Arctigenin attenuates paraquat-induced human lung epithelial A549 cell injury by suppressing ROS/p38 mitogen-activated protein kinases-mediated apoptosis

BACKGROUND:Paraquat(PQ)-induced acute lung injury(ALI)and pulmonary fi brosis are common diseases with high mortality but without eff ective antidotes in emergency medicine.Our previous study has proved that arctigenin suppressed pulmonary fibrosis induced by PQ.We wondered whether arctigenin could also have a protective eff ect on PQ-induced ALI.METHODS:A PQ-induced A549 cell injury model was used,and the effect of arctigenin was determined by a cell counting kit-8(CCK-8)cell viability assay.In addition,terminal deoxynucleotidyl transferase(TdT)-mediated dUTP nick-end labelling(TUNEL)staining assays and mitochondrial membrane potential assays were performed to evaluate the level of cell apoptosis.The generation of reactive oxygen species(ROS)was refl ected by dihydroethidium(DHE)staining and a 2’,7’-dichlorodihy drofluorescein diacetate(DCFH-DA)assay.Moreover,immunoblotting studies were used to assess the expression of mitogen-activated protein kinases(MAPKs)and p38 MAPK.RESULTS:Arctigenin attenuated PQ-induced inhibition of A549 cell viability in a dose-dependent manner.Arctigenin also significantly reduced PQ-induced A549 cell apoptosis,as refl ected by the TUNEL assay and mitochondrial membrane potential assay,which may result from suppressed ROS/p38 MAPK signaling because we found that arctigenin dramatically suppressed ROS generation and p38 MAPK phosphorylation.CONCLUSION:Arctigenin could attenuate PQ-induced lung epithelial A549 cell injury in vitro by suppressing ROS/p38 MAPK-mediated cell apoptosis,and arctigenin might be considered a potential candidate drug for PQ-induced ALI.

rmhTNF-α Combined with Cisplatin Inhibits Proliferation of A549 Cell Line In Vitro

Objective To explore the inhibitory effect of recombinant mutant human tumor necrosis factor-α(rmhTNF-α) in combination with cisplatin on human lung adenocarcinoma cell line A549. Methods Human lung adenocarcinoma cell line A549 was treated with varying concentrations of rmhTNF-α(0.38, 0.75, 1.50, 6.00 and 12.00 IU/ml) or cisplatin(3.91, 7.81, 15.63, 31.25 and 62.50 μg/ml) for 24 hours. Viable cell number was analyzed by using crystal violet staining. The inhibitory rates of A549 cells growth by the two drugs were calculated. For analyzing whether there was a synergistic effect of rmhTNF-α with cisplatin, A549 cells were treated with 0.75 IU/ml rmhTNF-α and increased concentrations of cisplatin. Results rmhTNF-α or cisplatin inhibited the growth of A549 cell lines in a dose-dependent manner. The inhibitory effect of rmhTNF-α combined with cisplatin was significantly greater than cisplatin alone at the same concentration(all P<0.01). Conclusion rmhTNF-α combined with cisplatin might have synergistic inhibitory effect on human lung adenocarcinoma cell line A549.

Digital gene expression profiling analysis of A549 cells cultured with PM10 in moxa smoke

Background:Moxibustion is a traditional Chinese medicine therapy to cure diseases by fumigating meridians or affected parts via burning of moxa floss.Moxa smoke(MS)is one of the key factors in moxibustion.In this study,we adopted digital gene expression profiling,a next-generation gene sequencing technology,to investigate the effect of MS,inhalable particulate matter(PM10),on human lung adenocarcinoma A549 cells.Methods:The effects of MS PM10 on A549 cells,over different treatment durations were investigated in different groups:the 4-h group(4-h MS group and 4-h control group)and the 20-h group(20-h MS group and 20-h control group).Samples collected from the four groups were stored at
80C for subsequent digital gene expression analysis.The differentially expressed genes(DEGs),identified after PM10 treatment,were screened,and their expression patterns analyzed by cluster analysis,Gene Ontology term enrichment,and Kyoto Encyclopedia of Genes and Genomes pathway analysis.Results:Compared with two control groups,1109 DEGs were identified after 4 h of MS intervention and 3565 DEGs were found after 20 h of MS intervention,respectively.Compared with that after 4-h intervention,2149 DEGs were identified after 20-h intervention.Cluster analysis demonstrated that PM10 can significantly inhibit cell cycle process with the prolongation of intervention time.Significant pathway enrichment analysis showed that MS PM10 can inhibit A549 cell cycle process at all phases.When MS PM10 exposure time prolongs,the inhibitory effect on cell cycle process becomes more obvious.Conclusion:MS PM10 has many biological activities,and may cause differential expression of genes involved in various biological processes.Nevertheless,further research on MS is warranted for better understanding of the mechanistic details.

