Characterization of Two Human Lung Adenocarcinoma Cell Lines by Reciprocal Chromosome Painting

Lung cancer is a leading cause of cancer death worldwide. Some lung cancer patients correlate with a gas of radon besides smoking. To search for common chromosomal aberrations in lung cancer cell lines established from patients induced by different factors, a combined approach of chromosome sorting, forward and reverse chromosome painting was used to characterize karyotypes of two lung adenocarcinoma cell lines： A549 and GLC-82 with the latter line derived from a patient who has suffered long-term exposure to environmental radon gas pollution. The chromosome painting results revealed that complex chromosomal rearrangements occurred in these two lung adenocarcinoma cell lines. Thirteen and twenty-four abnormal chromosomes were identified An A549 and GLC-82 cell lines, respectively. Almost half of abnormal chromosomes in these two cell lines were formed by non-reciprocal translocations, the others were derived from deletions and duplication/or amplification in some chromosomal regions. Furthermore, two apparently common breakpoints, HSA8q24 and 12q14 were found in these two lung cancer cell lines.

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.

Effects of PI3K Inhibitor NVP-BKM120 on Acquired Resistance to Gefitinib of Human Lung Adenocarcinoma H1975 Cells

The effects of class I PI3K inhibitor NVP-BKM120 on cell proliferation, cell cycle distri- bution, cellular apoptosis, phosphorylation of several proteins of the PI3K/AKT signaling pathway and the mRNA expression levels of HIFl-ct, VEGF and MMP9 in the acquired gefitinib resistant cell line H1975 were investigated, and whether NVP-BKM120 can overcome the acquired resistance caused by the EGFR T790M mutation and the underlying mechanism were explored. MTT assay was performed to detect the effect of gefitinib, NVP-BKM120, NVP-BKM120 plus 1 ~unol/L gefitinib on growth of H1975 cells. The distribution of cell cycle and apoptosis rate of H1975 cells were examined by using flow cytometry. The mRNA expression levels of tumor-related genes such as HIFI-a, VEGF and MMP9 were detected by using real-time quantitative PCR. Western blotting was used to detect the ex- pression level of phosphorylated proteins in the PI3K/AKT signaling pathway, such as Ser473-p-AKT, Ser235/236-p-S6 and Thr70-p-4E-BP1, as well as total AKT, $6 and 4E-BP1. The results showed that the NVP-BKM120 could inhibit the growth of H1975 cells in a concentration-dependent manner, and H1975 cells were more sensitive to NVP-BKM120 than gefitinib （IC50：1.385 vs. 15.09 ~mol/L respec- tively）, whereas combination of NVP-BKM120 and gefitinib （1 ~trnol/L） did not show more obvious ef- fect than NVP-BKM120 used alone on inhibition of cell growth （P〉0.05）. NVP-BKM120 （1 ~unol/L） increased the proportion ofH1975 cells in G0~G1 phase and the effect was concentration-dependent, and 2 ~maol/L NVP-BKM120 promoted apoptosis ofH1975 cells. There was no significant difference in the proportion of H1975 cells in G0-G1 phase and apoptosis rate between NVP-BKM120-treated alone group and NVP-BKM120 plus genfitinib （1 ~unol/L）-treated group or between DMSO-treated control group and gefitinib （1 Ixmol/L）-treated alone group （P〉0.05 for all）. It was also found that the mRNA expression levels of these genes were down-regulated by NVP-BKM120 （1 ~unol/L）, and NVP-BKM120 （1 ~tmol/L） or NVP-BKM120 （1 pmol/L） plus gefitinib （1 ~tmol/L） obviously inhibited the activation of Akt, $6 and 4E-BP1 as compared with control group, but single use of gefitinib （1 pmol/L） exerted no significant effect. These data suggested that NVP-BKM120 can overcome gefitinib resistance in H1975 cells, and the combination of NVP-BKM120 and gefitinib did not have additive or synergistic effects. It was also concluded that NVP-BKM120 could overcome the acquired resistance to gefitinib by down-regulating the phosphorylated protein in PI3K/AKT signal pathways in H1975 cells, but it could not enhance the sensitivity of H 1975 cells to gefitinib.

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...

