黄芩苷对肺腺癌细胞多药耐药性的影响及机制

目的探讨黄芩苷对人肺腺癌细胞株A549多药耐药性(MDR)的影响及作用机制。方法体外培养人肺腺癌细胞株A549后给予0、0.5、1、2、4、8mg/L黄芩苷对细胞进行处理,MTT法检测不同浓度黄芩苷对A549细胞生长的影响情况;逆转录-聚合酶链反应法(RT-PCR)检测各组细胞多药耐药基因1(MDR-1)、多药耐药相关蛋白-1(MRP1)、谷胱苷肽转移酶-π(GST-π)、拓扑异构酶(TOPO)ⅡαmRNA的表达情况。结果与对照组相比,黄芩苷组细胞生长明显受到抑制(P<0.05或<0.01);黄芩苷处理后肿瘤细胞MDR-1、MRP1、GST-π、TOPOⅡαmRNA表达水平降低,与对照组比较差异均有统计学意义(P<0.01)。结论 baicalin可以调节多种MDR基因的表达,具有明显的逆转肺腺癌细胞MDR的作用。     

The change of invasive ability of human pneumonic adenocarcinoma cell line in vitro before and after intervention with baicalin

目的 研究黄芩苷(baicalin)对人肺腺癌细胞株A549侵袭能力的影响,探讨baicalin抑制肺腺癌细胞侵袭能力的相关机制.方法 分别用0、0.5、1、2、4、8 mg/L baicalin处理A549细胞株,对照组加等量0.5% DMSO,孵育48 h后,细胞划痕试验测定细胞迁移抑制率;分别用上述浓度的baicalin处理A549细胞株16 h,Transwell小室侵袭试验测定细胞侵袭抑制率;2 mg/L baicalin处理细胞48 h,RT-PCR方法分别测定基质金属蛋白酶(MMP)-2、MMP-9、细胞间黏附分子(ICAM)-1的mRNA表达.结果 baicalin可明显抑制人肺腺癌细胞株A549的迁移和侵袭,与对照组(0 mg/L baicalin)比较,baicalin处理组MMP-2、MMP-9、ICAM-1 mRNA表达和活性均显著下降(均P<0.05).结论 baicalin对人肺腺癌细胞的侵袭能力有明显抑制作用,其机制可能与下调MMP-2、MMP-9、ICAM-1表达有关.     

芍药苷对肺腺癌细胞A549凋亡的诱导作用

目的:研究芍药苷对肺腺癌A549细胞凋亡的诱导作用。方法:以0(阴性对照)、5、10、20、40μmol/L芍药苷培养A549细胞48 h后,以MTT法检测A549细胞活力。上述浓度芍药苷培养A549细胞24 h后,酶标法检测半胱氨酸天冬氨酸蛋白水解酶3(Caspase-3)活性;Western blot法检测Bcl-xl、Bax、核因子κB p65(NF-κB p65)、磷酸化核因子κB p65(NF-κB pp65)蛋白表达。结果:与阴性对照比较,5~40μmol/L芍药苷培养A549细胞48 h后,细胞活力减弱;5~40μmol/L芍药苷培养细胞24 h后,细胞Caspase-3活性、Bax蛋白表达增强;10~40μmol/L芍药苷培养细胞24 h后,细胞Bcl-xl蛋白表达减弱,NF-κB pp65蛋白表达减弱;以上差异均具有统计学意义(P<0.01或P<0.05)。结论:芍药苷可诱导A549细胞凋亡,其机制可能与激活NF-κB信号通路、调节相关基因表达有关。     

