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.

The effect of antisense oligodeoxynucleotides targeting Aurora A kinase on cell proliferation and chemosensitivity to paclitaxel in human lung cancer cell line A549

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

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.

芝麻素对长春瑞滨诱导人肺腺癌A549细胞凋亡的影响

[目的]探讨芝麻素对长春瑞滨诱导人肺腺癌A549细胞株凋亡的影响.[方法]选择人肺腺癌A549细胞株进行体外传代培养,制备成浓度为1.0×105个/mL的细胞悬浮液并接种于96孔板中.实验设空白组(加入DMEM培养液)、对照组(加入DMEM培养液及人肺腺癌A549细胞株)、芝麻素组(芝麻素10、20、30、40、50、60、70、80、90、100μg/mL)、长春瑞滨组(长春瑞滨5、10、15、20、25、30、35μg/mL)及联合用药组(加入IC50浓度的芝麻素和长春瑞滨,IC50为后续联合用药组实验药物浓度).空白组加入160μL的DMEM培养液,对照组及实验组各加入160μL已制备好的人肺腺癌A549细胞株悬浮液,待细胞株贴壁后,空白组、对照组及实验组分别加入20μL的生理盐水和20μL各相关浓度的药物.采用MTT法检测细胞增殖能力,利用倒置显微镜及HE染色法观察细胞形态学变化,采用流式细胞仪检测细胞凋亡情况.[结果]在10~100μg/mL质量浓度范围内,低质量浓度的芝麻素具有抑制A549细胞株增殖的作用,IC50质量浓度为40μg/mL;在5~35μg/mL质量浓度范围内,长春瑞滨具有抑制A549细胞株增殖的作用,且随着药物质量浓度升高细胞抑制率升高,IC50质量浓度为20μg/mL;芝麻素与长春瑞滨联合用药对A549细胞株的抑制率明显高于单药用药(P<0.01).倒置显微镜及HE染色法观察结果显示,与对照组比较,芝麻素(40μg/mL)、长春瑞滨(20μg/mL)及联合用药(芝麻素40μg/mL+长春瑞滨20μg/mL)作用于A549细胞株48 h后贴壁细胞数量均明显减少,细胞间连接疏松,贴壁能力减弱,部分细胞体积变小、变圆或呈不规则形,核染色质凝集,细胞膜起泡形成凋亡小体,失去原有肿瘤细胞多角形或梭形形态,且联合用药组较单药组上述变化更为明显.流式细胞仪检测结果显示,药物作用48 h后,芝麻素组(40μg/mL)、长春瑞滨组(20μg/mL)及联合用药组(芝麻素40μg/mL+长春瑞滨20μg/mL)细胞凋亡率均明显高于对照组(P<0.01),且联合用药组早期凋亡率明显高于单独用药组(P<0.01).[结论]芝麻素可增强长春瑞滨诱导人肺腺癌A549细胞凋亡的作用.

DCLK1激活FAK/PI3K/AKT/mTOR信号通路促进A549细胞的恶性行为

目的探讨双肾上腺素皮质样激酶1(DCLK1)对A549细胞增殖、迁移、侵袭等恶性生物学行为的影响,并探究可能涉及的相关分子机制。方法慢病毒感染法建立稳定表达DCLK1分子的A549细胞系,反转录-聚合酶链技术和蛋白质印记法进行鉴定。CCK-8与平板克隆实验检测过表达DCLK1后细胞增殖能力变化。Transwell实验观察过表达DCLK1对细胞迁移与侵袭能力的影响。癌症基因组图谱(TCGA)数据库分析DCLK1对肺腺癌细胞的调控富集通路,蛋白质印记法进行验证。结果DCLK1在A549细胞中过表达可增加细胞的增殖、迁移与侵袭等能力,而抑制FAK/PI3K/AKT/mTOR信号通路可削弱DCLK1对A549细胞的恶性调控。结论DCLK1通过激活FAK/PI3K/AKT/mTOR信号通路,促进A549细胞的恶性生物学行为。

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.

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.

