β-榄香烯乳剂逆转多药耐药细胞株MCF-7/ADM对阿霉素耐药性研究

目的：测定中药β-榄香烯乳剂对MCF-7/ADM细胞的无毒剂量,并检测此无毒剂量是否有逆转MCF-7/ADM细胞对化疗药物阿霉素（ADM）的多药耐药（multidrug resistance,MDR）性。方法：采用四甲基偶氮唑蓝（MTT）法测定药物的细胞毒性及耐药细胞逆转倍数;荧光分光光度法测定细胞内药物浓度。结果：无毒剂量的β-榄香烯乳剂(6μg/ml）能显著降低化疗药物ADM对乳腺癌耐药细胞株MCF-7/ADM细胞的IC50,明显增加耐药细胞内药物浓度。结论：初步研究表明β-榄香烯乳剂具有逆转MCF-7/ADM细胞MDR的作用。     

沉默AEG-1基因逆转乳腺癌MCF-7/ADM细胞耐药性

本研究旨在分析星形细胞上调基因1(astrocyte elevated gene-1,AEG-1)对乳腺癌MCF-7/ADM细胞化疗药物耐药性的影响,并探讨其作用机制。将MCF-7/ADM细胞在含1.0 mg/L阿霉素(adriamycin,ADM)的培养液中培养以维持细胞的耐药性;应用shRNA技术沉默乳腺癌MCF-7/ADM细胞中AEG-1基因表达;采用MTT比色法检测ADM对MCF-7/ADM的细胞耐毒作用,据以计算ADM的半数抑制浓度(IC50);流式细胞术检测细胞凋亡;Western blot方法检测AEG-1、p53和多药耐药基因1(multidrug resistance gene 1,MDR1)蛋白的表达水平以及Akt、MDM2和Bad的磷酸化水平。结果显示,乳腺癌MCF-7/ADM细胞的AEG-1蛋白水平显著高于MCF-7细胞(P0.05),经shRNA干扰后AEG-1蛋白水平显著降低(P0.05);沉默AEG-1基因能显著降低ADM对MCF-7/ADM细胞的IC50(P0.05),促进MCF-7/ADM细胞凋亡(P0.05),并增强ADM对MCF-7/ADM细胞的促凋亡作用,抑制Akt、MDM2和Bad的磷酸化(P0.05),促进p53蛋白表达(P0.05),降低MDR1蛋白表达水平(P0.05)。结果表明,沉默AEG-1基因可通过促进MCF-7/ADM细胞凋亡和下调MDR1蛋白表达,以逆转MCF-7/ADM细胞对ADM的耐药性。     

龙葵碱通过抑制Med19表达增加多柔比星耐药乳腺癌细胞对多柔比星的敏感性

目的 探讨龙葵碱（solanine）对乳腺癌细胞多柔比星耐药性的影响及其机制。方法 选用乳腺癌细胞MCF-7细胞和对阿霉素（adriamycin,ADM）或多柔比星耐药的乳腺癌细胞MCF-7/ADR细胞,转染pcDAN-Med19过表达中介体19(mediator 19,Med19),应用CCK-8法检测细胞增殖水平,qRT-PCR和Western blot检测细胞中Med19和凋亡相关Bcl-2、Bax及Cleaved-Caspase-3表达水平,TUNEL染色和流式细胞术分析细胞凋亡水平。结果 龙葵碱显著降低MCF-7/ADM细胞活力。Med19高表达于MCF-7/ADM细胞,龙葵碱显著抑制MCF-7/ADM细胞中Med19表达,龙葵碱对多柔比星处理的MCF-7/ADM细胞活力的降低、凋亡的促进、凋亡相关蛋白Bax和Cleaved-Caspase-3水平的上调及Bcl-2水平的下调可被过表达Med19明显抑制。结论 龙葵碱通过抑制Med19表达而降低乳腺癌细胞MCF-7/ADM对多柔比星的耐药性。     

