high doses often occurred in the presence of cytotoxicity, likely as a result of externalization of preformed mediator. Our results are consistent with a local cytotoxic and pro-inflammatory mechanism of response to exposure to inhaled ambient PM2.5 and reinforce the continued relevance of in vitro assays for mechanistic research in PM toxicology. Our study furthers the goal of developing reference samples of environmentally relevant particulate matter of various sizes that can be used for hypothesis testing by multiple investigators. Published by Elsevier Ltd.

  12. Zinc transporters are differentially expressed in human non-small cell lung cancer

    PubMed Central

    Yang, Jingxuan; Li, Min

    2016-01-01

    Lung cancer is one of the most common human malignancies worldwide, but its oncogenesis process remains unclear. Recent studies demonstrated that zinc (Zn) and Zn transporters were associated with the development and progression of human cancers. The role of Zn transporters including ZIPs and ZnTs in lung cancer, however, has never been evaluated. Thus, we aimed to investigate the expression levels of all human Zn transporters, including 14 ZIPs and 10 ZnTs, in eight different lung cancer cell lines and paired human tumor tissues. We observed great variations in ZIPs and ZnTs mRNA levels across cell lines and human lung cancer specimens. ZIPs showed a tendency to be upregulated, while ZnTs exhibited a downward expression trend. ZIP4 was overexpressed in six lung cancer cell lines and 59% (26/44) of tumor tissues, which was consistent with results from lung cancer datasets including TCGA database. Our results indicated that the dysregulation of Zn transporters may contribute to lung tumorigenesis. PMID:27611948

  13. Nintedanib modulates surfactant protein-D expression in A549 human lung epithelial cells via the c-Jun N-terminal kinase-activator protein-1 pathway.

    PubMed

    Kamio, Koichiro; Usuki, Jiro; Azuma, Arata; Matsuda, Kuniko; Ishii, Takeo; Inomata, Minoru; Hayashi, Hiroki; Kokuho, Nariaki; Fujita, Kazue; Saito, Yoshinobu; Miya, Toshimichi; Gemma, Akihiko

    2015-06-01

    Idiopathic pulmonary fibrosis (IPF) is a progressive disease with a high mortality rate. Signalling pathways activated by several tyrosine kinase receptors are known to be involved in lung fibrosis, and this knowledge has led to the development of the triple tyrosine kinase inhibitor nintedanib, an inhibitor of vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), and fibroblast growth factor receptor (FGFR), for the treatment of IPF. Pulmonary surfactant protein D (SP-D), an important biomarker of IPF, reportedly attenuates bleomycin-induced pulmonary fibrosis in mice. In this study, we investigated whether nintedanib modulates SP-D expression in human lung epithelial (A549) cells using quantitative real-time reverse transcriptase polymerase chain reaction and western blotting. To investigate the mechanisms underlying the effects of nintedanib, we evaluated the phosphorylation of c-Jun N-terminal kinase (JNK) and its downstream target c-Jun. The effect of the JNK inhibitor SP600125 on c-Jun phosphorylation was also tested. Activation of activator protein-1 (AP-1) was examined using an enzyme-linked immunosorbent assay-based test, and cell proliferation assays were performed to estimate the effect of nintedanib on cell proliferation. Furthermore, we treated mice with nintedanib to examine its in vivo effect on SP-D levels in lungs. These experiments showed that nintedanib up-regulated SP-D messenger RNA expression in a dose-dependent manner at concentrations up to 5 μM, with significant SP-D induction observed at concentrations of 3 μM and 5 μM, in comparison with that observed in vehicle controls. Nintedanib stimulated a rapid increase in phosphorylated JNK in A549 cells within 30 min of treatment and stimulated c-Jun phosphorylation, which was inhibited by the JNK inhibitor SP600125. Additionally, nintedanib was found to activate AP-1. A549 cell proliferation was not affected by nintedanib at any of the tested

  14. Interleukin 7 signaling prevents apoptosis by regulating bcl-2 and bax via the p53 pathway in human non-small cell lung cancer cells.

    PubMed

    Liu, Zi-Hui; Wang, Ming-Hui; Ren, Hong-Jiu; Qu, Wei; Sun, Li-Mei; Zhang, Qing-Fu; Qiu, Xue-Shan; Wang, En-Hua

    2014-01-01

    Interleukin 7/Interleukin 7 receptor (IL-7/IL-7R) signaling induces the upregulation of cyclin D1 to promote cell proliferation in lung cancer, but its role in preventing the apoptosis of non-small cell lung cancer (NSCLC) cell lines remains unknown. To study the role of IL-7 in lung cancer cell apoptosis, normal HBE cells as well as A549 and H1299 NSCLC cells were examined using flow cytometry. The results showed that the activation of IL-7R by its specific ligand, exogenous interleukin-7, was associated with a significant decline in apoptotic cells. Western blot and real-time PCR assays indicated that the activation of IL-7/IL-7R significantly upregulated anti-apoptotic bcl-2 and downregulated pro-apoptotic bax and p53 at both protein and mRNA levels. The knockdown of IL-7R through small interfering RNAs significantly attenuated these effects of exogenous IL-7. However, there was no significant anti-apoptotic effect in H1299 (p53-) cells. Furthermore, the inhibition of p53 significantly abolished the effects of IL-7/IL-7R on lung cancer cell apoptosis. These results strongly suggest that IL-7/IL-7R prevents apoptosis by upregulating the expression of bcl-2 and by downregulating the expression of bax, potentially via the p53 pathway in A549 and HBE cells.

  15. Differential effect of grape seed extract against human non-small-cell lung cancer cells: the role of reactive oxygen species and apoptosis induction.

    PubMed

    Tyagi, Alpna; Raina, Komal; Gangar, Subhash; Kaur, Manjinder; Agarwal, Rajesh; Agarwal, Chapla

    2013-01-01

    The present study examines grape seed extract (GSE) efficacy against a series of non-small-cell lung cancer (NSCLC) cell lines that differ in their Kras and p53 status to establish GSE potential as a cytotoxic agent against a wide range of lung cancer cells. GSE suppressed growth and induced apoptotic death in NSCLC cells irrespective of their k-Ras status, with more sensitivity toward H460 and H322 (wt k-Ras) than A549 and H1299 cells (mutated k-Ras). Mechanistic studies in A549 and H460 cells, selected, based on comparative efficacy of GSE at higher and lower doses, respectively, showed that apoptotic death involves cytochrome c release associated caspases 9 and 3 activation, and poly (ADP-ribosyl) polymerase cleavage, strong phosphorylation of ERK1/2 and JNK1/2, downregulation of cell survival proteins, and upregulated proapoptotic Bak expression. Importantly, GSE treatment caused a strong superoxide radical-associated oxidative stress, significantly decreased intracellular reduced glutathione levels, suggesting, for the first time, the involvement of GSE-caused oxidative stress in its apoptotic inducing activity in these cells. Because GSE is a widely-consumed dietary agent with no known untoward effects, our results support future studies to establish GSE efficacy and usefulness against NSCLC control.

  16. Orally active microtubule-targeting agent, MPT0B271, for the treatment of human non-small cell lung cancer, alone and in combination with erlotinib.

    PubMed

    Tsai, A-C; Wang, C-Y; Liou, J-P; Pai, H-C; Hsiao, C-J; Chang, J-Y; Wang, J-C; Teng, C-M; Pan, S-L

    2014-04-10

    Microtubule-binding agents, such as taxanes and vinca alkaloids, are used in the treatment of cancer. The limitations of these treatments, such as resistance to therapy and the need for intravenous administration, have encouraged the development of new agents. MPT0B271 (N-[1-(4-Methoxy-benzenesulfonyl)-2,3-dihydro-1H-indol-7-yl]-1-oxy-isonicotinamide), an orally active microtubule-targeting agent, is a completely synthetic compound that possesses potent anticancer effects in vitro and in vivo. Tubulin polymerization assay and immunofluorescence experiment showed that MPT0B271 caused depolymerization of tubulin at both molecular and cellular levels. MPT0B271 reduced cell growth and viability at nanomolar concentrations in numerous cancer cell lines, including a multidrug-resistant cancer cell line NCI/ADR-RES. Further studies indicated that MPT0B271 is not a substrate of P-glycoprotein (P-gp), as determined by flow cytometric analysis of rhodamine-123 (Rh-123) dye efflux and the calcein acetoxymethyl ester (calcein AM) assay. MPT0B271 also caused G2/M cell-cycle arrest, accompanied by the up-regulation of cyclin B1, p-Thr161 Cdc2/p34, serine/threonine kinases polo-like kinase 1, aurora kinase A and B and the downregulation of Cdc25C and p-Tyr15 Cdc2/p34 protein levels. The appearance of MPM2 and the nuclear translocation of cyclin B1 denoted M phase arrest in MPT0B271-treated cells. Moreover, MPT0B271 induced cell apoptosis in a concentration-dependent manner; it also reduced the expression of Bcl-2, Bcl-xL, and Mcl-1 and increased the cleavage of caspase-3 and -7 and poly (ADP-ribose) polymerase (PARP). Finally, this study demonstrated that MPT0B271 in combination with erlotinib significantly inhibits the growth of the human non-small cell lung cancer A549 cells as compared with erlotinib treatment alone, both in vitro and in vivo. These findings identify MPT0B271 as a promising new tubulin-binding compound for the treatment of various cancers.

  17. Expression of human eukaryotic initiation factor 3f oscillates with cell cycle in A549 cells and is essential for cell viability

    PubMed Central

    2010-01-01

    Background Transcriptional and postranslational regulation of the cell cycle has been widely studied. However, there is scarce knowledge concerning translational control of this process. Several mammalian eukaryotic initiation factors (eIFs) seem to be implicated in controlling cell proliferation. In this work, we investigated if the human eIF3f expression and function is cell cycle related. Results The human eIF3f expression has been found to be upregulated in growth-stimulated A549 cells and downregulated in G0. Western blot analysis and eIF3f promotor-luciferase fusions revealed that eIF3f expression peaks twice in the cell cycle: in the S and the M phases. Deregulation of eIF3f expression negatively affects cell viability and induces apoptosis. Conclusions The expression pattern of human eIF3f during the cell cycle confirms that this gene is cell division related. The fact that eIF3f expression peaks in two cell cycle phases raises the possibility that this gene may exert a differential function in the S and M phases. Our results strongly suggest that eIF3f is essential for cell proliferation. PMID:20462454

  18. MicroRNA-222 promotes human non-small cell lung cancer H460 growth by targeting p27.

    PubMed

    Zhong, Chongjun; Ding, Shengguang; Xu, Yiming; Huang, Haitao

    2015-01-01

    Two highly homologous microRNAs (miRNAs, miRs), miR-222 and miR-221, act as a cluster in cellular regulation. We have previously reported that miR-221 promoted the growth of human non-small cell lung cancer cell line H460. However, the role of miR-222 in regulating the growth of H460 is unclear. H460 cells were transfected with miR-222 mimics, inhibitors or their negative controls and their effects were confirmed by Real-time quantitative reverse transcription polymerase chain reactions (qRT-PCRs). Cell viability was assessed by Cell Counting Kit-8 (CCK-8) while cell proliferation was determined by 5-Ethynyl-2'-deoxyuridine (EdU) assay. P27 and P57, two putative targets of miR-222, were checked by qRT-PCRs. We found that miR-222 overexpression increased cell viability and proliferative rate in H460 cells while opposite effects were obtained by down-regulation of miR-222. P27 but not P57 was identified as a potential target of miR-222 in H460 cells as P27 was negatively regulated by miR-222 in the protein level. In summary, the present study indicates that miR-222 controls the growth of H460 likely by targeting P27. Inhibition of miR-222 might be a novel therapy for human non-small cell lung cancer.

  19. Genotoxic potential of Polycyclic Aromatic Hydrocarbons-coated onto airborne Particulate Matter (PM 2.5) in human lung epithelial A549 cells.

    PubMed

    Billet, Sylvain; Abbas, Imane; Le Goff, Jérémie; Verdin, Anthony; André, Véronique; Lafargue, Paul-Eric; Hachimi, Adam; Cazier, Fabrice; Sichel, François; Shirali, Pirouz; Garçon, Guillaume

    2008-10-18

    To improve the knowledge of the underlying mechanisms of action involved in air pollution Particulate Matter (PM)-induced toxicity in human lungs, with a particular interest of the crucial role played by coated-organic chemicals, we were interested in the metabolic activation of Polycyclic Aromatic Hydrocarbons (PAH)-coated onto air pollution PM, and, thereafter, the formation of PAH-DNA adducts in a human lung epithelial cell model (A549 cell line). Cells were exposed to Dunkerque city's PM(2.5) at its Lethal Concentrations at 10% and 50% (i.e. LC(10)=23.72 microg/mL or 6.33 microg/cm2, and LC(50)=118.60 microg/mL or 31.63 microg/cm2), and the study of Cytochrome P450 (CYP) 1A1 gene expression (i.e. RT-PCR) and protein activity (i.e. EROD activity), and the formation of PAH-DNA adducts (i.e. 32P-postlabeling), were investigated after 24, 48, and/or 72 h. PAH, PolyChlorinated Dibenzo-p-Dioxins and -Furans (PCDD/F), Dioxin-Like PolyChlorinated Biphenyls (DLPCB), and PolyChlorinated Biphenyls (PCB)-coated onto collected PM were determined (i.e. GC/MS and HRGC/HRMS, respectively), Negative (i.e. TiO2 or desorbed PM, dPM; EqLC10=19.42 microg/mL or 5.18 microg/cm2, and EqLC50=97.13 microg/mL or 25.90 microg/cm2), and positive (i.e. benzo(a)pyrene; 1 microM) controls were included in the experimental design. Statistically significant increases of CYP1A1 gene expression and protein activity were observed in A549 cells, 24, 48 and 72 h after their exposure to dPM, suggesting thereby that the employed outgassing method was not efficient enough to remove total PAH. Both the CYP1A1 gene expression and EROD activity were highly induced 24, 48 and 72 h after cell exposure to PM. However, only very low levels of PAH-DNA adducts, also not reliably quantifiable, were reported 72 h after cell exposure to dPM, and, particularly, PM. The relatively low levels of PAH together with the presence of PCDD/F, DLPCB, and PCB-coated onto Dunkerque City's PM 2.5 could notably contribute to

  20. The fruit juice of Morinda citrifolia (noni) downregulates HIF-1α protein expression through inhibition of PKB, ERK-1/2, JNK-1 and S6 in manganese-stimulated A549 human lung cancer cells.

    PubMed

    Jang, Byeong-Churl

    2012-03-01

    High exposure of manganese is suggested to be a risk factor for many lung diseases. Evidence suggests anticancerous and antiangiogenic effects by products derived from Morinda citrifolia (noni) fruit. In this study, we investigated the effect of noni fruit juice (NFJ) on the expression of HIF-1α, a tumor angiogenic transcription factor in manganese-chloride (manganese)-stimulated A549 human lung carcinoma cells. Treatment with manganese largely induced expression of HIF-1α protein but did not affect HIF-1α mRNA expression in A549 cells, suggesting the metal-mediated co- and/or post-translational HIF-1α upregulation. Manganese treatment also led to increased phosphorylation of extracellular-regulated protein kinase-1/2 (ERK-1/2), c-Jun N-terminal kinase-1 (JNK-1), protein kinase B (PKB), S6 and eukaryotic translation initiation factor-2α (eIF-2α) in A549 cells. Of note, the exposure of NFJ inhibited the manganese-induced HIF-1α protein upregulation in a concentration-dependent manner. Importantly, as assessed by results of pharmacological inhibition and siRNA transfection studies, the effect of NFJ on HIF-1α protein downregulation seemed to be largely associated with the ability of NFJ to interfere with the metal's signaling to activate PKB, ERK-1/2, JNK-1 and S6 in A549 cells. It was further shown that NFJ could repress the induction of HIF-1α protein by desferoxamine or interleukin-1β (IL-1β), another HIF-1α inducer in A549 cells. Thus, the present study provides the first evidence that NFJ has the ability to strongly downregulate manganese-induced HIF-1α protein expression in A549 human lung cancer cells, which may suggest the NFJ-mediated beneficial effects on lung pathologies in which manganese and HIF-1α overexpression play pathogenic roles.

  1. Organic compounds in tire particle induce reactive oxygen species and heat-shock proteins in the human alveolar cell line A549.

    PubMed

    Gualtieri, Maurizio; Mantecca, Paride; Cetta, Francesco; Camatini, Marina

    2008-05-01

    Debris produced from the attrition of tires of motor vehicles constitutes 5-7% of the atmospheric particulate matter (PM10). Debris particles are indeed small enough to enter human lung and thus morphological and chemical characterization has been performed. We demonstrated that the organic fraction of tire debris induces a dose-dependent increase in cell mortality, DNA damage, as well as a significant modification of cell morphology at the dose of 60 microg/ml, which may correspond to the quantity present in the air humans inhale daily. The present research aims at investigating if reactive oxygen species (ROS) production and Hsp70 expression are involved in the cascade of toxic effects produced on the A549 cell line, as it has been suggested for the ultrafine atmospheric particles and diesel exhaust. To this end, cells were exposed at the doses of 10, 50, 60, 75 microg/ml of TD organic extract (TDOE) and analyzed at different exposure time. ROS were detected by the oxidation of 2'7'-dichlorodihydrofluorescein diacetate to dichlorofluorescein, and fluorescence was measured by flow cytometry. Hsp70 protein expression was determined by immunochemical analysis, and protein expression quantification performed by optical densitometry. ROS production was analysed after 2 h of treatment. A statistically significant increase in fluorescence was observed and the intensity of the stress response was parallel to the increasing concentrations used. An evident increase of Hsp70 expression at lower doses (10, 50 microg/ml) and at longer exposure times (72 h) was observed, during the time that our previous studies showed that cell viability, plasma membrane integrity, and DNA molecules were not affected. Thus it can be deduced that the increase in Hsp70 expression protected the cells from those damages, which became evident at the higher doses, and that this parameter might be used as a sensitive indicator of exposure. These data suggest that ROS production may be the first

  2. Octanal-induced inflammatory responses in cells relevant for lung toxicity: expression and release of cytokines in A549 human alveolar cells.

    PubMed

    Song, M-K; Lee, H-S; Choi, H-S; Shin, C-Y; Kim, Y-J; Park, Y-K; Ryu, J-C

    2014-07-01

    Inhalation is an important route of aldehyde exposure, and lung is one of the main targets of aldehyde toxicity. Octanal is distributed ubiquitously in the environment and is a component of indoor air pollutants. We investigated whether octanal exposure enhances the inflammatory response in the human respiratory system by increasing the expression and release of cytokines and chemokines. The effect of octanal in transcriptomic modulation was assessed in the human alveolar epithelial cell line A549 using oligonucleotide arrays. We identified a set of genes differentially expressed upon octanal exposure that may be useful for monitoring octanal pulmonary toxicity. These genes were classified according to the Gene Ontology functional category and Kyoto Encyclopedia of Genes and Genomes analysis to explore the biological processes related to octanal-induced pulmonary toxicity. The results show that octanal affects the expression of several chemokines and inflammatory cytokines and increases the levels of interleukin 6 (IL-6) and IL-8 released. In conclusion, octanal exposure modulates the expression of cytokines and chemokines important in the development of lung injury and disease. This suggests that inflammation contributes to octanal-induced lung damage and that the inflammatory genes expressed should be studied in detail, thereby laying the groundwork for future biomonitoring studies. © The Author(s) 2014.

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

    SciTech Connect

    Maruyama, I.; Majerus, P.W.

    1987-05-01

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

  4. MicroRNA-429 induces tumorigenesis of human non-small cell lung cancer cells and targets multiple tumor suppressor genes

    SciTech Connect

    Lang, Yaoguo; Xu, Shidong; Ma, Jianqun; Wu, Jun; Jin, Shi; Cao, Shoubo; Yu, Yan

    2014-07-18

    Highlights: • MiR-429 expression is upregulated in non-small cell lung cancer (NSCLC). • MiR-429 inhibits PTEN, RASSF8 and TIMP2 expression. • MiR-429 promotes metastasis and proliferation. • We report important regulatory mechanisms involved in NSCLC progression. • MiR-429 is a potential therapeutic target and diagnostic marker. - Abstract: Lung cancer is the major cause of cancer death globally. MicroRNAs are evolutionally conserved small noncoding RNAs that are critical for the regulation of gene expression. Aberrant expression of microRNA (miRNA) has been implicated in cancer initiation and progression. In this study, we demonstrated that the expression of miR-429 are often upregulated in non-small cell lung cancer (NSCLC) compared with normal lung tissues, and its expression level is also increased in NSCLC cell lines compared with normal lung cells. Overexpression of miR-429 in A549 NSCLC cells significantly promoted cell proliferation, migration and invasion, whereas inhibition of miR-429 inhibits these effects. Furthermore, we demonstrated that miR-429 down-regulates PTEN, RASSF8 and TIMP2 expression by directly targeting the 3′-untranslated region of these target genes. Taken together, our results suggest that miR-429 plays an important role in promoting the proliferation and metastasis of NSCLC cells and is a potential target for NSCLC therapy.

  5. Tumor Antivascular Effects of Radiotherapy Combined with Combretastatin A4 Phosphate in Human Non-Small-Cell Lung Cancer

    SciTech Connect

    Ng, Q.-S.; Goh, Vicky; Carnell, Dawn; Meer, Khalda; Padhani, Anwar R.; Saunders, Michele I.; Hoskin, Peter J. . E-mail: peterhoskin@nhs.net

    2007-04-01

    Purpose: The tumor vascular effects of radiotherapy and subsequent administration of the vascular disrupting agent combretastatin A4 phosphate (CA4P) were studied in patients with advanced non-small-cell lung cancer using volumetric dynamic contrast-enhanced computed tomography (CT). Patients and Methods: Following ethical committee approval and informed consent, 8 patients receiving palliative radiotherapy (27 Gy in six fractions, twice weekly) also received CA4P (50 mg/m{sup 2}) after the second fraction of radiotherapy. Changes in dynamic CT parameters of tumor blood volume (BV) and permeability surface area product (PS) were measured for the whole tumor volume, tumor rim, and center after radiotherapy alone and after radiotherapy in combination with CA4P. Results: After the second fraction of radiotherapy, 6 of the 8 patients showed increases in tumor PS (23.6%, p = 0.011). Four hours after CA4P, a reduction in tumor BV (22.9%, p < 0.001) was demonstrated in the same 6 patients. Increase in PS after radiotherapy correlated with reduction in BV after CA4P (r = 0.77, p = 0.026). At 72 h after CA4P, there was a sustained reduction in tumor BV of 29.4% (p < 0.001). Both increase in PS after radiotherapy and reduction in BV after CA4P were greater at the rim of the tumor. The BV reduction at the rim was sustained to 72 h (51.4%, p 0.014). Conclusion: Radiotherapy enhances the tumor antivascular activity of CA4P in human non-small-cell lung cancer, resulting in sustained tumor vascular shutdown.

  6. Expression and clinical significance of CXCR5/CXCL13 in human non-small cell lung carcinoma

    PubMed Central

    SINGH, RAJESH; GUPTA, PRANAV; KLOECKER, GOETZ H.; SINGH, SHAILESH; LILLARD, JAMES W.

    2014-01-01

    CXCR5 and/or CXCL13 expression is elevated in certain carcinomas and lymphomas. To determine if these factors are involved in progression of non-small cell lung cancer (LuCa), we evaluated their expression in patients with various forms of this disease. Lung biopsies from patients with non-neoplastic cells (n=8), squamous cell carcinoma (SCC; n=24), or adenocarcinoma (AC; n=54) were stained for CXCR5. Histopathological analysis of these samples showed significantly higher expression of CXCR5 (p<0.001) in carcinomas (i.e., SCCs and ACs) relative to non-neoplastic lung tissue. Nuclear and membrane CXCR5 intensities were highest in ACs, with median values of 185 and 130, respectively, followed by SCCs with median values of 170 and 110, respectively. The lowest nuclear and membrane expressions of CXCR5 were found in non-neoplastic tissues, having median values of 142 and 90, respectively. Sera from SCC patients (n=17), AC patients (n=14), and healthy controls (n=9) were tested for the presence of CXCL13. Serum CXCL13 levels in LuCa patients were higher than in healthy controls. CXCR5 expression in cell lines of human non-small cell lung carcinoma (NCI-H1915) and small cell lung carcinoma (SW-1271) were evaluated by flow cytometry. CXCR5 expression was higher in NCI-H1915 cells relative to SW-1271 cells. The functional significance of CXCR5 expression was tested in a migration assay. In response to CXCL13, more NCI-H1915 cells migrated than SW-1271 cells. These findings suggest that the CXCR5-CXCL13 axis influences LuCa progression. After validation in larger patient groups, CXCR5 and CXCL13 may prove useful as biomarkers for LuCa. Correspondingly, blockade of this axis could serve as an effective therapy for LuCa. PMID:25271023

  7. The histone demethylase PHF8 is an oncogenic protein in human non-small cell lung cancer

    SciTech Connect

    Shen, Yuzhou; Pan, Xufeng; Zhao, Heng

    2014-08-15

    Highlights: • PHF8 overexpresses in human NSCLC and predicts poor survival. • PHF8 regulates lung cancer cell growth and transformation. • PHF8 regulates apoptosis in human lung cancer cells. • PHF8 promotes miR-21 expression in human lung cancer. • MiR-21 is critically essential for PHF8 function in human lung cancer cells. - Abstract: PHF8 is a JmjC domain-containing protein and erases repressive histone marks including H4K20me1 and H3K9me1/2. It binds to H3K4me3, an active histone mark usually located at transcription start sites (TSSs), through its plant homeo-domain, and is thus recruited and enriched in gene promoters. PHF8 is involved in the development of several types of cancer, including leukemia, prostate cancer, and esophageal squamous cell carcinoma. Herein we report that PHF8 is an oncogenic protein in human non-small cell lung cancer (NSCLC). PHF8 is up-regulated in human NSCLC tissues, and high PHF8 expression predicts poor survival. Our in vitro and in vivo evidence demonstrate that PHF8 regulates lung cancer cell proliferation and cellular transformation. We found that PHF8 knockdown induces DNA damage and apoptosis in lung cancer cells. PHF8 promotes miR-21 expression in human lung cancer, and miR-21 knockdown blocks the effects of PHF8 on proliferation and apoptosis of lung cancer cells. In summary, PHF8 promotes lung cancer cell growth and survival by regulating miR-21.

  8. Transcriptome Sequencing Reveals Key Pathways and Genes Associated with Cisplatin Resistance in Lung Adenocarcinoma A549 Cells

    PubMed Central

    Fang, Yani; Zhang, Cheng; Wu, Tong; Wang, Qi; Liu, Jinhui; Dai, Penggao

    2017-01-01

    Acquired resistance to cisplatin-based chemotherapy frequently occurs in patients with non-small cell lung cancer, and the underlying molecular mechanisms are not well understood. The aim of this study was to investigate whether a distinct gene expression pattern is associated with acquired resistance to cisplatin in human lung adenocarcinoma. Whole-transcriptome sequencing was performed to compare the genome-wide gene expression patterns of the human lung adenocarcinoma A549 cisplatin-resistant cell line A549/DDP with those of its progenitor cell line A549. A total of 1214 differentially expressed genes (DEGs) were identified, 656 of which were upregulated and 558 were downregulated. Functional annotation of the DEGs in the Kyoto Encyclopedia of Genes and Genomes database revealed that most of the identified genes were enriched in the PI3K/AKT, mitogen-activated protein kinase, actin cytoskeleton regulation, and focal adhesion pathways in A549/DDP cells. These results support previous studies demonstrating that the pathways regulating cell proliferation and invasion confer resistance to chemotherapy. Furthermore, the results proved that cell adhesion and cytoskeleton regulation is associated with cisplatin resistance in human lung cancer. Our study provides new promising biomarkers for lung cancer prognosis and potential therapeutic targets for lung cancer treatment. PMID:28114404

  9. Human Lung Cancer Cell Line A-549 ATCC Is Differentially Affected by Supranutritional Organic and Inorganic Selenium

    PubMed Central

    Flores Villavicencio, Lérida Liss; Cruz-Jiménez, Gustavo; Barbosa-Sabanero, Gloria; Kornhauser-Araujo, Carlos; Mendoza-Garrido, M. Eugenia; de la Rosa, Guadalupe; Sabanero-López, Myrna

    2014-01-01

    The effects of organic and inorganic forms of selenium (Se) on human cells have been extensively studied for nutritional concentrations; however, to date, little is known about the potential toxicity at supranutritional levels. In the present study we determined the effects of sodium selenite (SSe) and selenomethionine (SeMet) on cell growth and intracellular structures in lung cancer cells exposed at Se concentrations between 0 and 3 mM. Our results showed that SSe affected cell growth more rapidly than SeMet (24 h and 48 h, resp.). After 24 h of cells exposure to 0.5, 1.5, and 3 mM SSe, cell growth was reduced by 10, 50, and 60%, as compared to controls. After 48 h, nuclear fragmentation was evident in cells exposed to SSe, suggesting an induction to cell death. In contrast, SeMet did not affect cell proliferation, and the cells were phenotypically similar to controls. Microtubules and microfilaments structures were also affected by both Se compounds, again SSe being more toxic than SeMet. To our knowledge, this is the first report on the differential effects of organic and inorganic Se in supranutritional levels in lung cancer cells. PMID:25477771

  10. Fabrication of nano-silver particles using Cymodocea serrulata and its cytotoxicity effect against human lung cancer A549 cells line

    NASA Astrophysics Data System (ADS)

    Palaniappan, P.; Sathishkumar, G.; Sankar, R.

    2015-03-01

    The present study reports, green synthesis of bioactive silver nanoparticles (AgNPs) under different temperature (60 °C, room temperature and 4° refrigerator) using the aqueous extract of sea grass Cymodocea serrulata as a potential bioreductant. Increased temperature fabricates more AgNPs compare to room temperature and refrigerator condition. At first the reduction of Ag+ ions were confirmed through color change which produces an absorbance spectra at 420 nm in UV-Visible spectrophotometer. Additionally various exclusive instrumentations such as X-ray diffraction (XRD), Dynamic light scattering (DLS), scanning electron microscope (SEM) analysis and Transmission electron microscope (TEM) were authorizes the biosynthesis and physio-chemical characterization of AgNPs. From Fourier transform infrared spectroscopy (FTIR) analysis, it was identified that the water soluble fractions of the sea grass mainly responsible for reduction of ionic silver (Ag+) into (Ag0) nano-ranged particles and also they act as stabilizing agent to sustain the durability of NPs for long period of time. Further, synthesized AgNPs shows potential cytotoxicity against human lung cancer A549 cells (LD50-100 μg/ml). The overall results suggest that C. serrulata is a valuable bioresource to generate rapid and eco-friendly bioactive AgNPs towards cancer therapy.

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

    PubMed

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

    2016-01-01

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

  12. Genotoxic effects of three selected black toner powders and their dimethyl sulfoxide extracts in cultured human epithelial A549 lung cells in vitro.

    PubMed

    Gminski, Richard; Decker, Katharina; Heinz, Christina; Seidel, Albrecht; Könczöl, Mathias; Goldenberg, Ella; Grobéty, Bernard; Ebner, Winfried; Gieré, Reto; Mersch-Sundermann, Volker

    2011-05-01

    Until now, the adverse effects of toner powders on humans have been considered to be minimal. However, several recent reports have suggested possible significant adverse health effects from toner dust inhalation. The aim of this study was to evaluate the genotoxic potential of black toner powders in vitro. For the study of DNA damage, A549 cells were exposed to toner-powder suspensions and to their DMSO extracts, and then subjected to the comet assay and to the in-vitro cytokinesis block micronucleus test (CB-MNvit). Cytotoxic effects of the toner samples were assessed by the erythrosin B assay. Furthermore, size, shape, and composition of the toner powders were investigated. None of the three toner powders or their DMSO extracts reduced cell viability; however, they did induce DNA damage and formed micronuclei at concentrations from 80 to 400 μg cm(-2) , although to a varying extent. All toner powders contain considerable amounts of the pigments carbon black and magnetite (Fe(3) O(4) ) as well as small amounts of polycyclic aromatic hydrocarbons (PAHs). The overall results of our in-vitro study suggest that the investigated toner-powder samples are not cytotoxic but genotoxic. From the results of the physical and chemical characterization, we conclude that metals and metalloids as components of magnetite, or PAHs as components of the carbon-bearing material, are responsible for the genotoxic effects. Further research is necessary to determine the relevance of these in-vitro observations for private and occupational toner powder exposure.

  13. Fabrication of nano-silver particles using Cymodocea serrulata and its cytotoxicity effect against human lung cancer A549 cells line.

    PubMed

    Palaniappan, P; Sathishkumar, G; Sankar, R

    2015-03-05

    The present study reports, green synthesis of bioactive silver nanoparticles (AgNPs) under different temperature (60°C, room temperature and 4° refrigerator) using the aqueous extract of sea grass Cymodocea serrulata as a potential bioreductant. Increased temperature fabricates more AgNPs compare to room temperature and refrigerator condition. At first the reduction of Ag(+) ions were confirmed through color change which produces an absorbance spectra at 420nm in UV-Visible spectrophotometer. Additionally various exclusive instrumentations such as X-ray diffraction (XRD), Dynamic light scattering (DLS), scanning electron microscope (SEM) analysis and Transmission electron microscope (TEM) were authorizes the biosynthesis and physio-chemical characterization of AgNPs. From Fourier transform infrared spectroscopy (FTIR) analysis, it was identified that the water soluble fractions of the sea grass mainly responsible for reduction of ionic silver (Ag(+)) into (Ag(0)) nano-ranged particles and also they act as stabilizing agent to sustain the durability of NPs for long period of time. Further, synthesized AgNPs shows potential cytotoxicity against human lung cancer A549 cells (LD50-100μg/ml). The overall results suggest that C. serrulata is a valuable bioresource to generate rapid and eco-friendly bioactive AgNPs towards cancer therapy.

  14. Evaluation of cytotoxic, oxidative stress, proinflammatory and genotoxic responses of micro- and nano-particles of dolomite on human lung epithelial cells A(549).

    PubMed

    Patil, Govil; Khan, Mohd Imran; Patel, Devendra Kumar; Sultana, Sarwat; Prasad, Rajendra; Ahmad, Iqbal

    2012-09-01

    Dolomite is a natural mineral of great industrial importance and used worldwide, thus millions of workers are at risk of occupational exposure. Its toxicity is however, meagerly documented. In the present investigation, a dolomite powder obtained from its milling unit was analyzed by some standard methods namely, optical microscopy, transmission electron microscopy and dynamic light scattering. Results showed that dolomite powder contained particles of different shapes and size both microparticles (MPs) and nanoparticles (NPs), suggesting potential occupational exposure of these particles. An attempt was therefore, made to investigate dolomite toxicity in a particle size-dependent manner in human lung epithelial cells A(549). The comparative toxicity evaluation of MPs and NPs was carried out by assessing their effects on cell viability, membrane damage, glutathione, reactive oxygen species (ROS), lipid peroxidation (LPO), micronucleus (MN) and proinflammatory cytokines, namely tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6). These markers of cytotoxicity, genotoxicity and inflammation were assayed in cells exposed to MPs and NPs in a dose-and time-dependent manner. Invariably, their toxic effects were dose-and time-dependent while NPs in general were significantly more toxic. Notably, NPs caused oxidative stress, genotoxicity and inflammatory responses, as seen by significant induction of ROS, LPO, MN, TNF-α, IL-1β and IL-6. Thus, the study tends to suggest that separate health safety standards would be required for micrometer and nanometer scale particles of dolomite.