肺岩宁方介导Atg5对肺癌A549细胞自噬调控抗肺癌侵袭转移的实验研究

目的探讨肺岩宁方(Feiyan Ning Decoction,FYN)通过介导Atg5(自噬相关靶向基因5,Autophagy-related gene 5)自噬相关基因调控人肺腺癌A549细胞自噬,及其对A549肺癌细胞增殖、迁移、侵袭等作用的影响。方法肺岩宁方干预培养肺癌A549细胞,通过运用CCK-8(Cell Counting kit-8)细胞增殖实验检测肺岩宁方对细胞增殖抑制率的影响,显微镜下观察肺岩宁方作用后的细胞形态学改变;采用划痕实验检测肺岩宁方及自噬抑制剂氯喹(Chloroquine diphosphate salt,CQ)对肺癌A549细胞迁移能力;运用Transwell细胞侵袭实验检测肺岩宁方及氯喹对肺癌A549细胞侵袭能力;运用Western Blot检测自噬相关蛋白Atg5、LC3表达情况;运用透射电子显微镜观察肺癌A549细胞在肺岩宁方干预下的自噬结构形态改变情况。结果CCK8检测试验结果:肺岩宁方能有效抑制A549细胞增值活力(P<0.01),其优化浓度为200μg mL-1;划痕实验结果:与对照组比,肺岩宁方干预A549细胞迁移能力减弱(P<0.05),A549细胞在FYN联合CQ的作用下迁移能力明显减弱(P<0.01);Transwell实验结果:与对照组比较,肺岩宁方抑制细胞侵袭转移能力(P<0.05),FYN联合CQ细胞侵袭力明显抑制(P<0.05);Western Blot实验结果:肺岩宁方下调自噬相关蛋白Atg5、LC3表达能力(P<0.01);透射电子显微镜观察结果:肺岩宁方抑制自噬结构的出现。结论肺岩宁方介导Atg5自噬相关基因,具有调控人肺腺癌A549细胞自噬能力,可抑制A549细胞增殖活力、迁移能力、侵袭转移水平。

益气养阴散结方对A549裸鼠肿瘤脂滴相关蛋白与脂代谢相关蛋白的影响

目的探讨益气养阴散结方对A549裸鼠肿瘤脂滴相关蛋白、脂代谢相关蛋白的影响。方法取4~6周龄BALB/c裸鼠30只,皮下接种A549细胞株以复制肺腺癌小鼠模型,按照随机数字表法分为五组,模型组、顺铂组和益气养阴散结方低、中、高剂量组,每组6只。接种次日起给药,模型组给予生理盐水0.3 mL/d;顺铂组给予顺铂4 mg/kg,每周2次;益气养阴散结方低、中、高剂量组给予5、10、20 g/(kg·d)益气养阴散结方,连续给药8周。采用免疫印迹、免疫组化检测脂周素-1(perilipin-1)、脂滴蛋白5(LSDP5)、47kDa尾连蛋白(TIP47)、脂肪分化相关蛋白(ADRP)、脂肪细胞型脂肪酸结合蛋白(A-FABP)、Delta样因子-1(DLK-1)、小窝蛋白-1(caveolin-1)等脂滴相关蛋白、脂代谢相关蛋白在肿瘤中的表达。结果30只裸鼠成瘤,成瘤率100%。在实验期内,模型组和益气养阴散结方中剂量组各死亡裸鼠1只。免疫印迹结果表明,与模型组比较,益气养阴散结方低、高剂量组显著下调脂滴相关蛋白、脂代谢相关蛋白的表达[perilipin-1:(0.76±0.23)、(0.58±0.18)比(0.93±0.22);LSDP5:(0.79±0.23)、(0.38±0.11)比(1.06±0.30);TIP47:(0.49±0.19)、(0.24±0.06)比(0.71±0.25);ADRP:(0.48±0.15)、(0.27±0.08)比(0.61±0.17);A-FABP:(0.52±0.14)、(0.31±0.09)比(0.59±0.19);DLK1:(0.75±0.23)、(0.64±0.08)比(1.07±0.21);caveolin-1:(0.60±0.25)、(0.41±0.09)比(1.09±0.31),P<0.05或P<0.01]。免疫组化结果表明,与模型组比较,益气养阴散结方中、高剂量组显著下调脂滴相关蛋白、脂代谢相关蛋白的表达[perilipin-1:(4.17±0.32)、(3.90±0.57)比(5.70±0.97);TIP47:(5.13±0.50)、(3.73±0.31)比(5.67±0.70);ADRP:(4.67±1.01)、(4.17±0.61)比(6.13±0.81);AFABP:(4.02±0.40)、(3.40±0.23)比(5.67±0.75);caveolin-1:(5.03±0.50)、(4.23±0.35)比(6.33±0.93),P<0.05或P<0.01],高剂量组下调DLK1表达[(5.10±0.40)比(7.93±0.91),P<0.01]。结论高剂量益气养阴散结方可显著降低perilipin-1、LSDP5、TIP47、ADRP、A-FABP、DLK1、caveolin-1等脂滴相关蛋白、脂代谢相关蛋白在A549裸鼠肿瘤中的表达。