Inhibitory effects of dobutamine on human gastric adenocarcinoma

AIM: To explore the inhibitory effects of dobutamine on gastric adenocarcinoma cells.

Free radical scavenging property and antiproliferative activity of Rhodiola imbricata Edgew extracts in HT-29 human colon cancer cells

Objective:To investigate the in vitro antioxidant and antiproliferative activity of rhizome extracts of Rhodiola imbricata(R.imbricata) in HT-29 human colon cancer cell line.Methods:The successively extracted rhizome of R.imbricata using various solvents was analyzed for their total phenolics,tannins and flavonoid contents.In vitro antioxidant activity was evaluated by employing different assays,including DPPH,ABTS radical scavenging assays,FRAP, phosphomolybdenum reduction assay,superoxide anion,hydroxyl radical scavenging activities and metal chelating ability.Results:Acetone and methanol extracts recorded higher phenolic content and showed comparable antioxidant activity with standard reference.Additionally,they also inhibited the proliferation of HT-29 cells upon treatment at higher concentration(200μg/mL) (acetone and methanol,84%and 84%.respectively).On examination acetone extract exhibited antiproliferative activity in a concentration dependent manner whereas,methanol extract showed both dose dependent and lime dependent inhibitory activity.Conclusions:The results obtained justify the traditional usage of R.imbricata from their promising antioxidant activity.

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.

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。

构建过表达TRPV1基因的Caco-2细胞株

采用慢病毒载体系统构建辣椒素受体基因TRPV1过表达的人结直肠腺癌细胞Caco-2稳定重组株.将双酶切后的慢病毒空载体pCDH和TRPV1全基因PCR产物通过T4 DNA Ligase连接,构建包含TRPV1基因的过表达载体pCDH-TRPV1.将过表达载体pCDH-TRPV1转化DH 5α感受态细菌,大量扩繁后提取过表达载体pCDH-TRPV1的质粒,与psPAX2和pMD两种含有慢病毒包装所必需元件的质粒混合,再与脂质体混合制备脂质体-载体混合液.将脂质体-载体混合液转染至单层的293T细胞中,培养48h进行病毒包装.收集富含慢病毒颗粒的293T细胞上清液,超离心纯化成浓缩病毒,然后再与polybrene一起感染单层Caco-2细胞,通过GFP绿荧光信号来筛选获得TRPV1基因过表达的稳定细胞株.通过Realtime PCR方法和Western-blot检测TRPV1的mRNA表达量及蛋白表达量,结果表明,Caco-2-TRPV1重组细胞株的TRPV1的mRNA表达量及蛋白表达量均显著高于Caco-2-GFP对照细胞(P<0.05).成功构建了TRPV1基因过表达的稳定细胞株,为后续辣椒素降脂机理的研究提供了正向调控细胞模型.

染料木黄酮对人胃癌细胞生长抑制作用研究

目的 : 探讨染料木黄酮对体外培养的人胃癌 SGC- 790 1细胞的抑制和诱导细胞发生凋亡的作用。方法 : 用 MTT法、集落形成实验、透射电镜及流式细胞仪的方法 ,观察染料木黄酮处理 SGC- 790 1后细胞生长和细胞凋亡。结果 : (1 ) MTT法和集落形成实验证实染料木黄酮对 SGC- 790 1细胞生长有抑制作用 ,抑制作用呈剂量 -效应关系 ; (2 )透射电镜可见 SGC- 790 1细胞在形态学上出现典型的细胞核固缩、碎裂等凋亡细胞的形态学改变 ; (3)流式细胞仪检测到凋亡峰。结论 : 染料木黄酮对 SGC- 790 1细胞有抑制作用 ,而这一作用的机制之一是通过诱导人胃癌细胞发生凋亡 。

