黄芩素联合亚高温热疗对人肺腺癌A549细胞抑制作用的体外研究

目的:观察黄芩素联合亚高温热疗对人肺腺癌A549细胞的体外抑制作用并探讨其可能的作用机制。方法:以体外培养的肺腺癌A549细胞为研究对象,CCK-8法检测黄芩素联合亚高温热疗对A549细胞增殖的抑制作用,流式细胞仪检测黄芩素联合亚高温热疗对A549细胞周期的影响,Western blot法检测黄芩素联合亚高温热疗对A549细胞HSP70(热休克蛋白70)蛋白表达的影响。结果:不同浓度的黄芩素均能有效地抑制A549的细胞增殖,黄芩素联合亚高温热疗具有更好的细胞抑制作用;联合亚高温热疗处理后,G2/M期细胞含量明显增加。黄芩素联合亚高温热疗组A549细胞的HSP70蛋白表达明显降低,与热疗组比较,差异显著(P<0.05)。结论:亚高温热疗能增强黄芩素对A549细胞的体外抑制作用。

In vitro study of radiosensitization by β-Elemene in A549 cell line from adenocarcinoma of lung

Objective： To explore the effect and possible mechanism in vitro of radiosensitization by β-Elemene in A549 cell line from adenocarcinoma of lung. Methods： The A549 cell line from adenocarcinoma of lung was chosen for the study to determine the inhibition ratio by using MTT assay. Morphologic change, growth curve, cloning efficiency, divisional index were observed. Change of cell cycle and apoptosis rate were analyzed by FCM and the expressions of gene P53 and Bcl-2 were detected. Results： Reproductive activity of the group which was under irradiation and β-Elemene was significantly suppressed and its cloning efficiency and divisional index also declined. The apoptosis rate of the group which was under irradiation and β-Elemene was significantly higher at 48 h and 72 h, which was analyzed by FCM. The expression of P53 without Bcl-2 was observed in the group under irradiation and β-Elemene and the group under β-Elemene only at the 48th hour point, while the expression of Bcl-2 without p53 was observed in the group under irradiation only and the control group. Conclusion： β-Elemene is good at radiosensitization and its mechanism may be relevant to the up-regulation of P53, down-regulation of Bcl-2 and inducing apoptosis.

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.

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

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

Aression of TLR_9 in human pulmonary adenocarcinoma cell line A549

Objective:Being considered as a bridge between the innate immunity and acquired immunity,Toll-like receptors(TRLs) are very important innate immunity moleculars.Recent researchs on the innate immunity have focused on the relationship between TLRs and human tumor.This paper investgated the expression and significance of TLR9 in human pulmonary adenocarcinoma cell(A549 cell) and human bronchial epithelial cell(HBE cell).Methods:After culturing A549 cell and HBE cell in vitro,the expression of TLR9 mRNA and protein in both cells were detected by immunocytochemistry,Real-time Quantitative Reverse Transcriptase-Polymerase Chain Reaction(Real-time Quantitative PCR) and Western blot,respectively.Results:By immunocytochemistry staining,TLR9 was mainly expressed in both cells' cell membrane and endochylema as brown-yellow material.It showed that the expressions of TLR9 mRNA and protein in A549 cell were stronger than those in HBE cell(P < 0.01).Conclusion:The results suggest TLR9 might cause the progression of human pulmonary adenocarcinoma,and the mechanism needs to be further investgatied.

Empirical studies about quercetin increasing chemosensitivity on human lung adenocarcinoma cell line A549

Objective： The present study was designed to investigate whether quercetin exerts increasing chemosensitivity on human lung adenocarcinoma cells when quercetin combined with cisplatin （DDP） and vincristine （VCR） in vitro respectively and its possible antitumor mechanism. To provide experimental proof for clinical combination application. Methods： Using intermittent administration of high dose VCR, human lung adenocarcinoma sensitive cell line （A549/S） was induced to VCR- resistant human lung adenocarcinoma cell line （A549NCR）. MTT assay was adapted for examing the 50% inhibition （IC50） value of DDP and VCR on A549/S and A549/VCR when quercetin combined with DDP and VCR respectively. Results： IC50 of DDP on A549/S and A549/VCR was 10.18 and 12.35 mg/L, and the IC50 of VCR on the two cell lines was 1.21 and 12.77 rag/L, respectively. The resistance fold of A549/VCR on VCR and DDP was 10.55 and 121, respectively. When quercetin at concentration of 50, 100 and 200 pmol/L in combination with DDP and VCR respectively, the IC50 of DDP and VCR on A549/S and A549/VCR were obvious decreased （P 〈 0.05 - P 〈 0.01）. Conclusion： The experiment results suggested that quercetin could increase the chemosensitivity and partly revise the resistance of A549NCR.