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

The experiment aims to investigate the influence of Tamoxifen and the combination of Tamoxifen and Cisplatin （DDP） on the growth of human lung adenocarcinoma A549 cells. Methods： We treated human lung adenocarcinoma A549 cells with different concentrations of Tamoxifen, DDP and combination of DDP and Tamoxifen with non-toxicity for 72 h. Then we calculated the inhibition rate through MTT approach and detected the apoptosis rate by flow cytometry. The statistical analysis was performed with SPSS 13.0 software and statistical differences were determined by one-way ANOVA. The data were expressed as the mean ＋ standard deviation and all experiments were performed in three times. The value of P 〈 0.05 was considered to indicate a statistically significant difference. Results： 1. The inhibition rates of Tamoxifen with 2.5 pmol/L, 5 tJmol/L, 10 μmol/L, and 20 μmol/L on the growth of the A549 cells were 18.7%, 25.8%, 54% and 98.8%, respectively （P = 0.000）. Tamoxifen with concentration of 1 μmol/L has no obvious cytoxicity on the A549 cells （P 〉 0.05）. 2. As the increase concentration of Tarnoxifen, the S stage and G2/M of the A549 cells decreased while the G0/G1 increased. The apoptosis rate of Tamoxifen with 0 μmol/L, 0.1 μmol/L, 1 μmol/L and 10 μmol/L on the A549 cells were 6.51%, 8.91%, 17.97% and 42.7%, respectively. 3. The inhibition rates of combination of Tamoxifen with 1 μmol/L and DDP with 1.25 μg/mL, 2.5 μg/mL, 5 μg/mL, 10 μg/mL and 20 μg/mL on the A549 cells were 40.4%, 54.4%, 72.9%, 86.1% and 92.4%, respectively （P 〈 0.05）. Conclusion： Tamoxifen can inhibit the proliferation of human lung adenocarcinoma A549 cells and induce the apoptosis of the A549 cells. The combination of Tamoxifen with non-toxicity and DDP can improve the sensitivity of chemotherapy on the A549 cells.

Oleanolic acid-induced apoptosis and its relation with intracellular calcium in human lung adenocarcinoma A549 cells

Objective To investigate the effect of oleanolic acid (OA) on apoptosis,correlation between apoptosis and intracellular calcium,and its mechanism in human lung adenocarcinoma cell line A549. Methods Human lung adenocarcinoma A549 cells were incubated in vitro and assigned with OA concentrations of 0,10,20 and 40μg/mL. The apoptosis status of A549 cell line was detected with Annexin V-FITC/PI by flow cytometry (FCM); fluorescence intensity (FI) of A549 cells was assessed and the level of intracellular calcium was calculated at 24 hour of OA intervention. The relation between apoptosis and calcium FI was illustrated by curve fitting. Results FCM showed that 10,20 and 40μg/mL of OA could induce A549 cell apoptosis,which followed a concentration-effect pattern; 24-hour intervention with 20μg/mL and 40μg/mL OA showed increased A549 cell apoptosis,and was significantly different from that with 0μg/mL OA (P<0.01). The FI of intracellular calcium concentration in 10,20 and 40μg/mL OA groups was significantly higher than that in 0μg/mL group after 24 hours’ intervention,and the FI showed a trend of increase with increased OA concentration (P<0.01). Curve fitting showed a significant correlation between apoptosis rate and intracellular calcium concentration in A549 cells (r=0.981,P<0.01). Regression equation was Y=0.508X-1.627. Conclusion OA plays a role in inducing apoptosis of human lung adenocarcinoma cells in a concentration-dependent manner. The OA-induced apoptosis is responsible for intracellular calcium overload of the tumor.