  15. E3 ubiquitin ligase Pirh2 enhances tumorigenic properties of human non-small cell lung carcinoma cells

    PubMed Central

    Fedorova, Olga; Shuvalov, Oleg; Merkulov, Valeriy; Vasileva, Elena; Antonov, Alexey; Barlev, Nikolai A.

    2016-01-01

    The product of RCHY1 human gene, Pirh2, is a RING-finger containing E3 ligase that modifies p53 with ubiquitin residues resulting in its subsequent degradation in proteasomes. Transcription of RCHY1 is regulated by p53 itself thus forming a negative regulatory feedback loop. Functionally, by eliminating p53, Pirh2 facilitates tumorigenesis. However, the role of Pirh2 in cancer cells lacking p53 is yet not well understood. Therefore, we decided to elucidate the role of Pirh2 in p53-negative human non-small cell lung carcinoma cells, H1299. We found that ectopic expression of Pirh2 enhanced cell proliferation, resistance to doxorubicin, and increased migration potential. Ablation of Pirh2 by specific shRNA reversed these phenotypes. Mechanistically, Pirh2 increased mRNA and protein levels of the c-Myc oncogene. The bioinformatics data indicate that co-expression of both c-Myc and Pirh2 strongly correlated with poor survival of lung cancer patients. Collectively, our results suggest that Pirh2 can be considered as a potential pharmacological target for developing anticancer therapies to treat p53-negative cancers. PMID:28191284

  16. The environmental carcinogen 3-nitrobenzanthrone and its main metabolite 3-aminobenzanthrone enhance formation of reactive oxygen intermediates in human A549 lung epithelial cells

    SciTech Connect

    Hansen, Tanja . E-mail: tanja.hansen@item.fraunhofer.de; Seidel, Albrecht; Borlak, Juergen

    2007-06-01

    The environmental contaminant 3-nitrobenzanthrone (3-NBA) is highly mutagenic and a suspected human carcinogen. We aimed to evaluate whether 3-NBA is able to deregulate critical steps in cell cycle control and apoptosis in human lung epithelial A549 cells. Increased intracellular Ca{sup 2+} and caspase activities were detected upon 3-NBA exposure. As shown by cell cycle analysis, an increased number of S-phase cells was observed after 24 h of treatment with 3-NBA. Furthermore, 3-NBA was shown to inhibit cell proliferation when added to subconfluent cell cultures. The main metabolite of 3-NBA, 3-ABA, induced statistically significant increases in tail moment as judged by alkaline comet assay. The potential of 3-NBA and 3-ABA to enhance the production of reactive oxygen species (ROS) was demonstrated by flow cytometry using 2',7'-dichlorofluorescein-diacetate (DCFH-DA). The enzyme inhibitors allopurinol, dicumarol, resveratrol and SKF525A were used to assess the impact of metabolic conversion on 3-NBA-mediated ROS production. Resveratrol decreased dichlorofluorescein (DCF) fluorescence by 50%, suggesting a role for CYP1A1 in 3-NBA-mediated ROS production. Mitochondrial ROS production was significantly attenuated (20% reduction) by addition of rotenone (complex I inhibition) and thenoyltrifluoroacetone (TTFA, complex II inhibition). Taken together, the results of the present study provide evidence for a genotoxic potential of 3-ABA in human epithelial lung cells. Moreover, both compounds lead to increased intracellular ROS and create an environment favorable to DNA damage and the promotion of cancer.

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

    PubMed

    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.

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

    PubMed Central

    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

  19. The environmental carcinogen 3-nitrobenzanthrone and its main metabolite 3-aminobenzanthrone enhance formation of reactive oxygen intermediates in human A549 lung epithelial cells.

    PubMed

    Hansen, Tanja; Seidel, Albrecht; Borlak, Jürgen

    2007-06-01

    The environmental contaminant 3-nitrobenzanthrone (3-NBA) is highly mutagenic and a suspected human carcinogen. We aimed to evaluate whether 3-NBA is able to deregulate critical steps in cell cycle control and apoptosis in human lung epithelial A549 cells. Increased intracellular Ca(2+) and caspase activities were detected upon 3-NBA exposure. As shown by cell cycle analysis, an increased number of S-phase cells was observed after 24 h of treatment with 3-NBA. Furthermore, 3-NBA was shown to inhibit cell proliferation when added to subconfluent cell cultures. The main metabolite of 3-NBA, 3-ABA, induced statistically significant increases in tail moment as judged by alkaline comet assay. The potential of 3-NBA and 3-ABA to enhance the production of reactive oxygen species (ROS) was demonstrated by flow cytometry using 2',7'-dichlorofluorescein-diacetate (DCFH-DA). The enzyme inhibitors allopurinol, dicumarol, resveratrol and SKF525A were used to assess the impact of metabolic conversion on 3-NBA-mediated ROS production. Resveratrol decreased dichlorofluorescein (DCF) fluorescence by 50%, suggesting a role for CYP1A1 in 3-NBA-mediated ROS production. Mitochondrial ROS production was significantly attenuated (20% reduction) by addition of rotenone (complex I inhibition) and thenoyltrifluoroacetone (TTFA, complex II inhibition). Taken together, the results of the present study provide evidence for a genotoxic potential of 3-ABA in human epithelial lung cells. Moreover, both compounds lead to increased intracellular ROS and create an environment favorable to DNA damage and the promotion of cancer.

  20. Astaxanthin down-regulates Rad51 expression via inactivation of AKT kinase to enhance mitomycin C-induced cytotoxicity in human non-small cell lung cancer cells.

    PubMed

    Ko, Jen-Chung; Chen, Jyh-Cheng; Wang, Tai-Jing; Zheng, Hao-Yu; Chen, Wen-Ching; Chang, Po-Yuan; Lin, Yun-Wei

    2016-04-01

    Astaxanthin has been demonstrated to exhibit a wide range of beneficial effects, including anti-inflammatory and anti-cancer properties. However, the molecular mechanism of astaxanthin-induced cytotoxicity in non-small cell lung cancer (NSCLC) cells has not been identified. Rad51 plays a central role in homologous recombination, and studies show that chemo-resistant carcinomas exhibit high levels of Rad51 expression. In this study, astaxanthin treatment inhibited cell viability and proliferation of two NSCLC cells, A549 and H1703. Astaxanthin treatment (2.5-20 μM) decreased Rad51 expression and phospho-AKT(Ser473) protein level in a time and dose-dependent manner. Furthermore, expression of constitutively active AKT (AKT-CA) vector rescued the decreased Rad51 mRNA and protein levels in astaxanthin-treated NSCLC cells. Combined treatment with phosphatidylinositol 3-kinase (PI3K) inhibitors (LY294002 or wortmannin) further decreased the Rad51 expression in astaxanthin-exposed A549 and H1703 cells. Knockdown of Rad51 expression by transfection with si-Rad51 RNA or cotreatment with LY294002 further enhanced the cytotoxicity and cell growth inhibition of astaxanthin. Additionally, mitomycin C (MMC) as an anti-tumor antibiotic is widely used in clinical NSCLC chemotherapy. Combination of MMC and astaxanthin synergistically resulted in cytotoxicity and cell growth inhibition in NSCLC cells, accompanied with reduced phospho-AKT(Ser473) level and Rad51 expression. Overexpression of AKT-CA or Flag-tagged Rad51 reversed the astaxanthin and MMC-induced synergistic cytotoxicity. In contrast, pretreatment with LY294002 further decreased the cell viability in astaxanthin and MMC co-treated cells. In conclusion, astaxanthin enhances MMC-induced cytotoxicity by decreasing Rad51 expression and AKT activation. These findings may provide rationale to combine astaxanthin with MMC for the treatment of NSCLC. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. Knockdown of Immature Colon Carcinoma Transcript 1 Inhibits Proliferation and Promotes Apoptosis of Non-Small Cell Lung Cancer Cells.

    PubMed

    Wang, Yiling; He, Jiantao; Zhang, Shenghui; Yang, Qingbo; Wang, Bo; Liu, Zhiyu; Wu, Xintian

    2016-07-13

    Non-small cell lung cancer, as the most frequent type lung cancer, has lower survival rate of 5 years, despite improvements in surgery and chemotherapy. Previous studies showed immature colon carcinoma transcript 1 is closely related to tumorigenesis of human cancer cells. In the present study, we found immature colon carcinoma transcript 1 was overexpressed in lung cancer tissues using Oncomine database mining, and the biological effect of immature colon carcinoma transcript 1 was investigated in non-small cell lung cancer cell lines 95D and A549. Lentivirus-mediated RNA interference was used to knock down immature colon carcinoma transcript 1 expression in 95D and A549 cells in vitro, and the knockdown efficiency was determined using quantitative real-time polymerase chain reaction and Western blot assay. Knockdown of immature colon carcinoma transcript 1 significantly suppressed non-small cell lung cancer cell proliferation and colony formation ability confirmed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and colony formation assay. Flow cytometry was applied to measure cell cycle arrest, and the result showed the cell cycle arrested in G2/M phase in 95D cells and arrested in G0/G1 phase in A549 cells. Furthermore, we measured the levels of cell cycle-associated proteins by Western blot analysis and found immature colon carcinoma transcript 1-mediated cell proliferation inhibition appeared due to downregulation of cell cycle activator cyclin D1 and upregulation of cell cycle inhibitor p21. In addition, immature colon carcinoma transcript 1 silencing significantly induced non-small cell lung cancer cell apoptosis by annexin V/7-amino-actinomycin D double-staining assay. All our data suggest that immature colon carcinoma transcript 1 may play an important role for non-small cell lung cancer cell proliferation and could be a potential molecular target for diagnosing and treating human non-small cell lung cancer.

  2. Oxidative damage to DNA and repair induced by Norwegian wood smoke particles in human A549 and THP-1 cell lines.

    PubMed

    Danielsen, Pernille Høgh; Loft, Steffen; Kocbach, Anette; Schwarze, Per E; Møller, Peter

    2009-03-31

    Genotoxic effects of traffic-generated particulate matter (PM) are well described, whereas little data are available on PM from combustion of biomass and wood, which contributes substantially to air pollution world wide. The aim of this study was to compare the genotoxicity of wood smoke particulate matter (WSPM), authentic traffic-generated particles, mineral PM and standard reference material (SRM2975) of diesel exhaust particles in human A549 lung epithelial and THP-1 monocytic cell lines. DNA damage was measured as strand breaks (SB) and formamidopyrimidine DNA glycosylase (FPG) sites by the comet assay, whereas cell cytotoxicity was determined as lactate dehydrogenase release. The exposure to WSPM generated SB and FPG sites in both cell lines at concentrations from 2.5 or 25 microg/ml, which were not cytotoxic. Compared to all other studied particles, WSPM generated greater responses in terms of both SB and FPG sites. Organic extracts of WSPM and SRM2975 elicited higher levels of SB than native and washed PM at 25 and 100 microg/ml, whereas assay saturation precluded reliable assessment of FPG sites. During a 6h post-exposure period, in which the medium with PM had been replaced by fresh medium, 60% of the DNA lesions generated by WSPM were removed. In conclusion, WSPM generated more DNA damage than traffic-generated PM per unit mass in human cell lines, possibly due to the high level of polycyclic aromatic hydrocarbons in WSPM. This suggests that exposure to WSPM might be more hazardous than PM collected from vehicle exhaust with respect to development of lung cancer.

  3. Clinical significance of aberrant Wnt7a promoter methylation in human non-small cell lung cancer in Koreans.

    PubMed

    Kim, Tae-Hyung; Moon, Ji-Yong; Kim, Sang-Heon; Paik, Seung Sam; Yoon, Ho Joo; Shin, Dong Ho; Park, Sung Soo; Sohn, Jang Won

    2015-02-01

    The Wnt signaling pathway has regulatory roles in cell proliferation, differentiation, and polarity. Aberrant Wnt pathway regulation can lead to abnormal cell proliferation and cancer, and loss of Wnt7a expression has been demonstrated in lung cancer cell lines. E-cadherin keeps intercellular integrity and prevents metastasis. Therefore, E-cadherin has been known as a prognostic factor in cancer. In the present study, we investigated the E-cadherin expression status by immunohistochemical stain and the Wnt7a promoter methylation status in human non-small cell lung carcinoma (NSCLC) by methylation-specific PCR. We also analyzed their correlations with clinicopathological factors. Methylation of the Wnt7a gene promoter was detected in the lung tissues of 32 of 121 (26.4%) patients with NSCLC. Wnt7a promoter methylation was correlated with advanced tumor stage (P = 0.036) and distant metastasis (P = 0.037). In addition, Wnt7a promoter methylation showed correlation with loss of E-cadherin expression (P < 0.001). However, Wnt7a promoter methylation was not closely related with gender, age, histological type, or smoking habit. Even though Wnt7a methylation could not show significant correlation with the long term survival of the patients with limited follow up data, these findings suggest that loss of the Wnt7a gene induced by promoter methylation might be another prognostic factor for NSCLC and that restoration of Wnt7a may be a promising treatment for NSCLC.

  4. Involvement of TP53 and TP16 expression in human papillomavirus-associated non-small cell lung cancer

    PubMed Central

    Li, Ming; Zhang, Xiao-Lei; Deng, Fang; Qian, Li-Ting; Meng, Shui-Ping; Shan, Wu-Lin; Wang, Bao-Long

    2016-01-01

    Human papilloma virus (HPV) infection has previously been reported to be associated with TP53 and TP16 expression in Japanese and Taiwanese patients with lung cancer, but data for advanced non-small cell lung cancer (NSCLC) patients is limited. The present study examined the association between HPV infection and TP53 and TP16 expression in Chinese patients with advanced NSCLC. HPV DNA was detected in 20 out of 83 (24%) lung tumors, and was observed more frequently in non-smokers, patients with lymph node metastasis, and patients with poorly differentiated tumors (P=0.048, P=0.044 and P=0.024, respectively). Thirteen (65%) out of 20 HPV-infected tumors were positive for TP53 expression while eight (40%) were positive for TP16 expression. Multivariate analysis revealed that poor differentiation alone (OR=0.163) was an independent predictive factor of HPV infection in NSCLC. TP16-positive patients had a significantly longer survival time when compared with TP16-negative patients (P<0.001, log-rank test), a trend a not observed for TP53. Our results suggest that TP53 and TP16 protein expression is not associated with the expression of HPV DNA, but that TP16 expression may be an independent prognostic factor of long survival in advanced NSCLC. PMID:27900000

  5. The significance of PIWI family expression in human lung embryogenesis and non-small cell lung cancer

    PubMed Central

    Navarro, Alfons; Tejero, Rut; Viñolas, Nuria; Cordeiro, Anna; Marrades, Ramon M.; Fuster, Dolors; Caritg, Oriol; Moises, Jorge; Muñoz, Carmen; Molins, Laureano; Ramirez, Josep; Monzo, Mariano

    2015-01-01

    The expression of Piwi-interacting RNAs, small RNAs that bind to PIWI proteins, was until recently believed to be limited to germinal stem cells. We have studied the expression of PIWI genes during human lung embryogenesis and in paired tumor and normal tissue prospectively collected from 71 resected non-small-cell lung cancer patients. The mRNA expression analysis showed that PIWIL1 was highly expressed in 7-week embryos and downregulated during the subsequent weeks of development. PIWIL1 was expressed in 11 of the tumor samples but in none of the normal tissue samples. These results were validated by immunohistochemistry, showing faint cytoplasmic reactivity in the PIWIL1-positive samples. Interestingly, the patients expressing PIWIL1 had a shorter time to relapse (TTR) (p = 0.006) and overall survival (OS) (p = 0.0076) than those without PIWIL1 expression. PIWIL2 and 4 were downregulated in tumor tissue in comparison to the normal tissue (p < 0.001) and the patients with lower levels of PIWIL4 had shorter TTR (p = 0.048) and OS (p = 0.033). In the multivariate analysis, PIWIL1 expression emerged as an independent prognostic marker. Using 5-Aza-dC treatment and bisulfite sequencing, we observed that PIWIL1 expression could be regulated in part by methylation. Finally, an in silico study identified a stem-cell expression signature associated with PIWIL1 expression. PMID:25742785

  6. Isogambogenic acid induces apoptosis-independent autophagic cell death in human non-small-cell lung carcinoma cells

    PubMed Central

    Yang, Jianhong; Zhou, Yongzhao; Cheng, Xia; Fan, Yi; He, Shichao; Li, Shucai; Ye, Haoyu; Xie, Caifeng; Wu, Wenshuang; Li, Chunyan; Pei, Heying; Li, Luyuan; Wei, Zhe; Peng, Aihua; Wei, Yuquan; Li, Weimin; Chen, Lijuan

    2015-01-01

    To overcome drug resistance caused by apoptosis deficiency in patients with non-small cell lung carcinoma (NSCLC), there is a need to identify other means of triggering apoptosis-independent cancer cell death. We are the first to report that isogambogenic acid (iso-GNA) can induce apoptosis-independent autophagic cell death in human NSCLC cells. Several features of the iso-GNA-treated NSCLC cells indicated that iso-GNA induced autophagic cell death. First, there was no evidence of apoptosis or cleaved caspase 3 accumulation and activation. Second, iso-GNA treatment induced the formation of autophagic vacuoles, increased LC3 conversion, caused the appearance of autophagosomes and increased the expression of autophagy-related proteins. These findings provide evidence that iso-GNA induces autophagy in NSCLC cells. Third, iso-GNA-induced cell death was inhibited by autophagic inhibitors or by selective ablation of Atg7 and Beclin 1 genes. Furthermore, the mTOR inhibitor rapamycin increased iso-GNA-induced cell death by enhancing autophagy. Finally, a xenograft model provided additional evidence that iso-GNA exhibited anticancer effect through inducing autophagy-dependent cell death in NSCLC cells. Taken together, our results demonstrated that iso-GNA exhibited an anticancer effect by inducing autophagy-dependent cell death in NSCLC cells, which may be an effective chemotherapeutic agent that can be used against NSCLC in a clinical setting. PMID:25571970

  7. KRAS-mutation status dependent effect of zoledronic acid in human non-small cell cancer preclinical models

    PubMed Central

    Kenessey, István; Kói, Krisztina; Horváth, Orsolya; Cserepes, Mihály; Molnár, Dávid; Izsák, Vera; Dobos, Judit; Hegedűs, Balázs

    2016-01-01

    Background In non-small cell lung cancer (NSCLC) KRAS-mutant status is a negative prognostic and predictive factor. Nitrogen-containing bisphosphonates inhibit prenylation of small G-proteins (e.g. Ras, Rac, Rho) and thus may affect proliferation and migration. In our preclinical work, we investigated the effect of an aminobisphosphonate compound (zoledronic acid) on mutant and wild type KRAS-expressing human NSCLC cell lines. Results We confirmed that zoledronic acid was unable to inhibit the prenylation of mutant K-Ras unlike in the case of wild type K-Ras. In case of in vitro proliferation, the KRAS-mutant human NSCLC cell lines showed resistance to zoledronic acid wild-type KRAS-cells proved to be sensitive. Combinatory application of zoledronic acid enhanced the cytostatic effect of cisplatin. Zoledronic acid did not induce significant apoptosis. In xenograft model, zoledronic acid significantly reduced the weight of wild type KRAS-EGFR-expressing xenograft tumor by decreasing the proliferative capacity. Futhermore, zoledronic acid induced VEGF expression and improved in vivo tumor vascularization. Materials and methods Membrane association of K-Ras was examined by Western-blot. In vitro cell viability, apoptotic cell death and migration were measured in NSCLC lines with different molecular background. The in vivo effect of zoledronic acid was investigated in a SCID mouse subcutaneous xenograft model. Conclusions The in vitro and in vivo inhibitory effect of zoledronic acid was based on the blockade of cell cycle in wild type KRAS-expressing human NSCLC cells. The zoledronic acid induced vascularization supported in vivo cytostatic effect. Our preclinical investigation suggests that patients with wild type KRAS-expressing NSCLC could potentially benefit from aminobisphosphonate therapy. PMID:27780929

  8. Smad2/3-Regulated Expression of DLX2 Is Associated with Radiation-Induced Epithelial-Mesenchymal Transition and Radioresistance of A549 and MDA-MB-231 Human Cancer Cell Lines

    PubMed Central

    Choi, Yeo-Jin; Baek, Ga-Young; Park, Hae-Ran; Jo, Sung-Kee; Jung, Uhee

    2016-01-01

    The control of radioresistance and metastatic potential of surviving cancer cells is important for improving cancer eradication by radiotheraphy. The distal-less homeobox2 (DLX2) gene encodes for a homeobox transcription factor involved in morphogenesis and its deregulation was found in human solid tumors and hematologic malignancies. Here we investigated the role of DLX2 in association with radiation-induced epithelial to mesenchymal transition (EMT) and stem cell-like properties and its regulation by Smad2/3 signaling in irradiated A549 and MDA-MB-231 human cancer cell lines. In irradiated A549 and MDA-MB-231 cells, EMT was induced as demonstrated by EMT marker expression, phosphorylation of Smad2/3, and migratory and invasive ability. Also, irradiated A549 and MDA-MB-231 cells showed increased cancer stem cells (CSCs) marker. Interestingly, DLX2 was overexpressed upon irradiation. Therefore, we examined the role of DLX2 in radiation-induced EMT and radioresistance. The overexpression of DLX2 alone induced EMT, migration and invasion, and CSC marker expression. The reduced colony-forming ability in irradiated cells was partially restored by DLX2 overexpression. On the other hand, the depletion of DLX2 using si-RNA abolished radiation-induced EMT, CSC marker expression, and phosphorylation of Smad2/3 in irradiated A549 and MDA-MB-231 cells. Also, depletion of DLX2 increased the radiation sensitivity in both cell lines. Moreover, knockdown of Smad2/3, a key activator of TGF-β1 pathway, abrogated the radiation-induced DLX2 expression, indicating that radiation-induced DLX2 expression is dependent on Smad2/3 signaling. These results demonstrated that DLX2 plays a crucial role in radioresistance, radiation-induced EMT and CSC marker expression, and the expression of DLX2 is regulated by Smad2/3 signaling in A549 and MDA-MB-231 cell lines. PMID:26799321

  9. Cardiac troponin I is abnormally expressed in non-small cell lung cancer tissues and human cancer cells.

    PubMed

    Chen, Chao; Liu, Jia-Bao; Bian, Zhi-Ping; Xu, Jin-Dan; Wu, Heng-Fang; Gu, Chun-Rong; Shi, Yi; Zhang, Ji-Nan; Chen, Xiang-Jian; Yang, Di

    2014-01-01

    Cardiac troponin I (cTnI) is the only sarcomeric protein identified to date that is expressed exclusively in cardiac muscle. Its expression in cancer tissues has not been reported. Herein, we examined cTnI expression in non-small cell lung cancer (NSCLC) tissues, human adenocarcinoma cells SPCA-1 (lung) and BGC 823 (gastric) by immunohistochemistry, western blot analysis and real-time PCR. Immunopositivity for cTnI was demonstrated in 69.4% (34/49) NSCLC tissues evaluated, and was strong intensity in 35.3% (6/17) lung squamous cell carcinoma cases. The non-cancer-bearing lung tissues except tuberculosis (9/9, 100%) showed negative staining for cTnI. Seven monoclonal antibodies (mAbs) against human cTnI were applied in immunofluorescence. The result showed that the staining pattern within SPCA-1 and BGC 823 was dependent on the epitope of the cTnI mAbs. The membrane and nucleus of cancer cells were stained by mAbs against N-terminal peptides of cTnI, and cytoplasm was stained by mAbs against the middle and C-terminal peptides of cTnI. A ~25 kD band was identified by anti-cTnI mAb in SPCA-1 and BGC 823 extracts by western blot, as well as in cardiomyocyte extracts. The cTnI mRNA expressions in SPCA-1 and BGC 823 cells were about ten thousand times less than that in cardiomyocytes. Our study shows for the first time that cTnI protein and mRNA were abnormally expressed in NSCLC tissues, SPCA-1 and BGC 823 cells. These findings challenge the conventional view of cTnI as a cardiac-specific protein, enabling the potential use of cTnI as a diagnostic marker or targeted therapy for cancer.

  10. MiR-153 inhibits migration and invasion of human non-small-cell lung cancer by targeting ADAM19

    SciTech Connect

    Shan, Nianxi; Shen, Liangfang; Wang, Jun; He, Dan; Duan, Chaojun

    2015-01-02

    Highlights: • Decreased miR-153 and up-regulated ADAM19 are correlated with NSCLC pathology. • MiR-153 inhibits the proliferation and migration and invasion of NSCLC cells in vitro. • ADAM19 is a direct target of miR-153. • ADAM19 is involved in miR-153-suppressed migration and invasion of NSCLC cells. - Abstract: MiR-153 was reported to be dysregulated in some human cancers. However, the function and mechanism of miR-153 in lung cancer cells remains unknown. In this study, we investigated the role of miR-153 in human non-small-cell lung cancer (NSCLC). Using qRT-PCR, we demonstrated that miR-153 was significantly decreased in clinical NSCLC tissues and cell lines, and downregulation of miR-153 was significantly correlated with lymph node status. We further found that ectopic expression of miR-153 significantly inhibited the proliferation and migration and invasion of NSCLC cells in vitro, suggesting that miR-153 may be a novel tumor suppressor in NSCLC. Further integrated analysis revealed that ADAM19 is as a direct and functional target of miR-153. Luciferase reporter assay demonstrated that miR-153 directly targeted 3′UTR of ADAM19, and correlation analysis revealed an inverse correlation between miR-153 and ADAM19 mRNA levels in clinical NSCLC tissues. Knockdown of ADAM19 inhibited migration and invasion of NSCLC cells which was similar with effects of overexpression of miR-153, while overexpression of ADAM19 attenuated the function of miR-153 in NSCLC cells. Taken together, our results highlight the significance of miR-153 and ADAM19 in the development and progression of NSCLC.

  11. Human papilloma virus in non-small cell lung cancer in never smokers: a systematic review of the literature.

    PubMed

    Hasegawa, Yoshikazu; Ando, Masahiko; Kubo, Akihito; Isa, Shun-Ichi; Yamamoto, Satomi; Tsujino, Kazuyuki; Kurata, Takayasu; Ou, Sai-Hong I; Takada, Minoru; Kawaguchi, Tomoya

    2014-01-01

    Non-small cell lung cancer (NSCLC) in never smokers has emerged as a global public health issue. The cause is still unclear, and few studies have focused on the prevalence of human papillomavirus (HPV) in the never smokers. We performed a systematic search of PubMed for articles of HPV infection in human subjects with NSCLC up to September 2012. Although smoking status was not fully reported in all studies, we contacted the authors by e-mail to supplement this information. Differences in the distribution of patients with and without HPV infection were tested with the Chi squared test. We identified 46 eligible articles, including 23 from Asian countries (N=2337 NSCLC cases), 19 from European countries (N=1553) and 4 from North and South America (N=160). The HPV prevalence was 28.1% (95% confidence interval (CI) 26.6-30.3%), 8.4% (95% CI 7.1-9.9%) and 21.3% (95% CI 15.2-28.4%), respectively. Eleven studies from East Asia (N=1110) and 4 from Europe (N=569) provided information on smoking status. The number of never smoker was 392 patients (33.9%) in East Asia and 54 patients (14.8%) in Europe. The HPV prevalence in East Asian countries was similar between never and ever smokers (33.9% vs 39.2%, P=0.080). Based on the literature confirming the presence of HPV in lung cancer in never smokers, the virus plays a role in carcinogenesis in the disease. There were different patterns of HPV prevalence between Asian and European countries in the never smokers as well as in ever smokers. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  12. Alpha-chaconine-reduced metastasis involves a PI3K/Akt signaling pathway with downregulation of NF-kappaB in human lung adenocarcinoma A549 cells.

    PubMed

    Shih, Yuan-Wei; Chen, Pin-Shern; Wu, Cheng-Hsun; Jeng, Ya-Fang; Wang, Chau-Jong

    2007-12-26

    Alpha-chaconine, isolated from Solanum tuberosum Linn., is a naturally occurring steroidal glycoalkaloid in potato sprouts. Some reports demonstrated that alpha-chaconine had various anticarcinogenic properties. The aim of this study is to investigate the inhibitory effect of alpha-chaconine on lung adenocarcinoma cell metastasis in vitro. We chose the highly metastatic A549 cells, which were treated with various concentrations of alpha-chaconine to clarify the potential of inhibiting A549 cells invasion and migration. Data showed that alpha-chaconine inhibited A549 cell invasion/migration according to wound healing assay and Boyden chamber assay. Our results also showed that alpha-chaconine could inhibit phosphorylation of c-Jun N-terminal kinase (JNK) and Akt, whereas it did not affected phosphorylation of extracellular signal regulating kinase (ERK) and p38. In addition, alpha-chaconine significantly decreased the nuclear level of nuclear factor kappa B (NF-kappaB) and the binding ability of NF-kappaB. These results suggested that alpha-chaconine inhibited A549 cell metastasis by a reduction of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) activities involving suppression of phosphoinositide 3-kinase/Akt/NF-kappaB (PI3K/Akt/NF-kappaB) signaling pathway. Inhibiting metastasis by alpha-chaconine might offer a pivotal mechanism for its effective chemotherapeutic action.

  13. Expression of cytochrome P450 2A13 in human non-small cell lung cancer and its clinical significance

    PubMed Central

    Sun, Li; Fan, Xiaoli

    2013-01-01

    Lung cancer is one of the most important causes of cancer-related mortality worldwide. Human cytochrome P450 2A13 enzyme (CYP2A13) is predominantly expressed in the respiratory tract and could catalyze various carcinogens. In this study, we quantified CYP2A13 expression in non-small cell lung cancer (NSCLC) tissues and examined the relation between CYP2A13 and clinicopathologic factors. Thirty-five paired lung cancer and normal tissues were studied for the expression of the CYP2A13 gene by using real-time PCR and Western blotting assays. We also investigated the relationship between CYP2A13 expression and clinicopathologic factors such as age, gender, histology and lymph node status in tumor tissues. SPSS (17.0) statistical software was applied for data analysis. The real-time PCR results showed that there was no significant difference in the CYP2A13 mRNA transcript levels between tumor and paired normal tissues in the 35 samples and in 12 paired squamous cell carcinomas. In adenocarcinoma, the expression of CYP2A13 mRNA in tumor tissues was 12.5% of that in adjacent tissues (P < 0.05) and it was not associated with age, gender, histology and lymph node status of the patients. The amounts of CYP2A13 proteins detected by Western blotting assays correlated well with those of the corresponding mRNAs. In conclusion, the expression of CYP2A13 was downregulated in lung adenocarcinoma. CYP2A13 may be involved in the development and progression of lung adenocarcinoma. PMID:23720675

  14. Suppression of reactive oxygen species-mediated ERK and JNK activation sensitizes dihydromyricetin-induced mitochondrial apoptosis in human non-small cell lung cancer.

    PubMed

    Kao, Shang-Jyh; Lee, Wei-Jiunn; Chang, Jer-Hwa; Chow, Jyh-Ming; Chung, Chi-Li; Hung, Wen-Yueh; Chien, Ming-Hsien

    2017-04-01

    Nonsmall cell lung cancer (NSCLC) is the most common type of lung cancer with a high mortality rate and still remains a therapeutic challenge. A strategy for targeting NSCLC is to identify agents that are effective against NSCLC cells while sparing normal cells. Dihydromyricetin (DHM) is the major flavonoid component derived from Ampelopsis grossedentata, which has a long history of use in medicine. Herein, the molecular mechanisms by which DHM exerts its anticancer effects against NSCLC cells were investigated. Results from MTS, colony formation, Western blot, flow cytometric, and JC-1 mitochondrial membrane potential assays revealed that DHM showed a selective cytotoxic effect against NSCLC cells (A549 and H1975), but not against normal lung (WI-38) fibroblasts, by inducing apoptosis. DHM-induced cell apoptosis occurred through Bcl-w suppression-mediated mitochondrial membrane depolarization, caspase-9/-7/-3 activation, and poly(ADP-ribose) polymerase (PARP) cleavage in A549 and H1975 cells. Moreover, treatment of A549 and H1975 cells with DHM induced increase of intracellular peroxide and sustained activation of extracellular signal-regulated kinase (ERK)1/2 and c-Jun N-terminal kinase (JNK)1/2, and the reactive oxygen species scavenger, N-acetylcysteine (NAC), reversed DHM-induced ERK and JNK activation. Furthermore, treatment of cells with specific inhibitors of ERK and JNK or NAC significantly promoted the DHM-induced activation of caspase-9/-7/-3 and PARP cleavage and also sensitized the antitumorigenic effect of DHM on NSCLC cells. These findings define and support a novel function of DHM of inducing mitochondrion-derived apoptosis in human NSCLC cells, and a combination of DHM with ERK and JNK inhibitors should be a good strategy for preventing NSCLC proliferation. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1426-1438, 2017. © 2016 Wiley Periodicals, Inc.

  15. Hedgehog Pathway Inhibition Radiosensitizes Non-Small Cell Lung Cancers

    SciTech Connect

    Zeng, Jing; Aziz, Khaled; Chettiar, Sivarajan T.; Aftab, Blake T.; Armour, Michael; Gajula, Rajendra; Gandhi, Nishant; Salih, Tarek; Herman, Joseph M.; Wong, John; Rudin, Charles M.; Tran, Phuoc T.; Hales, Russell K.

    2013-05-01

    Purpose: Despite improvements in chemoradiation, local control remains a major clinical problem in locally advanced non-small cell lung cancer. The Hedgehog pathway has been implicated in tumor recurrence by promoting survival of tumorigenic precursors and through effects on tumor-associated stroma. Whether Hedgehog inhibition can affect radiation efficacy in vivo has not been reported. Methods and Materials: We evaluated the effects of a targeted Hedgehog inhibitor (HhAntag) and radiation on clonogenic survival of human non-small cell lung cancer lines in vitro. Using an A549 cell line xenograft model, we examined tumor growth, proliferation, apoptosis, and gene expression changes after concomitant HhAntag and radiation. In a transgenic mouse model of Kras{sup G12D}-induced and Twist1-induced lung adenocarcinoma, we assessed tumor response to radiation and HhAntag by serial micro-computed tomography (CT) scanning. Results: In 4 human lung cancer lines in vitro, HhAntag showed little or no effect on radiosensitivity. By contrast, in both the human tumor xenograft and murine inducible transgenic models, HhAntag enhanced radiation efficacy and delayed tumor growth. By use of the human xenograft model to differentiate tumor and stromal effects, mouse stromal cells, but not human tumor cells, showed significant and consistent downregulation of Hedgehog pathway gene expression. This was associated with increased tumor cell apoptosis. Conclusions: Targeted Hedgehog pathway inhibition can increase in vivo radiation efficacy in lung cancer preclinical models. This effect is associated with pathway suppression in tumor-associated stroma. These data support clinical testing of Hedgehog inhibitors as a component of multimodality therapy for locally advanced non-small cell lung cancer.