防己水煮液逆转MCF-7/ADM多药耐药细胞的初步研究

目的：观察中药防己水煮液对MCF-7／ADM多药耐药细胞的逆转作用,并对防己水煮液的逆转MCF-7多药耐药细胞株的有效组分作初步追踪。方法：用SRB显色法观察防己水煮液对MCF-7野生型细胞的毒性作用及对MCF-7／ADM多药耐药细胞的逆转作用,并用相同方法跟踪防己水煮液不同分离组分逆转MCF-7多药耐药细胞株的作用,确定有效组分。结果：对耐2．0μg／mLADM的MCF-7／ADM多药耐药细胞,当阿霉素浓度降到0．25μg／mL时,防己水煮液对MCF-7／ADM多药耐药细胞的逆转效果比较明显,即0．25μg／mL阿霉素为防己水煮液逆转MCF-7／ADM多药耐药细胞的敏感浓度点;对MCF-7／ADM多药耐药细胞起逆转作用的有效成分大部分集中在经正丁醇萃取所得相和水相中,而极性低的有机溶剂相中基本不合可以逆转MCF-7／ADM多药耐药细胞的有效组分。     

两株毛筒腔菌属真菌逆转人乳腺癌细胞多药耐药性

高水平的多药耐药性（multidrug resistance,MDR）基因在肿瘤细胞中过量表达是肿瘤细胞耐药的内在原因,是导致肿瘤化疗失败的主要原素。寻求一种抑制MDR活性的抑制剂是提升抗肿瘤药物药效的重要途径。本研究采用低浓度持续诱导方法建立人乳腺癌细胞（MCF-7）耐药细胞系,结果显示,阿霉素（ADM）、紫杉醇和顺铂对MCF-7耐药细胞系有交叉耐药性,耐药指数（resistance index,RI）分别为5.11、3.55和1.79。菌株对肿瘤细胞的逆转活性筛选表明,红棕毛筒腔菌Tubeufia rubra PF02-2和河池毛筒腔菌T. hechiensis XSL05具有逆转肿瘤细胞多药耐药性为敏感性的活性,逆转倍数（reversion fold,RF）分别为3.79和1.07。结果表明,T. rubra和T. hechiensis具有开发为MDR逆转剂的潜能。     

粉防己碱逆转阿霉素的人乳腺癌MCF—7细胞的抗凋亡作用

肿瘤细胞的多药耐药性与抗细胞凋亡作用关系密切。本研究用抗肿瘤药物阿霉素 (5μmol· L- 1 )处理人乳腺癌敏感和耐药的 MCF- 7细胞 2 4hr后 ,观察到在敏感细胞中 ,有较多的漂浮细胞 ,阿霉素主要分布在细胞核中 ;而在耐药细胞中 ,细胞形态未发生变化 ,阿霉素主要分布在细胞质中 ,其含量明显减少。阿霉素诱导 MCF- 7细胞的凋亡作用进一步用 An-nexin V - FITC染色法证实。此外 ,用高效逆转耐药性的药物粉防己碱 (2 0μmol· L- 1 )与阿霉素合用处理敏感和耐药的细胞 ,用线粒体荧光染料 Mitosensor TM染色 ,证明合用组凋亡细胞明显增多。通过流式细胞术分析显示 :细胞凋亡的发生与细胞周期无关。本研究表明 :粉防己碱能逆转耐阿霉素的人乳腺癌 MCF- 7细胞的抗凋亡作用。     