芬苯达唑通过激活自噬抑制肺腺癌细胞增殖及侵袭、迁移能力

目的 探究芬苯达唑对肺腺癌细胞增殖、迁移、侵袭的影响以及自噬与迁移、侵袭的关系。方法 将人肺腺癌A549、H358细胞分为对照组及芬苯达唑（1、2.5、5、10、20 μmol/L）组。用CCK-8法及细胞计数法检测细胞增殖水平,用划痕实验及Transwell实验检测细胞迁移、侵袭能力,通过RT-qPCR及蛋白免疫印迹法检测上皮-间充质转化（EMT）相关蛋白的mRNA及蛋白表达水平,通过转录组测序并分析自噬相关基因及其表达量。用自噬抑制剂氯喹（5、10 μmol/L）与芬苯达唑（5 μmol/L）处理细胞后再通过划痕及Transwell实验检测细胞迁移、侵袭能力。结果 与对照组相比,芬苯达唑各浓度处理组能显著抑制A549和H358细胞活性及迁移、侵袭能力（P＜0.05、0.01、0.001）;随着芬苯达唑药物浓度增加,N-钙黏蛋白（N-cadherin）、细胞角蛋白（Cytokeratin）、纤维连接蛋白（FN1）mRNA相对表达逐渐下降（P＜0.05、0.01、0.001）;紧密连接蛋白（Occludin）、波形蛋白（Vimentin）mRNA表达水平在低浓度时较高,随着芬苯达唑药物浓度增加Occludin、Vimentin mRNA表达水平降低（P＜0.05、0.01、0.001）。与对照组相比,随着芬苯达唑药物浓度增加,N-cadherin、Cytokeratin、FN1、Occludin蛋白相对表达量显著降低,而Vimentin蛋白相对表达水平随药物浓度降低而增高（P＜0.05、0.01、0.001）。基因差异热图显示,芬苯达唑处理后自噬相关基因（MAP1LC3B、ATG5、ULK3、Bax、ATG16L1）表达量增高,芬苯达唑的浓度增加使自噬相关蛋白重组人自噬效应蛋白（Beclin-1）、微管相关蛋白轻链3（LC3）-II/LC3-I相对蛋白表达量升高,选择性自噬接头蛋白P62表达量明显降低（P＜0.05、0.01、0.001）。在使用了氯喹阻断自噬后,可以逆转芬苯达唑引起的细胞增殖、迁移及侵袭（P＜0.05、0.01、0.001）抑制作用。结论 芬苯达唑可以通过促进自噬从而抑制了肺腺癌细胞的增殖、迁移和侵袭能力。     

黄芩苷的抗肿瘤作用及对肿瘤细胞端粒酶的影响

目的 探讨黄芩苷的抗肿瘤作用及其对肿瘤细胞端粒酶的影响.方法 采用四氮唑盐法(MTT法)检测黄芩苷对体外培养的3种人肿瘤细胞增殖的抑制作用,计算抑瘤率;体内试验检测黄芩苷对2种肿瘤的生长抑制作用;采用端粒重复序列扩增-聚合酶链反应-酶联免疫吸附测定法(TRAP-PCR-ELISA)方法检测Eca-109细胞在黄芩苷作用后端粒酶活性.结果 黄芩苷在20～100μg/ml剂量范围内对3种肿瘤细胞均有剂量依赖性抑制作用(P＜0.01);体内试验结果显示黄芩苷对接种的实体瘤S180、肝癌H22细胞株的抑瘤率分别为29.95～71.5%和35.1%～80.6%;黄芩苷作用后,Eca-109细胞端粒酶活性明显抑制(P＜O.01).结论 黄芩苷对体外培养的人肿瘤细胞和接种的小鼠肿瘤具有明显抑制作用,黄芩苷对肿瘤细胞的端粒酶活性有抑制作用.     