钳蝎蝎毒组分Ⅱ对人直肠腺癌C1184细胞生长的影响

蝎毒组分Ⅱ（ＳｃｏｒｐｉｏｎＶｅｎｏｍｃｒｕｄｅⅡ，ＳＶＣⅡ）为河南马氏钳蝎粗毒（ＳＶＣ）经分离纯化后获得的有效成分之一。实验结果表明，以丝裂霉素作为阳性对照，ＳＶＣⅡ（４～２０μｇ／ｍｌ）对人直肠腺癌Ｃ１１８４细胞株有明显杀伤作用。与空白对照相比，ＳＶＣⅡ使细胞生长抑制率、细胞毒性和克隆形成抑制率增大（Ｐ＜０．０５或（Ｐ＜０．０１），并存在剂量效应关系，其半数抑制浓度分别为１１．２０μｇ／ｍｌ、１１．９３μｇ／ｍｌ和１７．３８μｇ／ｍｌ。从生长曲线可见，ＳＶＣⅡ对细胞生长呈剂量和时间依赖性抑制。ＳＶＣⅡ还能使细胞分裂指数下降。提示ＳＶＣⅡ具有抗癌细胞增殖作用。

肺癌细胞系XWLC-05的建立及其生物学特性研究

背景与目的:云南省宣威地区是我国肺癌高发地区之一,女性肺癌死亡率居全国首位,本研究旨在建立云南宣威女性肺腺癌细胞系,为肺癌高发区的防治研究提供理想的体外实验模型。方法:以手术切除的肺癌组织标本进行原代培养,通过光镜观察、电镜观察、绘制生长曲线、计算倍增时间、双层软琼脂培养、流式细胞仪、染色体及其显带分析、肿瘤标志物检测、异体移植实验及免疫组化检测等对其生物学特性进行分析鉴定,建立细胞系。结果:体外培养细胞生长稳定,细胞形态学、增殖动力学及浸润性生长结果表明该细胞系符合恶性细胞特征,染色体数目分布在55～69之间,众数为60～63;小鼠接种成瘤率为100%,瘤细胞形态与原患者的病理切片相似,命名为XWLC-05(XuanweiLungCancer-05)。结论:该细胞系符合建系标准,是一株新建的人肺腺癌细胞系。

大黄素对体外人肺腺癌细胞增殖抑制作用的影响

目的探讨大黄素对人肺腺癌Anip973细胞(简称肺癌细胞)作用的机制。方法采用四甲基偶氮唑蓝(MTT)比色法和生长曲线测定比较不同浓度的大黄素在不同作用时间内对体外培养的肺癌细胞增殖的影响,同时应用流式细胞术检测细胞凋亡率,并对此进行电镜分析。结果在一定范围内,大黄素的浓度越大、作用时间越长对肿瘤细胞生长的抑制作用越强,凋亡率也越高,60μmol/L大黄素处理肺癌细胞72h后增殖抑制率达90%。结论大黄素可明显抑制肺癌细胞的增殖,其抑制作用具有时效和量效关系。

一株人胰腺癌细胞系的建立及其特性

人胰腺癌是很难建系的癌细胞之一,特别是从原发瘤建立的细胞系。我们成功地从一个胰腺癌组织建立了一株人胰腺癌细胞系,命名为PC-3。PC-3细胞呈上皮样,贴壁生长,经过四年连续培养,细胞系稳定。通过免疫组化、电镜观察、染色体及DNA含量分析,细胞集落形成及裸鼠移植,生长因子对瘤细胞生长的影响和癌基因的表达证实PC-3细胞系为人胰腺癌细胞系。PC-3细胞系的建立进一步丰富了人胰腺癌细胞库,对深入了解人胰腺癌细胞生物学及分子生物学特性提供了有力的物质基础。

水飞蓟素可通过MAPK信号转导通路促进肺腺癌A549细胞凋亡

目的:探讨水飞蓟素(SM)对人肺腺癌细胞(A549)的抗癌作用及其分子机制.方法:采用倒置显微镜和电子显微镜等形态学检测、四甲基偶氮唑蓝(MTT)比色法、流式细胞仪(FCM)技术结合PI及Annexin V双标记染色以及MAPK的磷酸化活性检测方法.结果:SM对人肺腺癌A549细胞的增殖有抑制作用;SM作用A549细胞48h后,随着浓度的增加,倒置显微镜下可见细胞数目减少,有些细胞变小、变圆;透射电镜观察发现,随着SM作用浓度的增加,A549细胞中逐步出现增多的凋亡细胞,凋亡细胞表现出典型的超微结构特征;流式细胞仪检测的结果发现,随着药物作用时间的延长,A549细胞的G1期细胞比例增多,S期细胞明显减少,G2期细胞略有减少,并出现一明显的凋亡峰.SM可以抑制A549细胞中ERK的活性,并在一定程度上提高p38和JNK的活性.结论:SM可以在体外抑制人肺腺癌细胞A549的增殖作用,并诱导其凋亡.SM可能通过抑制A549细胞中ERK的活性,并提高p38和JNK的活性发挥其诱导作用.