  16. Hedgehog Pathway Inhibition Radiosensitizes Non-Small Cell Lung Cancers

    PubMed Central

    Zeng, Jing; Aziz, Khaled; Chettiar, Sivarajan T.; Aftab, Blake T.; Armour, Michael; Gajula, Rajendra; Gandhi, Nishant; Salih, Tarek; Herman, Joseph M.; Wong, John; Rudin, Charles M.; Tran, Phuoc T.; Hales, Russell K.

    2012-01-01

    Purpose Despite improvements in chemoradiation, local control remains a major clinical problem in locally advanced non-small cell lung cancer. The Hedgehog pathway has been implicated in tumor recurrence by promoting survival of tumorigenic precursors and through effects on tumor-associated stroma. Whether Hedgehog inhibition can affect radiation efficacy in vivo has not been reported. Methods and Materials We evaluated the effects of a targeted Hedgehog inhibitor (HhAntag) and radiation on clonogenic survival of human non-small cell lung cancer lines in vitro. Using an A549 cell line xenograft model, we examined tumor growth, proliferation, apoptosis, and gene expression changes after concomitant HhAntag and radiation. In a transgenic mouse model of KrasG12D-induced and Twist1-induced lung adenocarcinoma, we assessed tumor response to radiation and HhAntag by serial micro-computed tomography (CT) scanning. Results In 4 human lung cancer lines in vitro, HhAntag showed little or no effect on radio-sensitivity. By contrast, in both the human tumor xenograft and murine inducible transgenic models, HhAntag enhanced radiation efficacy and delayed tumor growth. By use of the human xenograft model to differentiate tumor and stromal effects, mouse stromal cells, but not human tumor cells, showed significant and consistent downregulation of Hedgehog pathway gene expression. This was associated with increased tumor cell apoptosis. Conclusions Targeted Hedgehog pathway inhibition can increase in vivo radiation efficacy in lung cancer preclinical models. This effect is associated with pathway suppression in tumor-associated stroma. These data support clinical testing of Hedgehog inhibitors as a component of multimodality therapy for locally advanced non-small cell lung cancer. PMID:23182391

  17. A novel quinazoline-based analog induces G2/M cell cycle arrest and apoptosis in human A549 lung cancer cells via a ROS-dependent mechanism.

    PubMed

    Shi, Hailong; Li, Yan; Ren, Xiaorong; Zhang, Yaohong; Yang, Zhen; Qi, Chenze

    2017-04-29

    6-amino-4-(4-phenoxyphenylethylamino)quinazoline (QNZ) is an excellent quinazoline-containing NF-κB inhibitor also acting as a novel anticancer agent. Considering both the medicinal significance of quinazoline scaffold and the tunable functionality of Michael acceptor-centric pharmacophores in the electrophilicity-based prooxidant strategy, we designed a novel QNZ-inspired electrophilic molecule QNZ-A by introducing a Michael acceptor unit at position-6 of quinazoline ring in QNZ. Our results identified QNZ-A as a promising selective cytotoxic agent against A549 cells. QNZ-A, by virtue of its Michael acceptor unit, induced reactive oxygen species (ROS) accumulation associated with collapse of the redox buffering system in A549 cells. This caused up-regulation of p53-inducible p21 and down-regulation of redox sensitive Cdc25C along with Cyclin B1/Cdk1, leading to a G2/M cell cycle arrest and final cell apoptosis. By contrast, QNZ-B, a reduction product of QNZ-A lacking the Michael acceptor unit failed to induce ROS generation and all these cell cycle-related events. In conclusion, this work provided a successful example of designing QNZ-directed anticancer agent by a ROS-promoting strategy and identified QNZ-A as a selective anticancer agent against A549 cells through G2/M cell cycle arrest and apoptosis via a ROS-dependent mechanism. Copyright © 2017. Published by Elsevier Inc.

  18. Cytotoxic activity, DNA damage, cellular uptake, apoptosis and western blot analysis of ruthenium(II) polypyridyl complex against human lung decarcinoma A549 cell.

    PubMed

    Lai, Shang-Hai; Jiang, Guang-Bin; Yao, Jun-Hua; Li, Wei; Han, Bing-Jie; Zhang, Cheng; Zeng, Chuan-Chuan; Liu, Yun-Jun

    2015-11-01

    A new ruthenium(II) polypyridyl complex [Ru(dmp)2(pddppn)](ClO4)2Ru1 was synthesized and characterized. The cytotoxic activity in vitro of the complex was evaluated by MTT method. Ru1 shows high effect on the inhibition of the cell growth against BEL-7402, HeLa, MG-63 and A549 cells with low IC50 values of 1.6±0.4, 9.0±0.8, 1.5±0.2 and 1.5±0.3 μM, respectively. The cellular uptake indicates that Ru1 can enter into the cytoplasm and accumulate in the cell nuclei. Ru1 can induce apoptosis in A549 cells and enhance the levels of reactive oxygen species (ROS) and induce the decrease of mitochondrial membrane potential. In addition, Ru1 can down-regulate the levels of Bcl-2, Bcl-x, Bak, and Bim expression and up-regulate the expression of Bag-1 and Bad. The complex induces apoptosis of A549 cells through an intrinsic ROS-mediated mitochondrial dysfunction pathway, which was accompanied by regulating the expression of caspases and Bcl-2 family proteins. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. Comparison of wood smoke PM2.5 obtained from the combustion of FIR and beech pellets on inflammation and DNA damage in A549 and THP-1 human cell lines.

    PubMed

    Corsini, Emanuela; Budello, Silvia; Marabini, Laura; Galbiati, Valentina; Piazzalunga, Andrea; Barbieri, Pierluigi; Cozzutto, Sergio; Marinovich, Marina; Pitea, Demetrio; Galli, Corrado L

    2013-12-01

    The aim of this study was to investigate the effect on the induction of interleukin-8 of particulate matter (PM) from fir and beech pellets burnt in domestic appliances on two human cells lines, namely the lung epithelial cell line A549 and the promyelocytic cell line THP-1. The effects of PM2.5 obtained from combustion of beech and fir pellets were compared to reference diesel exhaust particulates (DEP). In parallel, wood smoke PM-induced genotoxicity and oxidative stress were also investigated in A549 cells. Cells were treated for different times (3-72 h) with increasing concentrations of PM2.5 obtained from sequential combustions of fir and beech pellets or reference DEP. Cell viability was assessed by lactate dehydrogenase leakage, and the release of interleukin-8 or CXCL8 (IL-8) was measured to evaluate the pro-inflammatory effect. Oxidative stress was evaluated by the 5(6)-carboxy-2',7'dichlorofluorescein diacetate (DCFH-DA) assay and DNA damage by the alkaline comet assay and micronucleus frequency by flow cytometry. Both A549 and THP-1 cells responded in a dose- and time-related manner to wood smoke PM2.5 with IL-8 release, particles obtained from late combustions being the most active. THP-1 cells were more sensitive than A549 cells. On a mass base, similar effects were observed for both fir and beech PM2.5. However, the combustion of beech pellets generated approximately three times more PM2.5 than fir pellets. Regarding the mechanism of PM2.5 uptake, in both THP-1 and A549 cells, cytochalasin D prevented PM2.5-induced IL-8 mRNA expression and cytokine release, indicating a key role for actin polymerization in particles uptake and that the production of IL-8 correlated with particle phagocytosis. As signal transduction pathway involvement, in both THP-1 and A549 cells, PM2.5-induced IL-8 release could be completely blocked by the selective inhibitor SB203580, indicating a role of p38 MAPK activation. PM2.5 from both fir and beech pellets also induced

  20. Comparative evaluation of antiproliferative activity and induction of apoptosis by some fluoroquinolones with a human non-small cell lung cancer cell line in culture.

    PubMed

    Mondal, E R; Das, S K; Mukherjee, P

    2004-01-01

    Lung cancer is the leading cause of cancer- related death in the world today. Since the effective management of drug resistant lung cancer, and particularly non-small cell lung carcinomas is a major problem, attempts need to be made to identify new potential anticancer drugs that can kill non-small cell lung cancer cells efficiently. In the present study, a human non-small cell lung carcinoma NCI-H460 cell line was used to evaluate the antiproliferative activity of Fluoroquinolones like Enoxacin, Norfloxacin, Ciprofloxacin and Levofloxacin. As determined by Sulphorodhamine B assay (SRB assay), all Fluoroquinolones caused cellular growth inhibition in a concentration and time-dependent manner. Enoxacin was found to be the most effective Fluoroquinolone followed by Norfloxacin, Ciprofloxacin and Levofloxacin. Growth inhibitory effects were also found to be independent of the concentrations of serum growth factors in culture medium or variation of initial cell seeding density and proved to be irreversible in nature. Appearance of rounded cells with altered morphology and cell surface blebbing indicated cell killing by apoptosis. Cell shrinkage, nuclear condensation & fragmentation, and cytoplasmic blebbing as indicated by MGG staining confirmed this to be the case. Thus, this investigation clearly demonstrated that the NCI-H460 human non-small cell lung carcinoma cell line is highly sensitive to Fluoroquinolone treatment. The Fluoroquinolones used in this study which are clinically used as antibacterial agents, can also inhibit tumor cell growth suggesting their potential use in a strategy for cancer treatment which might help in controlling cancer.

  1. Regulation of different components from Ophiopogon japonicus on autophagy in human lung adenocarcinoma A549Cells through PI3K/Akt/mTOR signaling pathway.

    PubMed

    Chen, Juan; Yuan, Jiarui; Zhou, Liqiang; Zhu, Maomao; Shi, Ziqi; Song, Jie; Xu, Qingyu; Yin, Guowen; Lv, You; Luo, Yi; Jia, Xiaobin; Feng, Liang

    2017-03-01

    Autophagy plays a dual role in the development of cancer, acting as both a tumor suppressor and a cell survival inducer. Ophiopogon japonicus (L.f) Ker-Gawl (OJ), as a traditional Chinese medicine, specially possesses remarkable anti-cancer activity in the clinical. Previously, studies have indicated that flavonoids (FOJ) and steroidal saponins (SSOJ) are the main active substances of OJ. However, the effects of FOJ and SSOJ on autophagy of A549 cells have not been fully elucidated. In this study, we found that the expressions of autophagy-related mediators (LC3-II/LC3-I ratio, Atg-3, Atg-7 and Beclin-1) were increased in A549 cells by the treatment with FOJ (7.9mg crude drug/mL) and SSOJ (12.2mg crude drug/mL). Meanwhile, FOJ or SSOJ could induce the up-regulation of LC3-II at both protein and mRNA levels. Moreover, we observed the cytoplasmic vaculoes which formed double-layered membranes and only some cytoplasmic organelles or myelin figures remained in FOJ or SSOJ-treated A549 cells for 24h by Transmission Electron Microscopy (TEM). Further detection about the PI3K/Akt/mTOR signaling pathway showed that the levels of PI3K, Akt and mTOR were significantly suppressed with the FOJ or SSOJ treatment. The 3-MA (an autophagy inhibitor) and LY294002 (a PI3K inhibitor) further confirmed the underlying mechanism in the FOJ or SSOJ-induced autophagy of A549 cells. Additionally, the pretreatment with FOJ and SSOJ increased the level of p53, whereas decreased the expression of Ki67. These findings suggested that FOJ or SSOJ could activate the autophagy of A549 cells, wherein the mechanism might be associated with their inhibition of PI3K/Akt/mTOR signaling pathway. Thus, FOJ or SSOJ could be a potential autophagy inducer to prevent the process of lung cancer.

  2. CEP1612, a dipeptidyl proteasome inhibitor, induces p21WAF1 and p27KIP1 expression and apoptosis and inhibits the growth of the human lung adenocarcinoma A-549 in nude mice.

    PubMed

    Sun, J; Nam, S; Lee, C S; Li, B; Coppola, D; Hamilton, A D; Dou, Q P; Sebti, S M

    2001-02-15

    The ubiquitin proteasome system is responsible for the proteolysis of important cell cycle and apoptosis-regulatory proteins. In this paper we report that the dipeptidyl proteasome inhibitor, phthalimide-(CH2)8CH-(cyclopentyl) CO-Arg(NO2)-Leu-H (CEP1612), induces apoptosis and inhibits tumor growth of the human lung cancer cell line A-549 in an in vivo model. In cultured A-549 cells, CEP1612 treatment results in accumulation of two proteasome natural substrates, the cyclin-dependent kinase inhibitors p21WAF1 and p27KIP1, indicating its ability to inhibit proteasome activity in intact cells. Furthermore, CEP1612 induces apoptosis as evident by caspase-3 activation and poly(ADP-ribose) polymerase cleavage. Treatment of A-549 tumor-bearing nude mice with CEP1612 (10 mg/kg/day, i.p. for 31 days) resulted in massive induction of apoptosis and significant (68%; P < 0.05) tumor growth inhibition, as shown by terminal deoxynucleotidyltransferase-mediated UTP end labeling. Furthermore, immunostaining of tumor specimens demonstrated in vivo accumulation of p21WAF1 and p27KIP1 after CEP1612 treatment. The results suggest that CEP1612 is a promising candidate for further development as an anticancer drug and demonstrate the feasibility of using proteasome inhibitors as novel antitumor agents.

  3. Resveratrol raises in vitro anticancer effects of paclitaxel in NSCLC cell line A549 through COX-2 expression.

    PubMed

    Kong, Fanhua; Zhang, Runqi; Zhao, Xudong; Zheng, Guanlin; Wang, Zhou; Wang, Peng

    2017-09-01

    The aim of this study was to determine the raising anticancer effects of resveratrol (Res) on paclitaxel (PA) in non-small cell lung cancer (NSCLC) cell line A549. The 10 µg/ml of Res had no effect on human fetal lung fibroblast MRC-5 cells or on A549 cancer cells and the 5 or 10 µg/ml of PA also had no effect on MRC-5 normal cells. PA-L (5 µg/ml) and PA-H (10 µg/ml) had the growth inhibitory effects in NSCLC cell line A549, and Res increased these growth inhibitory effects. By flow cytometry experiment, after Res (5 µg/ml)+PA-H (10 µg/ml) treatment, the A549 cells showed the most apoptosic cells compared to other group treatments, and after additional treatment with Res, the apoptosic cells of both two PA concentrations were raised. Res+PA could reduce the mRNA and protein expressions of COX-2, and Res+PA could reduce the COX-2 related genes of VEGF, MMP-1, MMP-2, MMP-9, NF-κB, Bcl-2, Bcl-xL, procollagen I, collagen I, collagen III and CTGF, TNF-α, IL-1β, iNOS and raise the TIMP-1, TIMP-2, TIMP-3, IκB-α, p53, p21, caspase-3, caspase-8, caspase-9, Bax genes compared to the control cells and the PA treated cells. From these results, it can be suggested that Res could raise the anticancer effects of PA in A549 cells, thus Res might be used as a good sensitizing agent for PA.

  4. Ginsenoside Rg3 sensitizes human non-small cell lung cancer cells to γ-radiation by targeting the nuclear factor-κB pathway.

    PubMed

    Wang, Lei; Li, Xiankui; Song, Yi-Min; Wang, Bin; Zhang, Fu-Rui; Yang, Rui; Wang, Hua-Qi; Zhang, Guo-Jun

    2015-07-01

    At present, it is elusive how non-small cell lung cancer (NSCLC) develops resistance to γ-radiation; however, the transcription factor nuclear factor-κB (NF-κB) and NF-κB-regulated gene products have been proposed as mediators. Ginsenoside Rg3 is a steroidal saponin, which was isolated from Panax ginseng. Ginsenoside Rg3 possesses high pharmacological activity and has previously been shown to suppress NF-κB activation in various types of tumor cell. Therefore, the present study aimed to determine whether Rg3 could suppress NF-κB activation in NSCLC cells and sensitize NSCLC to γ-radiation, using an NSCLC cell line and NSCLC xenograft. A clone formation assay and lung tumor xenograft experiment were used to assess the radiosensitizing effects of ginsenoside Rg3. NF-κB/inhibitor of NF-κB (IκB) modulation was ascertained using an electrophoretic mobility shift assay and western blot analysis. NF-κB-regulated gene products were monitored by western blot analysis. The present study demonstrated that ginsenoside Rg3 was able to sensitize A549 and H1299 lung carcinoma cells to γ-radiation and significantly enhance the efficacy of radiation therapy in C57BL/6 mice bearing a Lewis lung carcinoma cell xenograft tumor. Furthermore, ginsenoside Rg3 suppressed NF-κB activation, phosphorylation of IκB protein and expression of NF-κB-regulated gene products (cyclin D1, c-myc, B-cell lymphoma 2, cyclooxygenase-2, matrix metalloproteinase-9 and vascular endothelial growth factor), a number of which were induced by radiation therapy and mediate radioresistance. In conclusion, the results of the present study suggested that ginsenoside Rg3 may potentiate the antitumor effects of radiation therapy in NSCLC by suppressing NF-κB activity and NF-κB-regulated gene products, leading to the inhibition of tumor progression.

  5. Downregulation of PAX6 by shRNA Inhibits Proliferation and Cell Cycle Progression of Human Non-Small Cell Lung Cancer Cell Lines

    PubMed Central

    Zhao, Xiaoting; Yue, Wentao; Zhang, Lina; Ma, Li; Jia, Wenyun; Qian, Zhe; Zhang, Chunyan; Wang, Yue

    2014-01-01

    Background The transcription factor PAX6 is primarily expressed in embryos. PAX6 is also expressed in several tumors and plays an oncogenic role. However, little is known about the role of PAX6 in lung cancer. Methods The function of PAX6 in lung cancer cells was evaluated by small interfering RNA-mediated depletion of the protein followed by analyses of cell proliferation, anchorage-independent growth, and cell cycle arrest. The changes of cyclin D1, pRB, ERK1/2, p38 expression caused by PAX6 inhibition were detected using western-blotting. The PAX6 mRNA level in 52 pairs of tumors and corresponding matched adjacent normal tissues from non-small cell lung cancer patients and lung cancer cell lines was detected by real-time PCR. Results Suppression of PAX6 expression inhibited cell growth and colony formation in A549 and H1299 cells. The percentage of cells in G1-phase increased when PAX6 expression was inhibited. The cyclin D1 protein level, as well as the pRB phosphorylation level, decreased as a result of PAX6 down-regulation. The activity of ERK1/2 and p38 was also suppressed in PAX6 knock-down cells. The PAX6 mRNA was highly expressed in lung cancer tissue and lung cancer cell lines. In most patients (about 65%), the relative ratio of PAX6 mRNA in primary NSCLC versus adjacent tissues exceeded 100. Conclusions Our data implicated that PAX6 accelerates cell cycle progression by activating MAPK signal pathway. PAX6 mRNA levels were significantly elevated in primary lung cancer tissues compared to their matched adjacent tissues. PMID:24454925

  6. (S)-crizotinib induces apoptosis in human non-small cell lung cancer cells by activating ROS independent of MTH1.

    PubMed

    Dai, Xuanxuan; Guo, Guilong; Zou, Peng; Cui, Ri; Chen, Weiqian; Chen, Xi; Yin, Changtian; He, Wei; Vinothkumar, Rajamanickam; Yang, Fan; Zhang, Xiaohua; Liang, Guang

    2017-09-07

    Non-small cell lung cancer (NSCLC) accounts for approximately 80-85% of all lung cancers and is usually diagnosed at an advanced stage with poor prognosis. Targeted therapy has produced unprecedented outcomes in patients with NSCLC as a number of oncogenic drivers have been found. Crizotinib, a selective small-molecule inhibitor, has been widely used for the treatment of NSCLC patients with ALK gene rearrangements. A recent study has also shown that (S)-enantiomer of crizotinib exhibits anticancer activity by targeting the protein mutT homologue (MTH1). Since this discovery, contradictory studies have cast a doubt on MTH1 as a therapeutic target of (S)-crizotinib. NCI-H460, H1975, and A549 cells and immunodeficient mice were chosen as a model to study the (S)-crizotinib treatment. The changes induced by (S)-crizotinib treatment in cell viability, apoptosis as well as ROS, and endoplasmic reticulum stress pathway in the cells were analyzed by MTT assay, FACSCalibur, Western blotting, ROS imaging and electron microscopy. Here, we report that MTH1 does not affect survival of NSCLC cells. We found that (S)-crizotinib induces lethal endoplasmic reticulum stress (ER) response in cultured NSCLC cells by increasing intracellular levels of reactive oxygen species (ROS). Blockage of ROS production markedly reversed (S)-crizotinib-induced ER stress and cell apoptosis, independent of MTH1. We confirmed these findings in NSCLC xenograft studies and showed that (S)-crizotinib-induced ER stress and cell apoptosis. Our results reveal a novel antitumor mechanism of (S)-crizotinib in NSCLC which involves activation of ROS-dependent ER stress apoptotic pathway and is independent of MTH1 inhibition.

  7. Honokiol exhibits enhanced antitumor effects with chloroquine by inducing cell death and inhibiting autophagy in human non-small cell lung cancer cells.

    PubMed

    Lv, Xiaoqin; Liu, Fang; Shang, Yue; Chen, Shu-Zhen

    2015-09-01

    Honokiol (HNK), a potential antitumor compound, has been widely studied in recent years. It induces apoptosis and affects autophagy in cancer cells, yet the mechanism of its antitumor efficacy remains obscure. Chloroquine (CQ), an autophagy inhibitor, is often applied to sensitize antitumor drugs in clinical trials. Here, we investigated the antitumor effect of HNK or CQ alone or in combination in non-small cell lung cancer (NSCLC) cells. Using an experimental approach, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) or sulforhodamine B (SRB) was used to determine the cytotoxicity of the agents. The expression levels of proteins were detected by western blotting. Apoptosis was examined via Annexin V-FITC and PI staining. H460 cell xenografts in nude mice were used to study the effects of HNK and/or CQ in vivo. Transfection with siRNA was applied to knock down cathepsin D. The results demonstrated the enhanced effects of HNK combined with CQ on the inhibition of proliferation, induction of apoptosis in vitro and the reduction in growth in vivo. It was confirmed that HNK and/or CQ triggered apoptosis via a caspase-dependent manner. Furthermore, HNK significantly increased the expression of p62 and LC3-Ⅱ in the A549 and H460 cells and inhibited autophagy and induced apoptosis in a cathepsin D-involved manner. In conclusion, an enhanced antitumor effect was demonstrated following treatment with HNK combined with CQ by inhibiting autophagy and inducing apoptosis via a caspase-dependent and cathepsin D-involved manner. This combination may be a novel and useful antitumor approach for chemotherapy in NSCLC.

  8. Minocycline enhances mitomycin C-induced cytotoxicity through down-regulating ERK1/2-mediated Rad51 expression in human non-small cell lung cancer cells.

    PubMed

    Ko, Jen-Chung; Wang, Tai-Jing; Chang, Po-Yuan; Syu, Jhan-Jhang; Chen, Jyh-Cheng; Chen, Chien-Yu; Jian, Yun-Ting; Jian, Yi-Jun; Zheng, Hao-Yu; Chen, Wen-Ching; Lin, Yun-Wei

    2015-10-01

    Minocycline is a semisynthetic tetracycline derivative; it has anti-inflammatory and anti-cancer effects distinct from its antimicrobial function. However, the molecular mechanism of minocycline-induced cytotoxicity in non-small cell lung cancer (NSCLC) cells has not been identified. Rad51 plays a central role in homologous recombination and high levels of Rad51 expression are observed in chemo- or radioresistant carcinomas. Our previous studies have shown that the MKK1/2-ERK1/2 signal pathway maintains the expression of Rad51 in NSCLC cells. In this study, minocycline treatment inhibited cell viability and proliferation of two NSCLC cells, A549 and H1975. Treatment with minocycline decreased Rad51 mRNA and protein levels through MKK1/2-ERK1/2 inactivation. Furthermore, expression of constitutively active MKK1 (MKK1-CA) vectors significantly rescued the decreased Rad51 protein and mRNA levels in minocycline-treated NSCLC cells. However, combined treatment with MKK1/2 inhibitor U0126 and minocycline further decreased the Rad51 expression and cell viability of NSCLC cells. Knocking down Rad51 expression by transfection with small interfering RNA of Rad51 enhanced the cytotoxicity and cell growth inhibition of minocycline. Mitomycin C (MMC) is typically used as a first or second line regimen to treat NSCLC. Compared to a single agent alone, MMC combined with minocycline resulted in cytotoxicity and cell growth inhibition synergistically in NSCLC cells, accompanied with reduced activation of phospho-ERK1/2, and reduced Rad51 protein levels. Overexpression of MKK1-CA or Flag-tagged Rad51 could reverse the minocycline and MMC-induced synergistic cytotoxicity. These findings may have implications for the rational design of future drug regimens incorporating minocycline and MMC for the treatment of NSCLC.

  9. Honokiol inhibits EMT-mediated motility and migration of human non-small cell lung cancer cells in vitro by targeting c-FLIP.

    PubMed

    Lv, Xiao-Qin; Qiao, Xin-Ran; Su, Ling; Chen, Shu-Zhen

    2016-12-01

    Honokiol (HNK) is a natural compound isolated from the magnolia plant with numerous pharmacological activities, including inhibiting epithelial-mesenchymal transition (EMT), which has been proposed as an attractive target for anti-tumor drugs to prevent tumor migration. In this study we investigated the effects of HNK on EMT in human NSCLC cells in vitro and the related signaling mechanisms. TNF-α (25 ng/mL) in combination with TGF-β1 (5 ng/mL) was used to stimulate EMT of human NSCLC A549 and H460 cells. Cell proliferation was analyzed using a sulforhodamine B assay. A wound-healing assay and a transwell assay were performed to examine cell motility. Western blotting was used to detect the expression levels of relevant proteins. siRNAs were used to knock down the gene expression of c-FLIP and N-cadherin. Stable overexpression of c-FLIP L (H157-FLIP L) or Lac Z (H157-Lac Z) was also performed. Treatment with TNF-α+TGF-β1 significantly enhanced the migration of A549 and H460 cells, increased c-FLIP, N-cadherin (a mesenchymal marker), snail (a transcriptional modulator) and p-Smad2/3 expression, and decreased IκB levels in the cells; these changes were abrogated by co-treatment with HNK (30 μmol/L). Further studies demonstrated that expression level of c-FLIP was highly correlated with the movement and migration of NSCLC cells, and the downstream effectors of c-FLIP signaling were NF-κB signaling and N-cadherin/snail signaling, while Smad signaling might lie upstream of c-FLIP. HNK inhibits EMT-mediated motility and migration of human NSCLC cells in vitro by targeting c-FLIP, which can be utilized as a promising target for cancer therapy, while HNK may become a potential anti-metastasis drug or lead compound.

  10. Anticancer activity of Noscapine, an opioid alkaloid in combination with Cisplatin in human non-small cell lung cancer.

    PubMed

    Chougule, Mahavir; Patel, Apurva R; Sachdeva, Pratik; Jackson, Tanise; Singh, Mandip

    2011-03-01

    The purpose of this study was to examine the efficacy of Noscapine (Nos) and Cisplatin (Cis) combination treatment in vitro in A549 and H460 lung cancer cells, in vivo in murine xenograft model and to investigate the underlying mechanism. The combination index values (< 0.6) suggested synergistic effects of Nos+Cis and resulted in the highest increase in percentage of apoptotic NSCLC cells and increased expression of p53, p21, caspase 3, cleaved caspase 3, cleaved PARP, Bax, and decreased expression of Bcl₂ and surviving proteins compared with treatment with either agent. Nos+Cis treatment reduced tumor volume by 78.1 ± 7.5% compared with 38.2 ± 6.8% by Cis or 35.4 ± 6.9% by Nos alone in murine xenograft lung cancer model. Nos+Cis treatment decreased expression of pAkt, Akt, cyclin D1, survivin, PARP, Bcl₂, and increased expression of p53, p21, Bax, cleaved PARP, caspase 3, cleaved caspase 3, cleaved caspase 8, caspase 8, cleaved caspase 9 and caspase 9 compared to single-agent treated and control groups. Our results suggest that Nos enhanced the anticancer activity of Cis in an additive to synergistic manner by activating multiple signaling pathways including apoptosis. These findings suggest potential benefit for use of Nos and Cis combination in treatment of lung cancer.

  11. Aspergillus fumigatus germ tube growth and not conidia ingestion induces expression of inflammatory mediator genes in the human lung epithelial cell line A549.

    PubMed

    Bellanger, Anne-Pauline; Millon, Laurence; Khoufache, Khaled; Rivollet, Danièle; Bièche, Ivan; Laurendeau, Ingrid; Vidaud, Michel; Botterel, Françoise; Bretagne, Stéphane

    2009-02-01

    Inhalation of conidia is the main cause of invasive pulmonary aspergillosis (IPA) and the respiratory epithelium is the first line of defence. To explore the triggering factor for the inflammatory response to Aspergillus fumigatus, the species mainly responsible for IPA, this study analysed the differential expression of three inflammatory genes in A549 cells after challenge with live and killed conidia. The influence of steroids, one of the main risk factors for developing IPA, was also investigated. Quantification of mRNAs of the inflammatory mediator genes encoding interleukin (IL)-8, tumour necrosis factor (TNF)-alpha and granulocyte-monocyte colony-stimulating factor (GM-CSF) was carried out using real-time PCR. Ingestion rates were studied for the conidia of A. fumigatus and Penicillium chrysogenum using a fluorescence brightener. Similar results were obtained for both species, with ingestion rates ranging from 35 to 40 %. Exposure of A549 cells to live A. fumigatus conidia only induced a four- to fivefold increase in the mRNA levels of the three genes, starting 8 h after the initial contact. Both inactivation of live A. fumigatus conidia and treatment by dexamethasone (10(-7) M) prevented the overexpression of TNF-alpha, IL-8 and GM-CSF. Fungal growth, rather than conidia ingestion, appears to be the main stimulus for the production of inflammatory mediators by epithelial cells, and this production is inhibited by steroid therapy. These results underline the role that the epithelium plays in the innate response against IPA.

  12. Synergistic induction of apoptosis by sulindac and simvastatin in A549 human lung cancer cells via reactive oxygen species-dependent mitochondrial dysfunction.

    PubMed

    Hwang, Ki-Eun; Park, Chul; Kwon, Su-Jin; Kim, Young-Suk; Park, Do-Sim; Lee, Mi-Kyung; Kim, Byoung-Ryun; Park, Seong-Hoon; Yoon, Kwon-Ha; Jeong, Eun-Taik; Kim, Hak-Ryul

    2013-07-01

    Prevention of lung cancer is more feasible and holds greater promise when different agents are used in combination to target multiple processes during carcinogenesis. The mechanisms by which non-steroidal anti-inflammatory drugs and statins inhibit cancer cell growth and induce apoptosis are not fully understood. This study was designed to investigate lung cancer chemoprevention through a mechanism-based approach using sulindac at low doses in combination with simvastatin. We found that sulindac-induced cytotoxicity was significantly enhanced in the presence of simvastatin. The combination of sulindac and simvastatin induced more extensive caspase-dependent apoptosis in A549 cells compared to that induced with either drug alone. The combination of sulindac and simvastatin also increased the loss of mitochondrial transmembrane potential (∆Ψm) and the cytosolic release of cytochrome c. In addition, ROS generation in cells treated with both sulindac and simvastatin was markedly increased compared to cells treated with either sulindac or simvastatin alone. The enhancement of ROS generation by sulindac and simvastatin was abrogated by pretreatment with NAC, which also prevented apoptosis and mitochondrial dysfunction induced by sulindac and simvastatin. These results suggest that sulindac and simvastatin-induced ROS generation in A549 lung cancer cells causes their accumulation in mitochondria, triggering the release of apoptogenic molecules from the mitochondria to the cytosol, and thus leading to caspase activation and cell death.

  13. Functional expression of the voltage-gated Na⁺-channel Nav1.7 is necessary for EGF-mediated invasion in human non-small cell lung cancer cells.

    PubMed

    Campbell, Thomas M; Main, Martin J; Fitzgerald, Elizabeth M

    2013-11-01

    Various ion channels are expressed in human cancers where they are intimately involved in proliferation, angiogenesis, invasion and metastasis. Expression of functional voltage-gated Na(+) channels (Nav) is implicated in the metastatic potential of breast, prostate, lung and colon cancer cells. However, the cellular mechanisms that regulate Nav expression in cancer remain largely unknown. Growth factors are attractive candidates; they not only play crucial roles in cancer progression but are also key regulators of ion channel expression and activity in non-cancerous cells. Here, we examine the role of epidermal growth factor receptor (EGFR) signalling and Nav in non-small cell lung carcinoma (NSCLC) cell lines. We show unequivocally, that functional expression of the α subunit Nav1.7 promotes invasion in H460 NSCLC cells. Inhibition of Nav1.7 activity (using tetrodotoxin) or expression (by using small interfering RNA), reduces H460 cell invasion by up to 50%. Crucially, non-invasive wild type A549 cells lack functional Nav, whereas exogenous overexpression of the Nav1.7 α subunit is sufficient to promote TTX-sensitive invasion of these cells. EGF/EGFR signalling enhances proliferation, migration and invasion of H460 cells but we find that, specifically, EGFR-mediated upregulation of Nav1.7 is necessary for invasive behaviour in these cells. Examination of Nav1.7 expression at mRNA, protein and functional levels further reveals that EGF/EGFR signalling via the ERK1/2 pathway controls transcriptional regulation of channel expression to promote cellular invasion. Immunohistochemistry of patient biopsies confirms the clinical relevance of Nav1.7 expression in NSCLC. Thus, Nav1.7 has significant potential as a new target for therapeutic intervention and/or as a diagnostic or prognostic marker in NSCLC.

  14. Synergistic antitumor effect of a combination of paclitaxel and carboplatin with nobiletin from Citrus depressa on non-small-cell lung cancer cell lines.

    PubMed

    Uesato, Shinichi; Yamashita, Hirofumi; Maeda, Ryu; Hirata, Yoshiyuki; Yamamoto, Maho; Matsue, Saki; Nagaoka, Yasuo; Shibano, Makio; Taniguchi, Masahiko; Baba, Kimiye; Ju-ichi, Motoharu

    2014-04-01

    Non-small-cell lung carcinomas do not sufficiently respond to cancer chemotherapeutic drugs. Combination effects of cancer chemotherapy drugs (paclitaxel and carboplatin) with nobiletin or powdered Shiikuwasha extract from Citrus depressa were examined by isobologram and combination index analyses. It was demonstrated that the combination generated a synergistic inhibitory effect against the proliferation of the human non-small-cell lung carcinoma cell lines A549 and H460 and that of the two chemotherapy drugs, paclitaxel was responsible for this synergistic effect. Furthermore, the percentage of apoptotic cells was decreased with increasing rates of nobiletin to paclitaxel and carboplatin. These findings were considered to be attributed to the ability of nobiletin to regulate cells in the G1 phase, which escaped cell death initiated by paclitaxel and carboplatin. An antitumor activity assay showed that this combination significantly suppressed the growth of subcutaneous A549 tumor xenografts in nude mice.

  15. Rubus idaeus L Inhibits Invasion Potential of Human A549 Lung Cancer Cells by Suppression Epithelial-to-Mesenchymal Transition and Akt Pathway In Vitro and Reduces Tumor Growth In Vivo.