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

Objective： To evaluate the effect of hypoxia on migration, invasion and adhesion to endothelium of human pulmo- nary adenocarcinoma A549 cells. Methods： Wound-healing and Transwell invasion assays were performed to study the effect of hypoxia on migration and invasion of A549 cells, and A549 cells were added to a monolayer of human umbilical vein endo-thelial cells （HUVECs） to test the ability to adhere to endothelium. Immunofluorescence assay and luciferase reporter gene assay were also used to evaluate the effect of hypoxia on distribution of E-cadherin, β-catenin, and actin, and hypoxia-inducible factor-1 （HIF-1）-dependent transcription, respectively. Results： Hypoxia facilitated A549 cell migration, invasion, and A549 cell-endothelial cells adhesion, and modulated the distribution of E-cadherin and β-catenin, and actin cytoskeleton rearrange-ment, and up-regulated HIF-l-dependent reporter gene expression in A549 cells. Conclusion： Promotion ofA549 cell migration, invasion, and adhesion on endothelium by hypoxia might be modulated through its up-regulating HIF-l-dependent gene expression, which then induced the redistribution of E-cadherin and β-catenin, and the actin cytoskeletal reorganization.

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

Objective： To investigate the effect of paclitaxel on cell proliferation and apoptosis of human lung adenocarcinoma A549 cells line and its mechanism in vitro. Methods ： Cell growth inhibition of paclitaxel on A549 cells was analyzed by MTT assay. Cell apoptosis was detected by DNA cytofluorometry, Hoechst33258 staining when treated with paclitaxel for 48 hours. Meanwhile, Cell cycle and apoptotic rate were analyzed by flow cytometry. The protein expressions of Bax and Bcl-2 were studied by Western Blot. Results： Paclitaxel inhibited the proliferation of A549 cells in a time-and dose-dependant manner. Hoechst33258 staining indicated that apoptosis was induced by paclitaxel. After treated for 48 hours, cell apoptosis rates of 25 nmo1/L, 50 nmol/L and 100 nmol/L paclitaxel groups were 11.52 ± 1.94% ,17.73 ±2.53%, and 29.32 ±5.51% respectively, which were significantly higher than those of control group 5.88 ±1.07%（all P 〈 0.01 ）, and apoptosis rate increased in dose-dependant manner. Meanwhile, G2/M stage cell percentage of 25 nmol/L, 50 nmol/L and 100 nmol/L paclitaxel groups were 42.52 ± 6.25%, 40.46 ± 5.81%, and 35.34 ±6.17% respectively,which were significantly higher than that of control group 22.32 ± 3.30%（all P 〈 0.01 ）; Western blot showed that paclitaxel increased the expression of Bax and decreased the expression of Bcl-2 in dose-dependant manner. Conclusion： Paclitaxel can inhibit A549 cell proliferation in a time-and dose-dependant manner. Its mechanism may be related to arresting cell cycle in G2/M stage and induce cell apoptosis by up-modulating Bax expression and down-modulating Bcl-2 expression.

COX-2 silencing inhibits cell proliferation in A549 cell

Objective: The aim of this study was to explore the effects on malignant proliferation of A549 cell by silencing cy-clooxygenase (COX)-2. Methods: In the present study, we constructed three siRNA vectors producing small interference RNA. The siRNA vectors and the vacant vectors were transfected into A549 cell with lipofectamine respectively and the transfected cell strains were constructed. The change of COX-2 expression levels was examined by Western blot and RT-PCR. The effects on the proliferation of lung cancer cells were studied by cell growth curve, clonogenic assay and xenograft assays. Results: The siRNA expression vectors produced marked effects in A549 cell but the inhibited effects were different. The effect of psi-10 was best and the mRNA and protein levels of COX-2 reduced 61.2% and 56.2% respectively in A549-si10 cell in contrast to the control. The growth of A549 cell slowed and the colony formation rate reduced after silencing COX-2. In xenograft assays, the growth speeds of tumor became slow and the numbers of tumor reduced after silencing COX-2. Conclusion: The si10 target of COX-2 has the best silencing effect in A549 cell and the best inhibition effect on malignant proliferation of A549 cell in vivo and in vitro.

