眼镜蛇毒神经生长因子诱导人鼻咽癌CNE-2细胞凋亡作用的研究

目的：研究广西眼镜蛇毒神经生长因子（Nerve growth factor，NGF）对人鼻咽癌CNE2细胞增殖的抑制作用和诱导细胞凋亡。方法：应用三磷酸腺苷-生物荧光肿瘤化疗药物敏感试验法（ATP—TCA法）测定不同质量浓度（0．0125，0．025，0．05，0．1，0．2g·L^-1）NGF处理24，48，72，96h对人鼻咽癌CNP2生长的抑制率；Hoechst 33342荧光染色观察凋亡细胞的形态；琼脂糖凝胶电泳测定DNA断裂状况；流式细胞术检测细胞周期变化和细胞凋亡率。结果：不同浓度的NGF能抑制CNE-2细胞增殖，并呈浓度时间效应关系，作用24，48，72，96h的半数抑制浓度（IC50）分别为0．213，0．095，0．048，0．033g·L^-1；经NGF（0．1g·L^-1）处理细胞48h后，荧光染色呈现典型的细胞凋亡形态学特征；DNA凝胶电泳可见凋亡细胞特有的DNA片段；0．（15，0．1，0．2g·L^-1 NGF作用48h后，CNB2细胞被阻滞于G0／G1期，细胞凋亡率分别为（28．2±2．0）％、（39．9±1．9）％、（50．3±1．3）％。结论：NGF通过诱导人鼻咽癌CNE-2细胞凋亡而产生抗鼻咽癌活性。     

土贝母皂苷诱导人鼻咽癌细胞株CNE-2Z细胞凋亡的研究

目的　研究土贝母皂苷 (下称皂苷 )对人鼻咽癌细胞株CNE 2Z细胞凋亡的影响。方法　MTT法检测皂苷对CNE 2Z细胞生长的影响 ;形态学方法 (荧光显微镜和透射电镜 )、流式细胞仪和DNA琼脂糖凝胶电泳观察和分析在皂苷作用下CNE 2Z细胞形态、DNA含量的变化和DNA断裂的情况 ;蛋白免疫印迹法检测凋亡相关基因bcl 2、bax和cas pase 3表达的变化。 结果　皂苷抑制CNE 2Z细胞的生长 ,其效果与皂苷的浓度和作用时间相关 ;诱导CNE 2Z细胞发生典型程序性死亡 ;凋亡抑制基因bcl 2表达逐渐减少 ;而凋亡诱导基因bax和caspase 3在 1、3、5h表达增高。 结论　诱导细胞凋亡是皂苷发挥抗瘤效果的一种重要机制 ;皂苷诱导的CNE 2Z细胞凋亡与bcl 2、bax和caspase 3基因有关     

新藤黄酸诱导HepG2细胞凋亡与Bax及Bcl-2的关系

<正>中药藤黄(gamboge)系藤黄科植物藤黄树(Garcinia hanburyi Hook.F.G)所分泌的干燥树脂,近年来,藤黄的抗肿瘤作用受到高度关注。已有研究证实,新藤黄酸(neo-gambogic acid,NGA)为藤黄抗肿瘤作用的主要有效成分之一[1-3]。肝细胞癌(hepatocellular carcinoma,HCC)是世界上最常见的恶性肿瘤之一[4],同时也是第三大引起患者死亡的肿瘤[5]。     

藤黄酸诱导SK-MES-1肺鳞癌细胞凋亡

机制可能与上调Caspase9、10及p53蛋白的表达有关.     