miR-34c逆转乳腺癌耐药细胞株MCF-7/DOX耐药性的研究

miR-34在肿瘤发生发展中起着至关重要的作用,然而,mi R-34在肿瘤耐药中的作用研究不多。该研究将合成的mi R-34c成熟序列转染乳腺癌阿霉素(doxorubicin,DOX)耐药细胞MCF-7/DOX,探讨mi R-34c体外逆转MCF-7/DOX细胞耐药性作用及其可能的机制。采用Real-time RT-PCR检测mi R-34c在乳腺癌耐药细胞株MCF-7/DOX中的表达,MTS法检测miR-34c对MCF-7/DOX细胞阿霉素耐药性的影响,流式细胞术检测miR-34c对MCF-7/DOX细胞周期和凋亡的影响,Real-time RT-PCR和Western blot法检测多药耐药相关蛋白MDR、MRP以及细胞周期与凋亡相关蛋白Bcl-2、E2F3的表达。结果显示,mi R-34c在乳腺癌MCF-7/DOX耐药细胞中低表达,转染mi R-34c可明显增加耐药细胞对阿霉素的敏感性;流式分析发现,miR-34c可以促进耐药细胞G2期细胞周期阻滞和凋亡;与对照组相比较,miR-34c转染组细胞MDR、MRP蛋白表达无明显变化,而Bcl-2、E2F3 mRNA和蛋白表达均明显下调。研究表明,miR-34c直接靶向抑制Bcl-2和E2F3的表达,诱导细胞周期G2期阻滞和凋亡,进而增强MCF-7/DOX耐药细胞对阿霉素的敏感性。     

环孢霉素A逆转人白血病耐药细胞系HL-60/ADM及其超微弱发光的观察

目的:探讨环孢霉素A(CSA)对抗阿霉素人急性早幼粒白血病细胞系HL-60/ADM的耐药逆转作用,并比较二者之超微弱发光强度的特点。方法:选择环孢霉素A(CSA)作为耐药逆转剂,分别采用MTT法、流式细胞仪和免疫组化法分析CSA对人白血病耐药细胞系的毒性及逆转效果;用IFFM-D型流动式化学发光仪检测细胞的超微弱发光强度。结果:当CSA浓度在4ug/mL以下时,对HL-60/ADM无明显毒性作用,超过此浓度,其毒性呈剂量效应(P＜0．001)。当CSA浓度为0．5ug/mL时就有明显逆转作用,随着CSA剂量增加,逆转作用逐渐增强(P＜0．01),当CSA剂量达8ug/mL以上时对细胞存活产生明显的影响;流式细胞仪分析细胞周期可见逆转耐药细胞系HL-60/ADM CSA较耐药细胞系HL-60/ADM的G2期细胞显著增多,而S期细胞明显减少(P＜0．01);免疫组化结果显示HL-60/ADM细胞膜上P-gp高表达,经CSA作用后其表达下降;在10-4mol/L鲁米诺及0．3%双氧水条件下HL-60/ADM细胞超微弱发光强度高于用CSA逆转后的细胞(P＜0．001)。结论:CSA能有效逆转HL-60/ADM的耐药性;逆转后细胞超微弱发光强度值的降低可能与其细胞增殖速度减慢,DNA复制减少,细胞内氧化代谢减弱以及自由基水平降低等因素有关。     

靶向干扰Chkl增强阿霉素抗人乳腺癌MCF-7/adr细胞的作用机制研究

Chkl的高表达可能是肿瘤对化疗药物的敏感性降低的重要因素之一,本研究的目的是观察siRNA干扰Chk1对人乳腺癌耐药细胞株MCF-7/adr(耐阿霉素)生长及细胞周期的影响,探讨Chk1在乳腺癌细胞耐药中的作用机制。采用RNAi技术抑制MCF-7/adr细胞中Chk1的表达。Westernblot检测转染前后细胞内Chk1蛋白表达情况,经阿霉素作用后,流式细胞术(FCM)检测其细胞周期分布及细胞凋亡率,MTT法检测细胞增殖。Western blot结果显示,Chk1 siRNA转染24h后,MCF-7/adr细胞中Chk1蛋白表达下降了67%,明显低于对照组和空载体转染组(P<0.05)。FCM法检测结果显示,同时,抑制Chk1的表达可解除阿霉素引起的G_2/M期阻滞;使阿霉素诱导的细胞凋亡率由转染前的(5.54±0.15)%上升到(22.24±0.13)%(P<0.05);在阿霉素浓度为0.4mg/L、4mg/L时,细胞的增殖活性分别下降13%、34%。提示siRNA干扰Chk1能够通过调控MCF-7/adr细胞周期及增殖从而增强乳腺癌细胞对阿霉素的敏感性,为临床上克服乳腺癌化疗耐药提供了新的作用靶点。     