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.

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.

β-榄香烯对人肺腺癌A549细胞株裸鼠移植瘤放疗增敏作用的研究

目的建立人肺腺癌A549细胞株裸鼠移植瘤模型,用增敏剂量的β-榄香烯联合放疗对移植瘤进行干预,探讨放疗增敏机制是否与抑制葡萄糖转运蛋白1(GLUT-1)表达有关。方法采用细胞悬液接种法建立裸鼠移植瘤模型,将肿瘤体积达到75 mm^(3)左右的20只裸鼠随机分为空白对照组(NS组)、β-榄香烯单药组(ELE组)、β榄香烯联合放疗组(ELE+RAD组)和单纯放疗组(RAD组)。监测干预后各组裸鼠肿瘤体积的变化,获得各组裸鼠的肿瘤生长曲线。通过计算增敏系数,检测45 mg·kg^(-1)β-榄香烯是否发挥增敏作用,采用Real-Time PCR、Western blotting及免疫组化染色检测各组移植瘤中GLUT-1的表达水平。结果成功建立A549细胞株裸鼠移植瘤模型,依据各组移植瘤体积变化绘制生长曲线。测得增敏系数(EF)为2.44,提示β-榄香烯的剂量已达到增敏效果。与NS组相比,实验组移植瘤GLUT-1 mRNA及蛋白表达水平均显著降低(P<0.01);ELE组与RAD组GLUT1 mRNA及蛋白表达水平无显著差异(P>0.05);与ELE组、RAD组相比,ELE+RAD组GLUT-1 mRNA及蛋白表达水平均显著降低(P<0.01)。免疫组化结果显示,与NS组比较,实验组GLUT-1的表达均被显著抑制(P<0.05);其中ELE组和RAD组GLUT-1的表达抑制作用相对较弱,RAD组的抑制作用略强于ELE组,但两组差异无统计学意义(P>0.05);而ELE+RAD组GLUT-1的表达抑制作用较强(P<0.05)。结论增敏剂量的β-榄香烯与放疗联合应用可显著抑制裸鼠移植瘤中GLUT-1 mRNA及蛋白表达,明显增强放疗对肺癌裸鼠移植瘤的生长抑制效果,提示β-榄香烯可能通过抑制GLUT-1表达发挥放疗增敏作用。