青天葵甲醇提取物通过ERK信号通路诱导鼻咽癌CNE-2细胞凋亡

目的 研究青天葵甲醇提取物（Nervilia fordii methanol extracts,NFME）体外对鼻咽癌CNE-2细胞的凋亡作用及其作用机制。方法 MTT法检测不同浓度（0,0.25,0.5,1,2,3 mg·mL^-1）的NFME处理CNE-2细胞24,48 h对CNE-2细胞生长抑制率的影响;克隆原形成试验观察NFME对CNE-2细胞克隆形成率的影响;Hoechst凋亡染色观察NFME对CNE-2细胞凋亡的影响;Western blotting测定NFME作用下caspase-3、ERK1/2和c-Raf蛋白磷酸化水平的变化。结果 MTT试验结果显示,与对照组相比,在给药24 h时0.5 mg·mL^-1的NFME就能抑制CNE-2细胞的增殖（P<0.05）,抑制率为12.64%。而在给药48 h时0.25 mg·mL^-1的NFME就能抑制CNE-2细胞增殖（P<0.05）,抑制率为22.43%;克隆原形成能力试验表明,0.25 mg·mL^-1的NFME能够抑制CNE-2细胞集落的形成（P<0.05）;Hoechst33258凋亡染色观察到0.25 mg·mL^-1的NFME作用24 h能够观察到CNE-2细胞发生凋亡（P<0.05）,凋亡率达到17.91%;Western blotting结果表明0.5 mg·mL^-1的NFME能使CNE-2细胞中caspase-3发生剪切,随着给药浓度增加其剪切作用越明显（P<0.01）,同时0.5 mg·mL^-1的NFME能够降低CNE-2细胞中ERK1/2和c-Raf蛋白的磷酸化水平（P<0.05）。结论 青天葵甲醇提取物能抑制鼻咽癌CNE-2细胞的增殖并诱导其发生凋亡,其作用机制可能与抑制ERK信号通路有关。     

新藤黄酸通过内质网应激诱导人鼻咽癌 CNE-2Z细胞凋亡的研究

目的 探讨新藤黄酸(gambogenic acid,GNA)对人鼻咽癌CNE-2Z细胞的影响和相关分子机制.方法 采用倒置显微镜观察药物处理组和对照组的CNE-2Z细胞形态的变化;MTT法检测药物处理组的CNE-2Z细胞增殖的抑制作用;DAPI染色和AV/PI 双染实验观察细胞凋亡情况;蛋白质印迹法检测GRP78、CHOP和ATF4蛋白的表达.结果 倒置显微镜和荧光显微镜观察,发现与对照组相比,GNA作用CNE-2Z细胞后,体积缩小变圆,细胞核发生典型核染色质固缩等细胞凋亡形态学的变化.GNA可以抑制人鼻咽CNE-2Z细胞的增殖,并有时间和浓度依赖性.AV/PI染色后早凋细胞膜呈苹果绿色,晚凋或坏死细胞质呈不同程度的黄-红色.蛋白质印迹法检测表明GRP78蛋白表达总体呈现浓度依赖性下调,上调了CHOP蛋白和ATF4蛋白.结论 GNA可以影响人鼻咽癌CNE-2Z细胞的增殖,引发细胞凋亡,这可能与内质网应激相关蛋白(GRP78、CHOP和ATF4蛋白)有关.     

紫草素通过ROS/p38信号通路诱导人宫颈癌HeLa细胞凋亡

目的探讨紫草素(shikonin)诱导人宫颈癌HeLa细胞凋亡的机制。方法以MTT法检测shikonin的细胞毒性;相差显微镜观察细胞形态变化;流式细胞仪检测细胞中活性氧(reactive oxygen species,ROS)的产生及凋亡率;Western blot检测相关蛋白的表达。结果 Shikonin时间和剂量依赖性的抑制HeLa细胞的生长;35μmol.L-1的shikonin作用于细胞24 h后可使细胞皱缩、变圆,并出芽形成明显的凋亡小体,且其可时间依赖性的诱导procaspase-3的剪切。35μmol.L-1的shikonin作用于细胞12 h和24 h均可以诱导细胞产生ROS。ROS清除剂NAC和p38抑制剂SB203580可以明显降低shikonin诱导的HeLa细胞的生长抑制和凋亡率;并且5 mmol.L-1 NAC可以上调由shikonin引起的pro-caspase-3的表达降低,下调由shikonin引起的p38蛋白的磷酸化。结论 Shikonin可以诱导HeLa细胞发生凋亡,ROS/p38信号通路参与了其凋亡的过程。     

钒化钠对人食管癌细胞系EC109 p-ERK1/2蛋白表达的影响

钒的药理作用报道最多的是它的降血糖作用[1].Ray等[2]研究显示,钒化钠可以通过促进乳腺癌MCF7细胞凋亡而抑制其增殖,显示了钒化钠作为有效的化疗制剂的潜能.我们前期研究显示,一定浓度的钒化钠能明显抑制食管癌细胞系EC109细胞株的生长,钒化钠的抗肿瘤机制可能与cyclin D1蛋白表达降低有关[3].本研究进一步揭示钒化钠抗肿瘤作用的分子机制,为钒化钠进一步发展成一种新型的抗肿瘤药物提供理论依据.     