川芎嗪与β-榄香烯联合应用对K562/ADM细胞的生长抑制作用

目的：探讨川芎嗪与β－榄香烯联合应用对K562/ADM的生长抑制作用。方法：以耐阿霉素细胞株K562／ADM为实验模型。结果：1．两者对K562／ADM及K562细胞的IC50接近,即耐药细胞K562／ADM对两种中药制剂不具有耐药性。2．非细胞毒性剂量的川芎嗪（TMP350μg/ml)及β－榄香精（β－elemene 4.0μg/ml)可显著降低ADM对K562／ADM细胞的IC50（P＜0．01）,提高细胞对ADM的敏感性,抗药性逆转分别为2．03倍及2．18倍。3．进一步将上述两种药物联合应用,发现其对ADM的抗药性逆转为4．65倍,明显高于二者单独应用,而且也高于两者单独应用之和,并且其对升高该细胞内ADM的浓度也具有协同作用。结论：川芭嗪与β－榄香烯联合应用能够抑制K562／ADM的的生长,并且具有协同性。     

Surfactin对人乳腺癌MCF-7细胞增殖、凋亡及细胞骨架的影响

以体外培养的人乳腺癌细胞株MCF-7为研究对象,探讨surfactin对肿瘤细胞增殖、凋亡及细胞骨架的影响。MTT实验表明,surfactin能抑制MCF-7的增殖,并且呈现出浓度和时间的依赖关系,作用48h时的IC50是27.3μmol/L。AO/EB荧光染色法及流式细胞术检测发现,surfactin可诱导MCF-7发生典型的凋亡形态学改变和G2/M期阻滞。免疫荧光和免疫印迹结果表明,surfactin显著抑制了细胞内vimentin的表达,诱导了α-tubulin的解聚和重排,使细胞的骨架系统发生了剧烈的变化。可见,surfactin具有抑制MCF-7细胞增殖,诱导细胞凋亡的作用,其机制可能与细胞骨架蛋白的表达水平有关。     

Apoptotic and antiproliferative effects of silk protein sericin conjugated-AgNO3 nanoparticles in human breast cancer cells

Bombyx mori silk sericin is a globular-like protein that is used as an antioxidant, antibacterial, and antitumor agent. In this current research, we isolated sericin by degumming process and formation of sericin-AgNO₃ NPs confirmed by UV–vis spectra, SEM, EDX, FTIR, and XRD patterns. The sericin and sericin-AgNO₃ NPs mediated changes in human breast cancer cells were determined. The antiproliferative activity of sericin-AgNO₃ NPs was analyzed by MTT dye reduction assay. Alterations at molecular levels were investigated by qRT-PCR, while apoptotic effects were studied by nuclear DNA staining. After 72 h treatment, sericin and sericin-AgNO₃ NPs showed significant antiproliferative effects in MDA-MB-231 (26 %) and MCF-7 (41 %) cells. Expression modification showed prominent stimulation of cell cycle arrest and stress related genes such as cyclin-dependent kinase inhibitors (CDKN1A, CDKN1B), and GADD family genes. RT-PCR results of the GADD family include GADD45A, B, G, 34, 153 and cyclin-dependent kinase inhibitors (CDKN1A, 1B) showed pronounced induction of 3.1 to 19.8-folds in MCF-7 cell line while induction in MDA-MB-231 cell line was 2.5 to 34.3-folds. Nuclear DAPI staining showed significant induction of apoptosis and nuclear fragmentation in MDA-MB-231 cells at a concentration of 1 mg/mL for both sericin and sericin-AgNO₃ NPs. Meanwhile, in case of MCF-7 cells, after treatment with sericin and sericin-AgNO₃ NPs (1 mg/mL), the cells changed into a round shape and lost their original spindle outlook in dose-dependent manners. We concluded that sericin-AgNO₃ NPs have significant antiproliferative, apoptosis, and genetic profiling effects in both breast cancer cell lines at the highest concentration.     

