Dietary Daidzein Enhances Antiapoptotic Effect of 17β-Estradiol (E_2) on Breast Cancer MCF-7 Cells

Objective： To investigate whether dietary daidzein interact with endogenous 17β-Estradiol （E2） to give rise to additive or inhibitory effects on proliferation and apoptosis in breast cancer cells. Methods： Cell cycle distribution and apoptosis induction were analyzed by using flow cytometry when breast cancer cell lines MCF-7 were cotreated with daidzein （1, 5 μmol/L） and E2 （0.1-10 nmol/L） for 5 days. Whether daidzein could alter E2-modulated mRNA expression of estrogen receptor alpha （ERα）, estrogen receptor beta （ERI3） and ERβ-estrogen response element （ERE） dependent transcription was investigated by RT-PCR and luciferase induction assays. The effects of daidzein on E2-modulated expression of proapoptotic p53, bax and antiapoptotic bcl-2 at both mRNA and protein levels were also investigated by RT-PCR and Western blot. Results： Daidzein enhanced the antiapoptotic effect in an Ea dose-dependent manner, but had no effect on E2-induced proliferation. Daidzein antagonized E2-induced ERβ mRNA expression and ERβ-ERE dependent transcription. In addition, daidzein only antagonized E2-upregulated expression of p53 and bax, but had no effect on E2-upregulated expression of bcl-2. Conclusion： Daidzein enhances the antiapoptotic effect of E2 on breast cancer cells by inhibiting E2-mediated p53-bax proapoptotic pathway. These results suggest that dietary daidzein may enhance deleterious effect of endogenous E2 in hormone-dependent breast cancer.

Breast Cancer MCF-7 Cell Spheroid Culture for Drug Discovery and Development

In vitro 3D cancer spheroids (tumoroids) exhibit a drug resistance profile similar to that found in solid tumors. 3D spheroid culture methods recreate more physiologically relevant microenvironments for cells. Therefore, these models are more appropriate for cancer drug screening. We have recently developed a protocol for MCF-7 cell spheroid culture, and used this method to test the effects of different types of drugs on this estrogen-dependent breast cancer cell spheroid. Our results demonstrated that MCF-7 cells can grow spheroid in medium using a low attachment plate. We managed to grow one spheroid in each well, and the spheroid can grow over a month, the size of the spheroid can grow over a hundred times in volume. Our targeted drug experimental results suggest that estrogen sulfotransferase, steroid sulfatase, and G protein-coupled estrogen receptor may play critical roles in MCF-7 cell spheroid growth, while estrogen receptors α and β may not play an essential role in MCF-7 spheroid growth. Organoids are the miniatures of in vivo tissues and reiterate the in vivo microenvironment of a specific organ, best fit for the in vitro studies of diseases and drug development. Tumoroid, developed from cancer cell lines or patients’ tumor tissue, is the best in vitro model of in vivo tumors. 3D spheroid technology will be the best future method for drug development of cancers and other diseases. Our reported method can be developed clinically to develop personalized drugs when the patient’s tumor tissues are used to develop a spheroid culture for drug screening.

QCM Detection of Adhesion, Spreading and Proliferation of Human Breast Cancer Cells (MCF-7) on a Gold Surface

The quartz crystal microbalance （QCM） was used to monitor the one-day incubation of human breast cancer cells （MCF-7） on the gold electrode. In combination with an optical microscope simulation experiment, the cell-population pictures at various stages, the QCM responses to the cells＇ adhesion, spreading and proliferation on the electrode surface were discussed. The △f0 and △R1 responses were found mainly from mixed effects of viscodensity and surface stress, and in proportion to the cell coverage, rather than to the number of cells at the electrode. The significant fore-and-aft changes in cyclic voltammetry and electrochemical impedance spectroscopy of the ferri-ferrocyanide redox couple also proved that the cells were adhesion to the gold surface.