鳖甲浸出液对人肠癌细胞（HR－8348）的毒性作用研究

报道鳖甲浸出液对人肠癌细胞系（ＨＲ－８３４８）的体外实验，分别从细胞抑制率、细胞集落形成率、细胞上清液中ＬＤＨ活性的测定，光、电镜下的细胞形态观察，探讨了鳖甲对人肠癌的作用机理。初步结果表明：单纯鳖甲２ｇ／Ｌ浓度以上对肠癌细胞有抑制生长作用，鳖甲加氟脲嘧啶联合用药对肠癌细胞的抑制生长作用较单纯氟脲嘧啶更显著。这一实验结果将为临床应用鳖甲抗肠癌治疗提供了实验依据，并提示：鳖甲加氟脲嘧啶联合用药对肠癌的治疗效果更佳，充分显示了协同抗癌作用。

对不同的卵巢上皮性腺癌细胞株建立裸鼠动物模型的实验研究

目的 :探讨卵巢上皮性腺癌细胞株建立裸鼠动物模型的最佳方案。 方法 :采用两种卵巢上皮性腺癌细胞株CAOV3(贴壁细胞 )、COC2 (悬浮细胞 )对裸鼠进行不同细胞密度皮下接种及不同方法的组织块接种。 结果 :在 5× 10 6/ (0 .2 0ml·只 )到 6× 10 7/ (0 .2 0ml·只 )细胞浓度内 ,两株细胞的成瘤率及肿瘤成形时间与接种细胞量无关。与COC2细胞株相比 ,CAOV3成瘤早 (以组织块接种者为甚 ) ,成瘤率高 ,成瘤后生长缓慢。而COC2细胞株一旦成瘤 ,则肿瘤生长速度极快 ,以至于实验尚未完成荷瘤鼠已死亡。 结论 :在建立卵巢上皮性腺癌裸鼠动物模型的实验中 ,以选用贴壁细胞株。

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。

大黄素对人肺腺癌细胞Anip973增殖周期及凋亡基因的影响

目的探讨大黄素对人肺腺癌Anip973细胞生长的抑制作用及其对细胞增殖周期和凋亡基因的影响。方法采用甲基噻唑(MTT)比色法和生长曲线测定不同浓度大黄素在不同作用时间内对在体外培养的Anip973细胞增殖的影响,同时应用流式细胞术检测细胞增殖周期及凋亡率的变化,用免疫印迹法检测Caspase-3蛋白表达。结果大黄素可在G0/G1期阻滞人肺腺癌Anip973细胞的增殖,使S期细胞比率降低,并激活Caspase-3蛋白;在一定范围内,大黄素的浓度越高、作用时间越长,对肿瘤细胞生长的抑制作用越强,凋亡率也越高。结论大黄素能抑制人肺腺癌Anip973细胞生长,其抗肿瘤机制是通过激活Caspase-3,诱导细胞凋亡而阻止细胞周期。

阿司匹林诱导人肺腺癌细胞株SPCA-1凋亡研究

目的 研究非甾体类抗炎药阿司匹林对肺腺癌SP CA 1体外增殖抑制作用及凋亡的影响。方法 用噻唑蓝(MTT)法测定细胞存活率 ;用流式细胞术测定细胞周期时相分布 ,瑞氏染色、Hoechest/PI双染、扫描电镜观察细胞表面形态改变 ,琼脂糖电泳法观察细胞凋亡。结果 阿司匹林对肺腺癌SPCA 1细胞有较强的抑制作用 ,有明显的时间和剂量依赖性 (1 0～ 12 5mmol·L-1) ;可使SPCA 1细胞G0 /G1期、G2 /M期比例明显升高 ,S期比例下降 ;凋亡细胞增多。结论 阿司匹林可能通过影响细胞周期时相分布、诱导凋亡以及引起细胞膜形态而抑制肺腺癌SPCA 1细胞增殖。