Bax translocation mediated mitochondrial apoptosis and caspase dependent photosensitizing effect of Ficus religiosa on cancer cells

The main aim of the present work was to investigate the potential effect of acetone extract of Ficus religosa leaf (FAE) in multiple apoptosis signalling in human breast cancer cells. FAE treatment significantly induced dose and time dependent, irreversible inhibition of breast cancer cell growth with moderate toxicity to normal breast epithelial cells. This observation was validated using Sulforhodamine B assay. Cell cycle analysis by Flow cytometry showed cell cycle arrest in G1 phase and induction of sub-G0 peak. FAE induced chromatin condensation and displayed an increase in apoptotic population in Annexin V-FITC/PI (Fluorescein isothiocyanate/Propidium iodide) double staining. FAE stimulated the loss of mitochondrial membrane potential in multiple breast cancer cell lines when compared to normal diploid cells. To understand the role of Bax in FAE induced apoptosis, we employed a sensitive cell based platform of MCF-7 cells expressing Bax-EGFP. Bax translocation to mitochondria was accompanied by the disruption of mitochondrial membrane potential and marked elevation in LEHDase activity (Caspase 9). Consistent with this data, FAE induced Caspase activation as evidenced by ratio change in FRET Caspase sensor expressing MCF-7 cell line and cleavage of prominent Caspases and PARP. Interestingly, FAE accelerated cell death in a mitochondrial dependent manner in continuous live cell imaging mode indicating its possible photosensitizing effect. Intracellular generation of reactive oxygen species (ROS) by FAE played a critical role in mediating apoptotic cell death and photosensitizing activity. FAE induced dose and time dependent inhibition of cancer cell growth which was associated with Bax translocation and mitochondria mediated apoptosis with the activation of Caspase 9 dependent Caspase cascade. FAE also possessed strong photosensitizing effect on cancer cell line that was mediated through rapid mitochondrial transmembrane potential loss and partial Caspase activation involving generation of intracellular ROS.     

灰树花多糖对乳腺癌细胞MCF-7的凋亡作用

采用MTT法测定不同给药浓度的灰树花多糖(PGF) (1、10、20、50、100和200 μg/mL)在24、48和72 h对乳腺癌细胞(MCF-7)增殖的抑制率,并采用Hoechst染色与流式细胞技术观察20、50和100 μg/mL PGF给药24 h后MCF-7的凋亡情况,同时采用Western blotting对20、50、100 μg/mL PGF给药24 h后MCF-7细胞中Bax、Bcl-2、Pro-Caspase-3以及Cleaved Caspase-3的蛋白表达水平进行检测。研究发现PGF给药24、48和72 h后对MCF-7的增殖均有显著的抑制作用。随着PGF给药浓度增加,MCF-7细胞核裂解增多,细胞凋亡数量增多。PGF 20、50和100 μg/mL给药对MCF-7细胞Bax、Bcl-2、Pro-Caspase-3以及Cleaved Caspase-3的蛋白表达水平可见显著性差异。     

MDM2反义寡核苷酸联合紫杉醇对乳腺癌MCF-7细胞株的作用

目的:探讨靶向MDM2反义寡核苷酸(ASON)联合紫杉醇对乳腺癌MCF-7细胞株的影响。方法:合成一段与MDM2 mRNA特异性结合的反义寡核苷酸和与反义寡核苷酸有4个碱基不同的的错义寡核苷酸(MON),脂质体2000介导不同浓度的MDM2ASON转染MCF-7乳腺癌细胞系,转染的乳腺癌细胞通过1μmol/L紫杉醇药物处理后,采用RT-PCR和Western Blot方法检测MDM2 ASON联合紫杉醇的协同作用及对乳腺癌MCF-7细胞株的抑制效率,MTT观察给药后MCF-7细胞的增殖能力和药物敏感性。结果:MDM2反义寡核苷酸联合紫杉醇明显下调MDM2 mRNA及MDM2蛋白表达水平,抑制MCF-7细胞的生长,随着MDM2 ASON浓度的增加,MDM2表达越来越低,协同作用越来越强,呈剂量依赖关系,A500联合紫杉醇的协同作用最明显,MTT显示紫杉醇处理的转染MCF-7细胞增殖抑制率明显增高,A500抑制增殖作用最明显,抑制率达(13.0±0.84)%。结论:不同浓度MDM2 ASON转染后的乳腺癌MCF-7细胞,等浓度紫杉醇处理后,乳腺癌MCF-7细胞MDM2表达明显降低,细胞凋亡增加,,MDM2 ASON联合紫杉醇对MCF-7细胞有协同作用,提高了乳腺癌MCF-7细胞对紫杉醇的药物敏感性。     