磁性纳米Fe3O4颗粒对藤黄酸诱导肝癌细胞凋亡的影响

目的研究磁性纳米Fe3O4颗粒(MNP-Fe3O4)对藤黄酸(GA)诱导肝癌HepG2细胞凋亡的作用。方法将HepG2细胞分为GA 0.5μmol/L单药组(A组)、GA 0.5μmol/L和MNP-Fe3O420μg/ml两药联合组(B组)及空白对照组(C组)。采用MTT法检测HepG2细胞增殖的抑制率,流式细胞术检测细胞的凋亡率,Western blot法检测B细胞淋巴瘤/白血病2(Bcl-2)及半胱天冬氨酸蛋白酶3(Caspase-3)蛋白的表达。结果 GA对HepG2细胞生长的抑制作用呈剂量依赖性。与C组相比,A、B组HepG2细胞凋亡率、Caspase-3蛋白表达增加,Bcl-2蛋白表达减少(P<0.05),且B组各指标变化更为显著(P<0.05)。结论 MNP-Fe3O4能增强GA对肝癌HepG2细胞的凋亡诱导作用;其机制可能与Bcl-2表达下调及Caspase-3表达上调有关。     

miR-218对新藤黄酸抑制人宫颈癌HeLa细胞增殖作用的影响及机制研究

目的研究miR-218对新藤黄酸抑制人宫颈癌He La细胞增殖作用的影响及其可能的分子机制。方法人宫颈癌He La细胞转染过表达真核表达载体pmiR-218,real-time PCR检测He La细胞miR-218的表达。将He La细胞分为空白对照组、新藤黄酸组、pmiR-218组、质粒对照组、新藤黄酸联合pmiR-218组。MTT法检测各组细胞增殖;流式细胞仪检测各组细胞凋亡;Western blot检测Bcl-2、Bax、E-cadherin表达;real-time PCR检测Bcl-2、Bax和E-cadherin的mRNA表达水平。结果 miR-218真核表达载体pmiR-218转染He La细胞后,细胞内miR-218表达水平明显增加(P<0.05)。miR-218可提高He La细胞对新藤黄酸的敏感性,高表达miR-218能促进新藤黄酸对He La细胞的增殖抑制作用,促进细胞凋亡,下调新藤黄酸对He La细胞Bcl-2/Bax蛋白的表达水平。结论 miR-218可提高He La细胞对新藤黄酸的敏感性,miR-218能增强新藤黄酸对He La细胞增殖抑制作用,并促进He La细胞凋亡,其作用机制可能与下调Bcl-2/Bax的表达有关。     

ERK1/2和p38MAPK介导BAPTA-AM抑制RANKL诱导小鼠骨髓巨噬细胞分化的机制研究

目的探讨BAPTA-AM对RANKL诱导的小鼠骨髓巨噬细胞分化成破骨细胞能力的影响以及其信号通路机制的研究。方法体外分离培养小鼠骨髓巨噬细胞,建立RANKL诱导的小鼠骨髓巨噬细胞分化成破骨细胞的实验模型。应用抗酒石酸酸性磷酸酶(TRAP)染色法检测RANKL诱导的小鼠破骨细胞分化和存活的能力及BAPTA-AM对其的抑制作用;采用免疫印迹法(Western blot)测定ERK1/2和p38MAPK蛋白的磷酸化水平。结果 RANKL诱导的小鼠骨髓巨噬细胞胞质内可见多核,并能分化成为破骨细胞。不同浓度的BAPTA-AM(1、2μmol·L-1)对破骨细胞的形成具有明显的抑制作用,且随着浓度增加能明显地降低TRAP阳性细胞数目。RANKL对小鼠骨髓巨噬细胞的ERK1/2和p38MAPK具有明显的激活作用,诱导胞质ERK1/2和p38MAPK磷酸化,而不同浓度的BAPTA-AM可明显地抑制这种磷酸化作用。结论 BAPTA-AM能明显地抑制RANKL诱导的小鼠骨髓巨噬细胞分化成破骨细胞,这种抑制作用可能是通过ERK1/2和p38MAPK信号蛋白介导。     