TIME-AND DOSE-DEPENDENT UP-REGULATION OF TNF-α mRNA AFTER IRRADIATION OF HUMAN NSCLC CELL LINES IN VITRO

Objective： Even though radiotherapy plays a major role in the local treatment of non-small cell lung cancer （NSCLC）, little is known about the molecular effects of irradiation in this tumor. In the present study, we examined two NSCLC cell lines for their endogenous production of TNF-α after irradiation. To investigate the radiation-induced TNF-α production in NSCLC cell lines. Methods： Two human NSCLC cell lines （A549： squamous; NCI-H596： adenosquamous） were investigated for their TNF-α mRNA （real-time RT-PCR） after exposure to different irradiation doses （2, 5, 10, 20, 30, 40 Gy） and time intervals （1, 3, 6, 12, 24, 48 or 72 h）. The TNF-α mRNA expression was quantified by real-time RT-PCR. The clonogenic survival was evaluated after irradiation with 2, 4, 6 and 8 Gy. Results： Non-irradiated NSCLC cells exhibited no or very low TNF-α expression. For the NCI-H596 cell line, TNF-α expression was significantly elevated 1～12 h （maximum 6h： 568fold increase relative to unirradiated cells） in a time-dependent manner. The radiation-induced increase could be observed after irradiation with 2 Gy reaching maximal at 40 Gy, with 83 times higher than normal controls. The clonogenic survival of these cell lines was nearly identical. Conclusion： NCI-H596 cells produce significant quantities of TNF-α following irradiation in a time- and dose-dependent manner. The pro-inflammatory cytokine TNF-α is a key mediator for the pathogenesis of radiation pneumonitis. Radiation-induced endogenous TNF-α expression in NSCLC cells may affect the normal lung adjacent to the tumor and may be associated with an adverse clinical outcome of the patient.

Reversal effect of recombinant human Endostatin on cisplatin resistance in A549/DDP human lung adenocarcinoma cells in vitro

Objective： Recombinant human Endostatin （rh-Endostatin, YH-16） can reverse cisplatin resistance in A549/DDP cells. However, the possible effect of rh-Endostatin in reversing DDP-resistance in A549/DDP cells and the mechanism are needed to be investigated. Methods： Lung adenocarcinoma cell line A549 and its DDP-resistant cell line A549/DDP were treated with DDP and/or recombinant human Endostatin. Difference in drug resistance was analyzed between different regi- mens and between different cell lines after a 72 h-treatment in vitro. And below the non-cytotoxic concentration of rh-End- ostatin, the possibility of rh-Endostatin in reversing DDP-resistance in A549/DDP was evaluated. The resistance protein which was detected in the study included P glycoprotein （P-gp） and topoisomerase II （Topo-II）. Results： Rh-Endostatin below 400 IJg/mL showed no cytotoxicity in either A549 or A549/DDP after 72 h-treatment with it. The inhibited concentration of 50% （IC50） observed for DDP was （0.79 _＋ 0.05） IJg/mL in A549 and （13.2 ＋ 1.1） in A549/DDP respectively. IC50 was reduced to 2.57 ＋ 0.05 #g/mL in A549/DDP treated by rh-Endostatin below the non-cytotoxic concentrations in combination with DDP, with a reversal fold （RF） of 5.14 and a relative reversal rate of 85.6%. Apoptotic rates were 2.01%, 13.47% and 29.26% re- spectively for cells treated with rh-Endostain, DDP, and the combination. The rate of the A549/DDP control group was 0.99%. The expression level of P-gp or Topo-II was higher in A549/DDP cells than in A549 cells. Rh-Endostatin may partially reverse DDP-resistance in A549/DDP cells in vitro, with a probable mechanism related to lowering expression of P-gp and Topo-II. Conclusien： Rh-Endostatin of non-cytotoxic dose partially reversed cisptatin resistance in cisplatin-resistant human lung adenocarcinoma cell line A549/DDP. Rh-Endostatin reversed the resistance of A549/DDP cells to DDP, which may be related to decreased protein expression of P-gp and Topo-II in A549/DDP cells.

E3泛素连接酶三结构域蛋白59对非小细胞肺癌细胞增殖和侵袭的影响

目的探讨三结构域蛋白59(TRIM59)对非小细胞肺癌细胞增殖、迁移和侵袭的影响及作用机制。方法将非小细胞肺癌细胞系A549分为siControl组、siTRIM59组、Flag-Vector组和Flag-TRIM59组,用CCK-8实验检测细胞增殖活力;用TUNEL染色实验检测细胞凋亡率;用划痕实验和Transwell实验检测细胞迁移和侵袭能力;用蛋白质免疫印记实验检测AKT表达水平和磷酸化水平。结果与siControl组相比,siTRIM59组细胞增殖活力下降(P<0.05),凋亡比例升高(P<0.05),细胞迁移率降低(P<0.05),侵袭到Transwell下层小室的细胞数减少(P<0.05),AKT磷酸化水平下降。与Flag-Vector组相比,Flag-TRIM59组细胞增殖活力增强(P<0.05),凋亡比例降低(P<0.05),细胞迁移率升高(P<0.05),侵袭到Transwell下层小室的细胞数增加(P<0.05),AKT磷酸化水平上升。结论TRIM59促进非小细胞肺癌细胞的增殖、迁移和侵袭,以及AKT的磷酸化。

