miR-185/YWHAZ轴通过糖酵解调控NSCLC A549细胞凋亡

目的 探讨miR-185/YWHAZ轴通过糖酵解对非小细胞肺癌(NSCLC)A549细胞凋亡的影响。方法 糖酵解抑制剂处理后,检测各组糖酵解酶、乳酸、NADH水平和细胞凋亡水平。过表达或敲低miR-185后,过表达或敲低YWHAZ后,均检测细胞乳酸、NADH和细胞凋亡水平。荧光素酶报告实验检测miR-185与YWHAZ mRNA的相互作用。结果 糖酵解抑制剂处理后,糖酵解酶、乳酸、NADH下降,细胞凋亡水平上升(P<0.05)。过表达miR-185时,细胞乳酸、NADH下降,YWHAZ mRNA和蛋白水平下降,细胞凋亡水平上升;敲低miR-185时上述变化逆转(P<0.05)。敲低YWHAZ时,细胞乳酸、NADH下降,细胞凋亡水平上升;过表达YWHAZ时上述变化逆转(P<0.05)。miR-185靶向YWHAZ mRNA的3′端非翻译区。结论 YWHAZ、miR-185分别是促进和抑制糖酵解的关键分子。miR-185靶向YWHAZ mRNA的3′端非翻译区,减少了YWHAZ表达,促进了NSCLC的细胞凋亡。

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.

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.

Effect of ulinastatin on paraquat-induced-oxidative stress in human type Ⅱ alveolar epithelial cells

BACKGROUND:Ulinastatin(UTI) is a urinary trypsin inhibitor extracted and purified from urine of males.This study aimed to explore the effects of UTI on paraquat-induced-oxidative stress in human type Ⅱ alveolar epithelial cells.METHODS:The human type II alveolar epithelial cells,A549 cells,were cultured in vitro.The A549 cells were treated with different concentrations of paraquat(200,400,600,800,1 000,1 200 umol/L) and ulinastatin(0,2 000,4 000,6 000,8 000 U/mL) for 24 hours,the cell viability was measured by cell counting kit-8 and the median lethal concentration was selected.In order to establish an in vitro model of paraquat intoxication and to determine the safe dose of ulinastatin,we calculated LD50 using cell counting kit-8 to determine the survival rate of the cells.A549 cells were divided into normal control group,paraquat group and paraquat+ulinastatin group.The levels of malondialdehyde(MDA) and myeloperoxidase(MPO) were detected by biochemistry colorimetry,while the level of reactive oxygen spies(ROS) was detected by DCFH-DA assay.RESULTS:The survival rate of A549 cells treated with different concentrations of paraquat decreased in a concentration-dependent manner.Whereas there was no decrease in the survival rate of cells treated with 0-4 000 U/mL ulinastatin.The levels of MDA,MPO,and ROS were significantly higher in the paraquat group than in the normal control group after 24-hour-exposure.And the survival rate of the paraquat+ulinastatin group was higher than that of the paraquat group,but lower than that of the normal control group.The levels of MDA,MPO,and ROS were lower than those of the paraquat group.CONCLUSION:Ulinastatin can alleviate the paraquat-induced A549 cell damage by reducing oxidative stress.

In vitro Study of Nucleostemin Gene as a Potential Therapeutic Target for Human Lung Carcinoma

Objective Nucleostemin （NS） is a GTP-conjugated protein located in the nucleoli of stem cells and some cancer cells, and maintains cell self-renewal. We aimed to evaluate NS as a potential target for lung carcinoma gene therapy by investigating NS gene expression and its effect on A549 cell proliferation. Methods NS mRNA and protein expression in A549, HepG2, SMMC-7721, HeLa, and U251 cells was analyzed by RT-PCR and western blotting following transfection of NS siRNAs and negative control siRNA （NC）. The effect on cell proliferation was also analyzed by MTF assays. Results NS mRNA and protein were both expressed in A549 cells and four other tumor cell lines; the relative expression levels were similar in all five cell lines. The three pairs of NS siRNA, either transfected alone or cotransfected into A549 cells, could effectively inhibit the expression of NS mRNA and protein. Moreover, the interference ratio showed an obvious concentration-dependent relationship. NS siRNA treatment resulted in significant inhibition of A549 cell proliferation by 35.7%. Conclusion NS gene was not only highly expressed but also played an important role in A549 cell proliferation. Thus, targeting of NS may be a promising novel strategy for the treatment of lung carcinoma.