Gambogic acid potentiates clopidogrel-induced apoptosis and attenuates irinotecan-induced apoptosis through down-regulating human carboxylesterase 1 and -2

1.?In this study, we report that gambogic acid (GA), a promising anticancer agent, potentiates clopidogrel-induced apoptosis and attenuates CPT-11-induced apoptosis by down-regulating human carboxylesterase (CES) 1 and -2 via ERK and p38 MAPK pathway activation, which provides a molecular explanation linking the effect of drug combination directly to the decreased capacity of hydrolytic biotransformation.

2.?The expression levels of CES1 and CES2 decreased significantly in a concentration- and time-dependent manner in response to GA in Huh7 and HepG2 cells; hydrolytic activity was also reduced.

3.?The results showed that pretreatment with GA potentiated clopidogrel-induced apoptosis by down-regulating CES1. Moreover, the GA-mediated repression of CES2 attenuated CPT-11-induced apoptosis.

4.?Furthermore, the ERK and p38 MAPK pathways were involved in the GA-mediated down-regulation of CES1 and CES2.

5.?Taken together, our data suggest that GA is a potent repressor of CES1 and CES2 and that combination with GA will affect the metabolism of drugs containing ester bonds.     

Gambogenic acid mediated apoptosis through the mitochondrial oxidative stress and inactivation of Akt signaling pathway in human nasopharyngeal carcinoma CNE-1 cells

In the present study, Gambogenic acid exhibits potential anti-tumor activity in several cancer cell lines. However, Gambogenic acid-induced apoptosis mechanism is not well understood. Here, we report that Gambogenic acid was capable to induce CNE-1 cells apoptosis and caused mitochondrial and endoplasmic reticulum injury, analyzed via transmission electron microscopy and acridine orange/ethidium bromide (AO/EB) double staining. To quantitatively analyze apoptosis, through the propidium iodide (PI)/Annexin V-FITC double staining to detect cell apoptosis, PI staining of the cell cycle distribution. To further explore the potential mechanism of Gambogenic acid mediated apoptosis in CNE-1 cells, we also examined mitochondrial oxidative stress in the levels of reactive oxygen species, the release of cytochrome c, intracellular Ca(2+) concentration and mitochondrial membrane potential by flow cytometry. Moreover, Gambogenic acid could result in a time and concentration-dependent decrease in Phospho-Akt expression, basal expression levels of Akt change was not obvious, In addition, we detected Bcl-2 family including Bcl-2, Bax and Bad expression in mRNA level. This resulted in a decrease of Bcl-2 and Bad increased in CNE-1 cells after Gambogenic acid treatment. Overall, our results indicated that Gambogenic acid mediated apoptosis through inactivation of Akt, accompanied with mitochondrial oxidative stress and cross-talk with Bcl-2 family in the process of apoptosis.     

白藜芦醇诱导鼻咽癌细胞CNE-2Z凋亡的线粒体机制(英文)