LncRNA FEZF1-AS1通过调控EZH2对肺间质细胞增殖、迁移及侵袭的作用

目的研究长链非编码RNA FEZ家族锌指1-反义RNA 1(lncRNA FEZF1-AS1)调控zeste同源物增强子2(EZH2)对肺间质细胞增殖、迁移、侵袭能力及上皮细胞-间质转化(EMT)的影响及其作用机制。方法将人肺腺癌细胞系A549分为对照组(control)和模型组[model,用转化生长因子β1(TGF-β1)20 ng/mL作用48 h,诱导成为肺间质细胞]。用Western blot检测细胞中E-钙黏蛋白(E-cadherin)、N-钙黏蛋白(N-cadherin)及波形蛋白(vimentin)的蛋白表达。RT-qPCR检测细胞中lncRNA FEZF1-AS1和EZH2基因表达。转染组细胞分为转染si NC组、si lncRNA FEZF1-AS1+OE vector组和si lncRNA FEZF1-AS1+OE EZH2组。CCK-8法检测细胞增殖、细胞划痕检测细胞迁移、Transwell小室法检测细胞侵袭;用Western blot检测细胞中E-cadherin、N-cadherin、vimentin及EZH2的蛋白表达,用RNA免疫沉淀(RIP)测定FEZF1-AS1与EZH2的直接结合作用。结果与对照组比较,模型组E-cadherin的蛋白表达水平减少(P<0.05);N-cadherin及vimentin的蛋白表达水平升高(P<0.05);与对照组比较,模型组lncRNA FEZF1-AS1与EZH2基因的表达水平明显升高(P<0.05);与si NC组相比,si lncRNA FEZF1-AS1+OE vector组细胞增殖、迁移、侵袭能力降低,E-cadherin蛋白表达升高,N-cadherin、vimentin、EZH2蛋白表达降低(P<0.05);与si lncRNA FEZF1-AS1+OE vector组比较,si lncRNA FEZF1-AS1+OE EZHZ组细胞增殖、侵袭、迁移能力升高,E-cadherin蛋白表达降低,N-cadherin、vimentin、EZH2蛋白表达升高(P<0.05);RIP实验进一步证实了lncRNA FEZF1-AS1与EZH2具有结合作用。结论LncRNA FEZF1-AS1通过调控EZH2促进肺间质细胞增殖、侵袭、转移和EMT过程。

丹参酮ⅡA诱导人肺腺癌A549细胞凋亡

目的探讨丹参酮ⅡA(TanshinoneⅡA,TanⅡA)抗人肺腺癌A549作用及其可能作用机制。方法通过细胞形态学和MTT法观察TanⅡA对A549细胞增殖的影响;应用Hoechest33258和PI双染法观察细胞凋亡;采用荧光分光光度计检测细胞内钙及线粒体膜电位;RT-PCR检测Bad和MT-1A mRNA的表达。结果 TanⅡA能抑制A549细胞增殖,且随TanⅡA剂量的增加和作用时间的延长而增强,TanⅡA作用A549细胞24、48和72h的IC50分别为117.85、14.87和6.89μmol·L-1。TanⅡA作用A549细胞24h后,A549细胞出现染色质聚集等典型的凋亡形态学改变,且随TanⅡA剂量的增加,A549细胞凋亡百分率逐渐增大。TanⅡA作用后,A549细胞的细胞内钙升高、线粒体膜电位降低、Bad mRNA表达增加、MT-1A mRNA表达下调。结论 TanⅡA具有抗A549作用,其诱导细胞凋亡可能与钙依赖性通路和MT-1A表达下调有关。