Edaravone attenuates paraquat-induced lung injury by inhibiting oxidative stress in human type Ⅱalveolar epithelial cells

BACKGROUND:Edaravone(3-methyl-1-penyl-2-pyrazolin-5-one) is a potent free-radical scavenger and has the antioxidant ability to inhibit lipid peroxidation.The study aimed to examine the effect of edaravone on protecting the acute injury of human type II alveolar epithelial cells(A549cells) induced by paraquat(PQ) and the change of production of reactive oxygen species(ROS),malondialdehyde(MDA),superoxide dismutase(SOD).METHODS:A549 cells were cultured and divided into PQ group(group P),edaravone-treated group(group E) and normal control group(group C).The cells in group P were exposed to paraquat(600 umol/L),and the cells in group E were treated with edaravone(100 umol/L) additionally,and no drug intervention was given to the cells in group C.Real-time monitoring by LSCM was used to detect the cell response and the intracellular dynamic change of ROS level in A549 cells after administration of PQ and edaravone.And the levels of SOD and MDA were detected respectively by biochemistry colorimetry.Data were expressed as mean ± standard error of the mean.Statistical analysis was carried out with the soft SPSS 16.0.RESULTS:The concentration of intracellular ROS significantly increased when PQ was given to A549 cells.But after administration of edaravone,the concentration of intracellular ROS was decreased.Compared to the PQ group,the levels of SOD in the edaravone group were significantly increased while the levels of MDA were markedly decreased.CONCLUSIONS:Paraquat can increase the oxidative stress,and induce the lipid peroxidation of A549 cells.Edaravone has the effect to scavenge reactive oxygen species,and to protect against the PQ-induced lung toxicity.

The Inhibitory Effects of Rh-endostatin(YH-16) in Combination with Radiotherapy on Lung Adenocarcinoma A549 in Mice and the Underlying Mechanisms

In order to investigate the inhibitory effects of Endostar(rh-endostatin,YH-16)in combination with radiotherapy on lung adenocarcinoma A549 in mice and the interaction mechanisms of combined therapy,the transplantation tumor models of A549 lung adenocarcinoma were established.When the largest diameter of tumor reached 1.0cm,all nude mice were randomly divided into 4 groups:Endostar group,radiotherapy group,radiotherapy plus Endostar(combined treatment)group,and control group(n=6 in each group).The largest d...

Highly Efficient Labeling of Human Lung Cancer Cells Using Cationic Poly-L-lysine-Assisted Magnetic Iron Oxide Nanoparticles

Cell labeling with magnetic iron oxide nanoparticles(IONPs)is increasingly a routine approach in the cellbased cancer treatment.However,cell labeling with magnetic IONPs and their leading effects on the biological properties of human lung carcinoma cells remain scarcely reported.Therefore,in the present study the magnetic c-Fe2O3nanoparticles(MNPs)were firstly synthesized and surface-modified with cationic poly-L-lysine(PLL)to construct the PLL-MNPs,which were then used to magnetically label human A549 lung cancer cells.Cell viability and proliferation were evaluated with propidium iodide/fluorescein diacetate double staining and standard 3-(4,5-dimethylthiazol-2-diphenyl-tetrazolium)bromide assay,and the cytoskeleton was immunocytochemically stained.The cell cycle of the PLL-MNPlabeled A549 lung cancer cells was analyzed using flow cytometry.Apoptotic cells were fluorescently analyzed with nuclear-specific staining after the PLL-MNP labeling.The results showed that the constructed PLL-MNPs efficiently magnetically labeled A549 lung cancer cells and that,at low concentrations,labeling did not affect cellular viability,proliferation capability,cell cycle,and apoptosis.Furthermore,the cytoskeleton in the treated cells was detected intact in comparison with the untreated counterparts.However,the results also showed that at high concentration(400 lg m L-1),the PLL-MNPs would slightly impair cell viability,proliferation,cell cycle,and apoptosis and disrupt the cytoskeleton in the treated A549 lung cancer cells.Therefore,the present results indicated that the PLL-MNPs at adequate concentrations can be efficiently used for labeling A549 lung cancer cells and could be considered as a feasible approach for magnetic targeted anti-cancer drug/gene delivery,targeted diagnosis,and therapy in lung cancer treatment.