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.

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

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

Role of integrin β1 in sensitivity to chemotherapy of pulmonary adenocarcinoma A549

Objective:The aim of our study was to investigate the effect of integrin β1 on influencing the sensitivity to chemotherapy of human pulmonary adenocarcinoma A549 cells.Methods:Human pulmonary adenocarcinoma A549 multicellular spheroids(MCS) were constructed with three dimensional cell culture methods.Cell counting using blood cell counter was employed to detect the sensitivity to ADM of A549 MCS before and after blocking integrin β1;integrin β1 expression of A549 MCS and cell apoptosis was detected by flow cytometry.Results:A549 MCS were successfully established.The integrin β1 expression of A549 MCS elevated with the concentration of ADM(< 0.02 μg/mL).Blocking of integrin β1 lead to higher sensitivity to ADM,and IC50 decreased from 0.19 μg/mL to 0.11 μg/mL,and apoptosis rate increased from(15.81 ± 1.87)% to(30.14 ± 2.89)%.Conclusion:The cell adhesion molecules integrin β1 could influence the sensitivity to chemotherapy of A549 MCS and inhibiting of cell apoptosis might be its mechanism.

Inhibitory effect of toremifene monotherapy or combined with gemcitabine on A549 human lung adenocarcinoma cells

Objective： The aim of this study was to investigate the effect of toremifene on A549 human lung adenocarci- noma cells, and its sensibilization with gemcitabine, so that to provide a new clinical approach for non-small-cell lung cancer （NSCLC）. Methods： A549 cells were seeded into 96-well plates and exposed to different agents （gemcitabine or gemcitabine with toremifene）. The cytotoxicity of each agent was evaluated by MTT, cell cycle and apoptotic rate were detected by flow cytometry （FCM）. Results： 1. By using FCM, we found A549 cells in S and G2/M phases with toremifene decreased but increased in G0/G1 phase. The higher concentration of toremifene, the more decreased was when compared with the control group. 2. FCM showed toremifene＇s apoptosis effect on A549 cells increased with its increasing dose. 3. By MTT, toremifene had no cytotoxic effect on A549 cells at the concentration of 5 or 2.5 pmol/L. The IC5o of gemcitabine to A549 was 34.51 tJmol/L, and the combined group was 13.59 pmol/L. Conclusion： Toremifene could inhibit the growth of A549 human lung adenocarcinoma cells. Toremifene combined with gemcitabine showed significantly remarkable chemotherapy sensibilization on A549 human lung adenocarcinoma cells.

Endostar enhances anti-tumor effects of radiation via inhibition of HIF-1α and bFGF in lung adenocarcinoma cell line A549

Objective： The aim of the study was to investigate whether enhanced anti-tumor effects of endostar （ES） on radiation involved hypoxia inducible factor-la （HIF-la） and basic ~broblast growth factor （bFGF）. Methods： A549 cells were divided into four groups： control group; endostar （ES） group; radiation （RT） group; endostar plus radiation （ES ＋ RT） group. The inhibition of proliferation rates ofA549 cells was measured by cell counting kit-8 （CCK-8）. HIF-la and bFGF expression levels were checked by enzyme linked immunosorbent assay （ELISA） and reverse transcription-polymerase chain reaction （RT-PCR）. Results： The proliferation inhibition rate in the ES ＋ RT group was higher than that in other groups. On the other hand, the expressions of HIF-1α and bFGF in the ES ＋ RT group were significantly reduced compared to other groups. HIF-1α and bFGF levels were positively correlated （r = 0.80, P 〈 0.01）. Conclusion： Our results suggest that endostar could enhance anti-tumor effect of radiation by reducing expressions of HIF-1α and bFGF.