REVERSION OF MULTIDRUG RESISTANCE IN THE P-GLYCOPROTEIN POSITIVE BREAST CANCER CELL LINE(MCF-7/ADR) BY INTRODUCTION OF HAMMERHEAD RIBOZYME

A hammerhead ribozyme which site-specifically cleaved the GUC position in canon 880 of the mdr1 mRNA was designed. The target site was chosen between the two ATP binding sites, which may be important for the function of the P-Gp as an ATP-dependent pump. A DNA sequence encoding the ribozyme gene was then incorporated into a eukaryotic expression vector (pH Apr-1 neo) and transfected into the breast cancer cell line MCF-7/Adr, which is resistant to adriamycin and expresses the MDR phenotype. The ribozyme was stably expressed in the cell line by the RNA dot blotting assay. The result of Northern blot assay showed that the expressed ribozyme could decrease the level of mdrl mRNA expression by 83. 5 %; and the expressed ribozyme could inhibite the formation of p-glycoprotein detected by immuno- cy-tochemistry assay and could reduce the cell’s resistance to adrimycin; this means that the resistant cells were 1 000-fold more resistant than the parental cell line(MCF-7), whereas those cell clones that showed ribozyme expression were only 6-fold more resistant than the parental cell line. These results show that a potentially useful tool is at hand which may inactivate MDR1 mRNA and revert the multidrug resistance phenotype.

Cytotoxic Activity of <i>Thelesperma megapotamicum</i>Organic Fractions against MCF-7 Human Breast Cancer Cell Line

Thelesperma megapotamicum (Asteraceae) is commonly used in Argentine to treat various diseases (renal, digestive affections, and as anaesthesia). The present study showed the mechanisms involved “in vitro” cytotoxicity of T. megapotamicum Fractions. Five Fractions (F1 - F5) were separated by column chromatography (Silica gel) using hexane:diethyl ether as eluents. Viability was evaluated in Human breast carcinoma cell line (MCF-7) by staining with crystal violet. With respect to F1 Fraction treatment, the cell survival was 49.14% ± 8.87%, while the F2 and F3 ones exhibited a strong reduction of cell viability to only 26.35% ± 1.63% and 23.3%1 ± 0.53% of the control cell at 50 μg/ml, respectively. Apoptotic effect of these Fractions was detected using FITC-labeled Annexin V and propidium iodide binding assays and was confirmed by a higher proportion of apoptotic cells due to F2 and F3 treatments. T. megapotamicum active Fractions could facilitate the tumoral cells death by decreasing the activity of the enzyme Gamma-glutamyltranspeptidase and causing alteration in cell membrane sialoglycoconjugates and others involved anticancer mechanisms including apoptosis.

Studies on mechanism of cis9,trans11-CLA and trans10,cis12-CLA inducing apoptosis of human breast cancer cell line MCF-7

Objective： The aim of the study was to explore the activities of cis9, trans11-CLA （C9, t11-CLA） and transl0, cis12-CLA （t10, c12-CLA） inhibiting tumor, and investigate their relationships with PPARy and apoptotic proteins, and mechanism of anti-cancer. Methods： The inhibitory rate, cell growth curve and apoptotic morphological observation of MCF-7 cells were obtained by MTT assay, trypan blue staining and Hoechst33342 fluorescence staining. The apoptotic rate and cell cycle were detected with flow cytometry. Transcriptional level of genes was detected with RT-PCR semi-quantitative method, and Western blot was performed to detect proteins levels. Results： The two CLA isomers could reduce cell proliferation （P 〈 0.05）, increase apoptotic rate （P 〈 0.05）, and increase obviously the transcriptional and protein levels of PPARy （P 〈 0.01）. The synchronism and correlation between the effects of CLA to PPARy and apoptotic proteins Bax, Bcl-2, Caspase 3 changes were found with the dose- and time-dependent manners. There was cooperative relation between the levels of PPARy and the rates of Bax/Bcl-2, Caspase 3 （small fragment） by experiments of PPARy inhibitor GW9662 and ligand Rosiglitazone. Conclusion： The apoptotic pathway of PPARy-Bcl-2-Caspase 3 signaling was found. The C9, t11-CLA and tl0, c12-CLA could inhibit MCF-7 cell proliferation and promote apoptosis via activating PPARy-Bcl-2-Caspase 3 pathway. CLA may be a kind of activator of PPARv.