The Three Main SCFAs Inhibit the Inflammatory Response of A549 Cells Induced by Acinetobacter baumannii

Objective: The objective is to explore the mechanism of inhibitory effect of three main SCFAs (acetate, propionate and butyrate) on inflammatory response of A549 cells. Methods: Human lung adenocarcinoma cells (A549 cells) were cultured, and were divided into normal control group (NC group), A. baumannii infection group (A. baumannii group), NF-κB inhibitor group (JSH group), A. baumannii infection + sodium acetate group (NaAc group), A. baumannii infection + sodium propionate group (NaPc group) and A. baumannii infection + sodium butyrate group (NaB group). Real-time quantitative PCR was used to detect the mRNA expression of NLRP3, Caspase-1, IL-1β, IL-6, and TGF-β in A549 cells. Western blotting assay was used to determine the expression of autophagy and “pyroptosis” related proteins of NRLP3, cleaved-Caspase-1 (P20), GSDMD (P30), LC-3 and Beclin-1. At the same time, the expression of NF-κB p65 protein in nucleus and cytoplasm of A549 cells was detected. The level of reactive oxygen species in A549 cells was detected by flow cytometry. Results: Compared with A. baumannii group, the mRNA expression of NLRP3, IL-1β and IL-6 in NaAc group, NaPc group and NaB group decreased significantly, the mRNA expression of Caspase-1 in NaPc group and NaB group decreased significantly, only the mRNA expression of TGF-β in NaB group increased significantly;LC3-II expression increased significantly in NaPc group and NaB group, only Beclin-1 expression increased and GSDMD (p30) expression decreased significantly in NaB group. All three kinds of SCFAs could significantly inhibit the expression of cleaved-Caspase-1 (p20) after A. baumannii infection, but there was no significant change in the protein expression of NLRP3. Compared with NC group, the production of reactive oxygen species in A. baumannii group increased significantly at 3 h after A. baumannii infection. Compared with A. baumannii group, NaB could significantly suppress the production of reactive oxygen species induced by A. baumannii. Compared with A. baumannii group, the expression of NF-κB p65 in nucleus was significantly decreased and the expression of NF-κB p65 in cytoplasm was significantly increased after 24 h pre-incubation with NaB, NaPc and NaAc, respectively. Conclusion: A. baumannii can induce inflammatory injury of pulmonary epithelial cells, and the three major SCFAs can inhibit the activation of NLRP3 inflammasome and the release of pro-inflammatory factors through NF-κB/ROS/NLRP3 pathway, which provides a new way for clinical prevention of severe inflammatory injury caused by A. baumannii infection.

Effects of sotetsuflavone on expression of endostatin, TGF-β, STAT3, β-catenin and ZO-1 in non-small cell lung cancer A549 cells

Objective To investigate the effects of sotetsuflavone on endostatin, transforming growth factor-β （TGF-β）, signal transducers and activators of transcription 3 （STAT3）, β-catenin and zonula occludens-1 protein （ZO-1） in non-small cell lung cancer A549 cells. Methods： STAT3, β-catenin, TGF-β and ZO-1 mRNA expression were detected by real-time PCR. Endostatin and TGF-β expression were detected by immunofluorescence assay. STAT3 and β-catenin protein expression were detected by western blot. Results： Compared with the control group, TGF-β, STAT3 and β-catenin expression were down-regulated, endostatin and ZO-1 expression were up-regulated by sotetsuflavone. Simultaneously, it showed a significant concentration-dependent. Conclusion： The mechanism of action of sotetsuflavone in the treatment of lung cancer may be via inhibiting the expression of TGF-β, STAT3, and β-catenin, increasing the expression of endostatin and ZO-1, thereby exerting an anti-tumor effect.