Effect of three suture lines on the proliferation and cell cycle of lung adenocarcinoma cell A549 in vitro

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

Correlation of STAT3, CEA in lung adenocarcinoma cell A549

Objective: The purpose of this study was to analyze the relationship between signal transducer and activator of transcription 3 (STAT3) and carcinoembryonic antigen (CEA) in lung adenocarcinoma cell A549, and to explore the value of STAT3 on early diagnosis of lung adenocarcinoma. Methods: The expression of CEA, STAT3 mRNA and it's protein in human lung adenocarcinoma cell A549 and normal human lung cells MRC-5 were tested by immunohistochemistry staining (PV) and quantitative real time fluorescent PCR. The correlation between STAT3 and CEA in human lung adenocarcinoma cell A549 was analyzed. Results: The protein and mRNA levels of STAT3, CEA in lung adenocarcinoma cell A549 were apparently higher than those in normal human lung cells MRC-5. The levels of STAT3 mRNA and it's protein were positively correlated with CEA in lung adenocarcinoma cell A549. Conclusion: STAT3 have the same value in diagnosis of lung adenocarcinoma.

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 difference between multi-drug resistant cell line A549/Gem and its parental cell A549

Objective:To discuss the difference between multi-drug resistant cell line A549/Gem and its parental cell A549 on the basis of establishment of human gemcitabine-resistant cell line A549/Gem so as to elaborate the possible mechanisms of gemcitabine resistance.Methods:Human gemcitabine-resistant non-small cell lung cancer cell line A549/Gem was estab-lished by the method of repeated clinical serous peak concentration plus gradually increasing concentration of gemcitabine from its parental cell human lung adenocarcinoma cell line A549 which was sensitive to gemcitabine.During the course of inducement,we had monitored their morphology,checked their resistance indexes and resistant pedigree by MTT method,gathered their growth curves and calculated their doubling time,examined their DNA contents and cell cycles by FCM;at the same time,we had measured their expressions of P53,EGFR,Cerb-B-2,PTEN,PCNA,c-myc,VEGF,MDR-1,Bcl-2,nm23,MMP-9,TIMP-1,and CD44v6 proteins via immunocytochemistry staining,RRM1 and ERCC1 mRNA by real-time fluorescent quantitative-PCR.Results:The resistance index of A549/Gem' cells(the deputy of cells in the process of inducement) 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 A549/Gem' was shorter and figures in G0-G1 phases were increased than A549 cells.Compared with A549 cells,A549/Gem' cells achieved EGFR and c-myc proteins expressions,nm23 protein expression enhanced,P53,Cerb-B-2 and Bcl-2 proteins expressions reduced,PTEN,PCNA and MDR-1 proteins expressions vanished,but those of MMP-9,VEGF,CD44v6 and TIMP-1 proteins changed trivially.Meanwhile,expressions of RRM1 and ERCC1 mRNA were augmented markedly.The resistance index of A549/Gem cells 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 and ERCC1 mRNA decreased visibly.The doubling time of A549/Gem cells was longer and figures in G0-G1 phases were decreased than A549/Gem' cells.In A549/Gem cells,expressions of P53,EGFR,PCNA and MDR-1 proteins was same to those of A549/Gem' cells.A549/Gem cells achieved TIMP-1 and PTEN proteins expressions,Cerb-B-2,MMP-9,c-myc and Bcl-2 proteins expressions enhanced,nm23 protein expressions vanished,but the expressions of VEGF and CD44v6 proteins changed trivially.Furthermore,Compared with its parental cell A549,A549/Gem cell was mixed with giant cells of different sizes and was larger and more irregular.Conclusion:The human gemcitabine-resistant non-small cell lung cancer cell line A549/Gem had achieved multi-drug resistance and great changes of biological characters compared with its parental cells A549.And these changes possibly participated in the formation of multidrug resistance.

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.