Study on Cisplatin Aggravating DNA Damage and Causing a High Apoptosis Rate on Breast Cancer MCF-7 Cells

[Objectives] To investigate the mechanism of DNA damage of cisplatin( DDP),a broad spectrum anticancer drug on breast cancer MCF-7 cells,and to study the mechanism of apoptosis induced by DDP.[Methods]MCF-7 cells were treated by DDP( 0 mg/L,2 mg/L,4 mg/L,6 mg/L,6 mg/L,and 10 mg/L) for 48 hours. MTT assay was used to detect the inhibitory effect of DDP on MCF-7 cells and IC50 value was calculated. Western blot was adopted to detect the expression of γ-H2 AX,which was the marker of DNA double stranded breaks( DSBs) and ATM( sensory molecules of DSBs),the apoptotic signal transduction molecule cleaved caspase-3,and the proteins associated with apoptosis calpain.[Results]DDP inhibited MCF-7 cell activity in a concentration-dependent manner and IC50 was 7. 57 mg/L. In contrast to the control group( without DDP treatment),MCF-7 cells with DDP treatment expressed more γ-H2 AX,ATM,cleaved caspase-3 and calpain.[Conclusions] DDP could inhibit the activity of breast cancer MCF-7 cells. Its mechanisms may be associated with inhibition of MCF-7 cell apoptosis,induction of DNA double strand breaking and the expression of pro-apoptotic protein up-regulation.

Retinoic acid affects basic cellular processes and SOX2 and SOX18 expression in breast carcinoma cells

Genetic and molecular heterogeneity,together with intrinsic and acquired resistance to therapy,represent the major obstacles to the successful treatment of different types of breast carcinoma.Increasing evidence demonstrates that SOX transcription factors in breast carcinomas could act both as oncogenes and tumor suppressors and have been associated with tumor stage and grade,poor prognosis,and therapy resistance.Both SOX2 and SOX18 overexpression has been correlated with poor prognosis in breast carcinomas,and these genes are recognized as potential antitumor targets.Our aim was to evaluate the effect of retinoic acid(RA),a well-known cyto-differentiating agent,on breast carcinoma cells in vitro and to investigate the potential of RA treatment to modify the expression of SOX2 and SOX18 genes.By applying various experimental approaches,we evaluated the effect of RA on basic cellular processes in SK-BR-3 and MCF7 breast carcinoma cell lines.We have shown that RA inhibits cell growth,reduces the number of Ki-67 positive cells,and causes cell-cycle arrest.RA effect was more prominent in SK-BR-3 cell line that lacks SOX2 expression,including a higher decrease in cell viability,reduction in colony formation,and significant remodeling of cellular structure.We have shown that RA treatment led to the downregulation of SOX2 expression in MCF7 cells and to the reduction of SOX18 expression in both cell lines.By functional analysis,we showed that the anti-proliferative effect of RA in both cell lines was not based on the activity of stemness marker SOX2,pointing to a SOX2-independent mechanism of action.The ability of RA to reduce SOX2/SOX18 expression raises the possibility that these genes can be used as biomarkers to distinguish RA-responders from non-responders.Together,our study shows that the response of breast carcinoma cell lines to RA treatment may vary,highlighting that the development of RA-based therapy should consider differences in breast carcinoma subtypes.

Synthesis and Characterization of Trithiocarbonate-Organoclays Nanohybrids and Their Interaction with MCF-7 Cancer Cells

This work presents a new approach for the fabrication of organic/inorganic nanohybrids as anticancer drugs by an intercalation method using S,S-bis（α,α′-dimethyl-α″-acetic acid） （trithiocarbonate） as a modifier and two organoclays, such as reactive octadecylamine/MMT （montmorillonite） and non-reactive dimethyldidodecyl ammonium/MMT. The chemical and physical structures and the surface morphology of these covalently and non-covalently linked nanohybrids were investigated by FT-IR （Fourier translbrm infrared） spectroscopy, ^13C and ^29Si solid state NMR （nuclear magnetic resonance） spectroscopy, XRD （X-ray powder diffraction） and SEM （scanning electron microscopy） analyses, respectively. To evaluate the anticancer activities of the novel BATC/organoclay hybrids against MCF-7 breast cancer cells, a combination of different biochemical and biophysical testing techniques were used. Cell proliferation and cytotoxicity were detected in vitro using a real-time analysis. Cell death was confirmed by using apoptotic and necrotic analyses, the effects of which were detennined by the double staining and Annexin-V-FLUOS testing method. The results demonstrate that intercalated hybrid complexes containing a combination of various anticancer sites, such as free and complexed carboxyl, trithiocarbonate, amine and ammonium cations significantly induced cell death in breast cancer via their interactions with the DNA macromolecules of cancer cells by destroying the self-assemb｜ed structure of growing cells. Fabricated hybrid complexes may represent a new generation of effective and selective anticancer drug systems with a synthetic/natural origin for cancer chemotherapy.