    PubMed

    Chu, Shu-Chen; Hsieh, Yih-Shou; Hsu, Li-Sung; Chen, Kuo-Shuen; Chiang, Chien-Cheng; Chen, Pei-Ni

    2014-05-01

    The metastasis of lung cancer is the most prevalent cause of patient death. Various treatment strategies have targeted the prevention of the occurrence of metastasis. The epithelial-mesenchymal transition (EMT) in lung cancer cells is considered a prerequisite to acquire the invasive/migratory phenotype and to subsequently achieve metastasis. However, the effects ofRubus idaeuson cancer invasion and the EMT of the human lung carcinoma remain unclear. In this article, we test the hypothesis thatR idaeusethyl acetate (RIAE) possesses an antimetastatic effect and reverses the EMT potential of human lung A549 cells. We extract the raspberryR idaeuswith methanol (RIME), chloroform (RICE), ethyl acetate (RIAE),n-butanol (RIBE), and water (RIWE). The RIAE treatment obviously inhibits the invasive (P< .001), motility (P< .001), spreading, and migratory potential (P< .001) of highly metastatic human lung cancer A549 cells. The zymography and promoter luciferase analysis reveals that RIAE decreases the proteinase and transcription activities of MMP-2 and u-PA. Molecular analyses show that RIAE increases the E-cadherin level that is mainly localized at the cellular membrane. This result was also verified through confocal analyses. RIAE also induces the upregulation of an epithelial marker, such as α-catenin, and decreases mesenchymal markers, such as snail-1 and N-cadherin, that promote cell invasion and metastasis. RIAE inhibits MMP-2 and u-PA by attenuating the NF-κB and p-Akt expression. The inhibition of RIAE on the growth of A549 cells in vivo was also verified using a cancer cell xenograft nude mice model. Our results show the anti-invasive/antitumor effects of RIAE and associated mechanisms, which suggest that RIAE should be further tested in clinically relevant models to exploit its potential benefits against metastatic lung cancer cells.

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

    SciTech Connect

    Yu, Zhenhai; Huang, Liangqian; Qiao, Pengyun; Jiang, Aifang; Wang, Li; Yang, Tingting; Tang, Shengjian; Zhang, Wei; Ren, Chune

    2016-05-13

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

  17. Overexpression of SAMD9 suppresses tumorigenesis and progression during non small cell lung cancer

    SciTech Connect

    Ma, Qing; Yu, Tao; Ren, Yao-Yao; Gong, Ting; Zhong, Dian-Sheng

    2014-11-07

    Highlights: • SAMD9 is down-regulated in human non-small cell lung cancer (NSCLC). • Knockdown of SAMD9 expression is increased the invasion, migration and proliferation in H1299 cells in vitro. • Overexpression of SAMD9 suppressed proliferation and invasion in A549 cells in vitro. • Depletion of SAMD9 increases tumor formation in vivo. - Abstract: The Sterile Alpha Motif Domain-containing 9 (SAMD9) gene has been recently emphasized during the discovery that it is expressed at a lower level in aggressive fibromatosis and some cases of breast and colon cancer, however, the underlying mechanisms are poorly understood. Here, we found that SAMD9 is down-regulated in human non-small cell lung cancer (NSCLC). Furthermore, knockdown of SAMD9 expression is increased the invasion, migration and proliferation in H1299 cells in vitro and overexpression of SAMD9 suppressed proliferation and invasion in A549 cells. Finally, depletion of SAMD9 increases tumor formation in vivo. Our results may provide a strategy for blocking NSCLC tumorigenesis and progression.

  18. Cisplatin-mediated radiosensitization of non-small cell lung cancer cells is stimulated by ATM inhibition.

    PubMed

    Toulany, Mahmoud; Mihatsch, Julia; Holler, Marina; Chaachouay, Hassan; Rodemann, H Peter

    2014-05-01

    Cisplatin activates ataxia-telangiectasia-mutated (ATM), a protein with roles in DNA repair, cell cycle progression and autophagy. We investigated the radiosensitizing effect of cisplatin with respect to its effect on ATM pathway activation. Non-small cell lung cancer cells (NSCLC) cell lines (A549, H460) and human fibroblast (ATM-deficient AT5, ATM-proficient 1BR3) cells were used. The effects of cisplatin combined with irradiation on ATM pathway activity, clonogenicity, DNA double-strand break (DNA-DSB) repair and cell cycle progression were analyzed with Western blotting, colony formation and γ-H2AX foci assays as well as FACS analysis, respectively. Cisplatin radiosensitized H460 cells, but not A549 cells. Radiosensitization of H460 cells was not due to impaired DNA-DSB repair, increased apoptosis or cell cycle dysregulation. The lack of radiosensitization demonstrated for A549 cells was associated with cisplatin-mediated stimulation of ATM (S1981) and AMPKα (T172) phosphorylation and autophagy. However, in both cell lines inhibition of ATM and autophagy by KU-55933 and chloroquine diphosphate (CQ) respectively resulted in a significant radiosensitization. Combined treatment with the AMPK inhibitor compound-C led to radiosensitization of A549 but not of H460 cells. As compared to the treatment with KU-55933 alone, radiosensitivity of A549 cells was markedly stimulated by the combination of KU-55933 and cisplatin. However, the combination of CQ and cisplatin did not modulate the pattern of radiation sensitivity of A549 or H460 cells. In accordance with the results that cisplatin via stimulation of ATM activity can abrogate its radiosensitizing effect, ATM deficient cells were significantly sensitized to ionizing radiation by cisplatin. The results obtained indicate that ATM targeting can potentiate cisplatin-induced radiosensitization. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  19. Polymeric Nanoparticles Containing Taxanes Enhance Chemoradiotherapeutic Efficacy in Non-small Cell Lung Cancer

    SciTech Connect

    Jung, Joohee; Park, Sung-Jin; Chung, Hye Kyung; Kang, Hye-Won; Lee, Sa-Won; Seo, Min Hyo; Park, Heon Joo; Song, Si Yeol; Jeong, Seong-Yun; Choi, Eun Kyung

    2012-09-01

    Purpose: To reduce the side effects and improve the efficacy of chemoradiation therapy, taxanes were incorporated into polymeric nanoparticles (PNP), and their synergic effect on radiation therapy in non-small cell lung cancer was evaluated. Methods and Materials: The properties of PNP-taxanes were characterized by transmission electron microscopy and dynamic light scattering. The chemoradiotherapeutic efficacy of PNP-taxanes was determined by clonogenic assay, cellular morphology, and flow cytometry in A549 cells. In mice bearing A549-derived tumors, the tumor growth delay was examined after the treatment of PNP-taxanes and/or ionizing radiation (IR). Results: The PNP-taxanes were found to be approximately 45 nm in average diameter and to have high solubility in water. They showed the properties of active internalization into cells and preserved the anticancer effect of free taxanes. The survival fraction of A549 cells by clonogenic assay was significantly reduced in the group receiving combined treatment of PNP-taxanes and IR. In addition, in vivo radiotherapeutic efficacy was markedly enhanced by the intravenous injection of PNP-taxanes into the xenograft mice. Conclusions: We have demonstrated the feasibility of PNP-taxanes to enhance the efficacy of chemoradiation therapy. These results suggest PNP-taxanes can hold an invaluable and promising position in treating human cancers as a novel and effective chemoradiation therapy agent.

  20. Silencing survivin expression inhibits the tumor growth of non-small-cell lung cancer cells in vitro and in vivo.

    PubMed

    Zhang, Kejian; Li, Yang; Liu, Wei; Gao, Xinliang; Zhang, Kewei

    2015-01-01

    Survivin is a promising anticancer therapeutic target due to its important role in the inhibition of apoptosis of tumor cells. However, little is currently known about its role in non small cell lung cancer (NSCLC). The present study evaluated whether the downregulation of survivin expression would affect cell proliferation, cell cycle distribution, apoptosis and colony formation of NSCLC. A recombinant lentiviral small hairpin RNA (shRNA) expression vector, which specifically targeted survivin, was constructed and transfected into the A549 human NSCLC cell line. Quantitative polymerase chain reaction and western blotting were used to determine the mRNA and protein expression levels of survivin, 48 h following the knockdown of survivin expression. Cell proliferation, apoptosis, cell cycle distribution and colony formation were determined following the downregulation of survivin by shRNA. In addition, A549 cells were injected into nude mice, and the effects of shRNA targeting the survivin gene on tumor growth were assessed. Downregulation of survivin expression, using the RNA silencing approach in A549 tumor cells, significantly suppressed the proliferation and colony formation ability of the cells, and induced tumor apoptosis in vitro. The nude mice inoculated with A549 cells developed cancer, and treatment with shRNA targeting survivin markedly inhibited the growth of these cancers, with no obvious side effects. The results of the present study suggest that suppression of survivin expression by RNA interference may induce NSCLC apoptosis, and provide a novel approach for anticancer gene therapy.

  1. Long noncoding RNA TCF7 promotes invasiveness and self-renewal of human non-small cell lung cancer cells.

    PubMed

    Wu, Jinhui; Wang, Dongshuang

    2017-01-01

    Lung cancer is the most common solid tumor and the leading cause of cancer-related death worldwide. Non-small cell lung cancer (NSCLC) represents the major histological subtype and accounts for about 80 % cases of lung cancer cases. Recently, lncRNA lncTCF7 was identified, which is highly expressed in hepatocellular carcinoma (HCC) tumors and liver cancer stem cells (CSCs). However, the role of lnTCF7 in NSCLC remains largely unknown. In this study, Gain- and loss-of-function studies demonstrated the critical role of lncTCF7 in promoting invasion and self-renewal in NSCLC cells. We showed that lncTCF7 increased slug expression to promote the invasive capability of NSCLC cells and upregulated EpCAM expression to promote the self-renewal. Collectively, these findings provide new insights into the potential role of lncTCF7 upregulation in NSCLC metastasis and suggest a promising potential to suppress lncTCF7 for NSCLC patients.

  2. Allelic imbalance and instability of microsatellite loci on chromosome 1p in human non-small-cell lung cancer.

    PubMed Central

    Gasparian, A. V.; Laktionov, K. K.; Belialova, M. S.; Pirogova, N. A.; Tatosyan, A. G.; Zborovskaya, I. B.

    1998-01-01

    The mapping of allelic loss on the short arm of chromosome 1 has been performed in non-small-cell lung cancer. We used a set of 11 microsatellite loci spanning 1p to examine the frequency of allelic imbalance in a panel of 58 tumours. Fifty-one of 58 (87.9%) cases have shown somatic allelic loss at one or more loci tested. The two shortest regions of the overlap (SRO) of the deletions have been identified: SRO 1 at 1p13.1 and SRO 2 at 1p32-pter. Allelic losses at these regions have been compared among adenocarcinoma and squamous cell carcinoma and no difference has been found. In contrast to SRO 1, deletions at SRO 2 significantly correlated with advanced stage of the disease as well as post-operative metastasizing and relapse. These data may suggest that SRO 1 and SRO 2 can harbour tumour-supressor genes (TSGs) involved in different stages of NSCLC development. SRO 2 is still quite large and its refined mapping should help attempts to clone and identify the putative TSG(s). Microsatellite instability (replication errors) affecting only 6 (10.3%) of 58 tumour samples is an infrequent genetic alteration at the loci tested. Images Figure 2 PMID:9635835

  3. Vitamin A (retinol) downregulates the receptor for advanced glycation endproducts (RAGE) by oxidant-dependent activation of p38 MAPK and NF-kB in human lung cancer A549 cells.

    PubMed

    de Bittencourt Pasquali, Matheus Augusto; Gelain, Daniel Pens; Zeidán-Chuliá, Fares; Pires, André Simões; Gasparotto, Juciano; Terra, Silvia Resende; Moreira, José Cláudio Fonseca

    2013-04-01

    As an essential component of the diet, retinol supplementation is often considered harmless and its application is poorly controlled. However, recent works demonstrated that retinol may induce a wide array of deleterious effects, especially when doses used are elevated. Controlled clinical trials have demonstrated that retinol supplementation increased the incidence of lung cancer and mortality in smokers. Experimental works in cell cultures and animal models showed that retinol may induce free radical production, oxidative stress and extensive biomolecular damage. Here, we evaluated the effect of retinol on the regulation of the receptor for advanced glycation end-products (RAGE) in the human lung cancer cell line A549. RAGE is constitutively expressed in lungs and was observed to be down-regulated in lung cancer patients. A549 cells were treated with retinol doses reported as physiologic (2 μM) or therapeutic (5, 10 or 20 μM). Retinol at 10 and 20 μM increased free radical production, oxidative damage and antioxidant enzyme activity in A549 cells. These doses also downregulated RAGE expression. Antioxidant co-treatment with Trolox®, a hydrophilic analog of α-tocopherol, reversed the effects of retinol on oxidative parameters and RAGE downregulation. The effect of retinol on RAGE was mediated by p38 MAPK activation, as blockade of p38 with PD169316 (10 μM), SB203580 (10 μM) or siRNA to either p38α (MAPK14) or p38β (MAPK11) reversed the effect of retinol on RAGE. Trolox also inhibited p38 phosphorylation, indicating that retinol induced a redox-dependent activation of this MAPK. Besides, we observed that NF-kB acted as a downstream effector of p38 in RAGE downregulation by retinol, as NF-kB inhibition by SN50 (100 μg/mL) and siRNA to p65 blocked the effect of retinol on RAGE, and p38 inhibitors reversed NF-kB activation. Taken together, our results indicate a pro-oxidant effect of retinol on A549 cells, and suggest that modulation of RAGE expression by

  4. Elevated expression of USP9X correlates with poor prognosis in human non-small cell lung cancer

    PubMed Central

    Wang, You; Liu, Yu; Yang, Bo; Cao, Hong; Yang, Chun-Xu; Ouyang, Wen; Zhang, Shi-Min; Yang, Gui-Fang; Zhou, Fu-Xiang; Zhou, Yun-Feng

    2015-01-01

    Background The aim of this study was to investigate the expression of ubiquitin-specific peptidase 9, X-linked (USP9X) in non-small cell lung cancer (NSCLC) patients and to evaluate the relevance of USP9X expression to tumor prognosis. Methods Ninety-five patients who underwent surgical resection for clinical stage I-IIIA NSCLC between July 2008 and July 2011 were included in this study. Immunohistochemical analysis of USP9X expression was performed on 95 NSCLC tissues and 32 adjacent normal lung parenchymal tissues from these patients. The Chi-squared test was used to compare the clinicopathological characteristics between different groups. Kaplan-Meier analysis and a Cox regression model were used to determine the independent prognostic factors. A P value <0.05 was considered to be significant. Results The expression of USP9X was found to be significantly higher in NSCLC tissue (44.2%) than in adjacent normal lung parenchymal tissue (6.3%) (P<0.001). High USP9X expression was significantly associated with positive lymph node metastasis (P<0.001), clinical stage (P<0.001) and a reduced overall survival rate (P=0.001) in patients with NSCLC. Based on the multivariate analysis, the elevated expression of the USP9X protein was a significant predictor of poor prognosis for NSCLC patients (HR =2.244, P=0.028). Conclusions The current study demonstrated that the expression of USP9X in NSCLC tissue was significantly higher than that in normal lung tissue and that this elevated expression level of USP9X was associated with poor prognosis among NSCLC patients, suggesting that USP9X might serve as a prognostic biomarker for NSCLC. PMID:25973233

  5. Non-small-cell lung cancer-induced immunosuppression by increased human regulatory T cells via Foxp3 promoter demethylation.

    PubMed

    Ke, Xing; Zhang, Shuping; Xu, Jian; Liu, Genyan; Zhang, Lixia; Xie, Erfu; Gao, Li; Li, Daqian; Sun, Ruihong; Wang, Fang; Pan, Shiyang

    2016-05-01

    Patients with non-small-cell lung cancer (NSCLC) have immune defects that are poorly understood. Forkhead box protein P3 (Foxp3) is crucial for immunosuppression by CD4(+) regulatory T cells (Tregs). It is not well known how NSCLC induces Foxp3 expression and causes immunosuppression in tumor-bearing patients. Our study found a higher percentage of CD4(+) Tregs in the peripheral blood of NSCLC compared with healthy donors. NSCLC patients showed demethylation of eight CpG sites within the Foxp3 promoter with methylation ratios negatively correlated with CD4(+)CD25(+)Foxp3(+) T levels. Foxp3 expression in CD4(+) Tregs was directly regulated by Foxp3 promoter demethylation and was involved in immunosuppression by NSCLC. To verify the effect of tumor cells on the phenotype and function of CD4(+) Tregs, we established a coculture system using NSCLC cell line and healthy CD4(+) T cells and showed that SPC-A1 induced IL-10 and TGF-β1 secretion by affecting the function of CD4(+) Tregs. The activity of DNA methyltransferases from CD4(+) T was decreased during this process. Furthermore, eight CpG sites within the Foxp3 promoter also appeared to have undergone demethylation. Foxp3 is highly expressed in CD4(+) T cells, and this may be caused by gene promoter demethylation. These induced Tregs are highly immunosuppressive and dramatically inhibit the proliferative activity of naïve CD4(+) T cells. Our study provides one possible mechanism describing Foxp3 promoter demethylation changes by which NSCLC down-regulates immune responses and contributes to tumor progression. Foxp3 represents an important target for NSCLC anti-tumor immunotherapy.

  6. Human non-small cell lung cancer expresses putative cancer stem cell markers and exhibits the transcriptomic profile of multipotent cells.

    PubMed

    Zakaria, Norashikin; Yusoff, Narazah Mohd; Zakaria, Zubaidah; Lim, Moon Nian; Baharuddin, Puteri J Noor; Fakiruddin, Kamal Shaik; Yahaya, Badrul

    2015-02-25

    Despite significant advances in staging and therapies, lung cancer remains a major cause of cancer-related lethality due to its high incidence and recurrence. Clearly, a novel approach is required to develop new therapies to treat this devastating disease. Recent evidence indicates that tumours contain a small population of cells known as cancer stem cells (CSCs) that are responsible for tumour maintenance, spreading and resistant to chemotherapy. The genetic composition of CSCs so far is not fully understood, but manipulation of the specific genes that maintain their integrity would be beneficial for developing strategies to combat cancer. Therefore, the goal of this study isto identify the transcriptomic composition and biological functions of CSCs from non-small cell lung cancer (NSCLC). We isolated putative lung CSCs from lung adenocarcinoma cells (A549 and H2170) and normal stem cells from normal bronchial epithelial cells (PHBEC) on the basis of positive expression of stem cell surface markers (CD166, CD44, and EpCAM) using fluorescence-activated cell sorting. The isolated cells were then characterised for their self-renewal characteristics, differentiation capabilities, expression of stem cell transcription factor and in vivo tumouregenicity. The transcriptomic profiles of putative lung CSCs then were obtained using microarray analysis. Significantly regulated genes (p < 0.05, fold change (FC) > 2.0) in putative CSCs were identified and further analysed for their biological functions using the Database for Annotation, Visualization, and Integrated Discovery (DAVID). The putative lung CSCs phenotypes of CD166(+)/CD44(+) and CD166(+)/EpCAM(+) showed multipotent characteristics of stem cells, including the ability to differentiate into adipogenic and osteogenic cells, self-renewal, and expression of stem cell transcription factors such as Sox2 and Oct3/4. Moreover, the cells also shows the in vivo tumouregenicity characteristic when transplanted into

  7. Honokiol inhibits EMT-mediated motility and migration of human non-small cell lung cancer cells in vitro by targeting c-FLIP

    PubMed Central

    Lv, Xiao-qin; Qiao, Xin-ran; Su, Ling; Chen, Shu-zhen

    2016-01-01

    Aim: Honokiol (HNK) is a natural compound isolated from the magnolia plant with numerous pharmacological activities, including inhibiting epithelial-mesenchymal transition (EMT), which has been proposed as an attractive target for anti-tumor drugs to prevent tumor migration. In this study we investigated the effects of HNK on EMT in human NSCLC cells in vitro and the related signaling mechanisms. Methods: TNF-α (25 ng/mL) in combination with TGF-β1 (5 ng/mL) was used to stimulate EMT of human NSCLC A549 and H460 cells. Cell proliferation was analyzed using a sulforhodamine B assay. A wound-healing assay and a transwell assay were performed to examine cell motility. Western blotting was used to detect the expression levels of relevant proteins. siRNAs were used to knock down the gene expression of c-FLIP and N-cadherin. Stable overexpression of c-FLIP L (H157-FLIP L) or Lac Z (H157-Lac Z) was also performed. Results: Treatment with TNF-α+TGF-β1 significantly enhanced the migration of A549 and H460 cells, increased c-FLIP, N-cadherin (a mesenchymal marker), snail (a transcriptional modulator) and p-Smad2/3 expression, and decreased IκB levels in the cells; these changes were abrogated by co-treatment with HNK (30 μmol/L). Further studies demonstrated that expression level of c-FLIP was highly correlated with the movement and migration of NSCLC cells, and the downstream effectors of c-FLIP signaling were NF-κB signaling and N-cadherin/snail signaling, while Smad signaling might lie upstream of c-FLIP. Conclusion: HNK inhibits EMT-mediated motility and migration of human NSCLC cells in vitro by targeting c-FLIP, which can be utilized as a promising target for cancer therapy, while HNK may become a potential anti-metastasis drug or lead compound. PMID:27593221

  8. Gracilaria edulis exhibit antiproliferative activity against human lung adenocarcinoma cell line A549 without causing adverse toxic effect in vitro and in vivo.

    PubMed

    Sakthivel, Ravi; Muniasamy, Samuthirapandi; Archunan, Govindaraju; Devi, Kasi Pandima

    2016-02-01

    In the present study, the antiproliferative potential of various solvent extracts of Gracilaria edulis (GE) was tested against various cancer cell lines. In the A549 lung cancer cell line model, GE ethyl acetate extract (GEEA) (100 μg mL(-1)) treated group showed the maximum and significant (P < 0.05) growth inhibition at 48 h. The IC50 value was found to be 24.5 ± 19.1 μg mL(-1) at 48 h. Moreover, a low level of LDH release was observed at 48 h at various concentrations of (40, 60, 80 and 100 μg mL(-1)) GEEA extract-treated group compared to a control group. Changes in the cell morphology and echinoid spikes formation were observed at 48 h. Safety evaluation of GEEA in a non-cancerous liver cell line, PBMC and in Wistar rats positively revealed that the extract did not show any adverse toxic effects. The GEEA extract was partially purified by column chromatography and the active fraction was characterized through LC-MS analysis. Furthermore, HPLC and FT-IR analysis of the active fractions confirmed the presence of phytol, a diterpene compound with potent antiproliferative activity, which positively suggests that the red alga G. edulis contains a potent anticancer active principle.

  9. Effect of Paclitaxel-Mesoporous Silica Nanoparticles with a Core-Shell Structure on the Human Lung Cancer Cell Line A549

    NASA Astrophysics Data System (ADS)

    Wang, Tieliang; Liu, Ying; Wu, Chao

    2017-01-01

    A nanodrug delivery system of paclitaxel-mesoporous silica nanoparticles with a core-shell structure (PAC-csMSN) was used to increase the dissolution of paclitaxel (PAC) and improve its treatment of lung cancer. PAC was loaded into the core-shell mesoporous silica nanoparticles (csMSN) by the adsorption equilibrium method and was in an amorphous state in terms of its mesoporous structure. In vitro and in vivo studies showed that csMSN increased the dissolution rate of PAC and improved its lung absorption. The area under concentration-time curve (AUC) value of PAC-csMSN used for pulmonary delivery in rabbits was 2.678-fold higher than that obtained with the PAC. After continuous administration for 3 days, a lung biopsy showed no signs of inflammation. Cell apoptosis results obtained by flow cytometry indicated that PAC-csMSN was more potent than pure PAC in promoting cell apoptosis. An absorption investigation of PAC-csMSN in A549 cells was carried out by transmission electron microscopy (TEM) and laser scanning confocal microscopy (LSCM). The obtained results indicated that the cellular uptake was time-dependent and csMSN was uptaken into the cytoplasm. All these results demonstrate that csMSN have the potential to achieve pulmonary inhalation administration of poorly water-soluble drugs for the treatment of lung cancer.

  10. Green tea induces annexin-I expression in human lung adenocarcinoma A549 cells: involvement of annexin-I in actin remodeling.

    PubMed

    Lu, Qing-Yi; Jin, Yu Sheng; Zhang, Zuo-Feng; Le, Anh D; Heber, David; Li, Frederick P; Dubinett, Steven M; Rao, Jian Yu

    2007-05-01

    Green tea polyphenols exhibit multiple antitumor activities in various in vitro and in vivo tumor models, and the mechanisms of action are not clear. Previously, we found that green tea extract (GTE) regulates actin remodeling in different cell culture systems. Actin remodeling plays an important role in cancer cell morphology, cell adhesion, motility, and invasion. Using proteomic approaches, we found GTE-induced expression of annexin-I, a multifunctional actin binding protein, in these cell lines. In this study, we aimed to further define the functional role of GTE-induced annexin-I expression in actin remodeling, cell adhesion, and motility in lung adenocarcinoma A549 cells. We found that GTE stimulates the expression of annexin-I in a dose-dependent fashion. The GTE-induced annexin-I expression appears to be at the transcription level, and the increased annexin-I expression mediates actin polymerization, resulting in enhanced cell adhesion and decreased motility. Annexin-I specific interference resulted in loss of GTE-induced actin polymerization and cell adhesion, but not motility. In fact, annexin-I specific interference itself inhibited motility even without GTE. Together, annexin-I plays an important role in GTE-induced actin remodeling, and it may serve as a potential molecular target associated with the anticancer activities of green tea.

  11. Effect of Paclitaxel-Mesoporous Silica Nanoparticles with a Core-Shell Structure on the Human Lung Cancer Cell Line A549.

    PubMed

    Wang, Tieliang; Liu, Ying; Wu, Chao

    2017-12-01

    A nanodrug delivery system of paclitaxel-mesoporous silica nanoparticles with a core-shell structure (PAC-csMSN) was used to increase the dissolution of paclitaxel (PAC) and improve its treatment of lung cancer. PAC was loaded into the core-shell mesoporous silica nanoparticles (csMSN) by the adsorption equilibrium method and was in an amorphous state in terms of its mesoporous structure. In vitro and in vivo studies showed that csMSN increased the dissolution rate of PAC and improved its lung absorption. The area under concentration-time curve (AUC) value of PAC-csMSN used for pulmonary delivery in rabbits was 2.678-fold higher than that obtained with the PAC. After continuous administration for 3 days, a lung biopsy showed no signs of inflammation. Cell apoptosis results obtained by flow cytometry indicated that PAC-csMSN was more potent than pure PAC in promoting cell apoptosis. An absorption investigation of PAC-csMSN in A549 cells was carried out by transmission electron microscopy (TEM) and laser scanning confocal microscopy (LSCM). The obtained results indicated that the cellular uptake was time-dependent and csMSN was uptaken into the cytoplasm. All these results demonstrate that csMSN have the potential to achieve pulmonary inhalation administration of poorly water-soluble drugs for the treatment of lung cancer.

  12. Exploring the aneugenic and clastogenic potential in the nanosize range: A549 human lung carcinoma cells and amorphous monodisperse silica nanoparticles as models.

    PubMed

    Gonzalez, Laetitia; Thomassen, Leen C J; Plas, Gina; Rabolli, Virginie; Napierska, Dorota; Decordier, Ilse; Roelants, Mathieu; Hoet, Peter H; Kirschhock, Christine E A; Martens, Johan A; Lison, Dominique; Kirsch-Volders, Micheline

    2010-12-01

    We explored how to assess the genotoxic potential of nanosize particles with a well validated assay, the in vitro cytochalasin-B micronucleus assay, detecting both clastogens and aneugens. Monodisperse Stöber amorphous silica nanoparticles (SNPs) of three different sizes (16, 60 and 104 nm) and A549 lung carcinoma cells were selected as models. Cellular uptake of silica was monitored by ICP-MS. At non-cytotoxic doses the smallest particles showed a slightly higher fold induction of micronuclei (MNBN). When considering the three SNPs together, particle number and total surface area appeared to account for MNBN induction as they both correlated significantly with the amplitude of the effect. Using nominal or cellular dose did not show statistically significant differences. Likewise, alkaline comet assay and FISH-centromeric probing of MNBN indicated a weak and not statistically significant induction of oxidative DNA damage, chromosome breakage and chromosome loss. This line of investigation will contribute to adequately design and interpret nanogenotoxicity assays.

  13. TGF-β1 Downregulates COX-2 Expression Leading to Decrease of PGE2 Production in Human Lung Cancer A549 Cells, Which Is Involved in Fibrotic Response to TGF-β1

    PubMed Central

    Takai, Erina; Tsukimoto, Mitsutoshi; Kojima, Shuji

    2013-01-01

    Transforming growth factor-ß1 (TGF-β1) is a multifunctional cytokine that is involved in various pathophysiological processes, including cancer progression and fibrotic disorders. Here, we show that treatment with TGF-β1 (5 ng/mL) induced downregulation of cyclooxygenase-2 (COX-2), leading to reduced synthesis of prostaglandin E2 (PGE2), in human lung cancer A549 cells. Treatment of cells with specific inhibitors of COX-2 or PGE2 receptor resulted in growth inhibition, indicating that the COX-2/PGE2 pathway contributes to proliferation in an autocrine manner. TGF-β1 treatment induced growth inhibition, which was attenuated by exogenous PGE2. TGF-β1 is also a potent inducer of epithelial mesenchymal transition (EMT), a phenotype change in which epithelial cells differentiate into fibroblastoid cells. Supplementation with PGE2 or PGE2 receptor EP4 agonist PGE1-alcohol, as compared with EP1/3 agonist sulprostone, inhibited TGF-β1-induced expression of fibronectin and collagen I (extracellular matrix components). Exogenous PGE2 or PGE2 receptor agonists also suppressed actin remodeling induced by TGF-β1. These results suggest that PGE2 has an anti-fibrotic effect. We conclude that TGF-β1-induced downregulation of COX-2/PGE2 signaling is involved in facilitation of fibrotic EMT response in A549 cells. PMID:24098479

  14. Irciniastatin A induces potent and sustained activation of extracellular signal-regulated kinase and thereby promotes ectodomain shedding of tumor necrosis factor receptor 1 in human lung carcinoma A549 cells.

    PubMed

    Quach, Hue Tu; Hirano, Seiya; Fukuhara, Sayuri; Watanabe, Tsubasa; Kanoh, Naoki; Iwabuchi, Yoshiharu; Usui, Takeo; Kataoka, Takao

    2015-01-01

    Irciniastatin A is a pederin-type marine product that potently inhibits translation. We have recently shown that irciniastatin A induces ectodomain shedding of tumor necrosis factor (TNF) receptor 1 with slower kinetics than other translation inhibitors. In human lung carcinoma A549 cells, irciniastatin A induced a marked and sustained activation of extracellular signal-regulated kinase (ERK) and induced little activation of p38 mitogen-activated protein (MAP) kinase and c-Jun N-terminal kinase (JNK). Moreover, the TNF receptor 1 shedding induced by irciniastatin A was blocked by the MAP kinase/ERK kinase inhibitor U0126, but not by the p38 MAP kinase inhibitor SB203580 or the JNK inhibitor SP600125. Thus unlike other translation inhibitors that trigger ribotoxic stress response, our results show that irciniastatin A is a unique translation inhibitor that induces a potent and sustained activation of the ERK pathway, and thereby promotes the ectodomain shedding of TNF receptor 1 in A549 cells.

  15. Sp1 inhibition-mediated upregulation of VEGF 165 b induced by rh-endostatin enhances antiangiogenic and anticancer effect of rh-endostatin in A549.

    PubMed

    Li, Zhen-yu; Zhu, Fang; Hu, Jian-li; Peng, Gang; Chen, Jing; Zhang, Sheng; Chen, Xu; Zhang, Rui-guang; Chen, Ling-juan; Liu, Pian; Luo, Ming; Sun, Zhi-hua; Ren, Jing-hua; Huang, Li-li; Wu, Gang

    2011-08-01

    Recombinant human endostatin (rh-endostatin), a potential antiangiogenic agent, is used in non-small cell lung carcinoma treatment and represses vascular endothelial cell growth factor (VEGF) levels in tumor cell. However, precise affection of rh-endostatin on the proangiogenic VEGF isoforms (VEGF(165)) or antiangiogenic VEGF isoforms (VEGF(165)b) is not clear. We therefore tested the hypothesis that rh-endostatin could alter expression of these isoforms to regulate tumor growth. A549 cells were exposed to rh-endostatin, and the expression of VEGF(165) and VEGF(165)b was detected. The role of SP1 as a regulator of isoform expression was investigated. We then examined the anticancer and antiangiogenic efficacy of rh-endostatin in combination with exogenous VEGF(165)b against A549 cells, EA.HY 926 cells and xenograft model of human lung cancer. rh-Endostatin reduced VEGF(165) and induced VEGF(165)b as well as inhibited SP1 in A549 cells. SP1 inhibitor (betulinic acid) also developed those changes. VEGF(165)b-rh-endostatin combination was highly synergistic and inhibited growth, survival, and migration of A549 cells, VEGF-mediated VEGFR2 phosphorylation in EA.HY 926 cells, and tumor growth in xenograft model of human lung cancer. rh-Endostatin downregulates proangiogenic vascular endothelial growth factor A (VEGFA) isoform and upregulates antiangiogenic VEGFA isoform, possibly through inhibition of SP1. Furthermore, VEGF(165)b sensitizes A549 to rh-endostatin treatment and enhances the anticancer effect of rh-endostatin.

  16. CCL21/CCR7 up-regulate vascular endothelial growth factor-D expression via ERK pathway in human non-small cell lung cancer cells.

    PubMed

    Sun, Limei; Zhang, Qingfu; Li, Yang; Tang, Na; Qiu, Xueshan

    2015-01-01

    Lymphangiogenesis has received considerable attention and become a new research hotspot of tumor metastasis. Recently, C-C chemokine receptor 7 (CCR7) is known to promote metastasis of non-small cell lung cancer (NSCLC) cells into lymph nodes. In this study, we investigated the relationship between CCL21/CCR7 and the lymphangiogenic factor vascular endothelial growth factor (VEGF)-D in human lung cancer cells and its impact on patients' prognosis. We found that CCL21/CCR7 increase the expression of VEGF-D in NSCLC Cell Lines through induced ERK1/2 and Akt phosphorylation. In addition, our study found that the expression levels of CCR7 and CCL21 were correlated with VEGF-D, lymphatic vessels density (LVD), clinical stages, lymph node metastasis, and patient Survival in 90 human non-small cell lung cancer (NSCLC) specimens. Taken together, our results provide evidence that CCL21/CCR7 induce VEGF-D up-regulation and promote lymphangiogenesis via ERK/Akt pathway in lung cancer.