目的　探讨白藜芦醇诱导鼻咽癌细胞CNE 2Z凋亡的线粒体机制。方法　用 10 0 μmol·L- 1白藜芦醇分别处理细胞 0 (对照 ) ,2 ,4 ,8,12 ,2 4h和分别用 0 ,2 5 ,5 0 ,10 0 ,2 0 0 μmol·L- 1白藜芦醇处理CNE 2Z细胞 8h ,采用Western印迹分析Bcl 2 ,Bax和胞浆中细胞色素C(CytC)的蛋白水平变化 ,罗丹明 12 3荧光染色后经流式细胞术检测线粒体膜电位(△ψm)变化 ,比色法测定半胱天冬酶 9的活性改变。结果　① 10 0 μmol·L- 1白藜芦醇处理细胞不同时间 ,Bcl 2蛋白表达和△ψm 减少、胞浆中的CytC和半胱天冬酶 9活性增高均呈时间依赖性 (P <0 .0 1) ,但Bax蛋白表达无改变。除Bax以外的其他指标均自白藜芦醇处理 2h即有变化。Bcl 2的蛋白表达受抑和△ψm 的丧失在 4～ 8h间最明显 (与对照组比较P <0 .0 1) ,在 2 4h时已无意义。胞浆中CytC水平和半胱天冬酶 9活性在 8h达高峰 (分别为 0h的 3.0 ,5 .4倍 ) ,2 4h时仍明显高于对照组(P <0 .0 1)。②细胞经不同浓度的白藜芦醇处理 8h后 ,Bcl 2的蛋白表达受抑、△ψm 的丧失、CytC的释放和半胱天冬酶 9活性的升高均具有剂量依赖性 (P <0 .0 1) ,但Bax的蛋白表达未受影响。结论白藜芦醇可能经一个线粒体 /半胱天冬酶 9的特定途径诱导CNE 2Z细胞凋亡 ,但此凋亡?     

Gambogenic acid induced mitochondrial-dependent apoptosis and referred to phospho-Erk1/2 and phospho-p38 MAPK in human hepatoma HepG2 cells

Gambogenic acid, identified from Gamboge, is responsible for anti-tumor effects, and has been shown to be a potential molecule against human cancers. In this study, the molecular mechanism of gambogenic acid-induced apoptosis in HepG2 cells was investigated. Gambogenic acid significantly inhibited cell proliferation and induced apoptosis. Acridine orange/ethidium bromide (AO/EB) staining was used to observe apoptosis, and then confirmed by transmission electron microscopy. Gambogenic acid induced apoptosis and morphological changes in mitochondria, and intracellular reactive oxygen species (ROS) and mitochondrial membrane permeabilization (MMP) in mitochondrial apoptosis pathway were also examined. Results showed that the levels of phospho-p38 and its downstream phospho-Erk1/2 of HepG2 cells increased in time- and concentration-dependent manners after gambogenic acid treatments. Additionally, gambogenic acid increased expression ratio of Bcl-2/Bax in mRNA levels, Western blotting analysis also further confirmed the reduced level of Bcl-2 and increase the expression level of Bax in HepG2 cells. These results indicated that gambogenic acid induced mitochondrial oxidative stress and activated caspases through a caspase-3 and caspase-9-dependent apoptosis pathway. Moreover, gambogenic acid mediated apoptosis and was involved in the phospho-Erk1/2 and phospho-p38 MAPK proteins expression changes in HepG2 cells.     

2-Methoxyestradiol induces cell cycle arrest and apoptosis of nasopharyngeal carcinoma cells

AIM: To investigate 2-methoxyestradiol induced apoptosis and its mechanism of action in CNE2 cell lines.METHODS: CNE2 cells were cultured in RPMI-1640 medium and treated with 2-methoxyestradiol in different concentrations. MTT assay was used to detect growth inhibition. Flow cytometry and DNA ladders were used to detect apoptosis. Western blotting was used to observe the expression of p53, p21WAF1, Bax, and Bcl-2 protein.RESULTS: 2-methoxyestradiol inhibited proliferation of nasopharyngeal carcinoma CNE2 cells with IC50 value of2.82 μmol/L. The results of flow cytometry showed an accumulation of CNE2 cells in G2/M phase in response to 2-methoxyestradiol. Treatment of CNE2 cells with 2-methoxyestradiol resulted in DNA fragmentation. The expression levels of protein p53 and Bcl-2 decreased following 2-methoxyestradiol treatment in CNE2 cells, whereas Bax and p21WAF1 protein expression were unaffected after treatment with 2-methoxyestradiol. CONCLUSION:These results suggest that 2-methoxyestradiol induced cell cycle arrest at G2/M phase and apoptosis of CNE2 cells which was associated to Bcl-2 down-regulation.