粉防己碱逆转人乳腺癌MCF-7多药耐药细胞的抗凋亡作用

目的 :研究粉防己碱与长春新碱合用能否逆转人乳腺癌MCF 7多药耐药细胞的抗凋亡作用。方法 :用荧光染料Hoechst 3334 2和碘化丙锭联染观察染色质凝集 ,流式细胞术检测G1亚峰 ,TUNEL法检测凋亡细胞 ,Fluo 3染色法测定细胞内游离钙。结果 :抗肿瘤药物长春新碱处理MCF 7敏感和耐药细胞后 2 4h ,可观察到 2种类型的染色质凝集 ;但在耐药细胞中 ,相同浓度处理下染色质凝集的细胞明显减少。无明显细胞毒性的粉防己碱(2 0 μmol·L-1)与长春新碱合用 ,荧光显示法和TUNEL法均证实敏感和耐药的凋亡细胞明显增多。粉防己碱与长春新碱合用明显升高细胞内的游离钙含量。结论 :粉防己碱能有效地逆转人乳腺癌MCF

MIF对耐ADM人乳腺癌细胞MCF-7/ADM体内外耐药逆转作用

目的:采用动物体内外结合方法探讨米非司酮(mifepristone,MIF)对耐阿霉素(adriamycin,ADM)人乳腺癌细胞MCF-7/ADM耐药逆转作用。方法:四甲基偶氮唑蓝法检测5μmol/LMIF对MCF-7/ADM体外耐药逆转作用。MCF-7/ADM接种裸鼠皮下构建裸鼠移植瘤模型,空白对照组(NS组)为0.2mL生理盐水腹腔注射+0.5mL食用油灌胃;ADM组为5mg/kgADM腹腔注射+0.5mL食用油灌胃;MIF组为30mg/kgMIF灌胃+0.2mL生理盐水腹腔注射;ADM+MIF组为5mg/kgADM腹腔注射+30mg/kgMIF灌胃。观察各组裸鼠移植瘤情况。结果:(1)5μmol/LMIF对MCF-7/ADM细胞的抑制率小于5%,与未用MIF组的抑制率比较差异无统计学意义(P>0.05)。(2)ADM对MCF-7/ADM细胞的半抑制率为17.21mg/L,而对非耐药乳腺癌细胞MCF-7细胞的半抑制率为0.42mg/L,ADM对MCF-7/ADM细胞的半抑制率明显高于MCF-7的半抑制率(P<0.05)。(3)5μmol/LMIF与ADM联合处理MCF-7/ADM细胞后,MCF-7/ADM半抑制率为1.96mg/L,明显低于单用ADM组的半抑制率(P<0.05)。逆转ADM耐药倍数为8.78。(4)ADM+MIF组瘤体积[(232.5149±309.2377)mm3]均低于NS组的瘤体积[(962.2309±261.1313)mm3](均P<0.05),也低于MIF组的瘤体积[(778.2846±42.6919)mm3],还低于ADM组的瘤体积[(508.9648±16.2609)mm3](均P<0.05)。MIF+ADM组的瘤质量抑制率为78.0%。结论:MIF对耐阿霉素的人乳腺癌细胞MCF-7/ADM体内外均有逆转耐药性的作用。

右美托咪定对MCF-7乳腺癌细胞增殖、迁移和凋亡的影响

目的探讨右美托咪定对MCF-7乳腺癌细胞增殖、迁移和凋亡的影响。方法将MCF-7乳腺癌细胞分为六组,右美托咪定组(D1组、D2组、D3组、D4组、D5组)和对照组(C组)。D1、D2、D3、D4、D5组加入右美托咪定,终浓度分别为1 000、100、10、1、0.1 ng/ml,C组加入等容积的生理盐水。通过噻唑蓝(MTT)法观察右美托咪定对MCF-7乳腺癌细胞增殖的影响,通过划痕实验观察右美托咪定对MCF-7乳腺癌细胞迁移的影响,应用凋亡试剂盒结合流式细胞仪检测右美托咪定对MCF-7乳腺癌细胞凋亡的影响。结果与C组比较,D1、D2、D3、D4、D5五组乳腺癌细胞增殖率、迁移距离和凋亡率差异均无统计学意义。结论右美托咪定对MCF-7乳腺癌细胞的增殖、迁移和凋亡无明显影响。