  17. Multifunctional gold nanocomposites designed for targeted CT/MR/optical trimodal imaging of human non-small cell lung cancer cells

    NASA Astrophysics Data System (ADS)

    Chen, Jingwen; Sun, Yingqi; Chen, Qian; Wang, Le; Wang, Suhe; Tang, Yun; Shi, Xiangyang; Wang, Han

    2016-07-01

    Multifunctional gold nanocomposites, which were designed as dendrimer-entrapped gold nanoparticles functionalized with gadolinium, cyanine dye (Cy5.5), and folic acid, were synthesized to be used as the first dendrimer-based clinical nanoprobes for targeted X-ray computed tomography/magnetic resonance/optical trimodal imaging in vitro and in vivo of human non-small cell cancer cells.Multifunctional gold nanocomposites, which were designed as dendrimer-entrapped gold nanoparticles functionalized with gadolinium, cyanine dye (Cy5.5), and folic acid, were synthesized to be used as the first dendrimer-based clinical nanoprobes for targeted X-ray computed tomography/magnetic resonance/optical trimodal imaging in vitro and in vivo of human non-small cell cancer cells. Electronic supplementary information (ESI) available: Synthesis and characterization data of the nanoprobes; biocompatibility results; confirmation of the tumor cell uptake of the nanoprobes in vitro and in vivo; biodistribution results in vivo. See DOI: 10.1039/c6nr03143a

  18. Human RNA polymerase II associated factor 1 complex promotes tumorigenesis by activating c-MYC transcription in non-small cell lung cancer

    SciTech Connect

    Zhi, Xiuyi; Giroux-Leprieur, Etienne; Wislez, Marie; Hu, Mu; Zhang, Yi; Shi, Huaiyin; Du, Kaiqi; Wang, Lei

    2015-10-02

    Human RNA polymerase II (RNAPII)-associated factor 1 complex (hPAF1C) plays a crucial role in protein-coding gene transcription. Overexpression of hPAF1C has been implicated in the initiation and progression of various human cancers. However, the molecular pathways involved in tumorigenesis through hPAF1C remain to be elucidated. The current study suggested hPAF1C expression as a prognostic biomarker for early stage non-small cell lung cancer (NSCLC) and patients with low hPAF1C expression levels had significantly better overall survival. Furthermore, the expression of hPAF1C was found to be positively correlated with c-MYC expression in patient tumor samples and in cancer cell lines. Mechanistic studies indicated that hPAF1C could promote lung cancer cell proliferation through regulating c-MYC transcription. These results demonstrated the prognostic value of hPAF1C in early-stage NSCLC and the role of hPAF1C in the transcriptional regulation of c-MYC oncogene during NSCLC tumorigenesis. - Highlights: • hPAF1C expression is a prognostic biomarker for early stage non-small cell lung cancer. • The expression of hPAF1C was positively correlated with c-MYC in tumor samples of patients and in several NSCLC cell lines. • hPAF1C could promote lung cancer cell proliferation through regulating c-MYC transcription.

  19. Autocrine activity of BDNF induced by the STAT3 signaling pathway causes prolonged TrkB activation and promotes human non-small-cell lung cancer proliferation

    PubMed Central

    Chen, Bo; Liang, Yan; He, Zheng; An, Yunhe; Zhao, Weihong; Wu, Jianqing

    2016-01-01

    Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin superfamily, which has been implicated in the pathophysiology of the nervous system. Recently, several studies have suggested that BDNF and/or its receptor, tropomyosin related kinase B (TrkB), are involved in tumor growth and metastasis in several cancers, including prostate cancer, neuroblastoma, pancreatic ductal carcinoma, hepatocellular carcinoma, and lung cancer. Despite the increasing emphasis on BDNF/TrkB signaling in human tumors, how it participates in primary tumors has not yet been determined. Additionally, little is known about the molecular mechanisms that elicit signaling downstream of TrkB in the progression of non-small-cell lung cancer (NSCLC). In this study, we report the significant expression of BDNF in NSCLC samples and show that BDNF stimulation increases the synthesis of BDNF itself through activation of STAT3 in lung cancer cells. The release of BDNF can in turn activate TrkB signaling. The activation of both TrkB and STAT3 contribute to downstream signaling and promote human non-small-cell lung cancer proliferation. PMID:27456333

  20. Novel hydrophilic docetaxel (CQMU-0519) analogue inhibits proliferation and induces apoptosis in human A549 lung, SKVO3 ovarian and MCF7 breast carcinoma cell lines.

    PubMed

    Fauzee, N J S; Wang, Y-L; Dong, Z; Li, Q-G; Wang, T; Mandarry, M T; Xu, L; Pan, J

    2012-08-01

    Objectives of this investigation were not merely to perform a comparative study with original docetaxel, but to define anti-proliferative and apoptotic effects of novel hydrophilic docetaxel (CQMU-0519) analogue on A549 lung, SKVO3 ovary and MCF7 breast carcinoma cell lines. The materials for the study consist of a completely new docetaxel analogue (CQMU-0519), synthesized by the Department of Pharmacology, Chongqing Medical University, China, which is completely soluble in water. 50 nm of drug concentration was utilized on all three cell lines where cell population growth was assessed using cell culture kit-8 and flow cytometry analysis, whereas apoptotic pathways were unveiled by use of annexin-V FITC, apoptosis DNA ladder, caspases-3, 6, 8 and 9; in the meanwhile, regulation of Bcl-2 family members was analysed by western blotting. The novel docetaxel analogue (CQMU-0519) suppressed cell proliferation in all three cell lines, inhibition of cell proliferation and cell cycle arrest being more evident in G(2) /M phase. Also, in both lung and ovarian cell lines, apoptotic levels were higher as measured by the various tests performed, and downregulation of Bcl-2 and Bcl-xL with increased expressions of Bad and Bax indicated the intrinsic pathway for apoptosis. Nevertheless, it was found that MCF7 cells, although also manifesting high levels of apoptosis, used the extrinsic pathway instead. Hence, it was shown that novel docetaxel analogue (CQMU-0519) may have some prospective use in future clinical trials. Novel hydrophilic docetaxel analogue (CQMU-0519) inhibited cell proliferation and enhanced the intrinsic apoptotic pathway in lung and ovarian carcinoma cells, whereas it used the extrinsic one in breast adenocarcinoma cells. © 2012 Blackwell Publishing Ltd.

  1. Modulation of intrinsic in vitro resistance to carboplatin by edatrexate in the A549 human nonsmall cell lung cancer cell line.

    PubMed

    Perez, E A; Hack, F M; Fletcher, T S; Chou, T C

    1994-01-01

    Edatrexate (10-ethyl-deazaaminopterin) is a methotrexate analog that has been shown to have greater antitumor activity and improved therapeutic index compared to its parent compound in preclinical systems. We have evaluated the ability of edatrexate to modulate the intrinsic resistance of the lung adenocarcinoma A549 cell line to carboplatin. Concentration effects, exposure time and schedule dependence were assessed. Modulation of resistance was observed with edatrexate treatment (0.2 microM for 1 h) prior to carboplatin. The concentrations of carboplatin to achieve IC50 at the 1-, 3-, and 24-h IC50 were decreased by a mean of 16.8 times (12.2-22.2) with edatrexate preexposure. In contrast, there was little modulation observed of carboplatin resistance when carboplatin was administered prior to edatrexate. In addition, schedule dependency experiments were performed using the method described by Chou and Talalay, in which the ratio of carboplatin to edatrexate was constant or nonconstant, and both the potency of effects and the shapes of the concentration-effect curves were taken into account in a computerized analysis. These experiments also demonstrated schedule dependency. Although both treatments resulted in a reduced IC50 vs. carboplatin alone, the reduction was much greater when edatrexate was added first (12.59 vs. 2.59 times). We conclude that the combination of edatrexate and carboplatin demonstrates schedule-dependent modulation of intrinsic carboplatin resistance in this in vitro model at clinically achievable edatrexate plasma levels (0.01 to 10 microM). The greatest modulatory synergism was observed in the setting of edatrexate treatment before carboplatin. Our findings suggest a potentially useful schedule when combining edatrexate and carboplatin for the treatment of malignant disease.

  2. Induction of human microsomal prostaglandin E synthase 1 by activated oncogene RhoA GTPase in A549 human epithelial cancer cells

    SciTech Connect

    Choi, Hye Jin; Lee, Dong-Hyung; Park, Seong-Hwan; Kim, Juil; Do, Kee Hun; An, Tae Jin; Ahn, Young Sup; Park, Chung Berm; Moon, Yuseok

    2011-09-30

    Highlights: {yields} As a target of oncogene RhoA-linked signal, a prostaglandin metabolism is assessed. {yields} RhoA activation increases PGE{sub 2} levels and its metabolic enzyme mPGES-1. {yields} RhoA-activated NF-{kappa}B and EGR-1 are positively involved in mPGES-1 induction. -- Abstract: Oncogenic RhoA GTPase has been investigated as a mediator of pro-inflammatory responses and aggressive carcinogenesis. Among the various targets of RhoA-linked signals, pro-inflammatory prostaglandin E{sub 2} (PGE{sub 2}), a major prostaglandin metabolite, was assessed in epithelial cancer cells. RhoA activation increased PGE{sub 2} levels and gene expression of the rate-limiting PGE{sub 2} producing enzymes, cyclooxygenase-2 and microsomal prostaglandin E synthase 1 (mPGES-1). In particular, human mPGES-1 was induced by RhoA via transcriptional activation in control and interleukin (IL)-1{beta}-activated cancer cells. To address the involvement of potent signaling pathways in RhoA-activated mPGES-1 induction, various signaling inhibitors were screened for their effects on mPGES-1 promoter activity. RhoA activation enhanced basal and IL-1{beta}-mediated phosphorylated nuclear factor-{kappa}B and extracellular signal-regulated kinase1/2 proteins, all of which were positively involved in RhoA-induced gene expression of mPGES-1. As one potent down-stream transcription factor of ERK1/2 signals, early growth response gene 1 product also mediated RhoA-induced gene expression of mPGES-1 by enhancing transcriptional activity. Since oncogene-triggered PGE{sub 2} production is a critical modulator of epithelial tumor cells, RhoA-associated mPGES-1 represents a promising chemo-preventive or therapeutic target for epithelial inflammation and its associated cancers.

  3. In vivo-in vitro comparison of acute respiratory tract toxicity using human 3D airway epithelial models and human A549 and murine 3T3 monolayer cell systems.

    PubMed

    Sauer, Ursula G; Vogel, Sandra; Hess, Annemarie; Kolle, Susanne N; Ma-Hock, Lan; van Ravenzwaay, Bennard; Landsiedel, Robert

    2013-02-01

    The usefulness of in vitro systems to predict acute inhalation toxicity was investigated. Nineteen substances were tested in three-dimensional human airway epithelial models, EpiAirway™ and MucilAir™, and in A549 and 3T3 monolayer cell cultures. IC(50) values were compared to rat four-hour LC(50) values classified according to EPA and GHS hazard categories. Best results were achieved with a prediction model distinguishing toxic from non-toxic substances, with satisfactory specificities and sensitivities. Using a self-made four-level prediction model to classify substances into four in vitro hazard categories, in vivo-in vitro concordance was mediocre, but could be improved by excluding substances causing pulmonary edema and emphysema in vivo. None of the test systems was outstanding, and there was no evidence that tissue or monolayer systems using respiratory tract cells provide an added value. However, the test systems only reflected bronchiole epithelia and alveolar cells and investigated cytotoxicity. Effects occurring in other cells by other mechanisms could not be recognised. Further work should optimise test protocols and expand the set of substances tested to define applicability domains. In vivo respiratory toxicity data for in vitro comparisons should distinguish different modes of action, and their relevance for human health effects should be ensured.

  4. [The effect of Bcl-2 gene silencing on the sensitivity of cell line A549 to chemotherapeutic drugs].

    PubMed

    Wang, Jiao-qi; Du, Zhen-wu; Gao, Xian-fei; Wu, Mei; Zhang, Yu-cheng; Pan, Ying; Wang, Qian; Zhang, Gui-zhen

    2013-03-01

    To investigate the effects of miRNA-mediated down-regulation of the Bcl-2 gene on the chemotherapeutic sensitivities and mRNA transcriptions of sensitivity associated genes in human lung adenocarcinoma cell line A549 cells, and therefore to provide experimental data for improving the chemotherapeutic effects on non-small cell lung cancer (NSCLC). The miRNA recombinant plasmid targeting to human Bcl-2 gene was designed, synthesized and stably transferred into A549 cells by lipofectin technique as the experiment group. The transcription of Bcl-2 mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR) by agarose gel electrophoresis, real-time PCR, and the protein level of Bcl-2 was measured by Western blot to confirm the function of miRNA plasmid. The cell proliferation was examined by methyl thiazolyl tetrazolium (MTT) assay. Cell cycle was measured by flow cytometry. Drug sensitivities of A549 cells to etoposide, 5-fluorouracil, cisplatin, adriamycin, vincristine, paclitaxel and navelbine were analyzed by MTT assay. The mRNA expressions of excision repair cross-complementing gene 1 (ERCC1), thymidylate synthase (TYMS), Class III β-tubulin, topoisomerase 2 alpha (TOP2α) genes were detected by RT-PCR and real-time PCR. The recombinant miRNA plasmid was successfully synthesized and stably transferred into A549 cells. The transcription of Bcl-2 mRNA dramatically decreased by 98.1% in the experiment group (RQ = 0.002 ± 0.001) compared to that in the negative control group (RQ = 0.104 ± 0.003) by real-time PCR (t = 98.70, P < 0.05); and the protein level of Bcl-2 in the experiment group decreased by 57.6% by Western blot (t = 7.66, P < 0.05). The cell cycle profile showed that the low expression of Bcl-2 gene led to A549 cell cycle arrest at G1-phase. The results of MTT showed that the growth of A549 cells in the experiment group was markedly inhibited. The sensitivities of A549 cells to etoposide, cisplatin, paclitaxel, and navelbine were

  5. Griffipavixanthone from Garcinia oblongifolia champ induces cell apoptosis in human non-small-cell lung cancer H520 cells in vitro.

    PubMed

    Shi, Jun-Min; Huang, Hui-Juan; Qiu, Sheng-Xiang; Feng, Shi-Xiu; Li, Xu-E

    2014-01-27

    Griffipavixanthone (GPX) is a dimeric xanthone which was isolated in a systematic investigation of Garcinia oblongifolia Champ. In this study, we investigate the effect of GPX on cell proliferation and apoptosis on human Non-small-cell lung cancer (NSCLC) cells in vitro and determine the mechanisms of its action. GPX inhibited the growth of H520 cells in dose- and time-dependent manners, with IC50 values of 3.03 ± 0.21 μM at 48 h. The morphologic characteristics of apoptosis and apoptotic bodies were observed by fluorescence microscope and transmission electron microscope. In addition, Annexin V/PI double staining assay revealed that cells in early stage of apoptosis were significantly increased upon GPX treatment dose-dependently. Rh123 staining assay indicated that GPX reduced the mitochondrial membrane potential. DCFH-DA staining revealed that intracellular ROS increased with GPX treatment. Moreover, GPX cleaved and activated caspase-3. In summary, this study showed that GPX inhibited H520 cell proliferation in dose- and time-dependent manner. Further mechanistic study indicated that GPX induced cell apoptosis through mitochondrial apoptotic pathway accompanying with ROS production. Our results demonstrate the potential application of GPX as an anti-non-small cell lung cancer agent.

  6. The apoptotic effect of 1’S-1’-Acetoxychavicol Acetate (ACA) enhanced by inhibition of non-canonical autophagy in human non-small cell lung cancer cells

    PubMed Central

    Sok, Sophia P. M.; Arshad, Norhafiza M.; Azmi, Mohamad Nurul; Awang, Khalijah; Ozpolat, Bulent

    2017-01-01

    Autophagy plays a role in deciding the fate of cells by inducing either survival or death. 1’S-1-acetoxychavicol acetate (ACA) is a phenylpropanoid isolated from rhizomes of Alpinia conchigera and has been reported previously on its apoptotic effects on various cancers. However, the effect of ACA on autophagy remains ambiguous. The aims of this study were to investigate the autophagy-inducing ability of ACA in human non-small cell lung cancer (NSCLC), and to determine its role as pro-survival or pro-death mechanism. Cell viability assay was conducted using MTT. The effect of autophagy was assessed by acridine orange staining, GFP-LC3 punctate formation assay, and protein level were analysed using western blot. Annexin V-FITC/PI staining was performed to detect percentage of cells undergoing apoptosis by using flow cytometry. ACA inhibits the cell viability and induced formation of cytoplasmic vacuoles in NSCLC cells. Acidic vesicular organelles and GFP-LC3 punctate formation were increased in response to ACA exposure in A549 and SK-LU-1 cell lines; implying occurrence of autophagy. In western blot, accumulation of LC3-II accompanied by degradation of p62 was observed, which further confirmed the full flux of autophagy induction by ACA. The reduction of Beclin-1 upon ACA treatment indicated the Beclin-1-independent autophagy pathway. An early autophagy inhibitor, 3-methyaldenine (3-MA), failed to suppress the autophagy triggered by ACA; validating the existence of Beclin-1-independent autophagy. Silencing of LC3-II using short interfering RNA (siRNA) abolished the autophagy effects, enhancing the cytotoxicity of ACA through apoptosis. This proposed ACA triggered a pro-survival autophagy in NSCLC cells. Consistently, co-treatment with lysosomal inhibitor, chloroquine (CQ), exerted a synergistic effect resulting in apoptosis. Our findings suggested ACA induced pro-survival autophagy through Beclin-1-independent pathway in NSCLC. Hence, targeting autophagy pathway

  7. Curcumin-ER Prolonged Subcutaneous Delivery for the Treatment of Non-Small Cell Lung Cancer.

    PubMed

    Ranjan, Amalendu P; Mukerjee, Anindita; Gdowski, Andrew; Helson, Lawrence; Bouchard, Annie; Majeed, Muhammed; Vishwanatha, Jamboor K

    2016-04-01

    Non-small-cell lung cancer therapy is a challenge due to poor prognosis and low survival rate. There is an acute need for advanced therapies having higher drug efficacy, low immunogenicity and fewer side effects which will markedly improve patient compliance and quality of life of cancer patients. The purpose of this study was to develop a novel hybrid curcumin nanoformulation (Curcumin-ER) and evaluate the therapeutic efficacy of this formulation on a non-small cell lung cancer xenograft model. Use of curcumin, a natural anticancer agent, is majorly limited due to its poor aqueous solubility and hence it's low systemic bioavailability. In this paper, we carried out the nanoformulation of Curcumin-ER, optimized the formulation process and determined the anticancer effects of Curcumin-ER against human A549 non-small cell lung cancer using in vitro and in vivo studies. Xenograft tumors in nude mice were treated with 20 mg/kg subcutaneous injection of Curcumin-ER and liposomal curcumin (Lipocurc) twice a week for seven weeks. Results showed that tumor growth was suppressed by 52.1% by Curcumin-ER treatment and only 32.2% by Lipocurc compared to controls. Tumor sections were isolated from murine xenografts and histology and immunohistochemistry was performed. A decrease in expression of NFκB-p65 subunit and proliferation marker, Ki-67 was observed in treated tumors. In addition, a potent anti-angiogenic effect, characterized by reduced expression of annexin A2 protein, was observed in treated tumors. These results establish the effectiveness of Curcumin-ER in regressing human non-small cell lung cancer growth in the xenograft model using subcutaneous route of administration. The therapeutic efficacy of Curcumin-ER highlights the potential of this hybrid nanoformulation in treating patients with non-small cell lung cancer.

  8. A unique cell-surface protein phenotype distinguishes human small-cell from non-small-cell lung cancer

    SciTech Connect

    Baylin, S.B.; Gazdar, A.F.; Minna, J.D.; Bernal, S.D.; Sharper, J.H.

    1982-08-01

    Radioiodination (/sup 125/I) and two-dimensional polyacrylamide gel electrophoresis was used to determine that small-(oat) cell lung carcinoma (SCC)-a tumor with neuroedocrine features-possesses a surface protein pattern distinct from the other types of lung cancer cells (squamous, adeno-, and large-cell undifferentiated carcinoma). Twelve distinguishing proteins, 40 to 70 kilodaltons (kDal), characterized four separate lines of SCC; three of these, designated E (60 kDal; pI = 7.3), S (30 kDal; pI = 6.0), and U 57 kDal; pI = 5.6), may be unique SCC gene products and were identified only in (/sup 35/S)methionine labeling of SCC and not in non-SCC or human fibroblasts. Two lines of adeno-, one of squamous, and one of undifferentiated large-cell lung carcinoma exhibited similar surface protein patterns to one another. Nine distinguishing proteins (40 to 100 kDal) and at least five large proteins (>100 kDal) were unique to these lines. The surface protein phenotypes for SCC and non-SCC were distinct from those for human lymphoblastoid cells and fibroblasts. However, the neuroendocrine features of SCC were further substantiated because 6 of the 12 distinguishing SCC surface proteins, including E and U, were identified on human neuroblastoma cells. The proteins identified should (i) help define differentiation steps for normal and neoplastic bronchial epithelial cells, (ii) prove useful in better classifying lung cancers, and (iii) be instrumental in tracing formation of neuroendocrine cells.

  9. Extracellular HSP70 Activates ERK1/2, NF-kB and Pro-Inflammatory Gene Transcription Through Binding with RAGE in A549 Human Lung Cancer Cells.

    PubMed

    Somensi, Nauana; Brum, Pedro Ozorio; de Miranda Ramos, Vitor; Gasparotto, Juciano; Zanotto-Filho, Alfeu; Rostirolla, Diana Carolina; da Silva Morrone, Maurilio; Moreira, José Claudio Fonseca; Pens Gelain, Daniel

    2017-08-22

    Heat shock protein 70 (HSP70) has been recently described with extracellular actions, where it is actively released in inflammatory conditions. Acting as DAMPs (damage associated molecular pattern), extracellular HSP70 (eHSP70) interacts with membrane receptors and activates inflammatory pathways. At this context, the receptor for advanced glycation endproducts (RAGE) emerges as a possible candidate for interaction with eHSP70. RAGE is a pattern-recognition receptor and its expression is increased in several diseases related to a chronic pro-inflammatory state. One of the main consequences of RAGE ligand-binding is the ERK1/2 (extracellular signal-regulated kinases)-dependent activation of NF-kB (nuclear factor kappa B), which leads to expression of TNF-α (tumor necrosis factor alpha) and other cytokines. The purpose of this work is to elucidate if eHSP70 is able to evoke RAGE-dependent signaling using A549 human lung cancer cells, which constitutively express RAGE. Immunoprecipitation and protein proximity assay were utilized to demonstrate the linkage between RAGE and eHSP70. To investigate RAGE relevance on cell response to eHSP70, siRNA was used to knockdown the receptor expression. Signaling pathways activation were evaluated by western blotting, gene reporter luciferase and real time quantitative PCR. Protein eHSP70 shown to be interacting physically with the receptor RAGE in our cell model. Treatment with eHSP70 caused ERK1/2 activation and NF-κB transactivation impaired by RAGE knockdown. Moreover, the stimulation of pro-inflammatory cytokines expression by eHSP70 was inhibited in RAGE-silenced cells. Finally, conditioned medium of eHSP70-treated A549 cells caused differential effects in monocytes cytokine expression when A549 RAGE expression is inhibited. Our results evidence eHSP70 as a novel RAGE agonist capable of influence the cross-talk between cancer and immune system cells. © 2017 The Author(s). Published by S. Karger AG, Basel.

  10. Ascorbic acid increases drug accumulation and reverses vincristine resistance of human non-small-cell lung-cancer cells.

    PubMed Central

    Chiang, C D; Song, E J; Yang, V C; Chao, C C

    1994-01-01

    A human lung-cancer PC-9 subline with acquired resistance to vincristine (VCR), a chemotherapeutic agent, was established with incremental increases of the drug. The resistant PC-9 subline (PC-9/VCR) shows a 12-fold increase in resistance to VCR and a unique cross-resistance pattern: high cross-resistance to the potent VCR analogue colchicine (6.9-fold) and vinblastine (2.5-fold); lower cross-resistance to actinomycin D (1.8-fold), cisplatin (1.2-fold) and adriamycin (1.3-fold) and a sensitivity to melphalan and VP-16 which is similar to that of the parental cell line. A reduced accumulation of VCR in the resistant cells was demonstrated. Interestingly, the VCR resistance of the PC-9/VCR cell line was partially reversed by ascorbic acid, and the drug uptake was enhanced. In contrast, ascorbic acid had no effect on drug tolerance and drug accumulation was not observed in either PC-9 parental cells or known multidrug-resistant (MDR) cells, suggesting that VCR resistance in PC-9/VCR cells results essentially from reduced drug accumulation. It is worth noting that, whereas reduced drug accumulation in the PC-9/VCR cells was susceptible to modulation by ascorbic acid, the increased efflux rate characteristic of the resistant cells was not. Further, there was a higher efflux rate in resistant cells than in parental cells. DNA Southern- and RNA Northern-blot hybridization analyses indicate that PC-9/VCR cells do not contain amplified mdr genes or overexpress P-glycoprotein. In addition, the calcium-channel blocker verapamil, which acts as a competitive inhibitor of drug binding and efflux, did not affect the resistant phenotype of PC-9/VCR cells. These findings suggest an ascorbic acid-sensitive drug uptake mechanism which is important in mediating VCR resistance per se in human lung-cancer cells; this differs from the P-glycoprotein-mediated MDR mechanism. Images Figure 5 PMID:7914401

  11. ETS2 mediated tumor suppressive function and MET oncogene inhibition in human non-small cell lung cancer

    PubMed Central

    Kabbout, Mohamed; Garcia, Melinda M.; Fujimoto, Junya; Liu, Diane D.; Woods, Denise; Chow, Chi-Wan; Mendoza, Gabriela; Momin, Amin A.; James, Brian P.; Solis, Luisa; Behrens, Carmen; Lee, J. Jack

    2013-01-01

    PURPOSE The ETS2 transcription factor is an evolutionarily conserved gene that is deregulated in cancer. We analyzed the transcriptome of lung adenocarcinomas and normal lung tissue by expression profiling and found that ETS2 was significantly down-regulated in adenocarcinomas. In this study, we probed the yet unknown functional role of ETS2 in lung cancer pathogenesis. EXPERIMENTAL DESIGN Lung adenocarcinomas (n=80) and normal lung tissues (n=30) were profiled using the Affymetrix Human Gene 1.0 ST platform. Immunohistochemical (IHC) analysis was performed to determine ETS2 protein expression in NSCLC histological tissue specimens (n=201). Patient clinical outcome, based on ETS2 IHC expression, was statistically assessed using the log-rank and Kaplan-Meier tests. RNA interference and over-expression strategies were employed to assess effects of ETS2 expression on the transcriptome and on various malignant phenotypes. RESULTS ETS2 expression was significantly reduced in lung adenocarcinomas compared to normal lung (p<0.001). Low ETS2 IHC expression was a significant predictor of shorter time to recurrence in NSCLC (p=0.009, HR=1.89) and adenocarcinoma (p=0.03, HR=1.86). Moreover, ETS2 was found to significantly inhibit lung cancer cell growth, migration and invasion (p<0.05), and microarray and pathways analysis revealed significant (p<0.001) activation of the HGF pathway following ETS2 knockdown. In addition, ETS2 was found to suppress MET phosphorylation and knockdown of MET expression significantly attenuated (p<0.05) cell invasion mediated by ETS2-specific siRNA. Furthermore, knockdown of ETS2 augmented HGF-induced MET phosphorylation, cell migration and invasion. CONCLUSION(S) Our findings point to a tumor suppressor role for ETS2 in human NSCLC pathogenesis through inhibition of the MET proto-oncogene. PMID:23659968

  12. ∆Np73beta induces caveolin-1 in human non-small cell lung cancer cell line H1299.

    PubMed

    Caiola, Elisa; Marrazzo, Eleonora; Alesci, Simona; Broggini, Massimo; Marabese, Mirko

    2016-02-01

    Caveolins have recently attracted attention for their possible involvement in signal transduction. Their role in cancer is debated, being reported both a suppressive and oncogenic role in different experimental conditions. Caveolin-1 is regulated by the tumor suppressor p53 which is able to bind its promoter and activate transcription. We had previous evidences indicating that a specific p73 isoform, namely ∆Np73β, when overexpressed in NCI-H1299 induced growth arrest and cell death. By gene expression analysis in cell transiently overexpressed with ∆Np73β, a strong induction of caveolin-1 was found. Caveolin was induced both at mRNA and protein level, and we characterised the promoter sequence of the gene encoding for caveolin-1 and found that the promoter region containing the putative p53 (and hence p73) binding sequence was responsive to ∆Np73β, but not to ∆Np73α and ∆Np73γ which do not induce growth arrest as ∆Np73β does. A reduction in cell adhesion was observed in ∆Np73β overexpressing cells, again supporting a possible role of caveolins in determining these effects. By using specific siRNA directed against human caveolin-1, we could not however antagonize the effects induced by ∆Np73β. Although caveolin-1 represents one of the genes whose expression is strongly activated by ∆Np73β, we could not define a role of caveolin-1 as a mediator of ∆Np73β associated growth arrest. It could well be that the expression of caveolin-1 is able to mediate other activities of ∆Np73β, and studies are in progress to determine whether its expression is mainly associated to metastatic spread.

  13. Hyperthermia in the febrile range induces HSP72 expression proportional to exposure temperature but not to HSF-1 DNA-binding activity in human lung epithelial A549 cells.

    PubMed

    Tulapurkar, Mohan E; Asiegbu, Benedict E; Singh, Ishwar S; Hasday, Jeffrey D

    2009-09-01

    Expression of heat shock proteins (HSPs) is classically activated at temperatures above the physiologic range (>or=42 degrees C) via activation of the stress-activated transcription factor, heat shock factor-1 (HSF-1). Several studies suggest that less extreme hyperthermia, especially within the febrile range, as occurs during fever and exertional/environmental hyperthemia, can also activate HSF-1 and enhance HSP expression. We compared HSP72 protein and mRNA expression in human A549 lung epithelial cells continuously exposed to 38.5 degrees C, 39.5 degrees C, or 41 degrees C or exposed to a classic heat shock (42 degrees C for 2 h). We found that expression of HSP72 protein and mRNA increased linearly as incubation temperature was increased from 37 degrees C to 41 degrees C, but increased abruptly when the incubation temperature was raised to 42 degrees C. A similar response in luciferase activity was observed using A549 cells stably transfected with an HSF-1-responsive luciferase reporter plasmid. However, activation of intranuclear HSF-1 DNA-binding activity was comparable at 38.5 degrees C, 39.5 degrees C, and 41 degrees C and only modestly greater at 42 degrees C but the mobility of HSF1 protein on a denaturing gel was altered with increasing exposure temperature and was distinctly different at 42 degrees C. These findings indicate that the proportional changes in HSF-1-dependent HSP72 expression at febrile-range temperatures are dependent upon exposure time and temperature but not on the degree of HSF-1 DNA-binding activity. Instead, HSF-1-mediated HSP expression following hyperthermia and heat shock appears to be mediated, in addition to HSF-1 activation, by posttranslational modifications of HSF-1 protein.

  14. 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

    PubMed Central

    Liu, Xiaoqun; Liu, Xiangdong; Qiao, Tiankui; Chen, Wei; Yuan, Sujuan

    2015-01-01

    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 rate were clearly increased in the CpG+X-ray group compared with in the other groups (all P<0.05). The multi-target single-hitting model showed that the sensitization enhancement ratio calculated by mean death dose was 1.39 in CpG+X-ray group (vs 1.04 and 1.03 in the ATM

  15. Synergistic effects of the purine analog sulfinosine and curcumin on the multidrug resistant human non-small cell lung carcinoma cell line (NCI-H460/R).

    PubMed

    Andjelkovic, Tijana; Pesic, Milica; Bankovic, Jasna; Tanic, Nikola; Markovic, Ivanka D; Ruzdijic, Sabera

    2008-07-01

    Multidrug resistance (MDR) is the main obstacle to a successful chemotherapy of lung cancer. We tested the potential of sulfinosine and curcumin, alone and in combination, for modulating MDR in the human resistant, non-small cell lung carcinoma cell line (NCI-H460/R). First, we determined the mutational status of the p53 gene in NCI-H460/R cells by PCR-SSCP and DNA sequencing and identified mutations which could at least partially contribute to the development of the MDR phenotype. The effects of sulfinosine and curcumin were studied, both separately and in combination, at the level of cytotoxicity, cell cycle distribution and gene expression. Sulfinosine displayed dose-dependent growth inhibition in both resistant and control sensitive cell lines, whereas curcumin considerably inhibited their growth only at relatively high doses. When sulfinosine was combined with a low dose of curcumin the drugs exerted a synergistic cytotoxic effect in NCI-H460/R cells. The expression of MDR-related genes mdr1, gst-pi and topo IIalpha, was altered by sulfinosine and curcumin. The most pronounced effect was observed when the agents were applied together. Sulfinosine and curcumin caused perturbations in cell cycle distribution in the NCI-H460/R cell line. The combination of the two drugs induced a more pronounced cell cycle arrest in S and G(2)/M in NCI-H460/R cells. Our results show that sulfinosine and curcumin overcome MDR in non-small cell lung carcinoma cell line (NSCLC), especially in combination despite the presence of a mutated p53 gene.

  16. Artesunate induces AIF-dependent apoptosis in A549 cells

    NASA Astrophysics Data System (ADS)

    Zhou, Chen-juan; Chen, Tong-Sheng

    2012-03-01

    Artesunate (ART), a semi-synthetic derivative of the sesquiterpene artemisinin extracted from the Chinese herb Artemisia annua, exerts a broad spectrum of clinical activity against human cancers. It has been shown that ART induces cancer cells death through apoptosis pathway. This study investigated whether ART treatment induced reactive oxygen species (ROS)-dependent cell death in the apoptosis fashion in human lung adenocarconoma A549 cell line and the proapoptotic protein apoptosis inducing factor (AIF) is involved in ART-induced apoptosis. Cells treated with ART exhibited typical apoptotic morphology as chromatin condensation, margination and shrunken nucleus. ART treatment also induced a loss of mitochondrial membrane potential and AIF release from mitochondria. Silencing AIF can remarkable attenuated ART-induced apoptosis. Collectively, ART induces apoptosis by caspase-independent intrinsic pathway in A549 cells.

  17. Cordycepin induces autophagy-mediated c-FLIPL degradation and leads to apoptosis in human non-small cell lung cancer cells

    PubMed Central

    Liu, Xianfang; Guo, Sen; Lin, Yidan; Liu, Xiangguo; Su, Ling

    2017-01-01

    Cordycepin, a main active composition extracted from Cordyceps militaris, has been reported to exert anti-tumor activity in a broad spectrum of cancer types. However, the function of cordycepin on human non-small cell lung cancer cells is still obscure. Our present work showed that cordycepin inhibited cell growth by inducing apoptosis and autophagy in human NSCLC cells. Further study revealed that cordycepin triggered extrinsic apoptosis associated with down-regulation of c-FLIPL which suppresses the activity of caspase-8. And ectopic expression of c-FLIPL dramatically prevented cordycepin-caused apoptosis. Meanwhile, cordycepin stimulated autophagy through suppressing mTOR signaling pathway in lung cancer cells. When autophagy was blocked by Atg5 siRNA or PI3K inhibitor LY294002, the levels of apoptosis caused by cordycepin were obviously attenuated. In addition, suppression of autophagy could also elevate the level of c-FLIPL which indicated cordycepin-triggered autophagy promoted the degradation of c-FLIPL. Therefore, we conclude that cordycepin induces apoptosis through autophagy-mediated downregulation of c-FLIPL in human NSCLC cells. Taken together, our findings provide a novel prospect on the anti-tumor property of cordycepin, which may further prompt cordycepin to serve as a promising therapeutic approach in NSCLC treatment. PMID:28035061

  18. [Expression of Gemcitabine-resistance-related gene and polymorphism of ribonucleotide reductase M1 gene promoter in Gemcitabine-resistant A549/Gem and NCI-H460/Gem cell lines].