Efficiency of combining pomegranate juice with low-doses of cisplatin and taxotere on A549 human lung adenocarcinoma cells

Objective: To test the coalescence effect of two chemotherapy drugs at low effective dose(cisplatin and taxotere) combined with pomegranate juice on A549 cancer cells. Methods: Infrared spectroscopy method is a qualitative test that was performed to ensure the existence of the phytochemicals providing the antioxidant activity through the presence of the hydroxyl group(-OH). The viability of A549 cell line and normal MCs was tested using the neutral red uptake, Clonogenic survival, XTT and Cell migration assays. Results: Our results showed that this combination firstly led to a greater decrease in the viability of cells comparing to those treated with chemotherapy drugs alone, and secondly led to a significant reduction in cell migration. Conclusions: These data suggest a synergistic effect between the pomegranate and cisplatin which makes probably this combination a powerful option for treating lung adenocarcinoma and in parallel minimizing the systemic side effects.

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.

Study of ATP borate ester effects on cell sensitization to radiation emitted by a nuclear reactor

Adenosine triphosphate(ATP)borate ester as a new boron agent for boron neutron capture therapy was tested.It was synthesized via a dehydration reaction induced by heating adenosine triphosphate disodium with boric acid.Next,ATP borate ester pretreatments were assessed to study their effects on cell sensitization from exposure to thermal neutron irradiation emitted by a nuclear reactor.Using cell viability assays(CCK8),survival rates of A549 cells pretreated with or without boroncontaining agents,including ATP borate ester and 4-dihydroxyborylphenylalanine(BPA),were measured.One week after feeding an ATP borate ester solution to tumorbearing nude mice,elemental B content values of tumor muscle and blood were measured using inductively coupled plasma mass spectrometry(ICP-MS).Meanwhile,other tumor tissue samples were placed in a culture medium,subjected to a 3-min neutron irradiation exposure,and then fixed in formalin 24 h later for the terminaldeoxynucleotidyl transferase(TDT)-mediated d UTP nick end labeling(TUNEL)immunohistochemical staining analysis.Results showed that A549 cell irradiation sensitization(irradiation dose of 0.33 Gy)varied with pretreatment.Sensitization values of the ATP borate ester pretreatment group were 1.3–14.1 with boron agent concentrations of 0.3–4.5 mM.Within 1.1–3.4 mM,ATP borate ester showed significantly higher sensitization values than BPA.Meanwhile,TUNEL results demonstrated that apoptosis rates of tumor tissue cells exposed to irradiation after ATP borate ester pretreatment significantly exceeded the corresponding rates for BPA-pretreated cells.In animal experiments,although the distribution ratio of ATP borate ester(tumor tissue/normal muscle,T/N)of 1.2 was not significantly different compared with that of BPA(1.3),the total ATP borate ester concentration in the tumor tissue(0.79±0.05μg/g)significantly exceeded that of BPA(0.58±0.05μg/g).Thus,compared with BPA,the greater enrichment of ATP borate ester in tumor tissues permits preferential targeting toward tumor cells for radiation sensitization.Therefore,ATP borate ester is superior to BPA for use in boron neutron capture therapy.

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.

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

Objective: To investigate the co-expression of drug resistance- and apoptosis-related genes of cisplatin (CDDP)-selected lung adenocarcinoma cell line A 549 DDP for compared to the parental cell line A549, and reverse of drug resistance by antisense s-oligodeoxynucleotides (S-ODNs) of differentially expressed genes. Methods: Sense and antisense S-ODN were transferred into A 549 DDP cells by lipofectin. The expression of drug resistance and apoptosis related genes was examined by RT-PCR, immunocytochemistry and flow cytometry, respectively. Apoptostic cells were identified by DNA electrophoresis and terminal deoxynucleotidyl transferase (TdT)-mediated biotin dUTP nick end-labeling(TUNEL). Drug resistance of tumor cells was detected by a cell viability (MTT) assay. Results: The expression of bcl-2 was positive and that of multidrug resistance-associated protein (MRP) at mRNA and protein level was increased in A 549 DDP compared to A549 cells. MDR1, c-myc and topoisomeras II (TOPO II) were similarly co-expressed in two cell lines. Both cell lines were negative for c-erbB-2 expression. In A 549 DDP cells, the expression of bcl-2 and MRP was significantly inhibited by their respective antisense S-ODNs. Antisense S-ODNs could also decrease significantly drug resistance of A 549 DDP cells to CDDP by promoting cell apoptosis. Conclusion: Both intrinsic and acquired drug resistance were involved in co-expression of multiple MDR-related genes in lung adenocarcinoma. Cooperation of bcl-2 and MRP genes appeared to play an important action to confer the resistance of A 549 DDP cells to CDDP. Their antisense S-ODNs are responsible for the decrease of drug resistance of this cell line by promoting apoptosis.

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.