HPK1在乳腺癌组织中的表达及HPK1过表达对人乳腺癌MCF-7细胞增殖、凋亡的影响

目的:检测造血祖细胞激酶1(HPK1)在人非特异型浸润性乳腺癌组织及乳腺癌细胞中的表达,并构建HPK1慢病毒载体以探究HPK1过表达对人乳腺癌细胞增殖、凋亡的影响。方法:采用Western blot检测48例乳腺癌组织和配对癌旁组织中HPK1蛋白的表达,采用Western blot、RT-PCR法检测正常乳腺上皮细胞(MCF10A)和乳腺癌MCF-7细胞中HPK1的表达水平。PCR扩增HPK1序列,构建慢病毒p CDH-HPK1-puro重组载体,包装病毒、转染MCF-7细胞(过表达组),以感染空载体病毒的MCF-7细胞作为对照组,分别采用Western blot、RT-PCR检测2组细胞HPK1的表达水平,MTT法检测细胞增殖能力的变化,流式细胞术检测细胞凋亡及细胞周期。结果:与癌旁组织相比,HPK1蛋白在乳腺癌组织中低表达(P=0.036)。与MCF10A细胞相比,HPK1蛋白及mRNA在MCF-7细胞中低表达;与对照组相比,过表达组中HPK1蛋白及mRNA的表达水平上调,细胞增殖能力下降,细胞凋亡率升高,G0/G1期细胞比例升高(P均<0.05)。结论:HPK1在乳腺癌组织及细胞中低表达,HPK1过表达可抑制MCF-7细胞增殖和诱导凋亡,并使细胞周期阻断在G0/G1期。

氨氯地平对人乳腺癌细胞MCF-7细胞的抑制作用及机制研究

目的观察氨氯地平对人乳腺癌细胞MCF-7周期、周期蛋白相关基因及产物表达的影响,探讨氨氯地平对人乳腺癌MCF-7细胞周期的影响及其机制。方法MTT检测细胞增殖;流式细胞仪分析细胞周期;RT-PCR技术检测细胞周期相关基因cyclinD1、p21mRNA的表达;Western blot检测细胞周期蛋白cyclinD1、p21的蛋白表达。结果氨氯地平剂量和时间依赖性的抑制人乳腺癌MCF-7细胞增殖,IC50为14.439μmol.L-1。经7.22μmol.L-1(1/2IC50)、14.439μmol.L-1(IC50)、28.88μmol.L-1(2IC50)的氨氯地平作用人乳腺癌MCF-7细胞48h,G0/G1期细胞较对照组明显增高(P<0.05);并使人乳腺癌MCF-7细胞中cyclinD1mRNA及蛋白表达降低;p21mRNA及蛋白表达升高。结论氨氯地平对人乳腺癌MCF-7细胞具有抗增殖作用,并使细胞阻滞于G1期。其G1阻滞机制可能与调控细胞周期相关基因cyclinD1、p21mRNA及蛋白的表达相关。