Inhibitory Effect of Cantharidin on Proliferation of A549 Cells

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

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

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

Antineoplastic effects of deoxyelephantopin,a sesquiterpene lactone from Elephantopusscaber, on lung adenocarcinoma (A549) cells

OBJECTIVE： Deoxyelephantopin, a sesquiterpene lactone from Elephantopus scaber, showed inhibition of the growth of various tumor cells in vitro. In the present study, we investigated the cytotoxicity and apoptosis-inducing capacity of deoxyelephantopin on lung adenocarcinoma （A549） cells. METHODS： The cytotoxic effect of deoxyelephantopin on A549 cells and normal lymphocytes was evaluated using 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay and 50% inhibitory concentration （IC50） value was determined. The self-renewal and proliferating potential of A549 cells after treatment with deoxyelephantopin were examined by colony formation assay. Cellular morphology of deoxyelephantopin-treated cells was observed using phase- contrast microscopy. The induction of apoptosis was evaluated using acddine orange and ethidium bromide staining, Hoechst 33342 staining, terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end-labeling （TUNEL） assay, DNA fragmentation analysis and Annexin V-fluorescein isothiocyanate staining by flow cytometry. Activation of caspases was detected using fluorogenic substrate specific to caspases 2, 3, 8 and 9 and flow cytometric analysis. The total cellular DNA content and expression of cleaved poly （ADP-ribose） polymerase was also analyzed. RESULTS： Deoxyelephantopin exhibited cytotoxicity to A549 cells （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 both extrinsic and intrinsic pathways. CONCLUSION： These results suggest that deoxyelephantopin has great potential as a new chemotherapeutic agent to be developed further for the treatment of lung cancer.

Agglutinin isolated from Arisema heterophyllum Blume induces apoptosis and autophagy in A549 cells through inhibiting PI3K/Akt pathway and inducing ER stress

Arisaema heterophyllum Blume is one of the three medicinal plants known as traditional Chinese medicine Rhizoma Arisaematis(RA). RA has been popularly used to treat patients with convulsions, inflammation, and cancer for a long time. However, the underlying mechanisms for RA effects are still unclear. The present study was designed to determine the cytotoxicity of agglutinin isolated from Arisema heterophyllum Blume(AHA) and explore the possible mechanisms in human non-small-cell lung cancer A549 cells. AHA with purity up to 95% was isolated and purified from Arisaema heterophyllum Blume using hydrophobic interaction chromatography. AHA dose-dependently inhibited the proliferation of A549 cells and induced G_1 phase cell cycle arrest. AHA induced apoptosis by up-regulating pro-apoptotic Bax, decreasing anti-apoptotic Bcl-2, and activating caspase-9 and caspase-3. In A549 cells treated with AHA, the PI3K/Akt pathway was inhibited. Furthermore, AHA induced increase in the levels of ER stress markers such as phosphorylated eukaryotic initiation factor 2α(p-eIF2α), C/EBP-homologous protein(CHOP), inositol-requiring enzyme 1α(IRE1α), and phosphorylated c-Jun NH_2-terminal kinase(p-JNK). AHA also induced autophagy in A549 cells. Staining of acidic vesicular organelles(AVOs) and increase in the levels of LC3II and ATG7 were observed in AHA-treated cells. These findings suggested that AHA might be one of the active components with anti-cancer effects in Arisaema heterophyllum Blume. In conclusion, cytotoxicity of AHA on cancer cells might be related to its effects on apoptosis and autophagy through inhibition of PI3K/Akt pathway and induction of ER stress.

Marsdenia tenacissima extract suppresses A549 cell migration through regulation of CCR5-CCL5 axis, Rho C, and phosphorylated FAK

Marsdenia tenacissima, a traditional Chinese medicine, is long been used to treat various diseases including asthma, cancer, trachitis, tonsillitis, pharyngitis, cystitis, and pneumonia. Although Marsdenia tenacissima has been demonstrated to have strong anti-tumor effects against primary tumors, its effect on cancer metastasis remains to be defined, and the molecular mechanism underlying the anti-metastatic effect is unknown. In the present study, we investigated the effects of XAP(an extract of Marsdenia tenacissima) on A549 lung cancer cell migration and explored the role of CCR5-CCL5 axis in the anti-metastatic effects of XAP. Our resutls showed that XAP inhibited A549 lung cancer cell migration and invasion in a dose-dependent manner. The protein levels of CCR5, but not CCR9 and CXCR4, were decreased by XAP. The secretion of CCL5, the ligand of CCR5, was reduced by XAP. XAP down-regulated Rho C expression and FAK phosphorylation. In conclusion, XAP inhibited A549 cell migration and invasion through down-regulation of CCR5-CCL5 axis, Rho C, and FAK.