    PubMed

    Liu, Xiao-qing; Wang, Wei-xia; Lin, Li; Song, San-tai

    2010-01-01

    To assay the expression of cytidine deaminase (CDA), ribonucleotide reductase subunit 1 (RRM1), phosphatase and tensin homologue deleted from chromosome 10 (PTEN), excision repair cross-complementation group 1 (ERCC1), deoxycytidine kinase (dCK) and RRM1(-)37A/C polymorphism, which have been shown relevant to gemcitabine resistance in two human gemcitabine-resistant non-small cell lung cancer cell lines A549/Gem and NCI-H460/Gem, so as to make clear how do they vary during the course of acquiring resistance to gemcitabine. The human gemcitabine-resistant non-small cell lung cancer cell lines A549/Gem and NCI-H460/Gem were established in our Department by repeated clinical serum peak concentration and gradually increasing doses. Real-time fluorescent quantitative PCR was used to examine the expression of CDA, RRM1, PTEN, ERCC1, dCK and RRM1(-)37A/C polymorphism in those cell lines at different time points during their induction process. The resistance indexes of A549/Gem and NCI-H460/Gem cells reached 163.228 and 181.684, and then remained stable at 115.297 and 129.783, respectively. The expression of CDA, RRM1, PTEN and ERCC1 varied along with the changing gemcitabine resistance indexes, but expression of dCK did not change apparently. The wild type promoter was able to amplify the genomic DNA in different induction stages of A549/Gem and NCI-H460/Gem cells, but allelotype did not, indicating that the gene type of A549/Gem, NCI-H460/Gem and their parental cells remaining still wild type. Compared with their parental cells, the expressions of CDA, RRM1, PTEN and ERCC1 in human gemcitabine-resistant non-small cell lung cancer cell lines A549/Gem and NCI-H460/Gem rise, the expression of dCK changes inapparently, therefore, their gene type are remaining wild type.

  19. Inhibition of TRPC6 reduces non-small cell lung cancer cell proliferation and invasion

    PubMed Central

    Lu, Xiao-Yu; Yan, Yan; Zhai, Yu-Jia; Bao, Qing; Doetsch, Paul W.; Deng, Xingming; Thai, Tiffany L.; Alli, Abdel A.; Eaton, Douglas C.; Shen, Bao-Zhong; Ma, He-Ping

    2017-01-01

    Recent studies indicate that the transient receptor potential canonical 6 (TRPC6) channel is highly expressed in several types of cancer cells. However, it remains unclear whether TRPC6 contributes to the malignancy of human non-small cell lung cancer (NSCLC). We used a human NSCLC A549 cell line as a model and found that pharmacological blockade or molecular knockdown of TRPC6 channel inhibited A549 cell proliferation by arresting cell cycle at the S-G2M phase and caused a significant portion of cells detached and rounded-up, but did not induce any types of cell death. Western blot and cell cycle analysis show that the detached round cells at the S-G2M phase expressed more TRPC6 than the still attached polygon cells at the G1 phase. Patch-clamp data also show that TRPC whole-cell currents in the detached cells were significantly higher than in the still attached cells. Inhibition of Ca2+-permeable TRPC6 channels significantly reduced intracellular Ca2+ in A549 cells. Interestingly, either blockade or knockdown of TRPC6 strongly reduced the invasion of this NSCLC cell line and decreased the expression of an adherent protein, fibronectin, and a tight junction protein, zonula occluden protein-1 (ZO-1). These data suggest that TRPC6-mediated elevation of intracellular Ca2+ stimulates NSCLC cell proliferation by promoting cell cycle progression and that inhibition of TRPC6 attenuates cell proliferation and invasion. Therefore, further in vivo studies may lead to a consideration of using a specific TRPC6 blocker as a complement to treat NSCLC. PMID:28030826

  20. Relationship between intercellular communication and adriamycin resistance in non-small cell lung cancer.

    PubMed

    Bradley, C; Freshney, R I; Pitts, J

    1994-01-01

    The adriamycin chemosensitivity and extent of gap junctional intercellular communication were assessed in a panel of seven human non-small cell lung cancer (NSCLC) cell lines. Communication was assessed by autoradiographic detection of transfer of 3H uridine nucleotides between coupled cells. The strength of coupling varied widely between the cell lines and they could be separated into 3 groups: those which exhibited strong coupling, L-DAN and A549; those which exhibited weak coupling, SK-MES-1, Calu-3 and NCI-H125; and an intermediate group, WIL and NCI-H23. Adriamycin chemosensitivity was assessed by both clonogenic and MTT assays. The range of IC50 values as measured by either assay was extremely narrow, with no important differences between the lines. Thus, despite the wide spectrum of intercellular communication observed in these lines, this did not correlate with their adriamycin resistance.

  1. Effect of Adjuvant Magnetic Fields in Radiotherapy on Non-Small-Cell Lung Cancer Cells In Vitro

    PubMed Central

    Feng, Jianguo; Sheng, Huaying; Zhu, Chihong; Jiang, Hao; Ma, Shenglin

    2013-01-01

    Objectives. To explore sensitization and possible mechanisms of adjuvant magnetic fields (MFs) in radiotherapy (RT) of non-small-cell lung cancer. Methods. Human A549 lung adenocarcinoma cells were treated with MF, RT, and combined MF-RT. Colony-forming efficiency was calculated, cell cycle and apoptosis were measured, and changes in cell cycle- and apoptosis-related gene expression were measured by microarray. Results. A 0.5 T, 8 Hz stationary MF showed a duration-dependent inhibitory effect lasting for 1–4 hours. The MF-treated groups had significantly greater cell inhibition than did controls (P < 0.05). Surviving fractions and growth curves derived from colony-forming assay showed that the MF-only, RT-only, and MF-RT groups had inhibited cell growth; the MF-RT group showed a synergetic effect. Microarray of A549 cells exposed for 1 hour to MF showed that 19 cell cycle- and apoptosis-related genes had 2-fold upregulation and 40 genes had 2-fold downregulation. MF significantly arrested cells in G2 and M phases, apparently sensitizing the cells to RT. Conclusions. MF may inhibit A549 cells and can increase their sensitivity to RT, possibly by affecting cell cycle- and apoptosis-related signaling pathways. PMID:24224175

  2. Overexpression of PP2A inhibitor SET oncoprotein is associated with tumor progression and poor prognosis in human non-small cell lung cancer.

    PubMed

    Liu, Hao; Gu, Yixue; Wang, Hongsheng; Yin, Jiang; Zheng, Guopei; Zhang, Zhijie; Lu, Minyin; Wang, Chenkun; He, Zhimin

    2015-06-20

    SET oncoprotein is an endogenous inhibitor of protein phosphatase 2A (PP2A), and SET-mediated PP2A inhibition is an important regulatory mechanism for promoting cancer initiation and progression of several types of human leukemia disease. However, its potential relevance in solid tumors as non-small cell lung cancer (NSCLC) remains mostly unknown. In this study, we showed that SET was evidently overexpressed in human NSCLC cell lines and NSCLC tissues. Clinicopathologic analysis showed that SET expression was significantly correlated with clinical stage (p < 0.001), and lymph node metastasis (p < 0.05). Kaplan-Meier analysis revealed that patients with high SET expression had poorer overall survival rates than those with low SET expression. Moreover, knockdown of SET in NSCLC cells resulted in attenuated proliferative and invasive abilities. The biological effect of SET on proliferation and invasion was mediated by the inhibition of the PP2A, which in turn, activation of AKT and ERK, increased the expression of cyclin D1 and MMP9, and decreased the expression of p27. Furthermore, we observed that restoration of PP2A using SET antagonist FTY720 impaired proliferative and invasive potential in vitro, as well as inhibited tumor growth in vivo of NSCLC cells. Taken together, SET oncoprotein plays an important role in NSCLC progression, which could serve as a potential prognosis marker and a novel therapeutic target for NSCLC patients.

  3. Adenovirus vector-mediated FAM176A overexpression induces cell death in human H1299 non-small cell lung cancer cells.

    PubMed

    Xie, Hong; Hu, Jia; Pan, Huan; Lou, Yaxin; Lv, Ping; Chen, Yingyu

    2014-02-01

    FAM176A (family with sequence similarity 176 member A) is a novel molecule related to programmed cell death. A decreased expression of FAM176A has been found in several types of human tumors in including lung cancers. In the present study, we investigated the biological activities of FAM176A on the human non-small cell lung cancer cell line H1299 cells. We constructed a recombinant adenovirus 5-FAM176A vector (Ad5-FAM176A) and evaluated the expression and anti-tumor activities in vitro. Cell viability analysis revealed that the adenovirus-mediated increase of FAM176A inhibited the growth of the tumor cells in a dose- and time-dependent manner. This inhibitory effect was mediated by both autophagy and apoptosis that involved caspase activation. In addition, cell cycle analysis suggested that Ad5-FAM176A could induce cell cycle arrest at the G2/M phase, all of which suggested that adenovirus-mediated FAM176A gene transfer might present a new therapeutic approach for lung cancer treatment.

  4. Human RNA polymerase II associated factor 1 complex promotes tumorigenesis by activating c-MYC transcription in non-small cell lung cancer.

    PubMed

    Zhi, Xiuyi; Giroux-Leprieur, Etienne; Wislez, Marie; Hu, Mu; Zhang, Yi; Shi, Huaiyin; Du, Kaiqi; Wang, Lei

    2015-10-02

    Human RNA polymerase II (RNAPII)-associated factor 1 complex (hPAF1C) plays a crucial role in protein-coding gene transcription. Overexpression of hPAF1C has been implicated in the initiation and progression of various human cancers. However, the molecular pathways involved in tumorigenesis through hPAF1C remain to be elucidated. The current study suggested hPAF1C expression as a prognostic biomarker for early stage non-small cell lung cancer (NSCLC) and patients with low hPAF1C expression levels had significantly better overall survival. Furthermore, the expression of hPAF1C was found to be positively correlated with c-MYC expression in patient tumor samples and in cancer cell lines. Mechanistic studies indicated that hPAF1C could promote lung cancer cell proliferation through regulating c-MYC transcription. These results demonstrated the prognostic value of hPAF1C in early-stage NSCLC and the role of hPAF1C in the transcriptional regulation of c-MYC oncogene during NSCLC tumorigenesis. Copyright © 2015. Published by Elsevier Inc.

  5. Down-regulation of the DNA-repair endonuclease 8-oxo-guanine DNA glycosylase 1 (hOGG1) by sodium dichromate in cultured human A549 lung carcinoma cells.

    PubMed

    Hodges, N J; Chipman, J K

    2002-01-01

    Hexavalent chromium is a genotoxic human pulmonary carcinogen that elevates DNA oxidation, apparently through the generation of reactive DNA-damaging intermediates including Cr(V), Cr(IV) and reactive oxygen species. We tested the hypothesis that elevation of DNA oxidation may also be through inhibition of the expression of the repair glycosylase for 8-oxo deoxyguanine (hOGG1) in cultured A549 human lung epithelial cells. Treatment with sodium dichromate (0-100 microM, 16 h) resulted in a concentration-dependent decrease in the levels of OGG1 mRNA as measured by both RT-PCR and RNase protection assay. Sodium dichromate at 25 microM and above gave a marked reduction of OGG1 mRNA expression which was not seen at 1 microM and below. No effect on the expression of the apurinic endonuclease hAPE or the house-keeping gene GAPDH was observed at any of the concentrations of sodium dichromate investigated. Treatment of cells with the pro-oxidant H(2)O(2) (0-200 microM) for 16 h had no detectable effect on the levels of OGG1 mRNA or protein expression suggesting that the effect of sodium dichromate is not mediated by H(2)O(2). Western blotting demonstrated that sodium dichromate (100 microM; 16 h and >25 microM; 28 h) markedly reduced levels of OGG1 protein in nuclear cell extracts. Additionally, treatment of cells with sodium dichromate (>25 microM, 28 h) resulted in a concentration-dependent decrease in the ability of nuclear extracts to nick a synthetic oligonucleotide containing 8-oxo deoxyguanine (8-oxo dG). We conclude that the elevation of 8-oxo dG levels observed in A549 cells treated with sodium dichromate may be, at least in part, due to a reduced capacity to repair endogenous and hexavalent chromium-induced 8-oxo dG.

  6. Delivery of curcumin by directed self-assembled micelles enhances therapeutic treatment of non-small-cell lung cancer

    PubMed Central

    Zhu, Wen-Ting; Liu, Sheng-Yao; Wu, Lei; Xu, Hua-Li; Wang, Jun; Ni, Guo-Xin; Zeng, Qing-Bing

    2017-01-01

    Background It has been widely reported that curcumin (CUR) exhibits anticancer activity and triggers the apoptosis of human A549 non-small-cell lung cancer (NSCLC) cells. However, its application is limited owing to its poor solubility and bioavailability. Therefore, there is an urgent need to develop a new CUR formulation with higher water solubility and better biocompatibility for clinical application in the future. Materials and methods In this study, CUR-loaded methoxy polyethylene glycol–polylactide (CUR/mPEG–PLA) polymeric micelles were prepared by a thin-film hydration method. Their characteristics and antitumor effects were evaluated subsequently. Results The average size of CUR/mPEG–PLA micelles was 34.9±2.1 nm with its polydispersity index (PDI) in the range of 0.067–0.168. The encapsulation efficiency and drug loading were 90.2%±0.78% and 9.1%±0.07%, respectively. CUR was constantly released from the CUR/mPEG–PLA micelles, and its cellular uptake in A549 cells was significantly increased. It was also found that CUR/mPEG–PLA micelles inhibited A549 cell proliferation, increased the cell cytotoxicity, induced G2/M stage arrest and promoted cell apoptosis. Moreover, the CUR/mPEG–PLA micelles suppressed the migration and invasion of A549 cells more obviously than free CUR. Additionally, CUR/mPEG–PLA micelles inhibited human umbilical vein endothelial cells migration, invasion and corresponding tube formation, implying the antiangiogenesis ability. Its enhanced antitumor mechanism may be related to the reduced expression of vascular endothelial growth factor, matrix metalloproteinase (MMP)-2, MMP-9 and Bcl-2 as well as the increased expression of Bax. Conclusion The mPEG–PLA copolymer micelles can serve as an efficient carrier for CUR. The CUR/mPEG–PLA micelles have promising clinical potential in treating NSCLC. PMID:28435247

  7. NADPH:cytochrome c (P450) reductase activates tirapazamine (SR4233) to restore hypoxic and oxic cytotoxicity in an aerobic resistant derivative of the A549 lung cancer cell line

    PubMed Central

    Saunders, M P; Patterson, A V; Chinje, E C; Harris, A L; Stratford, I J

    2000-01-01

    Tirapazamine (TPZ, SR4233, WIN 59075) is a bioreductive drug that is activated in regions of low oxygen tension to a cytotoxic radical intermediate. This labile metabolite shows high selective toxicity towards hypoxic cells, such as those found in solid tumours. Under aerobic conditions, redox cycling occurs with subsequent generation of superoxide radicals, which are also cytotoxic. NADPH:cytochrome c (P450) reductase (P450R) is a one-electron reducing enzyme that efficiently activates TPZ. Recently a derivative of the A549 non-small cell lung cancer cell line (A549c50) was generated that showed substantially reduced P450R activity compared to its parental line (Elwell et al (1997) Biochem Pharmacol54: 249–257). Here, it is demonstrated that the A549c50 cells are markedly more resistant to TPZ under both aerobic and hypoxic conditions. In addition, these cells have a dramatically impaired ability to metabolize TPZ to its two-electron reduction product, SR4317, under hypoxic conditions when compared to wild-type cells. P450R activity in the A549c50 cells was reintroduced to similar levels as that seen in the parental A549 cells by transfection of the full-length cDNA for human P450R. These P450R over-expressing cells exhibit restored sensitivity to TPZ under both aerobic and hypoxic conditions, comparable to that found in the original parental A549 cells. Further, the ability of the transfected cells to metabolize TPZ to SR4317 under hypoxic conditions is also shown to be restored. This provides further evidence that P450R can play an important role in the activation, metabolism and toxicity of this lead bioreductive drug. © 2000 Cancer Research Campaign PMID:10682679

  8. HGF induces EMT in non-small-cell lung cancer through the hBVR pathway.

    PubMed

    Liu, Fang; Song, Shasha; Yi, Zhi; Zhang, Min; Li, Jiali; Yang, Fang; Yin, Hongtao; Yu, Xiufeng; Guan, Chao; Liu, Ying; Liu, Zizhen; Wang, Jing; Zhu, Daling

    2017-09-15

    The epithelial-to-mesenchymal transition (EMT) is a crucial event during non-small-cell lung cancer (NSCLC) invasion and metastasis. However, the mechanisms involved in NSCLC EMT have not been fully clarified. Hepatocyte growth factor (HGF) and human biliverdin reductase (hBVR) are reported to contribute to EMT in several diseases. Here, we show that compared with transforming growth factor beta (TGF-β), fibroblast growth factor (FGF), and epidermal growth factor (EGF), HGF is an important cell factor for EMT in NSCLC cell lines A549 and H460. Met protein, HGF receptors, and hBVR were found to be highly expressed and positively correlated with EMT in NSCLC tissue sections. In addition, HGF and hBVR induced a decrease in epithelial protein marker expression and an increase in mesenchymal protein marker expression as well as increased cellular migration and invasion, indicating that both HGF and hBVR mediate EMT in A549 and H460 cell lines. Furthermore, HGF-induced EMT and migration and invasion in both cell lines was inhibited by si-hBVR. Taken together, our data show that HGF induces EMT in NSCLC through the hBVR pathway. Copyright © 2017. Published by Elsevier B.V.

  9. Fisetin induces apoptosis and endoplasmic reticulum stress in human non-small cell lung cancer through inhibition of the MAPK signaling pathway.

    PubMed

    Kang, Kyoung Ah; Piao, Mei Jing; Madduma Hewage, Susara Ruwan Kumara; Ryu, Yea Seong; Oh, Min Chang; Kwon, Taeg Kyu; Chae, Sungwook; Hyun, Jin Won

    2016-07-01

    Fisetin (3,3',4',7-tetrahydroxyflavone), a dietary flavonoid compound, is currently being investigated for its anticancer effect in various cancer models, including lung cancer. Recent studies show that fisetin induces cell growth inhibition and apoptosis in the human non-small cell lung cancer line NCI-H460. In this study, we investigated whether fisetin can induce endoplasmic reticulum (ER) stress-mediated apoptosis in NCI-H460 cells. Fisetin induced mitochondrial reactive oxygen species (ROS) and characteristic signs of ER stress: ER staining; mitochondrial Ca(2+) overload; expression of ER stress-related proteins; glucose-regulated protein (GRP)-78, phosphorylation of protein kinase RNA (PKR)-like endoplasmic reticulum kinase (PERK) and phosphorylation of eukaryotic initiation factor-2 α subunit; cleavage of activating transcription factor-6; phosphorylation of inositol-requiring kinase-1 and splicing of X-box transcription factor-1; induction of C/EBP homologous protein and cleaved caspase-12. siRNA-mediated knockdown of CHOP and ATF-6 attenuated fisetin-induced apoptotic cell death. In addition, fisetin induced phosphorylation of ERK, JNK, and p38 MAPK. Moreover, silencing of the MAPK signaling pathway prevented apoptotic cell death. In summary, our results indicate that, in NCI-H460 cells, fisetin induces apoptosis and ER stress that is mediated by induction of the MAPK signaling pathway.

  10. Morphological changes and nuclear translocation of DLC1 tumor suppressor protein precede apoptosis in human non-small cell lung carcinoma cells

    SciTech Connect

    Yuan Baozhu Jefferson, Amy M.; Millecchia, Lyndell; Popescu, Nicholas C.; Reynolds, Steven H.

    2007-11-01

    We have previously shown that reactivation of DLC1, a RhoGAP containing tumor suppressor gene, inhibits tumorigenicity of human non-small cell lung carcinoma cells (NSCLC). After transfection of NSCLC cells with wild type (WT) DLC1, changes in cell morphology were observed. To determine whether such changes have functional implications, we generated several DLC1 mutants and examined their effects on cell morphology, proliferation, migration and apoptosis in a DLC1 deficient NSCLC cell line. We show that WT DLC1 caused actin cytoskeleton-based morphological alterations manifested as cytoplasmic extensions and membrane blebbings in most cells. Subsequently, a fraction of cells exhibiting DLC1 protein nuclear translocation (PNT) underwent caspase 3-dependent apoptosis. We also show that the RhoGAP domain is essential for the occurrence of morphological alterations, PNT and apoptosis, and the inhibition of cell migration. DLC1 PNT is dependent on a bipartite nuclear localizing sequence and most likely is regulated by a serine-rich domain at N-terminal part of the DLC1 protein. Also, we found that DLC1 functions in the cytoplasm as an inhibitor of tumor cell proliferation and migration, but in the nucleus as an inducer of apoptosis. Our analyses provide evidence for a possible link between morphological alterations, PNT and proapoptotic and anti-oncogenic activities of DLC1 in lung cancer.

  11. Programmed death-1 upregulation is correlated with dysfunction of tumor-infiltrating CD8+ T lymphocytes in human non-small cell lung cancer.

    PubMed

    Zhang, Yan; Huang, Shengdong; Gong, Dejun; Qin, Yanghua; Shen, Qian

    2010-09-01

    T-cell tolerance is an important mechanism for tumor escape, but the molecular pathways involved in T-cell tolerance remain poorly understood. It remains unknown whether the inhibitory immunoreceptor programmed death-1 (PD-1) plays a role in conditions of human non-small cell lung cancer (NSCLC). In this study, we detected PD-1 expression on CD8+ T cells from healthy control peripheral blood mononuclear cells (PBMCs) and the PBMCs of NSCLC patients as well as NSCLC tissues. Results showed that tumor-infiltrating CD8+ T cells had increased PD-1 expression and impaired immune function, including reducing cytokine production capability and impairing capacity to proliferate. Blockade of the PD-1/PD-L1 pathway by the PD-L1-specific antibody partially restored cytokine production and cell proliferation. These data provide direct evidence that the PD-1/PD-L1 pathway is involved in CD8+ T-cell dysfunction in NSCLC patients. Moreover, blocking this pathway provides a potential therapy target in lung cancer.

  12. Siamese crocodile bile induces apoptosis in NCI-H1299 human non-small cell lung cancer cells via a mitochondria-mediated intrinsic pathway and inhibits tumorigenesis.

    PubMed

    Tian, Ling; Deng, Yi-Tao; Dong, Xin; Fan, Jia-Yi; Li, Hua-Liang; Ding, Yu-Mei; Peng, Wei-Xi; Chen, Qing-Xi; Shen, Dong-Yan

    2017-02-16

    Non-small-cell lung cancer (NSCLC) is a widespread and particularly aggressive form of cancer. Patients with NSCLC and early metastases typically have poor prognosis, highlighting the critical need for additional drugs to improve disease outcome following surgical resection. The present study aimed to determine if Siamese crocodile bile (SCB) had an anti‑cancer effect on NCI‑H1299 human NSCLC cells. The inhibitory mechanism of SCB was examined in cell culture and nude mice. In vitro experimental results revealed that SCB inhibited the proliferation and colony‑forming ability of NCI‑H1299 cells by arresting cell cycle and inducing apoptosis. The loss of the mitochondrial membrane potential and the release of cytochrome c indicated that SCB treatment may lead to mitochondrial dysfunction in NCI‑H1299 cells. At the molecular level, SCB altered the ratio of protein expression of Bax/Bcl‑2 and activated associated caspases, suggesting that intrinsic pathway involvement in the SCB‑induced apoptosis of NCI‑H1299 cells. In the in vivo experiments, intraperitoneal injection of SCB for 4 weeks inhibited xenograft tumor growth by 46.8% without observable toxicity in nude mice. Immunohistochemistry analysis of proliferating cell nuclear antigen and vascular endothelial growth factor also revealed that SCB inhibited cell proliferation and metastasis in NSCLC xenograft tumors. Overall, SCB exerted an anti-cancer effect on NCI‑H1299 human NSCLC cells in vitro and in vivo and may have therapeutic potential for the treatment of human NSCLC.

  13. Flavonoids from Gynostemma pentaphyllum exhibit differential induction of cell cycle arrest in H460 and A549 cancer cells.

    PubMed

    Tsui, Ko-Chung; Chiang, Tzu-Hsuan; Wang, Jinn-Shyan; Lin, Li-Ju; Chao, Wei-Chih; Chen, Bing-Huei; Lu, Jyh-Feng

    2014-10-31

    Flavonoids, containing mainly kaempferol rhamnohexoside derivatives, were extracted from Gynostemma pentaphyllum (G. pentaphyllum) and their potential growth inhibition effects against H460 non-small cell lung cancer cells was explored and compared to that on A549 cells. The extracted flavonoids were found to exhibit antiproliferation effects against H460 cells (IC50 = 50.2 μg/mL), although the IC50 of H460 is 2.5-fold that of A549 cells (IC50 = 19.8 μg/mL). Further investigation revealed that H460 cells are more susceptible to kaempferol than A549, whereas A549 cell growth is better inhibited by kaempferol rhamnohexoside derivatives as compared with H460. In addition, flavonoids from G. pentaphyllum induced cell cycle arrest at both S and G2/M phases with concurrent modulated expression of the cellular proteins cyclin A, B, p53 and p21 in A549 cells, but not H460. On the contrary, apoptosis and concomitant alteration in balance of BCL-2 and BAX expression as well as activation of caspase-3 were equally affected between both cells by flavonoid treatment. These observations strongly suggest the growth inhibition discrepancy between H460 and A549 following flavonoid treatment can be attributed to the lack of cell cycle arrest in H460 cells and the differences between H460 and A549 cells may serve as contrasting models for further mechanistic investigations.

  14. Lycium europaeum fruit extract: antiproliferative activity on A549 human lung carcinoma cells and PC12 rat adrenal medulla cancer cells and assessment of its cytotoxicity on cerebellum granule cells.

    PubMed

    Ghali, Wafa; Vaudry, David; Jouenne, Thierry; Marzouki, Mohamed Nejib

    2015-01-01

    Cancer is a major worldwide health problem and one of the leading causes of death either in developed or developing countries. Plant extracts and derivatives have always been used for various disease treatments and many anticancer agents issued from plants and vegetables are clinically recognized and used all over the world. Lycium europaeum (Solanaceae) also called "wolfberry" was known since ancient times in the Mediterranean area as a medicinal plant and used in several traditional remedies. The Lycium species capacity of reducing the incidence of cancer and also of halting or reserving the growth of cancer was reported by traditional healers. In this study, the antiproliferative capacity, protective properties, and antioxidant activity of the hydro-alcoholic fruit extract of Lycium europaeum were investigated. Results showed that Lycium extract exhibits the ability to reduce cancer cell viability, inhibits proliferation, and induces apoptosis in A549 human lung cancer cells and PC12 rat adrenal medulla cancer cells, in a concentration- and time-dependent manner. Cytotoxic effect on normal rat cerebellum granule cells was assessed to be nonsignificant. Results also showed that Lycium fruit extract protected lipids, proteins, and DNA against oxidative stress damages induced by H2O2 via scavenging reactive oxygen species.

  15. Comparative Cytotoxicity of Glycyrrhiza glabra Roots from Different Geographical Origins Against Immortal Human Keratinocyte (HaCaT), Lung Adenocarcinoma (A549) and Liver Carcinoma (HepG2) Cells.

    PubMed

    Basar, Norazah; Oridupa, Olayinka Ayotunde; Ritchie, Kenneth J; Nahar, Lutfun; Osman, Nashwa Mostafa M; Stafford, Angela; Kushiev, Habibjon; Kan, Asuman; Sarker, Satyajit D

    2015-06-01

    Glycyrrhiza glabra L. (Fabaceae), commonly known as 'liquorice', is a well-known medicinal plant. Roots of this plant have long been used as a sweetening and flavouring agent in food and pharmaceutical products, and also as a traditional remedy for cough, upper and lower respiratory ailments, kidney stones, hepatitis C, skin disorder, cardiovascular diseases, diabetes, gastrointestinal ulcers and stomach ache. Previous pharmacological and clinical studies have revealed its antitussive, antiinflammatory, antiviral, antimicrobial, antioxidant, immunomodulatory, hepatoprotective and cardioprotective properties. While glycyrrhizin, a sweet-tasting triterpene saponin, is the principal bioactive compound, several bioactive flavonoids and isoflavonoids are also present in the roots of this plant. In the present study, the cytotoxicity of the methanol extracts of nine samples of the roots of G. glabra, collected from various geographical origins, was assessed against immortal human keratinocyte (HaCaT), lung adenocarcinoma (A549) and liver carcinoma (HepG2) cell lines using the in vitro 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazoliumbromide cell toxicity/viability assay. Considerable variations in levels of cytotoxicity were observed among various samples of G. glabra. Copyright © 2015 John Wiley & Sons, Ltd.

  16. Cell (A549)-particle (Jasada Bhasma) interactions using Raman spectroscopy.

    PubMed

    Pyrgiotakis, G; Bhowmick, T K; Finton, K; Suresh, A K; Kane, S G; Bellare, J R; Moudgil, B M

    2008-06-01

    Current methods for the evaluation of cell interactions with particles are nonspecific, slow, and invasive to the cells. Raman spectroscopy is a noninvasive technique, and is used in the present study to investigate particle-cell interactions. The main focus of the present study is to employ Raman spectroscopy for investigating the interaction of human lung adenocarcinoma cell line (A549) with the particulate system Jasada Bhasma, a traditional Indian medicine. Jasada Bhasma is a unique preparation of zinc and is traditionally used for the treatment of various diseases like diabetes, age-related eye diseases, and as a health promotional tonic. The Raman spectral analysis is executed by identifying the difference in intracellular DNA/RNA, and proteins and lipids concentration between particles--treated and untreated cells. Comparison between Bhasma-treated and -untreated cells indicates that vibrational peaks corresponding to the DNA/RNA molecule show a significant increase in cells treated with the Jasada Bhasma. Apart from the DNA molecule, several other vibrational peaks related to the protein molecules also show a significant increase in A549 cells after treatment with Bhasma. These results indicate that Bhasma treatment of A549 possibly delays DNA degradation and enables retention of higher amount of protein molecules in the cells.

  17. Synergistic cytotoxicity of ampelopsin sodium and carboplatin in human non-small cell lung cancer cell line SPC-A1 by G1 cell cycle arrested.

    PubMed

    Lu, Li; Yang, Li-Ning; Wang, Xue-Xi; Song, Chun-Li; Qin, Hong; Wu, Yong-Jie

    2017-02-01

    To evaluate the cytotoxic effects of ampelopsin sodium (Amp-Na) and carboplatin (CBP) used alone or in combination on human non-small cell lung cancer (NSCLC) cells SPC-A1 in vitro and its related mechanism. Cytotoxic effects were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. The synergistic effects of the drugs were calculated with coefficient of drug interaction (CDI). Cell cycle was determined by flow cytometry (FCM). The levels of p53, p21, cyclinE, cyclinD1, and phosphorylated cyclin-dependent kinase-2 (p-CDK2) were evaluated by Western blot. Amp-Na (6.25-200 μg/mL) and CBP (3.13-100 μg/mL) alone exhibited prominent cytotoxic activity in a concentration-dependent manner on SPC-A1 cells with 50% inhibitive concentration values of 57.07±14.46 and 34.97±6.30 μg/mL, respectively. Drug combinations were associated with significantly higher cytotoxic effects than each drug alone (P<0.05 or 0.01). The CDI analysis confirmed the synergy of Amp-Na and CBP on inhibiting cancer cell viability across a wide concentration range (CDI <1). FCM and Western blot showed that synergistic cytotoxic effects of Amp-Na and CBP were related to G1 arrested which mainlym ediated by p 21 through the inhibition of CDK2 activity independent of the p53 tumor suppressor pathway. Amp-Na exhibits anticancer activities and enhances the antitumor activities of CBP through up-regulation of p21 and inhibition of CDK2 activity in human NSCLC cells SPC-A1. These results suggest that Amp-Na may be applied to enhance the anticancer action of CBP.

  18. Pharmacokinetic correlation between experimental and clinical effects on human non-small cell lung cancers of cis-diammineglycolatoplatinum (254-S) and cis-diamminedichloroplatinum.

    PubMed

    Koenuma, M; Kasai, H; Uchida, N; Wada, T; Hattori, M; Oguma, T; Totani, T; Inaba, M

    1995-01-01

    We attempted to correlate the in vitro and in vivo antitumor activities of cis-diammineglycolatoplatinum (254-S), a novel platinum complex, and cis-diamminedichloro-platinum (CDDP) against the established culture cell lines and xenografts of human non-small cell lung cancer (NSCLC) with their clinical effects, based on the previous finding that the cytotoxicity of CDDP depends on the area under the curve (AUC). The concentration of 254-S and CDDP inhibiting the in vitro growth of 4 cultured NSCLC lines by 50% (IC50) was 0.82-7.8 and 0.53-4.2 micrograms/ml, respectively, showing a similar level. Of the 4 cell lines, only the most sensitive line, RERF-LC-AI, showed an IC50 close to a specific concentration (0.50 for 254-S and 0.32 micrograms/ml for CDDP) that reproduces in vitro the clinical AUCfree (24.8 and 5.34 micrograms-hr/ml) of the respective drugs. We treated 6 lines of human NSCLC xenografts implanted in nude mice with 254-S and CDDP at a particular dose (13.2 and 3.7 mg/kg) that is equivalent to the clinical doses with respect to the plasma AUCfree. 254-S and CDDP exhibited significant antitumor effects on 2 and 1 of the 6 lines, respectively. These in vitro and in vivo findings were considered to be relatively well correlated with the reported clinical response rates of 15-19% for 254-S and 14-15% for CDDP.