毛蚶抗癌蛋白对人乳腺癌细胞MCF-7的生长抑制作用

目的:从毛蚶软体部位中分离出具有一定抗癌活性的毛蚶蛋白组分,研究其对人乳腺癌细胞MCF-7的生长抑制作用。方法:以新鲜毛蚶软体部分为原材料,分离获得毛蚶抗癌蛋白组分(命名为NS);倒置相差显微镜观察不同浓度(50、100、200、400μg/mL)NS组分对MCF-7癌细胞生长的影响,将200μg/mL NS组分作用MCF-7细胞不同时间(12、24、48 h)后,Giemsa染色观察癌细胞及核形态的变化,流式细胞术检测NS组分对MCF-7细胞周期及细胞凋亡的影响,Western blot检测细胞周期及细胞凋亡相关蛋白的表达。结果:与阴性对照组比较,各浓度NS组分对人乳腺癌MCF-7细胞生长均具有明显的抑制作用(P<0.05或P<0.01);倒置相差显微镜下可见200μg/mL NS组分作用MCF-7细胞12 h后,部分细胞收缩变圆、脱落,24、48 h后细胞脱落现象更加明显;Giemsa染色发现,NS组分处理12 h后细胞出现有丝分裂相增加,继而出现多核或核碎裂等现象;流式细胞术检测发现NS组分主要引起细胞G2-M期阻滞;NS处理组凋亡率均较对照组明显升高(P均<0.01),且随着药物作用时间的延长,细胞凋亡比例逐渐增加。Western blot检测发现NS组分作用MCF-7细胞后p53及procaspase-3蛋白表达量均上调。结论:毛蚶抗癌蛋白NS组分体外对人乳腺癌MCF-7细胞的细胞毒作用主要通过引起细胞发生G2-M期阻滞、诱导细胞凋亡实现的,期间伴随p53及procaspase-3蛋白表达上调。

甲异靛对人乳腺癌MCF-7细胞增殖和凋亡影响的实验研究

目的:研究甲异靛对人乳腺癌MCF-7细胞增殖抑制和诱导凋亡作用。方法:采用MTT法检测甲异靛对MCF-7乳腺癌细胞的增殖抑制;流式细胞仪测定细胞凋亡率;光学显微镜观察细胞形态的变化;westernblot法测定caspase-3、PARP及bcl-2表达。结果:甲异靛能明显抑制MCF-7乳腺癌细胞增殖。甲异靛诱导MCF-7细胞凋亡,呈现典型细胞凋亡的形态变化,并呈时间和剂量依赖性,凋亡率最高可达(68.40±4.87)%,在细胞凋亡过程中出现PARP分子断裂和bcl-2下调,未检测到caspase-3。结论:甲异靛能诱导MCF-7乳腺癌细胞凋亡抑制细胞增殖,其发生机制可能与下调bcl-2有关。

JNK/MAPK在LPS诱导乳腺癌细胞MCF-7上皮间质转化过程中的作用

目的:探讨JNK/MAPK在LPS诱导乳腺癌细胞上皮间质转化(EMT)过程中的作用。方法:将乳腺癌细胞MCF-7分为3组:正常对照组、LPS(10μg/ml)处理组、LPS+SP-600125诱导实验组。应用realtime RT-PCR与Western blot法检测上皮细胞表面标志E-钙黏蛋白(E-cadherin)和间质细胞表面标志N-钙黏蛋白(N-cadherin)的表达以及C-Jun氨基末端激酶(JNK)表达。结果:LPS促进乳腺癌细胞系MCF-7的上皮间质转化(EMT)发生。MCF-7细胞的EMT过程伴随JNK1/2活性增加。应用SP-600125(10μmol/L)阻断JNK后,MCF-7细胞的EMT消失。结论:JNK在LPS诱导乳腺癌细胞MCF-7上皮间质转化过程中发挥调控作用。

软坚消瘤片药物血清对乳腺癌MCF-7细胞生长的机制研究

目的:通过观察软坚消瘤片药物血清对乳腺癌MCF-7细胞的芳香化酶和雌激素受体基因的表达、细胞增殖周期及细胞凋亡的影响,探讨软坚消瘤片治疗乳腺肿块性疾病的可能机制。方法:制备软坚消瘤片含药动物血清,以体外培养的MCF-7细胞为研究对象,以qRT-PCR法检测乳腺癌细胞芳香化酶(CYP19)及雌激素受体(ER)mRNA表达水平;流式细胞术(FCM)测定细胞增殖周期和凋亡水平;MTS实验测定细胞生长曲线。结果:与空白对照组比较,含药血清组作用细胞24h后能明显抑制乳腺癌细胞株MCF-7的CYP19mRNA、ERmRNA表达水平,表达水平分别降低0.32和0.09倍;药物血清作用48h后S期和G2/M期细胞所占比例降低,尤以G2/M期最为显著,相应的G0/G1期细胞增加,药物血清抑制了细胞增殖的正常进行;凋亡实验示细胞早期凋亡率增加,药物血清组总体凋亡率高于对照组;药物血清作用细胞48h以后细胞生长曲线幅度开始明显低于对照组,在96h时抑制效果最明显。结论:软坚消瘤片药物血清影响乳腺癌MCF-7细胞的雌激素合成及其作用发挥,同时抑制一定时段的细胞生长。