In Vitro Evaluation of Cytotoxicity and Oxidative Stress Induced by Multiwalled Carbon Nanotubes in Murine RAW 264.7 Macrophages and Human A549 Lung Cells

Objective To investigate in vitro cytotoxicity and oxidative stress response induced by multiwalled carbon nanotubes （MWCNTs）. Methods Cultured macrophages （murine RAW264.7 cells） and alveolar epithelium cells type II （human A549 lung cells） were exposed to the blank control, DNA salt control, and the MWCNTs suspensions at 2.5, 10, 25, and 100 ug/mL for 24 h. Each treatment was evaluated by cell viability, cytotoxicity and oxidative stress. Results Overall, both cell lines had similar patterns in response to the cytotoxicity and oxidative stress of MWCNTs. DNA salt treatment showed no change compared to the blank control. In both cell lines, significant changes at the doses of 25 and 100 ug/mL treatments were found in cell viabilities, cytotoxicity, and oxidative stress indexes. The reactive oxygen species （ROS） generation was also found to be significantly higher at the dose of 10 ug/mL treatment, whereas no change was seen in most of the indexes. The ROS generation in both cell lines went up in minutes, reached the climax within an hour and faded down after several hours. Conclusion Exposure to MWCNTs resulted in a dose-dependent cytotoxicity in cultured RAW264.7 cells and A549 cells, that was closely correlated to the increased oxidative stress.

Xiaoji Decoction(消积饮) Inhibited Cell Proliferation and Induced Apoptosis through Akt Signaling Pathway in Human Lung Cancer A549 Cells

Objective： To investigate the inhibitive effect and the underlying mechanism of Xiaoji Decoction （消极饮 XJD） in human lung cancer A549 cells. Methods： A549 cells in logarithmic proliferation were cultivated in RPMI-1640 containing 10% low, medium or high dosages of XJD serum. The inhibitive effect of XJD in A549 cell proliferation was assessed by methylthiazolyldiphenyl-tetrazolium bromide （MTT） assay. The pro-apoptotic effect of XJD in A549 cells was observed by fluorescence microscope via Hoechst 33258 staining. The role of the Akt signaling pathway was observed by examining the presence of p-Akt protein by Western blot and the mRNA expression of downstream proteins such as Bcl-2/BcI-XL-associated death promoter （BAD） and caspase-9 by real time polymerase chain reaction. Results： MTT assay revealed that XJD could inhibit A549 proliferation in a dose- and time-dependent manner. Hoechst 33258 staining showed that XJD induced the typical nuclear apoptotic morphology after XJD treatment. Moreover, XJD could reduce the phosphorylation of Akt and increase the mRNA expression of BAD and caspase-9. Conclusions： XJD can inhibit the proliferation of A549 cells in a dose- and time-dependent manner through signaling Akt pathway via up-regulating the expression of BAD and caspase-9. XJD may provide a novel therapeutic model for lung cancer and deserve further study.