Down-regulation of TSG101 by small interfering RNA inhibits the proliferation of breast cancer cells through the MAPK/ERK signal pathway

We designed to investigate the effects of down-regulating the tumor susceptibility gene 101 (TSG101) on the proliferation and apoptosis of the human breast cancer MCF-7 cell line, and the role of the MAPK/ERK signal pathway in this process. The siRNA against TSG101 was transfected into the breast cancer MCF-7 cell line using Lipofectamine 2000. After TSG101 knockdown, the proliferation of MCF-7 cells was measured by the MTT assay. The cell cycle distribution and apoptosis were examined by using flow cytometry while cell migration was measured using a transwell assay. The protein level of p-ERK was further assessed by immunofluorescence and western blotting. Our results are as following, the MCF-7 cells transfected with TSG101 siRNA proliferated significantly slower and exhibited significantly increased rates of apoptosis compared to the control cells. In the TSG101 siRNA transfected cells, the percentage of cells in the G?/G? and S phase of the cell cycle was significantly higher and lower, respectively, compared to the control cells. Moreover, the migration ability of TSG101 siRNA transfected cells was lower than the control groups. Lastly, the level of p-ERK protein in TSG101 siRNA transfected cells was significantly decreased compared with the control cells. In conclusion, TSG101 knockdown in breast cancer cells induces apoptosis and inhibits proliferation. The TSG101 depleted cells are arrested at the G?/S transition of the cell cycle. The migration of breast cancer cells is also impaired by TSG101 siRNA. TSG101 may play a biological role through modulation of the MAPK/ERK signaling pathway in breast cancer.     

Glargine promotes proliferation of breast adenocarcinoma cell line MCF-7 via AKT activation

Glargine is widely used as a long-acting insulin analogue in the treatment of diabetes mellitus. However, this insulin analogue has been recently suspected to be associated with an increased risk of cancer. The aim of this study was to investigate the influence of glargine on proliferation of breast adenocarcinoma cell line (MCF-7) and its possible mechanism. Effects of glargine and regular human insulin on the cell proliferation were tested in ER-positive MCF-7 cells by MTT assay. Apoptosis in MCF-7 cells was measured by flow cytometry. The protein levels of p-AKT, Bcl-2, and Bax were also determined by Western blotting and immunohistochemistry, respectively. The result showed that glargine (100, 200?nmol/l) stimulated proliferation of ER-positive MCF-7 cells compared with regular human insulin. At the same time, glargine decreased the percentage of early apoptosis in MCF-7 cells. Otherwise, glargine (100?nmol/l) stimulated the p-AKT in a time-dependent manner in MCF-7 cells. Furthermore, we found that glargine downregulated the level of Bax protein and upregulated that of Bcl-2 (p <0.05). These data show that glargine promote the proliferation of breast adenocarcinoma cells in vitro, probably by preventing apoptosis.     

槟榔碱对人乳腺癌MCF-7细胞增殖和凋亡的影响

目的：观察槟榔碱对人乳腺癌细胞（MCF-7）增殖和凋亡的影响,并探讨其机制。方法：采用四甲基偶氮唑盐（MTT）法检测不同浓度（0、10、30、50、100、300、500μmol/L）槟榔碱对MCF-7细胞增殖的影响,Hoechst 33342染色和流式细胞术检测细胞凋亡,Western blot法检测Bax,Bcl-2和P53蛋白表达。结果：低浓度（0、10、30、50μmol/L）槟榔碱不影响细胞的增殖和凋亡;而高浓度（100、300、500 μmol/L）槟榔碱呈浓度依赖性抑制MCF-7细胞增殖、诱导MCF-7细胞凋亡、提高P53和Bax蛋白表达、降低Bcl-2蛋白表达。结论：高浓度槟榔碱抑制MCF-7细胞增殖、诱导凋亡,其机制可能与提高P53和Bax蛋白表达,降低Bcl-2蛋白表达有关。     