  19. Metformin inhibits growth of human non-small cell lung cancer cells via liver kinase B-1-independent activation of adenosine monophosphate-activated protein kinase

    PubMed Central

    GUO, QIANQIAN; LIU, ZHIYAN; JIANG, LILI; LIU, MENGJIE; MA, JIEQUN; YANG, CHENGCHENG; HAN, LILI; NAN, KEJUN; LIANG, XUAN

    2016-01-01

    Metformin, the most widely administered oral anti-diabetic therapeutic agent, exerts its glucose-lowering effect predominantly via liver kinase B1 (LKB1)-dependent activation of adenosine monophosphate-activated protein kinase (AMPK). Accumulating evidence has demonstrated that metformin possesses potential antitumor effects. However, whether the antitumor effect of metformin is via the LKB1/AMPK signaling pathway remains to be determined. In the current study, the effects of metformin on proliferation, cell cycle progression, and apoptosis of human non-small cell lung cancer (NSCLC) H460 (LKB1-null) and H1299 (LKB1-positive) cells were assessed, and the role of LKB1/AMPK signaling in the anti-growth effects of metformin were investigated. Cell viability was determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, cell cycle distribution and apoptosis were assessed by flow cytometry, and protein expression levels were measured by western blotting. Metformin inhibited proliferation, induced significant cell cycle arrest at the G0–G1 phase and increased apoptosis in NSCLC cells in a time- and concentration-dependent manner, regardless of the level of LKB1 protein expression. Furthermore, knockdown of LKB1 with short hairpin RNA (shRNA) did not affect the antiproliferative effect of metformin in the H1299 cells. Metformin stimulated AMPK phosphorylation and subsequently suppressed the phosphorylation of mammalian target of rapamycin and its downstream effector, 70-kDa ribosomal protein S6 kinase in the two cell lines. These effects were abrogated by silencing AMPK with small interfering RNA (siRNA). In addition, knockdown of AMPK with siRNA inhibited the effect of metformin on cell proliferation in the two cell lines. These results provide evidence that the growth inhibition of metformin in NSCLC cells is mediated by LKB1-independent activation of AMPK, indicating that metformin may be a potential therapeutic agent for the treatment of

  20. Chlorin e6 – polyvinylpyrrolidone mediated photosensitization is effective against human non-small cell lung carcinoma compared to small cell lung carcinoma xenografts

    PubMed Central

    Chin, William WL; Heng, Paul WS; Olivo, Malini

    2007-01-01

    Background Photodynamic therapy (PDT) is an effective local cancer treatment that involves light activation of a photosensitizer, resulting in oxygen-dependent, free radical-mediated cell death. Little is known about the comparative efficacy of PDT in treating non-small cell lung carcinoma (NSCLC) and small cell lung carcinoma (SCLC), despite ongoing clinical trials treating lung cancers. The present study evaluated the potential use of chlorin e6 – polyvinylpyrrolidone (Ce6-PVP) as a multimodality photosensitizer for fluorescence detection and photodynamic therapy (PDT) on NSCLC and SCLC xenografts. Results Human NSCLC (NCI-H460) and SCLC (NCI-H526) tumor cell lines were used to establish tumor xenografts in the chick chorioallantoic membrane (CAM) model as well as in the Balb/c nude mice. In the CAM model, Ce6-PVP was applied topically (1.0 mg/kg) and fluorescence intensity was charted at various time points. Tumor-bearing mice were given intravenous administration of Ce6-PVP (2.0 mg/kg) and laser irradiation at 665 nm (fluence of 150 J/cm2 and fluence rate of 125 mW/cm2). Tumor response was evaluated at 48 h post PDT. Studies of temporal fluorescence pharmacokinetics in CAM tumor xenografts showed that Ce6-PVP has a selective localization and a good accuracy in demarcating NSCLC compared to SCLC from normal surrounding CAM after 3 h post drug administration. Irradiation at 3 h drug-light interval showed greater tumor necrosis against human NSCLC xenografts in nude mice. SCLC xenografts were observed to express resistance to photosensitization with Ce6-PVP. Conclusion The formulation of Ce6-PVP is distinctly advantageous as a diagnostic and therapeutic agent for fluorescence diagnosis and PDT of NSCLC. PMID:18053148

  1. Mitochondrial DNA-depleted A549 cells are resistant to bleomycin.

    PubMed

    Brar, Sukhdev S; Meyer, Joel N; Bortner, Carl D; Van Houten, Bennett; Martin, William J

    2012-09-01

    Alveolar epithelial cells are considered to be the primary target of bleomycin-induced lung injury, leading to interstitial fibrosis. The molecular mechanisms by which bleomycin causes this damage are poorly understood but are suspected to involve generation of reactive oxygen species and DNA damage. We studied the effect of bleomycin on mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) in human alveolar epithelial A549 cells. Bleomycin caused an increase in reactive oxygen species production, DNA damage, and apoptosis in A549 cells; however, bleomycin induced more mtDNA than nDNA damage. DNA damage was associated with activation of caspase-3, cleavage of poly(ADP-ribose) polymerase, and cleavage and activation of protein kinase D1 (PKD1), a newly identified mitochondrial oxidative stress sensor. These effects appear to be mtDNA-dependent, because no caspase-3 or PKD1 activation was observed in mtDNA-depleted (ρ(0)) A549 cells. Survival rate after bleomycin treatment was higher for A549 ρ(0) than A549 cells. These results suggest that A549 ρ(0) cells are more resistant to bleomycin toxicity than are parent A549 cells, likely in part due to the depletion of mtDNA and impairment of mitochondria-dependent apoptotic pathways.

  2. Mitochondrial DNA-depleted A549 cells are resistant to bleomycin

    PubMed Central

    Brar, Sukhdev S.; Meyer, Joel N.; Bortner, Carl D.; Van Houten, Bennett

    2012-01-01

    Alveolar epithelial cells are considered to be the primary target of bleomycin-induced lung injury, leading to interstitial fibrosis. The molecular mechanisms by which bleomycin causes this damage are poorly understood but are suspected to involve generation of reactive oxygen species and DNA damage. We studied the effect of bleomycin on mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) in human alveolar epithelial A549 cells. Bleomycin caused an increase in reactive oxygen species production, DNA damage, and apoptosis in A549 cells; however, bleomycin induced more mtDNA than nDNA damage. DNA damage was associated with activation of caspase-3, cleavage of poly(ADP-ribose) polymerase, and cleavage and activation of protein kinase D1 (PKD1), a newly identified mitochondrial oxidative stress sensor. These effects appear to be mtDNA-dependent, because no caspase-3 or PKD1 activation was observed in mtDNA-depleted (ρ0) A549 cells. Survival rate after bleomycin treatment was higher for A549 ρ0 than A549 cells. These results suggest that A549 ρ0 cells are more resistant to bleomycin toxicity than are parent A549 cells, likely in part due to the depletion of mtDNA and impairment of mitochondria-dependent apoptotic pathways. PMID:22773697

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

    PubMed

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

    2016-02-01

    To explore the effect of grape seed proanthocyanidins (GSPs) on the invasion and migration of A549 lung cancer cells and the underlying mechanism. 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. (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. 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.

  4. Synergistic induction of the Fas (CD95) ligand promoter by Max and NFkappaB in human non-small lung cancer cells.

    PubMed

    Wiener, Zoltan; Ontsouka, Edgar C; Jakob, Sabine; Torgler, Ralph; Falus, Andras; Mueller, Christoph; Brunner, Thomas

    2004-09-10

    Fas (CD95/APO-1) ligand is a member of the Tumor Necrosis Factor family and a potent inducer of apoptosis. Fas ligand is expressed in activated T cells and represents a major cytotoxic effector mechanism by which T cells kill their target cells. Activation-induced Fas ligand expression in T cells is under the stringent control of various transcription factors, including nuclear factor kappaB (NFkappaB) and c-Myc/Max. There is accumulating evidence that Fas ligand is also expressed by various non-hematopoietic tumor cells, however, little is known about Fas ligand regulation in tumor cells. In this study, we have analyzed the regulation of the Fas ligand gene promoter induction in two non-small cell lung cancer cell lines, with a major focus on the role of the c-Myc/Max transcription factor. Our results revealed that inhibition of c-Myc/Max did not substantially reduce basal levels of Fas ligand promoter activity, nor did overexpression of c-Myc significantly induce promoter activity. In contrast, we observed that overexpression of Max resulted in a marked increase in basal promoter activity and synergistically enhanced phorbolester- and doxorubicin-induced NFkappaB-mediated Fas ligand promoter activity. These results were confirmed by analyzing endogenous Fas ligand transcription. We conclude that high levels of Max and stress-induced NFkappaB activation may result in elevated expression of Fas ligand in human lung cancer cells and possibly contribute to Fas ligand-associated immune escape mechanisms.

  5. Mer or Axl receptor tyrosine kinase inhibition promotes apoptosis, blocks growth and enhances chemosensitivity of human non-small cell lung cancer.

    PubMed

    Linger, R M A; Cohen, R A; Cummings, C T; Sather, S; Migdall-Wilson, J; Middleton, D H G; Lu, X; Barón, A E; Franklin, W A; Merrick, D T; Jedlicka, P; DeRyckere, D; Heasley, L E; Graham, D K

    2013-07-18

    Non-small cell lung cancer (NSCLC) is a prevalent and devastating disease that claims more lives than breast, prostate, colon and pancreatic cancers combined. Current research suggests that standard chemotherapy regimens have been optimized to maximal efficiency. Promising new treatment strategies involve novel agents targeting molecular aberrations present in subsets of NSCLC. We evaluated 88 human NSCLC tumors of diverse histology and identified Mer and Axl as receptor tyrosine kinases (RTKs) overexpressed in 69% and 93%, respectively, of tumors relative to surrounding normal lung tissue. Mer and Axl were also frequently overexpressed and activated in NSCLC cell lines. Ligand-dependent Mer or Axl activation stimulated MAPK, AKT and FAK signaling pathways indicating roles for these RTKs in multiple oncogenic processes. In addition, we identified a novel pro-survival pathway-involving AKT, CREB, Bcl-xL, survivin, and Bcl-2-downstream of Mer, which is differentially modulated by Axl signaling. We demonstrated that short hairpin RNA (shRNA) knockdown of Mer or Axl significantly reduced NSCLC colony formation and growth of subcutaneous xenografts in nude mice. Mer or Axl knockdown also improved in vitro NSCLC sensitivity to chemotherapeutic agents by promoting apoptosis. When comparing the effects of Mer and Axl knockdown, Mer inhibition exhibited more complete blockade of tumor growth while Axl knockdown more robustly improved chemosensitivity. These results indicate that Mer and Axl have complementary and overlapping roles in NSCLC and suggest that treatment strategies targeting both RTKs may be more effective than singly-targeted agents. Our findings validate Mer and Axl as potential therapeutic targets in NSCLC and provide justification for development of novel therapeutic compounds that selectively inhibit Mer and/or Axl.

  6. Human Leukocyte Antigen G Polymorphism and Expression Are Associated with an Increased Risk of Non-Small-Cell Lung Cancer and Advanced Disease Stage

    PubMed Central

    Ben Amor, Amira; Beauchemin, Karine; Faucher, Marie-Claude; Hamzaoui, Agnes; Hamzaoui, Kamel; Roger, Michel

    2016-01-01

    Human leukocyte antigen (HLA)-G acts as negative regulator of the immune responses and its expression may enable tumor cells to escape immunosurveillance. The purpose of this study was to investigate the influence of HLA-G allelic variants and serum soluble HLA-G (sHLA-G) levels on risk of non-small-cell lung cancer (NSCLC). We analyzed 191 Caucasian adults with NSCLC and 191 healthy subjects recruited between January 2009 and March 2014 in Ariana (Tunisia). Serum sHLA-G levels were measured by immunoassay and HLA-G alleles were determined using a direct DNA sequencing procedures. The heterozygous genotypes of HLA-G 010101 and -G 010401 were associated with increased risks of both NSCLC and advanced disease stages. In contrast, the heterozygous genotypes of HLA-G 0105N and -G 0106 were associated with decreased risks of NSCC and clinical disease stage IV, respectively. Serum sHLA-G levels were significantly higher in patients with NSCLC and particularly in those with advanced disease stages compared to healthy subjects. The area under the receiver-operating characteristic (ROC) curves was 0.82 for controls vs patients. Given 100% specificity, the highest sensitivity achieved to detect NSCLC was 52.8% at a cutoff value of 24.9 U/ml. Patients with the sHLA-G above median level (≥ 50 U/ml) had a significantly shorter survival time. This study demonstrates that HLA-G allelic variants are independent risk factors for NSCLC. Serum sHLA-G levels in NSCLC patients could be useful biomarkers for the diagnostic and prognosis of NSCLC. PMID:27517300

  7. Mer or Axl Receptor Tyrosine Kinase Inhibition Promotes Apoptosis, Blocks Growth, and Enhances Chemosensitivity of Human Non-Small Cell Lung Cancer

    PubMed Central

    Linger, Rachel M.A.; Cohen, Rebecca A.; Cummings, Christopher T.; Sather, Susan; Migdall-Wilson, Justine; Middleton, Deryck H.G.; Lu, Xian; Barón, Anna E.; Franklin, Wilbur A.; Merrick, Daniel T.; Jedlicka, Paul; DeRyckere, Deborah; Heasley, Lynn E.; Graham, Douglas K.

    2012-01-01

    Non-small cell lung cancer (NSCLC) is a prevalent and devastating disease that claims more lives than breast, prostate, colon, and pancreatic cancers combined. Current research suggests that standard chemotherapy regimens have been optimized to maximal efficiency. Promising new treatment strategies involve novel agents targeting molecular aberrations present in subsets of NSCLC. We evaluated 88 human NSCLC tumors of diverse histology and identified Mer and Axl as receptor tyrosine kinases (RTKs) overexpressed in 69% and 93%, respectively, of tumors relative to surrounding normal lung tissue. Mer and Axl were also frequently overexpressed and activated in NSCLC cell lines. Ligand-dependent Mer or Axl activation stimulated MAPK, AKT, and FAK signaling pathways indicating roles for these RTKs in multiple oncogenic processes. In addition, we identified a novel pro-survival pathway—involving AKT, CREB, Bcl-xL, survivin, and Bcl-2—downstream of Mer, which is differentially modulated by Axl signaling. We demonstrated that shRNA knockdown of Mer or Axl significantly reduced NSCLC colony formation and growth of subcutaneous xenografts in nude mice. Mer or Axl knockdown also improved in vitro NSCLC sensitivity to chemotherapeutic agents by promoting apoptosis. When comparing the effects of Mer and Axl knockdown, Mer inhibition exhibited more complete blockade of tumor growth while Axl knockdown more robustly improved chemosensitivity. These results indicate that Mer and Axl play complementary and overlapping roles in NSCLC and suggest that treatment strategies targeting both RTKs may be more effective than singly-targeted agents. Our findings validate Mer and Axl as potential therapeutic targets in NSCLC and provide justification for development of novel therapeutic compounds that selectively inhibit Mer and/or Axl. PMID:22890323

  8. Overexpression of collagen triple helix repeat containing 1 (CTHRC1) is associated with tumour aggressiveness and poor prognosis in human non-small cell lung cancer.

    PubMed

    Ke, Zunfu; He, Weiling; Lai, Yuanhui; Guo, Xuefeng; Chen, Sharon; Li, Shuhua; Wang, Yuefeng; Wang, Liantang

    2014-10-15

    Collagen triple helix repeat-containing 1 (CTHRC1), a novel oncogene, was identified to be aberrantly overexpressed in several malignant tumors. However, the expression profile of CTHRC1 and its clinical significance in non-small cell lung cancer (NSCLC) remain unknown. In this study, we showed that CTHRC1 was evidently overexpressed in human NSCLC tissues and NSCLC cell lines at the protein and mRNA level. Ectopic up-regulation of CTHRC1 in cancer cells resulted in elevated invasive and proliferative abilities, which were attenuated by the specific CTHRC1 siRNA. The biological effect of CTHRC1 on metastasis and proliferation was mediated by the activation of the Wnt/β-catenin pathway. Furthermore, CTHRC1 immunoreactivity was evidently overexpressed in paraffin-embedded NSCLC tissues (212/292, 72.60%) in comparison to corresponding adjacent non-cancerous tissues (6/66, 9.09%) (p<0.001). Clinicopathologic analysis showed that CTHRC1 expression was significantly correlated with differentiation degree (p<0.001), clinical stage (p<0.001), T classification (p<0.001), lymph node metastasis (p=0.013) and distant metastasis (p<0.001). Kaplan-Meier analysis revealed that patients with high CTHRC1 expression had poorer overall survival rates than those with low CTHRC1 expression. Multivariate analysis indicated that CTHRC1 expression was an independent prognostic factor for the overall survival of NSCLC patients. Collectively, CTHRC1 plays important roles in NSCLC progression, and the evaluation of CTHRC1 expression could serve as a potential marker for metastasis progression and prognosis in NSCLC patients.

  9. Curcumin induces apoptosis in human non-small cell lung cancer NCI-H460 cells through ER stress and caspase cascade- and mitochondria-dependent pathways.

    PubMed

    Wu, Shin-Hwar; Hang, Liang-Wen; Yang, Jai-Sing; Chen, Hung-Yi; Lin, Hui-Yi; Chiang, Jo-Hua; Lu, Chi-Cheng; Yang, Jiun-Long; Lai, Tung-Yuan; Ko, Yang-Ching; Chung, Jing-Gung

    2010-06-01

    It has been reported that curcumin inhibited various types of cancer cells in vitro and in vivo. However, mechanisms of curcumin-inhibited cell growth and -induced apoptosis in human non-small cell lung cancer cells (NCI-H460) still remain unclear. In this study, NCI-H460 cells were treated with curcumin to determine its anticancer activity. Different concentrations of curcumin were used for different durations in NCI-H460 cells and the subsequent changes in the cell morphology, viability, cell cycle, mRNA and protein expressions were determined. Curcumin induced apoptotic morphologic changes in NCI-H460 cells in a dose-dependent manner. After curcumin treatment, BAX and BAD were up-regulated, BCL-2, BCL-X(L) and XIAP were down-regulated. In addition, reactive oxygen species (ROS), intracellular Ca(2+) and endoplasmic reticulum (ER) stress were increased in NCI-H460 cells after exposure to curcumin. These signals led to a loss of mitochondrial membrane potential (Delta Psi(m)) and culminated in caspase-3 activation. Curcumin-induced apoptosis was also stimulated through the FAS/caspase-8 (extrinsic) pathway and ER stress proteins, growth arrest- and DNA damage-inducible gene 153 (GADD153) and glucose-regulated protein 78 (GRP78) were activated in the NCI-H460 cells. Apoptotic cell death induced by curcumin was significantly reversed by pretreatment with ROS scavenger or caspase-8 inhibitor. Furthermore, the NCI-H460 cells tended to be arrested at the G(2)/M cell cycle stage after curcumin treatment and down-regulation of cyclin-dependent kinase 1 (CDK1) may be involved. In summary, curcumin exerts its anticancer effects on lung cancer NCI-H460 cells through apoptosis or cell cycle arrest.

  10. Human Leukocyte Antigen G Polymorphism and Expression Are Associated with an Increased Risk of Non-Small-Cell Lung Cancer and Advanced Disease Stage.

    PubMed

    Ben Amor, Amira; Beauchemin, Karine; Faucher, Marie-Claude; Hamzaoui, Agnes; Hamzaoui, Kamel; Roger, Michel

    2016-01-01

    Human leukocyte antigen (HLA)-G acts as negative regulator of the immune responses and its expression may enable tumor cells to escape immunosurveillance. The purpose of this study was to investigate the influence of HLA-G allelic variants and serum soluble HLA-G (sHLA-G) levels on risk of non-small-cell lung cancer (NSCLC). We analyzed 191 Caucasian adults with NSCLC and 191 healthy subjects recruited between January 2009 and March 2014 in Ariana (Tunisia). Serum sHLA-G levels were measured by immunoassay and HLA-G alleles were determined using a direct DNA sequencing procedures. The heterozygous genotypes of HLA-G 010101 and -G 010401 were associated with increased risks of both NSCLC and advanced disease stages. In contrast, the heterozygous genotypes of HLA-G 0105N and -G 0106 were associated with decreased risks of NSCC and clinical disease stage IV, respectively. Serum sHLA-G levels were significantly higher in patients with NSCLC and particularly in those with advanced disease stages compared to healthy subjects. The area under the receiver-operating characteristic (ROC) curves was 0.82 for controls vs patients. Given 100% specificity, the highest sensitivity achieved to detect NSCLC was 52.8% at a cutoff value of 24.9 U/ml. Patients with the sHLA-G above median level (≥ 50 U/ml) had a significantly shorter survival time. This study demonstrates that HLA-G allelic variants are independent risk factors for NSCLC. Serum sHLA-G levels in NSCLC patients could be useful biomarkers for the diagnostic and prognosis of NSCLC.

  11. MicroRNA-346 facilitates cell growth and metastasis, and suppresses cell apoptosis in human non-small cell lung cancer by regulation of XPC/ERK/Snail/E-cadherin pathway

    PubMed Central

    Sun, Cheng-Cao; Li, Shu-Jun; Yuan, Zhan-Peng; Li, De-Jia

    2016-01-01

    Determinants of growth and metastasis in cancer remain of great interest to define. MicroRNAs (miRNAs) have frequently emerged as tumor metastatic regulator by acting on multiple signaling pathways. Here we report the definition of miR-346 as a novel oncogenic microRNA that facilitates non-small cell lung cancer (NSCLC) cell growth and metastasis. XPC, an important DNA damage recognition factor in nucleotide excision repair was defined as a target for down-regulation by miR-346, functioning through direct interaction with the 3′-UTR of XPC mRNA. Blocking miR-346 by an antagomiR was sufficient to inhibit NSCLC cell growth and metastasis, an effect that could be phenol-copied by RNAi-mediated silencing of XPC. In vivo studies established that miR-346 overexpression was sufficient to promote tumor growth by A549 cells in xenografts mice, relative to control cells. Overall, our results defined miR-346 as an oncogenic miRNA in NSCLC, the levels of which contributed to tumor growth and invasive aggressiveness. PMID:27777383

  12. Nobiletin inhibits epithelial-mesenchymal transition of human non-small cell lung cancer cells by antagonizing the TGF-β1/Smad3 signaling pathway.

    PubMed

    Da, Chunli; Liu, Yuting; Zhan, Yiyi; Liu, Kai; Wang, Ruozheng

    2016-05-01

    Epithelial-mesenchymal transition (EMT) is a critical cellular process in cancer metastasis, during which epithelial polarized cells become motile mesenchymal cells. Since transforming growth factor-β (TGF-β) is a potent inducer of EMT, blocking of TGF-β/Smad signaling has become a promising cancer therapy. Nobiletin, a polymethoxy flavonoid from Citrus depressa, has been shown to be valuable for cancer treatment, yet the mechanism remains unclear. In the present study, lung adenocarcinoma A549 and H1299 cells were used to evaluate the effect of nobiletin on EMT induced by TGF-β1. Nobiletin successfully inhibited TGF-β1-induced EMT, migration, invasion and adhesion in vitro, accompanied by attenuation of MMP-2, MMP-9, p-Src, p-FAK, p-paxillin, Snail, Slug, Twist and ZEB1 expression. Nobiletin inhibited the transcriptional activity of Smads without changing the phosphorylation status or translocation of Smads induced by TGF-β1. Moreover, Smad3 is requisite in TGF-β1-stimulated EMT. Smad3 overexpression meaningfully impaired the ability of nobiletin to reverse TGF-β1-induced EMT. In vivo, nobiletin prohibited the growth of metastatic nodules in the lungs of nude mice. Moreover, nobiletin inhibited tumor growth and reversed EMT in mice bearing A549-Luc xenografts, as revealed by IVIS imaging and immunohistochemical analysis. Collectively, the data suggest that nobiletin prevents EMT by inactivating TGF-β1/Smad3 signaling.

  13. [Mechanism of Chlorogenic Acid in Apoptotic Regulation through Notch1 
Pathway in Non-small Cell Lung Carcinoma in Animal Level].

    PubMed

    Li, Wei; Liu, Xu; Zhang, Guoqian; Zhang, Linlin

    2017-08-20

    It has been proven that chlorogenic acids can produce anticancer effects by regulating cell cycle, inducing apoptosis, inhibiting cell growth, Notch signaling pathways are closely related to many human tumors. The aim of this study is to study the mechanism of chlorogenic acid on apoptosis of non-small lung cancer through Notch1 pathway in animal level, and hope to provide theory basis on clinical treatment and research aimed at targeting Notch1 signaling in non-small cell carcinoma (NSCLC). MTT assay was used to evaluate the A549 cell proliferation under the treatment of chlorogenic acid. The effect of chlorogenic acid on apoptotic and cell cycle were detected by flow cytometry. The animal model of A549 cell transplanted in nude was established, tumer size and weight were detected. The mRNA level of Notch1 signal pathway related facter were detected by RT-PCR; the expression of Notch1 signal pathway related facter in tumor tissue was detected by western blot. Chlorogenic acid inhibited the A549 cell proliferation. incresed cell apoptotic and cell percentagein G2/M (P<0.05), and in a dose-dependent manner. In animal model, tumer size and weight were lower than control group, the difference was statistically significant (P<0.05). The relative expression of mRNA of Notch1, VEGF, Delta4, HES1 and HEY1 were decreaced (P<0.05) in tumor tissue which treated with chlorogenic. The expression of Notch1 were decreaced, PTEN, p-PTEN, p-AKT were increced significantly in tumor tissue which treated with chlorogenic (P<0.05). Chlorogenic acid can regulate theapoptosis of non-small lung cancer through Notch pathway in animal level, which may be associated with the down-regulating the expression of VEGF and Delta4. Notch pathway may cross talk with PI3K/AKT pathway through PTEN in NSCLC.

  14. Ski prevents TGF-β-induced EMT and cell invasion by repressing SMAD-dependent signaling in non-small cell lung cancer.

    PubMed

    Yang, Haiping; Zhan, Lei; Yang, Tianjie; Wang, Longqiang; Li, Chang; Zhao, Jun; Lei, Zhe; Li, Xiangdong; Zhang, Hong-Tao

    2015-07-01

    Epithelial-mesenchymal transition (EMT) is a key event in cancer metastasis, which confers cancer cells with increased motility and invasiveness, and EMT is characterized by loss of epithelial marker E-cadherin and gain of mesenchymal marker N-cadherin. Transforming growth factor-β (TGF-β) signaling is a crucial inducer of EMT in various types of cancer. Ski is an important negative regulator of TGF-β signaling, which interacts with SMADs to repress TGF-β signaling activity. Although there is accumulating evidence that Ski functions as a promoter or suppressor in human types of cancer, the molecular mechanisms by which Ski affects TGF-β-induced EMT and invasion in non-small cell lung cancer (NSCLC) are not largely elucidated. In the present study, we investigated the mechanistic role of Ski in NSCLC metastasis. Ski was significantly reduced in metastatic NSCLC cells or tissues when compared with non-metastatic NSCLC cells or tissues. Moreover, following TGF-β stimulation Ski-silenced A549 cells had more significant features of EMT and a higher invasive activity when compared with A549 cells overexpressing Ski. Mechanistically, Ski-silenced and overexpressed A549 cells showed an increase and a reduction in the SMAD3 phosphorylation level, respectively. This was supported by plasminogen activator inhibitor-1 (PAI-1) promoter activity obtained in Ski-silenced and overexpressed A549 cells. However, after treatment of SIS3 (inhibitor of SMAD3 phosphorylation) followed by TGF-β1 stimulation, we did not observe any effect of Ski on TGF-β-induced EMT, and invasion in Ski-silenced and overexpressed A549 cells. In conclusion, our findings suggest that Ski represses TGF-β-induced EMT and invasion by inhibiting SMAD-dependent signaling in NSCLC.

  15. Hypoxia Potentiates the Radiation-Sensitizing Effect of Olaparib in Human Non-Small Cell Lung Cancer Xenografts by Contextual Synthetic Lethality.

    PubMed

    Jiang, Yanyan; Verbiest, Tom; Devery, Aoife M; Bokobza, Sivan M; Weber, Anika M; Leszczynska, Katarzyna B; Hammond, Ester M; Ryan, Anderson J

    2016-06-01

    Poly(ADP-ribose) polymerase (PARP) inhibitors potentiate radiation therapy in preclinical models of human non-small cell lung cancer (NSCLC) and other types of cancer. However, the mechanisms underlying radiosensitization in vivo are incompletely understood. Herein, we investigated the impact of hypoxia on radiosensitization by the PARP inhibitor olaparib in human NSCLC xenograft models. NSCLC Calu-6 and Calu-3 cells were irradiated in the presence of olaparib or vehicle under normoxic (21% O2) or hypoxic (1% O2) conditions. In vitro radiosensitivity was assessed by clonogenic survival assay and γH2AX foci assay. Established Calu-6 and Calu-3 subcutaneous xenografts were treated with olaparib (50 mg/kg, daily for 3 days), radiation (10 Gy), or both. Tumors (n=3/group) were collected 24 or 72 hours after the first treatment. Immunohistochemistry was performed to assess hypoxia (carbonic anhydrase IX [CA9]), vessels (CD31), DNA double strand breaks (DSB) (γH2AX), and apoptosis (cleaved caspase 3 [CC3]). The remaining xenografts (n=6/group) were monitored for tumor growth. In vitro, olaparib showed a greater radiation-sensitizing effect in Calu-3 and Calu-6 cells in hypoxic conditions (1% O2). In vivo, Calu-3 tumors were well-oxygenated, whereas Calu-6 tumors had extensive regions of hypoxia associated with down-regulation of the homologous recombination protein RAD51. Olaparib treatment increased unrepaired DNA DSB (P<.001) and apoptosis (P<.001) in hypoxic cells of Calu-6 tumors following radiation, whereas it had no significant effect on radiation-induced DNA damage response in nonhypoxic cells of Calu-6 tumors or in the tumor cells of well-oxygenated Calu-3 tumors. Consequently, olaparib significantly increased radiation-induced growth inhibition in Calu-6 tumors (P<.001) but not in Calu-3 tumors. Our data suggest that hypoxia potentiates the radiation-sensitizing effects of olaparib by contextual synthetic killing, and that tumor hypoxia may be a

  16. Enhanced Replication of Hepatitis E Virus Strain 47832c in an A549-Derived Subclonal Cell Line.

    PubMed

    Schemmerer, Mathias; Apelt, Silke; Trojnar, Eva; Ulrich, Rainer G; Wenzel, Jürgen J; Johne, Reimar

    2016-09-29

    Hepatitis E virus (HEV) is a human pathogen with increasing importance. The lack of efficient cell culture systems hampers systematic studies on its replication cycle, virus neutralization and inactivation. Here, several cell lines were inoculated with the HEV genotype 3c strain 47832c, previously isolated from a chronically infected transplant patient. At 14 days after inoculation the highest HEV genome copy numbers were found in A549 cells, followed by PLC/PRF/5 cells, whereas HepG2/C3A, Huh-7 Lunet BLR and MRC-5 cells only weakly supported virus replication. Inoculation of A549-derived subclone cell lines resulted in most cases in reduced HEV replication. However, the subclone A549/D3 was susceptible to lower virus concentrations and resulted in higher virus yields as compared to parental A549 cells. Transcriptome analysis indicated a downregulation of genes for carcinoembryonic antigen-related cell adhesion molecules (CEACAM) 5 and 6, and an upregulation of the syndecan 2 (SDC2) gene in A549/D3 cells compared to A549 cells. However, treatment of A549/D3 cells or A549 cells with CEACAM- or syndecan 2-specific antisera did not influence HEV replication. The results show that cells supporting more efficient HEV replication can be selected from the A549 cell line. The specific mechanisms responsible for the enhanced replication remain unknown.

  17. Enhanced Replication of Hepatitis E Virus Strain 47832c in an A549-Derived Subclonal Cell Line

    PubMed Central

    Schemmerer, Mathias; Apelt, Silke; Trojnar, Eva; Ulrich, Rainer G.; Wenzel, Jürgen J.; Johne, Reimar

    2016-01-01

    Hepatitis E virus (HEV) is a human pathogen with increasing importance. The lack of efficient cell culture systems hampers systematic studies on its replication cycle, virus neutralization and inactivation. Here, several cell lines were inoculated with the HEV genotype 3c strain 47832c, previously isolated from a chronically infected transplant patient. At 14 days after inoculation the highest HEV genome copy numbers were found in A549 cells, followed by PLC/PRF/5 cells, whereas HepG2/C3A, Huh-7 Lunet BLR and MRC-5 cells only weakly supported virus replication. Inoculation of A549-derived subclone cell lines resulted in most cases in reduced HEV replication. However, the subclone A549/D3 was susceptible to lower virus concentrations and resulted in higher virus yields as compared to parental A549 cells. Transcriptome analysis indicated a downregulation of genes for carcinoembryonic antigen-related cell adhesion molecules (CEACAM) 5 and 6, and an upregulation of the syndecan 2 (SDC2) gene in A549/D3 cells compared to A549 cells. However, treatment of A549/D3 cells or A549 cells with CEACAM- or syndecan 2-specific antisera did not influence HEV replication. The results show that cells supporting more efficient HEV replication can be selected from the A549 cell line. The specific mechanisms responsible for the enhanced replication remain unknown. PMID:27690085

  18. Identification of microRNA profiles in docetaxel-resistant human non-small cell lung carcinoma cells (SPC-A1)

    PubMed Central

    Rui, Wang; Bing, Feng; Hai-Zhu, Song; Wei, De; Long-Bang, Chen

    2010-01-01

    Abstract Docetaxel has been used as first-line chemotherapy in advanced non-small cell lung carcinoma (NSCLC), but further extensive and effective application is prevented by drug resistance. MicroRNAs (miRNAs) have recently been identified as important posttranscriptional regulators, which are involved in various biological processes. The aim of this study was to identify microRNA expression profiles involved in the development of docetaxel resistance in NSCLC. Here, microarray chip technology was employed to identify miRNA expression profiles in docetaxel-resistant human NSCLC cell line (SPC-A1/docetaxel). Then, the changes of miRNAs expression (>2-fold compared with control SPC-A1 cell line) were testified by quantitative real-time RT-PCR (qRT-PCR) assay. Furthermore, the potential target genes regulated by selected miRNAs were analysed by various target prediction tools. The expression of a total of 52 miRNAs showed significant difference between SPC-A1/docetaxel cells and control SPC-A1 cells (P < 0.01). Six miRNAs (miR-192, 200b, 194, 424, 98 and 212) exhibited more than 2-fold changes in their expression levels, which were validated by qRT-PCR. The expression of three miRNAs (miR-200b, 194 and 212) was significantly down-regulated in SPC-A1/docetaxel cells, while the expression of other three miRNAs (miR-192, 424 and 98) was significantly up-regulated in SPC-A1/docetaxel cells (P < 0.01). Potential target genes controlled by six selected miRNAs were divided into four groups according to various functions: apoptosis and proliferation (71 genes), cell cycle (68 genes), DNA damage (26 genes) and DNA repair (59 genes). The expression of a few target genes in SPC-A1/docetaxel and SPC-A1 cells were further confirmed by qRT-PCR and Western blot. Taken together, the identification of microRNA expression profiles in docetaxel-resistant NSCLC cells could provide a better understanding of mechanisms involved in drug sensitivity or resistance, which would be helpful to

  19. Identification of microRNA profiles in docetaxel-resistant human non-small cell lung carcinoma cells (SPC-A1).

    PubMed

    Rui, Wang; Bing, Feng; Hai-Zhu, Song; Wei, De; Long-Bang, Chen

    2010-01-01

    Docetaxel has been used as first-line chemotherapy in advanced non-small cell lung carcinoma (NSCLC), but further extensive and effective application is prevented by drug resistance. MicroRNAs (miRNAs) have recently been identified as important posttranscriptional regulators, which are involved in various biological processes. The aim of this study was to identify microRNA expression profiles involved in the development of docetaxel resistance in NSCLC. Here, microarray chip technology was employed to identify miRNA expression profiles in docetaxel-resistant human NSCLC cell line (SPC-A1/docetaxel). Then, the changes of miRNAs expression (>2-fold compared with control SPC-A1 cell line) were testified by quantitative real-time RT-PCR (qRT-PCR) assay. Furthermore, the potential target genes regulated by selected miRNAs were analysed by various target prediction tools. The expression of a total of 52 miRNAs showed significant difference between SPC-A1/docetaxel cells and control SPC-A1 cells (P < 0.01). Six miRNAs (miR-192, 200b, 194, 424, 98 and 212) exhibited more than 2-fold changes in their expression levels, which were validated by qRT-PCR. The expression of three miRNAs (miR-200b, 194 and 212) was significantly down-regulated in SPC-A1/docetaxel cells, while the expression of other three miRNAs (miR-192, 424 and 98) was significantly up-regulated in SPC-A1/docetaxel cells (P < 0.01). Potential target genes controlled by six selected miRNAs were divided into four groups according to various functions: apoptosis and proliferation (71 genes), cell cycle (68 genes), DNA damage (26 genes) and DNA repair (59 genes). The expression of a few target genes in SPC-A1/docetaxel and SPC-A1 cells were further confirmed by qRT-PCR and Western blot. Taken together, the identification of microRNA expression profiles in docetaxel-resistant NSCLC cells could provide a better understanding of mechanisms involved in drug sensitivity or resistance, which would be helpful to develop

  20. Leptin promotes metastasis by inducing an epithelial-mesenchymal transition in A549 lung cancer cells.

    PubMed

    Feng, Helin; Liu, Qingyi; Zhang, Ning; Zheng, Lihua; Sang, Meixiang; Feng, Jiangang; Zhang, Jinming; Wu, Xiangyun; Shan, Baoen

    2013-01-01

    Leptin, an adipocyte-derived cytokine associated with obesity, has been reported to participate in carcinogenesis. Epithelial-mesenchymal transition (EMT) is also considered as a key event in tumor metastasis. The aim of this study is to investigate the mechanism of leptin in the promotion of EMT leading to metastasis in A549 lung cancer cells. We investigated the effect of leptin on migration of A549 cells using wound healing and transwell assays. The incidence of EMT in A549 cells was examined by real-time PCR and immunofluorescence staining. The expression of TGF-β in A549 cells was detected by real-time PCR, and blocking of TGF-β in A549 cells was achieved by siRNA techniques. Additional work was performed using 100 patient samples, which included samples from 50 patients diagnosed with lung cancer and an additional 50 patients diagnosed with lung cancer with metastatic bone lesions. Leptin expression was measured using immunohistochemistry techniques. We demonstrated that leptin can effectively enhance the metastasis of human lung cancer A549 cell line using both wound healing and transwell assays. We also found the incidence of EMT in A549 cells after leptin exposure. Furthermore, we detected the expression of TGF-β in A549 cells, which had been reported to play an important role in inducing EMT. We showed that leptin can significantly upregulate TGF-β at both the mRNA and protein levels in A549 cells. Using siRNA to block the expression of TGF-β in A549 cells, we confirmed the role of TGF-β in the promotion of metastasis and induction of EMT. Furthermore, we found that in patient samples leptin was present at higher levels in samples associated with diagnosis of lung cancer bone metastases tissue than lung cancer tissue. Our results indicated that leptin promoted the metastasis of A549 human lung cancer cell lines by inducing EMT in a TGF-β-dependent manner.