SRS27,a semisynthetic diterpenoid lactone,inhibits airway inflammation and hyper-responsiveness via NF-κB pathway in an allergic mouse asthma model

Andrographis paniculata contains two main diterpenoid constituents,andrographolide(AGP)and 14-deoxy-11,12-didehydroandrographolide(DDAG),which were found to exhibit antiasthma effects in a mouse asthma model.However,due to inadequacies of both compounds in terms of drug-likeness,DDAG analogues were semisynthesised to tackle these shortcomings.The objective of the study was to investigate the potential of DDAG analogues as new antiasthma agents.Among the analogues,(SRS27)was proven to inhibit cysteinyl leukotrienes(CysLT)and nitric oxide(NO)synthesis in mouse macrophages,like AGP.DDAG,on the other hand,failed to exhibit such activities.SRS27 is less cytotoxic than AGP,suggests that a simple chemical modification of DDAG produces a compound with CysLT and NO inhibitory activites similar to AGP while maintaining toxicity profile similar to DDAG.It is interesting to note that other analogues such as SRS28,SRS49,SRS76 and SRS83 with chemical modifications on the same carbon numbers 3 and 19 of DDAG were unable to show inhibition of CysLT and NO synthesis.Consequently,the potential anti-inflammatory effect of SRS27 was investigated in ovalbumin(OVA)-induced mouse asthma model.The compound was administered in a prophylactic manner and showed a substantial decrease in mouse asthma model parameters.SRS27 at 3mg·kg-1 significantly reduced OVA-induced total cell such as macrophages,eosinophils,lymphocytes and neutrophils,as well as inflammatory cytokines such as IL-4,IL-5,IL-13 and eotaxin in bronchoalveolar lavage BAL fluid.The compound also suppressed serum IgE production.In addition,SRS27 suppressed mucus hyper-secretion and expression of inflammatory mediators such as TNF-α,MCP-1,Muc5 ac,RANTES,IL-33 and iNOS.SRS27 is the first known DDAG derivative tested positive in mouse asthma model and as such SRS27 could serve as a prototype prophylactic agent.

Enhanced cytotoxic effect on human lung carcinoma cell line(A549) by gold nanoparticles synthesized from Justicia adhatoda leaf extract

Objective: To synthesize bio-inspired gold nanoparticles(AuNPs) using the leaf extract of Justicia adhatoda and evaluate the anti-cancer activity on human lung cancer cell line(A549).Methods: Synthesis of AuNPs was done using an aqueous leaf extract of Justicia adhatoda as a green route. The bio-synthesized AuNPs were confirmed and characterized by using various spectral studies such as UV-Vis spectrum, Scanning Electron Microscope with EDAX, Transmission Electron Microscope, Fourier Transmission Infrared Spectroscope analysis and Surface Enhanced Raman Spectroscopy. The cell viability was determined by MTT reduction assay. In addition, cytomorphology and the nuclear morphological study of A549 cell line was observed under fluorescence microscope. Results: UV-Vis spectrum showed surface plasmon resonance peak at 547 nm, scanning electron microscope and transmission electron microscope studies showed the monodispersed spherical shape and its average size in the range of 40.1 nm was noticed. Fourier Transmission Infrared Spectroscope analysis confirmed that the C=O group of amino acids of proteins had strong ability to bind with the surface of nanoparticle. Interestingly, our results also demonstrated inhibited proliferation of A549 cell line by MTT(IC50 value: 80 μg/mL). Cell morphology was observed and cell death was caused by apoptosis as revealed by propidium iodide staining. Conclusions: The current study proves the anticancer potential of bio-synthesized AuNPs. Thus, synthesized AuNPs can be used for the treatment of human lung cancer cell(A549) and it can be exploited for drug delivery in future.

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.

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.

IDENTIFICATION OF DRUG RESISTANT RELATED cDNA IN LUNG ADENOCARCINOMA CELL LINES

Objective: To clone multidrug resistance (MDR) related genes in lung adenocarcinoma cell lines. Methods: The differentially expressed cDNA fragments between A549 and A549 DDP cells were analyzed by mRNA differential display PCR(DD RT-PCR). The fragments thus obtained were further analyzed by DNA sequencing and Northern blotting. Results: Three differentially expressed cDNA fragments were obtained and confirmed by Northern blot. Sequence analysis revealed that two of them were novel and one was 100% identical with ICE gene. Conclusion: Analyzing differentially expressed fragment between A549 and A549 DDP cells may be helpful for finding new MDR related genes. The drug resistance of A549 DDP cells may be related to the inhibition or down-regulation of ICE gene.

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.