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

The low dose hyper-radiosensitivity （HRS） in human lung cancer cell line A549 was investigated, the changes of ATM kinase, cell cycle and apoptosis of cells at different doses of radiation were observed, and the possible mechanisms were discussed. A549 cells in logarithmic growth phase were irradiated with ^60Co y-rays at doses of 0-2 Gy. Together with flow cytometry for precise cell sorting, cell survival fraction was measured by means of conventional colony-formation assay. The expression of ATM1981 Ser-P protein was examined by Western blot 1 h after radiation. Apoptosis was detected by Hoechst 33258 fluorescent staining, and Annexin V-FITC/PI staining flow cytometry 24 h after radiation. Cell cycle distribution was observed by flow cytometry 6, 12 and 24 h after radiation. The results showed that the expression of ATM1981Ser-P protein was observed at 0.2 Gy, followed by an increase at 〉0.2 Gy, and reached the peak at 0.5 Gy, with little further increase as the dose exceeded 0.5 Gy. Twenty-four h after radiation, partial cells presented the characteristic morphological changes of apoptosis, and the cell apoptosis curve was coincident with the survival curve. As compared with control group, the cell cycle almost had no changes after exposure to 0.1 and 0.2 Gy radiation （P〉0.05）. After exposure to 0.3, 0.4 and 0.5 Gy radiation, G2/M phase arrest occurred 6 and 12 h after radiation （P〈0.05）, and the ratio of G2/M phase cells was decreased 24 h after radiation （P〈0.05）. It was concluded that A549 cells displayed the phenomenon of HRS/IRR. The mode of cell death was mainly apoptosis. The activity of ATM and cell cycle change may take an important role in HRS/IRR.

Herbal formula Renshenwuweizi decoction induces p53-mediated cell cycle arrest and apoptosis in A549 cells

OBJECTIVE:To investigate the effect of Renshenwuweizi decoction(RSWWZ decoction)on the growth of non-small cell lung cancer cells in vitro.METHODS:A549 non-small cell lung cancer cells were divided into two groups:control and RSWWZ decoction treatment groups.Cell Counting Kit-8 was used to measure the inhibitory effect of RSWWZ decoction on the growth of A549 cells.4’,6-diamidino-2-phenylindole staining and Annexin V-fluorescein isothiocyanate/propidium iodide double staining were used to investigate apoptosis in A549 cells following RSWWZ decoction treatment,and the mitochondrial membrane potential of treated cells was detected with Rhodamine 123.Cell cycle progression was analyzed by flow cytometry.The m RNA levels of p53,Bax,B-cell lymphoma-2(Bcl-2)and p21 were measured by quantitative real-time reverse transcription polymerase chain reaction.The protein expressions of p53,Bax,Bcl-2,p21,cyclin-dependent kinases 2(CDK2),and cyclin A were detected by Western blot.RESULTS:RSWWZ decoction reduced the viability of A549 cells in a dose-dependent manner by inducing apoptosis and decreased mitochondrial membrane potential.RSWWZ decoction increased p53 and Bax expression and decreased Bcl-2 expression in a dose-dependent manner.RSWWZ decoction also induced an S-phase cell cycle arrest by increasing p21 and decreasing cyclin A and CDK2 expression.CONCLUSION:In vitro experiments revealed that the Renshenwuweizi decoction-induced decrease in A549 cell proliferation was achieved by inducing apoptosis and S-phase cell cycle arrest via the p53 pathway.These findings provide the experimental basis for Renshenwuweizi decoction treatment of lung cancer.

Analysis of Expression Profiles of Long Noncoding RNAs and mRNAs in A549 Cells Infected with H3N2 Swine Influenza Virus by RNA Sequencing

Long noncoding RNAs(lncRNAs)participate in regulating many biological processes.However,their roles in influenza A virus(IAV)pathogenicity are largely unknown.Here,we analyzed the expression profiles of lncRNAs and mRNAs in H3N2-infected cells and mock-infected cells by high-throughput sequencing.The results showed that 6129 lncRNAs and 50,031 mRNA transcripts in A549 cells displayed differential expression after H3N2 infection compared with mock infection.Among the differentially expressed lncRNAs,4963 were upregulated,and 1166 were downregulated.Functional annotation and enrichment analysis using gene ontology and Kyoto Encyclopedia of Genes and Genomes databases(KEGG)suggested that target genes of the differentially expressed lncRNAs were enriched in some biological processes,such as cellular metabolism and autophagy.The up-or downregulated lncRNAs were selected and further verified by quantitative real-time polymerase chain reaction(RT-qPCR)and reverse transcription PCR(RT-PCR).To the best of our knowledge,this is the first report of a comparative expression analysis of lncRNAs in A549 cells infected with H3N2.Our results support the need for further analyses of the functions of differentially expressed lncRNAs during H3N2 infection.