  1. The influence of ciprofloxacin on viability of A549, HepG2, A375.S2, B16 and C6 cell lines in vitro.

    PubMed

    Kloskowski, Tomasz; Gurtowska, Natalia; Nowak, Monika; Joachimiak, Romana; Bajek, Anna; Olkowska, Joanna; Drewa, Tomasz

    2011-01-01

    Ciprofloxacin is a chemotherapeutic agent mainly used in the treatment of the pulmonary and urinary tract infections but is also known for its anticancer properties. The aim of these study was to check the anticancer effect of ciprofloxacin on selected five cell lines. Human non-small cell lung cancer line A549, human hepatocellular carcinoma line HepG2, human and mouse melanoma lines (A375.S2 and B16) and rat glioblastoma line C6 were used for evaluation of cytotoxic properties of ciprofloxacin (in concentration range: 10-1000 microg/mL). Viability was established using trypan blue assay and MTT. Ciprofloxacin induced morphological changes and decreased viability of A549 cells in a concentration and time dependent manner. In case of A375.S2 and B16 cell lines, cytotoxicyty of ciprofloxacin was observed but we were not able to eradicate all cells from A375.S2 and B16 cultures. HepG2 line was sensitive to ciprofloxacin, but this effect was independent from concentration and incubation time. The C6 cells were insensitive to ciprofloxacin. Our results showed that ciprofloxacin can be potentially used for the experimental adjunctive therapy of lung cancer.

  2. Rab27A regulates exosome secretion from lung adenocarcinoma cells A549: involvement of EPI64.

    PubMed

    Li, Wenhai; Hu, Yunsheng; Jiang, Tao; Han, Yong; Han, Guoliang; Chen, Jiakuan; Li, Xiaofei

    2014-11-01

    Exosomes are small membrane vesicles secreted into the extracellular compartment by exocytosis. The unique composition of exosomes can be transported to other cells which allow cells to exert biological functions at distant sites. However, in lung cancer, the regulation of exosome secretion was poorly understood. In this study, we employed human lung adenocarcinoma A549 cells to determine the exosome secretion and involved regulation mechanism. We found that Rab27A was expressed in A549 cells and the reduction of Rab27A by Rab27A-specific shRNA could significantly decrease the secretion of exosome by A549 cells. EPI64, a candidate GAP that is specific for Rab27, was also detected in A549 cells. By pull-down assay, we found that EPI64 participated in the exosome secretion of A549 cells by acting as a specific GAP for Rab27A, not Rab27B. Overexpression of EPI64 enhanced exosome secretion. Taken together, in A549 cells, EPI64 could regulate the exosome secretion by functioning as a GAP specific for Rab27A.

  3. Role of gambogic acid and NaI131 in A549/DDP cells

    PubMed Central

    Huang, Jing; Zhu, Xiaoli; Wang, Huan; Han, Shuhua; Liu, Lu; Xie, Yan; Chen, Daozhen; Zhang, Qiang; Zhang, Li; Hu, Yue

    2017-01-01

    Resistance to platinum in tumor tissue is a considerable barrier against effective lung cancer treatment. Radionuclide therapy is the primary adjuvant treatment, however, the toxic side effects limit its dosage in the clinical setting. Therefore, the present study aimed to determine whether an NaI131 radiosensitizer could help reduce the toxic side effects of radionuclide therapy. In vitro experiments were conducted to determine whether NaI131 can inhibit platinum resistance in A549/DDP cells, which are cisplatin-resistant non-small cell lung cancer cells, and whether gambogic acid (GA) is an effective NaI131 radiosensitizer. Cell proliferation following drug intervention was analyzed using MTT and isobolographic analysis. Apoptosis was assessed by flow cytometry. In addition, the mechanisms of drug intervention were analyzed by measuring the expression of P-glycoprotein (P-gP), B cell lymphoma 2 (Bcl-2), Bcl2-associated X protein (Bax) and P53 using western blot analysis and immunocytochemistry. According to isobolographic analysis, a low concentration of NaI131 combined with GA had a synergistic effect on the inhibition of A549/DDP cell proliferation, which was consistent with an increased rate of apoptosis. Furthermore, the overexpression of Bax, and the downregulation of P-gP, P53 and Bcl-2 observed demonstrated the potential mechanism(s) of NaI131 and GA intervention. NaI131 may induce apoptosis in A549/DDP cells by regulating apoptosis-related proteins. A low concentration combination of NaI131 and GA was able to significantly inhibit A549/DDP cell proliferation and induce cell apoptosis. Thus, the two drugs appear to have a synergistic effect on apoptosis of A549/DDP cells. PMID:28123519

  4. Open reading frame 3 of genotype 1 hepatitis E virus inhibits nuclear factor-κappa B signaling induced by tumor necrosis factor-α in human A549 lung epithelial cells.

    PubMed

    Xu, Jian; Wu, Fan; Tian, Deying; Wang, Jingjing; Zheng, Zizheng; Xia, Ningshao

    2014-01-01

    Hepatitis E virus (HEV) is one of the primary causative agents of acute hepatitis, and represents a major cause of severe public health problems in developing countries. The pathogenesis of HEV is not well characterized, however, primarily due to the lack of well-defined cell and animal models. Here, we investigated the effects of genotype 1 HEV open reading frame 3 (ORF3) on TNF-α-induced nucleus factor-κappa B (NF-κB) signaling. Human lung epithelial cells (A549) were transiently transfected with ORF3 containing plasmids. These cells were then stimulated with TNF-α and the nucleus translocation of the p65 NF-κB subunit was assessed using western blot and laser confocal microscopy. DNA-binding activity of p65 was also examined using electrophoretic mobility shift assay (EMSA), and the suppression of NF-κB target genes were detected using real-time RT-PCR and ELISA. These results enabled us to identify the decreased phosphorylation levels of IKBα. We focused on the gene of negative regulation of NF-κB, represented by TNF-α-induced protein 3 (TNFAIP3, also known as A20). Reducing the levels of A20 with siRNAs significantly enhances luciferase activation of NF-κB. Furthermore, HEV ORF3 regulated A20 primarily via activating transcription factor 6 (ATF6), involved in unfolded protein response (UPR), resulting in the degradation or inactivation of the receptor interacting protein 1 (RIP1), a major upstream activator of IKB kinase compounds (IKKs). Consequently, the phosphorylation of IKBα and the nucleus translocation of p65 are blocked, which contributes to diminished NF-κB DNA-binding activation and NF-κB-dependent gene expression. The findings suggest that genotype 1 HEV, through ORF3, may transiently activate NF-κB through UPR in early stage, and subsequently inhibit TNF-α-induced NF-κB signaling in late phase so as to create a favorable virus replication environment.

  5. Three tumor-suppressor regions on chromosome 11p identified by high-resolution deletion mapping in human non-small-cell lung cancer

    SciTech Connect

    Bepler, G.; Garcia-Blanco, A. )

    1994-06-07

    Non-small-cell lung cancer is the leading cause of cancer death for men and women in the industrialized nations. Identification of regions for genes involved in its pathogenesis has been difficult. Data presented here show three distinct regions identified on chromosome 11p. Two regions on 11p13 distal to the Wilms tumor gene WT1 and on 11p15.5 between the markers HBB and D11S860 are described. The third region on the telomere of 11p15.5 has been previously described and is further delineated in this communication. By high-resolution mapping the size of each of these regions was estimated to be 2-3 megabases. The frequency of somatic loss of genetic information in these regions (57%, 71%, and 45%, respectively) was comparable to that seen in heritable tumors such as Wilms tumor (55%) and retinoblastoma (70%) and suggests their involvement in pathogenesis of non-small-cell lung cancer. Gene dosage analyses revealed duplication of the remaining allele in the majority of cases in the 11p13 and the proximal 11p15.5 region but rarely in the distal 11p15.5 region. In tumors with loss of heterozygosity in all three regions any combination of duplication or simple deletion was observed, suggesting that loss of heterozygosity occurs independently and perhaps at different points in time. These results provide a basis for studies directed at cloning potential tumor-suppressor genes in these regions and for assessing their biological and clinical significance in non-small-cell lung cancer.

  6. Liver X receptors agonist T0901317 downregulates matrix metalloproteinase-9 expression in non-small-cell lung cancer by repressing nuclear factor-κB.

    PubMed

    Chen, Qiong-Ju; Shi, Ying; Shi, Jun-Feng; Yuan, Zhen-Hua; Ma, Ji-Yong; Fang, Su-Rong; Gu, Wei

    2017-10-01

    The liver X receptors (LXRs) is an important component of the nuclear receptor (NR) superfamily. Previous studies have shown that the LXRs possessed antitumor activity in various types of tumor cells. However, the complicated mechanisms underlying the antitumor activity remain largely unexplored. In this study, we incubated A549 cells with the compound T0901317, a specific LXRs agonist, for 24 h. The MTT assay was used to assess cell viability. Transwell assays were used to evaluate cell migration and invasion. The shRNA was utilized for RNA interference. The target gene and protein expression levels were assessed using reverse transcription-PCR and western blot assay. The DNA-binding activity of nuclear factor κB (NF-κB) was examined using electrophoretic mobility shift assays. Luciferase reporter assay was used to detect the binding of NF-κB to the matrix metalloproteinase-9 (MMP-9) promoter. We found that T0901317 inhibited the invasion and migration of A549 cells in a dose-dependent manner. Meanwhile, we further indicated that activation of LXRβ, one subtype of LXRs, can downregulate MMP-9 expression. More importantly, activation of LXRβ triggered by T0901317 inhibited the invasion and metastasis of A549 cells by repressing NF-κB/MMP-9 signaling pathway. Taken together, our study shows that activation of LXRs triggered by T0901317 inhibits the invasion and metastasis of human non-small-cell lung cancer by repressing the NF-κB/MMP-9 signaling pathway.

  7. Green tea inhibits cycolooxygenase-2 in non-small cell lung cancer cells through the induction of Annexin-1.

    PubMed

    Lu, Qing-Yi; Jin, Yusheng; Mao, Jenny T; Zhang, Zuo-Feng; Heber, David; Dubinett, Steven M; Rao, Jianyu

    2012-11-02

    Elevated cyclooygenase-2 (COX-2) expression is frequently observed in human non-small cell lung cancer (NSCLC) and associated with poor prognosis, indicating critical involvement of the inflammatory pathway in lung carcinogenesis. Recently, we found that green tea extract (GTE) induced Annexin-1 (ANX1) in the lung adenocarcinoma A549 cells. ANX1 is a glucocorticoid-inducible 37kDa protein involved in a wide range biological function and is an important anti-inflammatory mediator. The present study further examines the interplay between the expressions and production of ANX1, COX-2, phospholipase A(2) (cPLA(2)) and prostaglandin E(2) (PGE(2)) following the treatment of NSCLC cell lines with GTE. We found that GTE induced ANX1 and inhibited COX-2 expression in lung cancer A549, H157 and H460 cell lines. Addition of pro-inflammatory cytokine IL-1β diminished GTE-induced ANX1. Silence of ANX1 in cells abrogates the inhibitory activity on COX-2, indicating that the anti-inflammatory activity of GTE is mediated at least partially by the up-regulation of ANX1. However, differential pattern of inhibitory effects of ANX1 on cPLA(2) expression was observed among various cell types, suggesting that the anti-inflammatory activity mediated by ANX1 is cell type specific. Our study may provide a new mechanism of GTE on the prevention of lung cancer and other diseases related to inflammation.

  8. Downregulated TIPE2 is associated with poor prognosis and promotes cell proliferation in non-small cell lung cancer

    SciTech Connect

    Li, Yuexia; Li, Xiaohui; Liu, Gang; Sun, Rongqing; Wang, Lirui; Wang, Jing; Wang, Hongmin

    2015-01-30

    Highlights: • TIPE2 is down-regulated in NSCLC tissues. • TIPE2 inhibits NSCLC cell proliferation, colony formation and invasion. • TIPE2 reduces the anti-apoptotic Bcl-XL protein and mesenchymal marker N-cadherin expression. - Abstract: The present study aims to investigate the expression pattern of TIPE2 protein and its clinical significance in human non-small cell lung cancer (NSCLC). We investigated the expression levels of TIPE2 in 96 NSCLC tumor samples by immunohistochemistry and then analyzed its clinical significance. Furthermore, the role of TIPE2 on the biological properties of the NSCLC cell line H1299 and A549 was experimentally tested in vitro and in vivo. We found that the expression level of TIPE2 was significantly higher in normal lung tissues compared with NSCLC tissues (P < 0.001), and TIPE2 downregulation was significantly correlated with advanced TNM stage (P = 0.006). TIPE2 expression was lower in lung cancer cell lines than normal bronchial cell line HBE. Transfection of TIPE2 plasmid was performed in H1299 and A549 cells. TIPE2 overexpression inhibited lung cancer cell proliferation, colony formation and cell invasive in vitro, and prevented lung tumor growth in vivo. In addition, TIPE2 transfection reduced the anti-apoptotic Bcl-XL protein and mesenchymal marker N-cadherin expression. Taken together, our results demonstrate that TIPE2 might serve as a tumor suppressor in NSCLC progression.

  9. HCRP1 is downregulated in non-small cell lung cancer and regulates proliferation, invasion, and drug resistance.

    PubMed

    Du, Yaming; Wang, Peng; Sun, Hongzhi; Yang, Jing; Lang, Xianping; Wang, Zhongbin; Zang, Sheng; Chen, Lei; Ma, Junjun; Sun, Daohan

    2016-10-13

    HCRP1 has been reported to have tumor suppressive function. However, its expression pattern and function in human non-small cell lung cancer (NSCLC) remain obscure. This study aims to explore clinical significance of HCRP1 in NSCLC. Immunohistochemical results showed high HCRP1 protein in normal bronchial epithelial tissue and downregulated HCRP1 expression in 47/98 lung cancer specimens. HCRP1 downregulation correlated with clinical stage (p = 0.0203), nodal status (p = 0.0168), and poor patient prognosis (log-rank, p = 0.0076). Univariate analysis showed that TNM stage (p < 0.0001) and HCRP1 (p = 0.0098) were significant prognostic factors; Cox regression model showed that TNM stage serves as an independent prognostic factor (p = 0.0011). We also found that HCRP1 was downregulated in lung cancer cells compared with normal HBE cells. HCRP1 plasmid transfection in H1299 cells inhibited proliferation, cell cycle progression, and invasion. HCRP1 depletion in A549 cells showed the opposite biological effects. In addition, we found that HCRP1 could inhibit MAPK and AKT signaling with downregulation of ERK and AKT phosphorylation, cyclin proteins, Bcl2 and MMP9, while HCRP1 depletion activated ERK and AKT signaling. The level of EGFR phosphorylation was also inhibited by HCRP1. In addition, we found that HCRP1 depletion confers multidrug resistance in H1299 cells. We employed paclitaxel and cisplatin in A549 cells with HCRP1 depletion. HCRP1 depletion decreased the effect of paclitaxel and cisplatin in A549 cells. Treatment with EGFR inhibitor AG1478 and AKT inhibitor LY249004 abolished the effect of HCRP1 depletion on drug resistance. In conclusion, the present study demonstrate that HCRP1 is downregulated in NSCLC and regulates proliferation, invasion, and drug resistance through modulation of EGFR signaling.

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

    PubMed

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

    2013-07-01

    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. 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 phase-contrast 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. Deoxyelephantopin exhibited cytotoxicity to A549 cells (IC50 = 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

  11. Anticancer effects of novel thalidomide analogs in A549 cells through inhibition of vascular endothelial growth factor and matrix metalloproteinase-2.

    PubMed

    El-Aarag, Bishoy; Kasai, Tomonari; Masuda, Junko; Agwa, Hussein; Zahran, Magdy; Seno, Masaharu

    2017-01-01

    Lung cancer is one of the major causes of cancer-related mortality worldwide, and non-small-cell lung cancer is the most common form of lung cancer. Several studies had shown that thalidomide has potential for prevention and therapy of cancer. Therefore, the current study aimed to investigate the antitumor effects of two novel thalidomide analogs in human lung cancer A549 cells. The antiproliferative, antimigratory, and apoptotic effects in A549 cells induced by thalidomide analogs were examined. In addition, their effects on the expression of mRNAs encoding vascular endothelial growth factor165 (VEGF165) and matrix metalloproteinase-2 (MMP-2) were evaluated. Their influence on the tumor volume in nude mice was also determined. Results revealed that thalidomide analogs exhibited antiproliferative, antimigratory, and apoptotic activities with more pronounced effect than thalidomide drug. Furthermore, analogs 1 and 2 suppressed the expression levels of VEGF165 by 42% and 53.2% and those of MMP-2 by 45% and 52%, respectively. Thalidomide analogs 1 and 2 also reduced the tumor volume by 30.11% and 53.52%, respectively. Therefore, this study provides evidence that thalidomide analogs may serve as a new therapeutic option for treating lung cancer.

  12. Enhanced DNA double-strand break repair of microbeam targeted A549 lung carcinoma cells by adjacent WI38 normal lung fibroblast cells via bi-directional signaling.

    PubMed

    Kobayashi, Alisa; Tengku Ahmad, Tengku Ahbrizal Farizal; Autsavapromporn, Narongchai; Oikawa, Masakazu; Homma-Takeda, Shino; Furusawa, Yoshiya; Wang, Jun; Konishi, Teruaki

    2017-10-01

    Understanding the mechanisms underlying the radiation-induced bystander effect (RIBE) and bi-directional signaling between irradiated carcinoma cells and their surrounding non-irradiated normal cells is relevant to cancer radiotherapy. The present study investigated propagation of RIBE signals between human lung carcinoma A549 cells and normal lung fibroblast WI38 cells in bystander cells, either directly or indirectly contacting irradiated A549 cells. We prepared A549-GFP/WI38 co-cultures and A549-GFP/A549 co-cultures, in which A549-GFP cells stably expressing H2BGFP were co-cultured with either A549 cells or WI38 cells, respectively. Using the SPICE-NIRS microbeam, only the A549-GFP cells were irradiated with 500 protons per cell. The level of γ-H2AX, a marker for DNA double-strand breaks (DSB), was subsequently measured for up to 24h post-irradiation in three categories of cells: (1) "targeted"/irradiated A549-GFP cells; (2) "neighboring"/non-irradiated cells directly contacting the "targeted" cells; and (3) "distant"/non-irradiated cells, which were not in direct contact with the "targeted" cells. We found that DSB repair in targeted A549-GFP cells was enhanced by co-cultured WI38 cells. The bystander response in A549-GFP/A549 cell co-cultures, as marked by γ-H2AX levels at 8h post-irradiation, showed a decrease to non-irradiated control level when approaching 24h, while the neighboring/distant bystander WI38 cells in A549-GFP/WI38 co-cultures was maintained at a similar level until 24h post-irradiation. Surprisingly, distant A549-GFP cells in A549-GFP/WI38 co-cultures showed time dependency similar to bystander WI38 cells, but not to distant cells in A549-GFP/A549 co-cultures. These observations indicate that γ-H2AX was induced in WI38 cells as a result of RIBE. WI38 cells were not only involved in rescue of targeted A549, but also in the modification of RIBE against distant A549-GFP cells. The present results demonstrate that radiation-induced bi

  13. Elevated pressure, a novel cancer therapeutic tool for sensitizing cisplatin-mediated apoptosis in A549

    SciTech Connect

    Oh, Sangnam; Kim, Yanghee; Kim, Joonhee; Kwon, Daeho; Lee, Eunil

    2010-08-13

    Research highlights: {yields} Sensitized apoptosis in cancer cells stimulated by EP precondition with p53 dependence. {yields} EP attenuates several CDDP-resistance mechanisms. {yields} No harmful effect of EP on normal fibroblasts. -- Abstract: Intensive cancer therapy strategies have thus far focused on sensitizing cancer cells to anticancer drug-mediated apoptosis to overcome drug resistance, and this strategy has led to more effective cancer therapeutics. Cisplatin (cis-diamminedichloroplatinum(II), CDDP) is an effective anticancer drug used to treat many types of cancer, including non-small cell lung carcinoma (NSCLC), and can be used in combination with various chemicals to enhance cancer cell apoptosis. Here, we introduce the use of elevated pressure (EP) in combination with CDDP for cancer treatment and explore the effects of EP on CDDP-mediated apoptosis in NSCLC cells. Our findings demonstrate that preconditioning NSCLC cells with EP sensitizes cells for CDDP-induced apoptosis. Enhanced apoptosis was dependent on p53 and HO-1 expression, and was associated with increased DNA damage and down-regulation of genes involved in nucleotide excision repair. The transcriptional levels of transporter proteins indicated that the mechanism by which EP-induced CDDP sensitization was intracellular drug accumulation. The protein levels of some antioxidants, such as hemeoxygenase-1 (HO-1), glutathione (GSH) and glutathione peroxidase (Gpx), were decreased in A549 cells exposed to EP via the down-regulation of the transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf-2). Furthermore, normal human fibroblasts were resistant to EP treatment, with no elevated DNA damage or apoptosis. Collectively, these data show that administration of EP is a potential adjuvant tool for CDDP-based chemosensitivity of lung cancer cells that may reduce drug resistance.

  14. Metuzumab enhanced chemosensitivity and apoptosis in non-small cell lung carcinoma

    PubMed Central

    Feng, Fei; Wang, Bin; Sun, Xiuxuan; Zhu, Yumeng; Tang, Hao; Nan, Gang; Wang, Lijuan; Wu, Bo; Huhe, Muren; Liu, Shuangshuang; Diao, Tengyue; Hou, Rong; Zhang, Yang; Zhang, Zheng

    2017-01-01

    ABSTRACT Targeted therapeutics is used as an alternative treatment of non-small cell lung cancer (NSCLC); however, treatment effect is far from being satisfactory, and therefore identification of new targets is needed. We have previously shown that metuzumab inhibit tumor growth in vivo. The present study was performed to investigate the anti-tumor efficacy of metuzumab combined with gemcitabine and cisplatin (GP), paclitaxel and cisplatin (TP) or navelbine and cisplatin (NP) regimens in multiple NSCLC cell lines. Our results demonstrate that, in comparison to single agent metuzumab or GP treated cells, metuzumab combined with GP display inhibitory effects on tumor growth. Furthermore, we found that metuzumab elevated the sensitivity of cell lines to gemcitabine, which was identified by MTT assay. Flow cytometric analysis showed that metuzumab combined with gemcitabine (GEM) treatment led to an obvious G1 arrest and an elevated apoptosis in A549, NCI-H460 and NCI-H520 cells. Western blot analysis also demonstrated a significantly reduced level of cyclin D1, Bcl-2, and an obviously increase level of Bax and full-length caspase-3 in A549, NCI-H460 and NCI-H520 cells treated with metuzumab/gemcitabine combination in comparison with single agent treated cells. In addition, metuzumab/gemcitabine treated A549, NCI-H460 and NCI-H520 cells also demonstrated a significantly increase in deoxycytidine kinase (dCK) protein level compared with single agent metuzumab or gemcitabine treated cells. Xenograft models also demonstrated that this metuzumab/gemcitabine combination led to upregulation of dCK. Taken together, the mechanisms of metuzumab combined with GP repress tumor growth were that the combined treatment significantly inhibited the tumor cell proliferation, apoptosis and cell cycle in vitro and in vivo and at least partially by induction of dCK expression. Our results suggested that metuzumab could significantly enhance chemosensitivity of human NSCLC cells to

  15. Role of microRNA-4458 in patients with non-small-cell lung cancer

    PubMed Central

    Bao, Lidao; Wang, Linlin; Wei, Guomin; Wang, Yuehong; Wuyun, Gerile; Bo, Agula

    2016-01-01

    Incidence and progression of non-small-cell lung cancer (NSCLC) is a multi-factor, multi-step process. The present study investigated the association between the expression level of microRNA (miR)-4458 in NSCLC and paracarcinoma liver tissues and survival rates, and studied the biological functions of miR-4458 at the cellular and protein level. NSCLC and paracarcinoma tissues were sequenced using a miR expression chip. The association between miR-4458 expression and tumor-node-metastasis staging, total survival rate and relapse-free survival rate was analyzed. miR-4458 was subjected to target gene prediction. The target protein of cyclin D1 (CCND1) was verified with western blot analysis, immunohistochemistry and a luciferase reporter assay. The relative level of miR-4458 in paracarcinoma tissues of 9 NSCLC patients decreased from 2.38 to 0.65 (P<0.001). Total five-year survival rates of the high-expression miR-4458 group (29.21%) significantly exceeded that of the low-expression group (14.37%) (P=0.025). The viability of human lung carcinoma A549 and H460 cells transfected with miR-4458 decreased significantly compared with cells transfected with a normal control (blank control plasmid) within 72 h (P<0.001). The percentage of A549 and H460 cells transfected with a miR-4458 mimic at the cell cycle stage G0/G1 was 69.94±8.05 and 68.15±7.75%, respectively. The percentages increased significantly compared with the control group (46.06±6.93 for A549 cells; 45.22±7.24 for H640 cells; P<0.001). CCND1 mRNA was downregulated significantly in H460 cells 72 h subsequent to the addition of miR-4458 mimics (P<0.001). The activity of mutant-CCND1 altered slightly, while the fluorescence intensity of the wild-type-CCND1 group decreased significantly following the addition of miR-4458 mimics. In conclusion, miR-4458 was expressed at low levels in lung cancer tissues, and it arrested cells in vitro at stage G0/G1 and inhibited cell proliferation. Therefore, miR-4458 may

  16. Cathepsin L upregulation-induced EMT phenotype is associated with the acquisition of cisplatin or paclitaxel resistance in A549 cells.

    PubMed

    Han, Mei-Ling; Zhao, Yi-Fan; Tan, Cai-Hong; Xiong, Ya-Jie; Wang, Wen-Juan; Wu, Feng; Fei, Yao; Wang, Long; Liang, Zhong-Qin

    2016-12-01

    Cathepsin L (CTSL), a lysosomal acid cysteine protease, is known to play important roles in tumor metastasis and chemotherapy resistance. In this study we investigated the molecular mechanisms underlying the regulation of chemoresistance by CTSL in human lung cancer cells. Human lung cancer A549 cells, A549/PTX (paclitaxel-resistant) cells and A549/DDP (cisplatin-resistant) cells were tested. The resistance to cisplatin or paclitaxel was detected using MTT and the colony-formation assays. Actin remodeling was observed with FITC-Phalloidin fluorescent staining or immunofluorescence. A wound-healing assay or Transwell assay was used to assess the migration or invasion ability. The expression of CTSL and epithelial and mesenchymal markers was analyzed with Western blotting and immunofluorescence. The expression of EMT-associated transcription factors was measured with Western blotting or q-PCR. BALB/c nude mice were implanted subcutaneously with A549 cells overexpressing CTSL, and the mice were administered paclitaxel (10, 15 mg/kg, ip) every 3 d for 5 times. Cisplatin or paclitaxel treatment (10-80 ng/mL) induced CTSL expression in A549 cells. CTSL levels were much higher in A549/PTX and A549/DDP cells than in A549 cells. Silencing of CTSL reversed the chemoresistance in A549/DDP and A549/TAX cells, whereas overexpression of CTSL attenuated the sensitivity of A549 cells to cisplatin or paclitaxel. Furthermore, A549/DDP and A549/TAX cells underwent morphological and cytoskeletal changes with increased cell invasion and migration abilities, accompanied by decreased expression of epithelial markers (E-cadherin and cytokeratin-18) and increased expression of mesenchymal markers (N-cadherin and vimentin), as well as upregulation of EMT-associated transcription factors Snail, Slug, ZEB1 and ZEB2. Silencing of CTSL reversed EMT in A549/DDP and A549/TAX cells; In contrast, overexpression of CTSL induced EMT in A549 cells. In xenograft nude mouse model, the mice implanted

  17. Cathepsin L upregulation-induced EMT phenotype is associated with the acquisition of cisplatin or paclitaxel resistance in A549 cells

    PubMed Central

    Han, Mei-ling; Zhao, Yi-fan; Tan, Cai-hong; Xiong, Ya-jie; Wang, Wen-juan; Wu, Feng; Fei, Yao; Wang, Long; Liang, Zhong-qin

    2016-01-01

    Aim: Cathepsin L (CTSL), a lysosomal acid cysteine protease, is known to play important roles in tumor metastasis and chemotherapy resistance. In this study we investigated the molecular mechanisms underlying the regulation of chemoresistance by CTSL in human lung cancer cells. Methods: Human lung cancer A549 cells, A549/PTX (paclitaxel-resistant) cells and A549/DDP (cisplatin-resistant) cells were tested. The resistance to cisplatin or paclitaxel was detected using MTT and the colony-formation assays. Actin remodeling was observed with FITC-Phalloidin fluorescent staining or immunofluorescence. A wound-healing assay or Transwell assay was used to assess the migration or invasion ability. The expression of CTSL and epithelial and mesenchymal markers was analyzed with Western blotting and immunofluorescence. The expression of EMT-associated transcription factors was measured with Western blotting or q-PCR. BALB/c nude mice were implanted subcutaneously with A549 cells overexpressing CTSL, and the mice were administered paclitaxel (10, 15 mg/kg, ip) every 3 d for 5 times. Results: Cisplatin or paclitaxel treatment (10–80 ng/mL) induced CTSL expression in A549 cells. CTSL levels were much higher in A549/PTX and A549/DDP cells than in A549 cells. Silencing of CTSL reversed the chemoresistance in A549/DDP and A549/TAX cells, whereas overexpression of CTSL attenuated the sensitivity of A549 cells to cisplatin or paclitaxel. Furthermore, A549/DDP and A549/TAX cells underwent morphological and cytoskeletal changes with increased cell invasion and migration abilities, accompanied by decreased expression of epithelial markers (E-cadherin and cytokeratin-18) and increased expression of mesenchymal markers (N-cadherin and vimentin), as well as upregulation of EMT-associated transcription factors Snail, Slug, ZEB1 and ZEB2. Silencing of CTSL reversed EMT in A549/DDP and A549/TAX cells; In contrast, overexpression of CTSL induced EMT in A549 cells. In xenograft nude mouse

  18. Over-expression of Orai1 mediates cell proliferation and associates with poor prognosis in human non-small cell lung carcinoma.

    PubMed

    Zhan, Zheng-Yu; Zhong, Lu-Xing; Feng, Miao; Wang, Jian-Feng; Liu, Di-Bin; Xiong, Jian-Ping

    2015-01-01

    Orai1 and STIM1 mediate calcium release-activated calcium current (CRAC) which is the best characterized store-operated calcium current involving in a wide range of cell progresses, such as cell proliferation, metastasis, apoptosis. Orai1 has been studied as a carcinogenic biomarker in some cancers such as esophageal cancer. However, its function and clinical significance in non-small cell lung cancer (NSCLC) have not been well studied. The present study was aimed at discussing the relationship between Orai1 and lung cancer malignant behavior with its clinical significance. We used quantitative real-time-PCR and Western blot to detect the expression of Orai1 in NSCLC cell lines and fresh cancer tissues. Immunohistochemistry were performed to test the location and expression of Orai1 in paraffin sections. We found that Orai1 was markedly overexpressed in both NSCLC cell lines and fresh cancer tissues. Immunohistochemistry data also revealed that overexpression of Orai1 was present in 42.4% of NSCLC tissues, compared with the corresponding adjacent nontumorous tissues. Furthermore, NSCLC patients with high Orai1 expression survived shorter than those with low Orai1 expression. In addition, when knockdown Orai1 by RNAi technic, we found the PI3k/AKT/ERK pathway was inhibited which may indicated that Orai1 could influence cell proliferation. Taken together, our study demonstrated that Orai1 was remarkably overexpressed in NSCLC and could be served as a potential prognostic marker for patients with this deadly disease.

  19. Synergistic and attenuated effect of HSS in combination treatment with docetaxel plus cisplatin in human non-small-cell lung SPC-A-1 tumor xenograft.

    PubMed

    Jia, Yuping; Zhou, Dongshun; Jia, Qingwen; Ying, Yong; Chen, Shuntai

    2016-04-01

    Platinum based combination regimens are first-line treatment option in treatment of non-small cell lung cancer (NSCLC) but the clinical utility has been limited because of their toxicities. Many reports indicated that patients with tumors can benefit from adjuvant chemotherapy drugs. The aim of this study was to confirm adjuvant chemotherapy of HSS with docetaxel plus cisplatin (DP) against NSCLC by evaluating antitumor activity and attenuated effect. In vivo SPC-A-1 xenograft model was established to evaluate antitumor activity and toxicity of HSS along or combination with DP. Evaluation indexes include the relative tumor proliferation rate, tumor growth inhibition rate, body weight, food consumption, hematological and biochemical analysis. HSS treatment showed inhibited tumor growth and increased tumor inhibition of DP treatment at doses of 250 mg/kg and 500 mg/kg. No significant toxicity was found in HSS-treated mice, but significant toxicity was found in DP-treated mice. HSS combinatio