荷包牡丹碱对人肺癌A549细胞生长及端粒酶活性的抑制作用

目的探讨荷包牡丹碱对人肺癌A549细胞生长的抑制作用及其作用机制。方法 A549细胞加入荷包牡丹碱0～200μmol.L-1分别作用24,48和72 h,MTT法测定A549细胞的生长抑制作用。荷包牡丹碱0～20μmol.L-1作用72 h,端粒重复序列扩增(TRAP)法测定端粒酶活性。变温紫外法检测荷包牡丹碱9μmol.L-1对端粒酶G-四链体的稳定作用。结果荷包牡丹碱25,50,100和200μmol.L-1作用细胞72 h后的抑制率分别为33.4%,88.2%,88.6%和89.4%,明显高于正常对照组细胞(P<0.05),并具有量效(r=0.906,P<0.05)和时效(r=0.949,P<0.05)性。与正常对照组相比,荷包牡丹碱5,10,15和25μmol.L-1可有效抑制A549细胞端粒酶的活性(P<0.05),相对TRAP端粒酶活性从正常对照组的1.471±0.102分别降低为1.093±0.054,1.013±0.016,0.554±0.034,0.365±0.081(P<0.05)。荷包牡丹碱9μmol.L-1使G-四链体的熔点值从正常对照组的48℃提高到54℃。结论荷包牡丹碱可以通过稳定G-四链体结构,抑制端粒酶活性,有效抑制人肺腺癌细胞A549的生长。

四嗪二甲酰胺对肺癌细胞株A549的体内外作用

为了观察四嗪二甲酰胺(ZGDHu-1)体外抑制肺癌细胞株A549增殖、诱导细胞凋亡和体内抗肿瘤活性的作用及其机制,将不同浓度的ZGDHu-1与A549细胞在体外培养,用台盼蓝染色、SRB法、5′-溴-2′脱氧尿苷-ELISA法,观察ZGDHu-1对A549细胞增殖的作用;用细胞形态学、DNA凝胶电泳、DNA含量及细胞周期分析、AnnexinV/PI双标记、Hoechst33258荧光染色等技术检测细胞凋亡。腹腔注射ZGDHu-1后观察其对裸鼠移植瘤生长的抑制作用。用RT-PCR和流式细胞术观察A549细胞bcl-2,bax,p53基因和蛋白质的表达改变。结果表明,ZGDHu-1能抑制A549细胞的增殖和活力,呈现作用时间和剂量的依赖关系。A549细胞经ZGDHu-1作用后,大部分细胞阻滞于G2-M期;出现DNA片段化,亚G1峰显著增加,AnnexinV+/PI-表达升高,Hoechst33258荧光染色后出现凋亡细胞的特征性改变等。ZGDHu-1以10,20及40mg.kg-1剂量给裸鼠体内用药14d后,移植瘤生长抑制率分别为43.7%,56.9%和60.0%。A549细胞经ZGDHu-1作用后,bcl-2基因和蛋白有所下调,但主要是上调bax基因和蛋白,导致bax/bcl-2比值明显增高,p53基因和蛋白表达也上调,均呈现剂量依赖性。ZGDHu-1在体内能明显抑制移植瘤的生长,体外通过诱导细胞凋亡抑制A549细胞增殖,其机制可能与上调bax和p53基因的表达有关。

转化生长因子-β1诱导A549细胞向间充质细胞转化的作用研究

目的:观察重组人转化生长因子-β1(rhTGF-β1)对人肺上皮细胞株(A549)的转化作用。方法:将体外培养的A549用不同浓度的rhTGF-β1干预后,倒置显微镜观察细胞形态学的变化,用MTT法检测rhTGF-β1对A549增殖的影响。48 h收集蛋白用蛋白质印迹和间接免疫化学方法检测间充质细胞表型Fibronectin-EDA(Fn-EDA)表达变化。结果:rhTGF-β1诱导A549向间充质细胞形态转化,在体外对A549增殖无明显影响,上调间充质细胞表型Fn-EDA表达。结论:rhTGF-β1可诱导A549发生上皮细胞-间质转化(ephethlial-mesenchymal transition,EMT),支持肺上皮细胞发生EMT的观点。