HPLC同时测定黄芩花中野黄芩苷和黄芩苷的含量

目的 建立HPLC同时测定黄芩花中野黄芩苷和黄芩苷含量的方法,并控制其质量。方法 收集不同时期（初花期、盛花期、凋花期）黄芩花样品,采用HPLC测定其中的野黄芩苷和黄芩苷含量。色谱柱ZORBAX Eclipse XDB-C₁₈（4.6 mm×250 mm,5 μm）,流动相为甲醇-水-磷酸（47：53：0.2）,流速1.0 mL·min^-1,检测波长280 nm;柱温30℃。结果 黄芩花中,野黄芩苷平均含量为1.690%,黄芩苷平均含量为0.306%;8月中旬盛花期时,2种指标成分含量最高,分别为1.857%,0.360%。结论 本法测定黄芩花中野黄芩苷和黄芩苷的含量简便、准确、快捷,可用于黄芩花的质量控制。     

HPLC法同时测定通宣理肺颗粒中黄芩苷和柚皮苷的含量

探讨高效液相色谱法同时测定通宣理肺颗粒中黄芩苷和柚皮苷的含量。方法：色谱柱：Agilent HC-C18 ( 4.6 mm×250 mm,5 μm );流动相：乙腈：0.1%磷酸 ( 22:78 );流速：1.0 mL?min-1;柱温：25 ℃;检测波长：281 nm;进样量：10 μL。结果：黄芩苷和柚皮苷分别在0.359-5.322 mg?mL-1和0.156-2.408 mg?mL-1范围内呈良好的线性关系。测定通宣理肺颗粒中黄芩苷和柚皮苷的含量分别为2.7225 mg?g-1和0.7963 mg?g-1。黄芩苷平均回收率95.58%,RSD为1.56%;柚皮苷平均回收率94.26%,RSD为1.68%。结论：本方法简单方便、准确可靠、专属性强,可用于通宣理肺颗粒药物的质量控制。     

RP-HPLC法测定肺泰胶囊中黄芩苷的含量

目的：用反相高效液相色谱法测定黄芩苷的含量。方法：反相高效液相色谱法,色谱柱为Inertsil-ODS-3（250mm×4.6mm,5μm）,流动相：甲醇-0.2%磷酸溶液（45∶55）,流速：1.0mL/min;检测波长为280nm[1]。结果：黄芩苷在0.1036～1.036μg范围内呈良好的线性关系,r=0.9998,回收率为98.7%,RSD=1.1%（n=5）。结论：本法简便、准确、重现性好,专属性强,可用于肺泰胶囊的质量控制。     

Caco-2细胞模型比较黄芩苷和黄芩苷滴丸在小肠的吸收

目的：比较黄芩苷和黄芩苷滴丸在小肠的吸收。方法：采用Caco-2细胞单层模型研究黄芩苷和黄芩苷滴丸由绒毛面到基底面的跨膜转运过程。通过测定黄芩苷和黄芩苷滴丸在Caco-2细胞模型的转运百分率及表观渗透系数（Papp）,比较二者的跨膜转运能力。结果：在细胞转运实验中,135min时,黄芩苷和黄芩苷滴丸的转运百分率分别为1.00%和6.25%,Papp分别为（0.664±0.103）×10-6cm·s-1和（4.462±1.10）×10-6cm·s-1,黄芩苷滴丸的Papp是黄芩苷的6倍。在跨膜转运180min内,黄芩苷滴丸的转运百分率与时间成正比。结论：将黄芩苷制成滴丸可能提高黄芩苷在小肠的吸收。     

黄芩苷的抗肿瘤作用研究进展

黄芩苷(baicalin)是从唇形科植物黄芩(Scutellaria baicalensis Georgi)中提取的主要生物活性成份,属于黄酮类化合物,黄芩苷药用价值高,具有抗菌、抗炎、抗氧化和保护神经等多种药理作用,并显示出巨大的抗肿瘤潜力.近几年研究发现,黄芩苷对人多种肿瘤具有较好的抑制作用,其作用机制包括诱导肿瘤...     