人乳腺癌组织和细胞中TNFR1表达及其对MCF-7细胞增殖、凋亡的影响

目的观察人乳腺癌组织和细胞中肿瘤坏死因子受体1(TNFR1)的表达,以及抑制人乳腺癌细胞MCF-7中TNFR1表达对细胞增殖及凋亡的影响,并探讨相关机制。方法取新鲜乳腺癌组织及癌旁组织各30例、人乳腺癌细胞株(MCF-7、MDA-MB-231、T47D)和人正常乳腺细胞,采用Real-time PCR法检测组织和细胞中的TNFR1mRNA;将MCF-7细胞分为3组,A组不转染,B组转染阴性对照质粒,C组转染siRNA-TNFR1质粒,转染72 h后分别采用MTT法及流式细胞仪观察细胞增殖及凋亡,Western blot法检测细胞中半胱氨酸天冬氨酸蛋白水解酶8(Caspase-8)及核转录因子κB(NF-κB)。结果人乳腺癌组织和癌旁组织中TNFR1 mRNA相对表达量分别为1.41±0.08和0.38±0.05,癌组织与癌旁组织相比P<0.05;人乳腺癌细胞MCF-7、MDA-MB-231、T47D中TNFR1mRNA相对表达量分别为2.48±0.06、1.51±0.02、1.66±0.01,人正常乳腺细胞中TNFR1 mRNA相对表达量为1.00±0.09,人乳腺癌细胞与人正常乳腺细胞相比P均<0.05。A、B、C组细胞增殖能力(吸光度值)分别为0.96±0.09、0.93±0.11、0.62±0.05,细胞凋亡率分别为8.23%±3.50%、9.92%±3.19%、24.61%±2.46%,C组与A、B组比较P均<0.05。A、B、C组细胞中Caspase-8蛋白相对表达量分别为0.40±0.03、0.42±0.05、0.82±0.01,NF-κB蛋白表达相对量分别为0.79±0.03、0.76±0.04、0.32±0.06,C组与A、B组比较P均<0.05。结论人乳腺癌组织和细胞中TNFR基因高表达,其可能通过激活NF-κB信号通路抑制Caspase-8的表达来促进乳腺癌细胞增殖、抑制细胞亡。

低氧环境下人乳腺癌细胞系MCF-7转录调节因子-1表达变化及其机制

目的观察人乳腺癌细胞系MCF-7 E26转录特异性序列-1(Ets-1)表达变化,并探讨其机制。方法取对数生长期MCF-7细胞,使用氯化钴(CoCl2)诱导的化学性低氧培养环境以及缺氧小室培养环境模拟肿瘤细胞低氧微环境,通过过表达和敲降缺氧诱导因子-1α(HIF-1α)研究Ets-1基因的表达情况,通过荧光定量PCR(q-PCR)和蛋白印记实验(Western blot)方法分别研究低氧环境下Ets-1 mRNA和蛋白表达情况,使用免疫共沉淀(Co-IP)和免疫荧光染色(IF)方法研究不同情况下蛋白互作,使用免疫沉淀法研究Ets-1蛋白翻译过程中的乙酰化、泛素化和磷酸化。结果低氧刺激下,乳腺癌细胞系中Ets-1蛋白水平显著上升;低氧环境下Ets-1 mRNA相对表达量升高;过表达HIF-1α导致乙酰基转移酶p300活性增强;并且p300与Ets-1在细胞质中共定位;低氧刺激下,Ets-1的乙酰化水平升高,Ets-1乙酰化后与E3泛素连接酶COP-1相互作用减弱;p300抑制剂C646处理MCF-7细胞降低Ets-1蛋白水平,并增强其与COP-1相互作用。结论低氧微环境下培养的MCF-7细胞中Ets-1蛋白和mRNA表达升高,Ets-1表达升高的调控机制可能为低氧促进了Ets-1转录,同时HIF-1α使乙酰基转移酶p300活性增强,引起Ets-1乙酰化水平升高,乙酰化Ets-1与E3泛素连接酶COP-1相互作用受阻,进而降低其降解。