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

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

The anti-cancer effect of Huaier aqueous extract with rh-Endostatin and DDP

Objective: The aim of our study was to explore the inhibition and apoptosis-inducing effect of the combination of Huaier aqueous extract with recombinant human Endostatin and DDP in human lung adenocarcinoma A549 cells. We also investigated the reversal effect of Huaier aqueous extract in reversing cisplatin resistance in human lung adenocarcinoma A549/DDP cells. Methods: We treated A549 cells with Huaier aqueous extract and the combination of Huaier aqueous extract and DDP or rh-Endostatin for 24 h, 36 h and 48 h. And then we calculated the inhibition rate through MTT approach and detected the apoptosis rate by flow cytometry. We also treated A549 and A549/DDP cells with DDP, Huaier aqueous extract, DDP and Huaier aqueous extract for 72 h, respectively. Results: Huaier aqueous extract can inhibit the growth of A549 cells and the inhibition rate improved with the increase of the concentration. The inhibition rate of the combination of rh-Endostatin and 4 mg/mL of Huaier aqueous extract in three time points and the combination of rh-Endostatin and 2 mg/mL of Huaier aqueous extract in the time point of 48 h on the growth of A549 cells all improved(P < 0.005). The inhibition rate of the combination of DDP and Huaier aqueous extract with the concentration of 2 mg/mL or 4 mg/mL on the growth of A549 cells all improved(P < 0.005). The combination of Huaier aqueous extract and DDP and the combination of Huaier aqueous extract with rh-Endostatin and DDP can improve the inhibition on the growth of A549 cells(P < 0.005). Conclusion: Huaier aqueous extract has the inhibition and apoptosis-inducing effects on the A549 cells. And the combination of Huaier aqueous extract and rh-Endostatin or DDP has the synergistic effects on the inhibition of A549 cells. The combination of Huaier aqueous extract with rh-Endostatin and DDP has the synergistic effects on the inhibition of A549 cells. Huaier aqueous extract can reverse the cisplatin resistance in human lung adenocarcinoma A549/DDP cells.

Inhibition of lung cancer stem cells self-renewal and tumorigenicity by lentivirus-delivered Bmi1 shRNA

Objective： The aim of the study was to observe the effect of Bmil reduction on the self-renewal and tumorigenicity ability of lung cancer stem cells （LCSCs） in human lung adenocarcinoma. Methods： Human lung adenocarcinoma cells A549 were consecutively passaged in NOD/SCID mice treated with Paclitaxel weekly. The proportions of LCSCs in A549 cells and the cells from the third passage （A549-3rd） were compared. The expression of Bmil in LCSCs was silenced by intratumoral injection with lentivirus-delivered Broil small hairpin RNA （shRNA）. RT-PCR and Western blot were used to test the mRNA and protein expressions of Broil in LCSCs. The protein level of p16INK4A was analyzed by Western blotting. The self- renewal and tumorigenicity ability of LCSCs were evaluated by counting the sphere formation rate in serum-free medium and the tumor formation rate in NOD/SCID mice. Results： In vivo passaging ofA549 cells under chemotherapy pressure enriched for LCSCs. The expression of Broil in LCSCs increased. Down-regulation of Bmil by RNA interference resulted in reduced self-renewal and tumorigenicity ability of LCSCs and paralleled the increased expression of p16INK4A, a Bmil target. Conclu- sion： Broil regulates self-renewal and tumorigenicity of LCSCs by silencing some target genes, including p16INK4A.

Anti-angiogenesis effect of Demethyl bryoanthrathiophene(DBT) in vitro

Objective:The aim of the study was to investigate the effect of Demethyl bryoanthrathiophene(DBT) on proliferations of human umbilical vein endothelial cells(HUVECs) and human lung adenocarcinoma cell line A549,and antiangiogenic effect of DBT on HUVECs in vitro.Methods:MTT assay was used to observe the effect of DBT on proliferations of HUVECs and A549 cells,flat plate scarification assay and tube formation in vitro test were used to observe the impact of DBT on migration and vaso-formed ability of HUVECs.The effects of DBT on apoptosis and cell cycle of HUVECs were calculated by flow cytometry.Results:MTT assay showed that treatment with DBT resulted in strong inhibition to the growth of HUVECs and A549 cells.The inhibition effects of DBT on HUVECs and A549 cells were related to dosage and times of dependency.In different doses of DBT(0.16,0.32 and 0.48 μmol/L) of flat plate scarification for 24 h,inhibition rates of DBT to migration of HUVECs were 14.70%,38.23% and 58.82%,respectively.In dose of DBT from 0.04,0.20 to 0.40 μmol/L for 24 h in tube formation,there were significance differences(P < 0.01) in the decreasing number of angiogenesis and incomplete blood vessel compared with control groups.All results showed that DBT promoted the apoptosis rate of HUVECs,and the increase of concentration of DBT accompanied the acceleration of apoptosis rate.Conclusion:DBT could inhibit the proliferations of HUVECs and A549 cells,and effectively suppress angiogenesis in vitro.