Induction of apoptosis by eugenol in human breast cancer cells

In the present study, potential anticancer effect of eugenol on inhibition of cell proliferation and induction of apoptosis in human MCF-7 breast cancer cells was investigated. Induction of cell death by eugenol was evaluated following MTT assay and monitoring lactate dehydrogenase released into the culture medium for cell viability and cytotoxicity, giemsa staining for morphological alterations, fluorescence microscopy analysis of cells using ethidium bromide and acridine orange and quantitation of DNA fragments for induction of apoptosis. Effect of eugenol on intracellular redox status of the human breast cancer cells was assessed by determining the level of glutathione and lipid peroxidation products (TBARS). Eugenol treatment inhibited the growth and proliferation of human MCF-7 breast cancer cells through induction of cell death, which was dose and time dependent. Microscopic examination of eugenol treated cells showed cell shrinkage, membrane blebbing and apoptotic body formation. Further, eugenol treatment also depleted the level of intracellular glutathione and increased the level of lipid peroxidation. The dose dependent increase in the percentage of apoptotic cells and DNA fragments suggested that apoptosis was involved in eugenol induced cell death and apoptosis might have played a role in the chemopreventive action of eugenol.     

ILP-2-ECM1(P85)促进乳腺癌细胞的生长

凋亡抑制蛋白-2（inhibitor of apoptosis protein-like protein-2, ILP-2）是新发现的凋亡抑制蛋白质,其抑制肿瘤细胞凋亡促进其生长的分子机制有待阐明,而细胞外基质蛋白1(extracellular matrix protein 1, ECM1)所介导的信号通路与肿瘤细胞的生长密切相关。本研究通过免疫共沉淀法,检测到乳腺癌MCF-7细胞中ILP-2与ECM1（P85）存在相互作用。分别用化学合成的ILP-2-siRNA及ECM1-siRNA干扰处理MCF-7细胞。以未转染的MCF-7细胞和转染阴性对照siRNA的细胞分别作为空白和阴性对照,利用蛋白质印迹法,检测ILP-2-siRNA干扰后ECM1、FAK、Akt蛋白的表达,以及ECM1-siRNA干扰后ILP-2蛋白的表达。其结果显示,与空白对照组相比,ILP-2-siRNA-5 (0.32 ± 0.095)及ECM1-siRNA-1 (0.42 ± 0.024)干扰效率较高(均P<0.001);ILP-2-siRNA-5组待测蛋白质的相对表达量均显著下调 (ECM1, 0.19 ± 0.013, P<0.001), FAK (0.64 ± 0.069, P<0.01), Akt (0.35 ± 0.120, P<0.01)),ECM1-siRNA-1组ILP-2 (0.48 ± 0.060) 蛋白表达也显著下调,表明ILP-2与ECM1-mTOR信号通路联系密切。分别在ILP-2-siRNA和ECM1-siRNA转染24、48和72 h时,使用CCK-8法检测乳腺癌细胞的增殖,并用TUNEL标记荧光法和吖啶橙/溴化乙啶双荧光染色法(AO/EB)检测其凋亡。结果显示,与空白对照组相比,ILP-2-siRNA-5组和ECM1-siRNA-1组的存活率均显著下降（P<0.001）,凋亡率均明显升高（P<0.001）。利用共转染技术同时敲低ILP-2和ECM1表达,检测细胞的凋亡情况。结果显示,在干扰处理后24 h（0.55±0.122）,48 h（0.80 ± 0.107）和72h（0.73 ± 0.091）的凋亡率显著均高于阴性对照组（P＜0.05）。但与只敲低ILP-2或ECM1相比,无显著性差异（P＞0.05）。表明ILP-2可能是通过与ECM1作用激活FAK-mTOR信号通路,影响MCF-7细胞的增殖和凋亡,对乳腺癌细胞MCF-7的生长发挥了积极的作用。