顺铂与氯霉素联用增强对人肺腺癌A549细胞的抑制作用

目的体外观察顺铂和氯霉素对人肺腺癌A549细胞的相互作用。方法采用MTT法,利用中效原理判定联合应用顺铂及氯霉素对人肺腺癌A549细胞的效应。结果顺铂与氯霉素联用可以明显抑制人肺腺癌A549细胞的生长,两药单用和联用时随着药物浓度增加,其效应也增加;两药联用时的中效浓度明显小于两药单独使用时的中效浓度（顺铂减少43.8%,氯霉素减少79.1%）;两药大剂量合用时为拮抗效应（CI〉1）,小剂量合用时为协同效应（CI〈1）;并且两药合用时的效应与给药的时间先后次序无关（P〉0.05）。结论顺铂与氯霉素的联用可以明显抑制人肺腺癌A549细胞的生长,两药联用时的中效浓度较单用时明显减小;上述两种药合用时大剂量为拮抗,小剂量为协同;两药合用时的效应与给药的时间先后次序无关。     

黄芩苷体外调控乳腺癌细胞表达TIMP2的实验研究

目的:探讨黄芩苷对Bcap37细胞生长及TIMP2表达的影响。方法:体外细胞培养、MTT法测定不同浓度黄芩苷对Bcap37细胞生长曲线的影响,RTPCR、Westernblot方法研究不同浓度黄芩苷对Bcap37细胞TIMP2的影响。结果:黄芩苷对Bcap37细胞的生长抑制呈浓度依赖关系;黄芩苷对Bcap37细胞TIMP2在mRNA水平、蛋白水平呈浓度依赖关系,小剂量上调,高剂量下调。结论:黄芩苷对人乳腺癌Bcap37细胞生长有抑制作用,可能与肿瘤细胞TIMP2的下调有关。     

雷公藤内酯醇对肺腺癌细胞系A549的体外抑制作用的研究

目的观察雷公藤内酯醇对肺腺癌细胞系A549体外生长特性的影响。方法分别采用MTT法、流式细胞仪、琼脂糖凝胶电泳以及荧光染色观察雷公藤内酯醇肺腺癌细胞系A549增殖、细胞周期以及凋亡的影响。结果14nmol.L-1～896nmol.L-1的雷公藤内酯醇均能抑制A549细胞的生长且生长抑制作用呈剂量以及时间依赖性。雷公藤内酯醇在低浓度(14nmol.L-1)条件下,能诱导A549细胞发生细胞周期阻滞,阻滞部位在S期;高浓度(55,112mol.L-1)的雷公藤内酯醇使A549阻滞在G2/M期,并诱导其发生凋亡。结论雷公藤内酯醇能抑制A549细胞的生长,使其阻滞在S期和G2/M期,并诱导其发生凋亡。     

Anti-tumor activity of CrTX in human lung adenocarcinoma cell line A549

Aim:

To assess the cytotoxic effect of crotoxin (CrTX), a potent neurotoxin extracted from the venom of the pit viper Crotalus durissus terrificus, in human lung adenocarcinoma A549 cells and investigated the underlying mechanisms.

Methods:

A549 cells were treated with gradient concentrations of CrTX, and the cell cycle and apoptosis were analyzed using a flow cytometric assay. The changes of cellular effectors p53, caspase-3 and cleaved caspase-3, total P38MAPK and pP38MAPK were investigated using Western blot assays. A549 xenograft model was used to examine the inhibition of CrTX on tumor growth in vivo.

Results:

Treatment of A549 cells with CrTX (25–200 μg/mL) for 48 h significantly inhibited the cell growth in a dose-dependent manner (IC₅₀=78 μg/mL). Treatment with CrTX (25 μg/mL) for 24 h caused G1 arrest and induced cell apoptosis. CrTX (25 μg/mL) significantly increased the expression of wt p53, cleaved caspase-3 and phospho-P38MAPK. Pretreatment with the specific P38MAPK inhibitor SB203580 (5 μmol/L) significantly reduced CrTX-induced apoptosis and cleaved caspase-3 level, but G₁ arrest remained unchanged and highly expressed p53 sustained. Intraperitoneal injection of CrTX (10 μg/kg, twice a week for 4 weeks) significantly inhibited A549 tumor xenograft growth, and decreased MVD and VEGF levels.

Conclusion:

CrTX produced significant anti-tumor effects by inducing cell apoptosis probably due to activation of P38MAPK and caspase-3, and by cell cycle arrest mediated by increased wt p53 expression. In addition, CrTX displayed anti-angiogenic effects in vivo.     

Exposure to parabens at the concentration of maximal proliferative response increases migratory and invasive activity of human breast cancer cells in vitro

Alkyl esters of p–hydroxybenzoic acid (parabens) are widely used as preservatives in personal care products, foods and pharmaceuticals. Their oestrogenic activity, their measurement in human breast tissue and their ability to drive proliferation of oestrogen‐responsive human breast cancer cells has opened a debate on their potential to influence breast cancer development. As proliferation is not the only hallmark of cancer cells, we have investigated the effects of exposure to parabens at concentrations of maximal proliferative response on migratory and invasive properties using three oestrogen‐responsive human breast cancer cell lines (MCF‐7, T‐47‐D, ZR‐75‐1). Cells were maintained short‐term (1 week) or long‐term (20 ± 2 weeks) in phenol‐red‐free medium containing 5% charcoal‐stripped serum with no addition, 10^–8 M 17β‐oestradiol, 1–5 × 10^–4 M methylparaben, 10^‐5 M n‐propylparaben or 10^–5 M n‐butylparaben. Long‐term exposure (20 ± 2 weeks) of MCF‐7 cells to methylparaben, n‐propylparaben or n‐butylparaben increased migration as measured using a scratch assay, time‐lapse microscopy and xCELLigence technology: invasive properties were found to increase in matrix degradation assays and migration through matrigel on xCELLigence. Western immunoblotting showed an associated downregulation of E‐cadherin and β‐catenin in the long‐term paraben‐exposed cells which could be consistent with a mechanism involving epithelial to mesenchymal transition. Increased migratory activity was demonstrated also in long‐term paraben‐exposed T‐47‐D and ZR‐75‐1 cells using a scratch assay and time‐lapse microscopy. This is the first report that in vitro, parabens can influence not only proliferation but also migratory and invasive properties of human breast cancer cells. Copyright © 2014 John Wiley & Sons, Ltd.     

Effects of environmental organochlorine pesticides on human breast cancer: Putative involvement on invasive cell ability

Human exposure to persistent organic pollutants (POPs) is a certainty, even to long banned pesticides like o,p′‐dichlorodiphenyltrichloroethane (o,p′‐DDT), and its metabolites p,p′‐dichlorodiphenyldichloroethylene (p,p′‐DDE), and p,p′‐dichlorodiphenyldichloroethane (p,p′‐DDD). POPs are known to be particularly toxic and have been associated with endocrine‐disrupting effects in several mammals, including humans even at very low doses. As environmental estrogens, they could play a critical role in carcinogenesis, such as in breast cancer. With the purpose of evaluating their effect on breast cancer biology, o,p′‐DDT, p,p′‐DDE, and p,p′‐DDD (50–1000 nM) were tested on two human breast adenocarcinoma cell lines: MCF‐7 expressing estrogen receptor (ER) α and MDA‐MB‐231 negative for ERα, regarding cell proliferation and viability in addition to their invasive potential. Cell proliferation and viability were not equally affected by these compounds. In MCF‐7 cells, the compounds were able to decrease cell proliferation and viability. On the other hand, no evident response was observed in treated MDA‐MB‐231 cells. Concerning the invasive potential, the less invasive cell line, MCF‐7, had its invasion potential significantly induced, while the more invasive cell line MDA‐MB‐231, had its invasion potential dramatically reduced in the presence of the tested compounds. Altogether, the results showed that these compounds were able to modulate several cancer‐related processes, namely in breast cancer cell lines, and underline the relevance of POP exposure to the risk of cancer development and progression, unraveling distinct pathways of action of these compounds on tumor cell biology. © 2013 Wiley Periodicals, Inc. Environ Toxicol 30: 168–176, 2015.