苦瓜多糖对人乳腺癌MDA-MB-231细胞生物学行为的影响

目的探讨苦瓜多糖对人乳腺癌MDA-MB-231细胞生物学行为的影响及其机制。方法采取体外培养MDA-MB-231细胞进行分组,苦瓜多糖组给予不同浓度苦瓜多糖(0μg/ml、30μg/ml、60μg/ml、120μg/ml),空白组(苦瓜多糖0μg/ml)培养基中含有与苦瓜多糖组等浓度的DMSO,将各组细胞孵育分别培养至12 h、24 h、48 h处理。MTT法检测细胞增殖率,FMC法检测后细胞周期分布与细胞凋亡率,Hoechst 33342观察凋亡小体变化,Trans well法检测细胞迁移率,免疫组化检测Ki67与Caspase-3阳性率,Western-blot检测MMP-9、Bcl-2、Bax表达水平,RT-PCR检测细胞周期蛋白(CyclinD1)、血管内皮生长因子(VEGF)表达量;体内建立移植瘤裸鼠模型,试验组裸鼠采用苦瓜多糖(120μg/ml)灌胃治疗,早晚一次,1 ml/次,对照组裸鼠灌胃等体积的生理盐水,两组裸鼠治疗6周后测量肿瘤质量、观察肿瘤体积变化。结果与空白组相比,苦瓜多糖组癌细胞增值率与迁移能力随苦瓜多糖剂量增加而降低(P 0. 05),细胞凋亡率、凋亡小体数量随着药用剂量增加而升高(P 0. 05); Western-blot检测结果显示苦瓜多糖组MMP-9、Bcl-2表达水平低于空白组(P 0. 05),Bax表达水平高于空白组(P 0. 05); RT-PCR结果显示苦瓜多糖组CyclinD1、VEGF表达量显著低于空白组(P 0. 05)。试验组肿瘤组织中的Ki67蛋白水平、肿瘤体积、肿瘤质量显著低于对照组(P 0. 05),而试验组肿瘤组织中Casppase-3蛋白水平显著高于对照组(P 0. 05)。结论苦瓜多糖可以通过调控细胞周期、凋亡、迁移侵袭相关蛋白及基因表达,进而调控人乳腺癌MDA-MB-231细胞生物学行为。     

非甾体类抗炎药对乳腺癌细胞MDA-MB-231增殖影响的实验室研究

目的 通过研究阿司匹林、塞来昔布及两者分别联合阿那曲唑对乳腺癌细胞MDA-MB-231的抑制作用,探讨并比较阿司匹林和塞来昔布的抗肿瘤作用.方法 采用MTT法检测不同浓度的阿司匹林、塞来昔布及两者分别联合阿那曲唑对乳腺癌细胞MDA-MB-231增殖的影响.结果 阿司匹林、塞来昔布单药对MDA-MB-231细胞生长均有抑制作用,表现为浓度、时间依赖性,统计学差异具有统计学意义(P<0.05);不同浓度的阿司匹林、塞来昔布分别联合阿那曲唑后对MDA-MB-231细胞的抑制作用较单用阿那曲唑增强,亦表现为浓度依赖性,差别具有统计学意义(P<0.05).结论 阿司匹林和塞来昔布均有抑制乳腺癌MDA-MB-231细胞的作用,两者分别联合阿那曲唑后有协同抗肿瘤的作用.     

沉默K970基因逆转人乳腺癌耐药细胞MCF-7／ADR耐药性

目的探讨沉默K070基因对耐表阿霉素人乳腺癌细胞MCF-7／ADR耐药逆转作用。方法以脂质体包裹的SiRNA-K070转染细胞,RT-PCR验证K070沉默效率。CCK8试剂盒测定细胞增殖活性,通过Caspase-3分光光度法、AnnexinV-PI染色检测细胞凋亡。结果SiRNA-K070转染组K070-mRNA表达明显下降,细胞对表阿霉素的敏感性明显增加,药物半数致死浓度（IC50）由34．38±6．75tzg／ml降低为13．06±2．62μg／ml,耐药指数（RI）由8．26减至3．14。以表阿霉素5btg／ml的培养液培养细胞24h,SiRNA-K070转染组（12pm01）与阴性对照组及空白对照组相比,caspase-3活性明显增高;凋亡细胞数明显增多。结论siRNA-K070通过下调K070的表达,逆转耐表阿霉素人乳腺癌细胞MCF-7／ADR对表阿霉素的耐药性,降低凋亡域值,从而增强了人乳腺癌细胞对表阿霉素的敏感性。     

IL-17在乳腺癌中的表达及意义

目的：探讨白细胞介素17（IL-17）在乳腺癌患者血清和T细胞中的表达及与乳腺癌功能指标的相关性。方法采用流式细胞仪分析乳腺癌患者外周血 CD3＋ CD4＋ IL-17＋ T 细胞百分数;ELISA 法测定血清 IL-17水平;化学发光免疫分析法检测 CA153和 CA125的水平,并分析 IL-17水平与各指标的相关性。结果乳腺癌患者外周血中 CD3＋ CD4＋ IL-17＋ T 细胞百分数与健康对照组差异有统计学意义（P ＜0．05）;血清 IL-17水平与健康对照组差异有统计学意义（P ＜0．05）;血清 IL-17水平与CD3＋ CD4＋ IL-17＋ T 细胞百分数及 CA153水平均呈正相关;血清 IL-17水平与 CA125水平无显著相关性。结论IL-17水平与乳腺癌有关。     

探究乌司他丁和多西他赛对人乳腺癌细胞MDA-MB-231增殖、侵袭的影响

目的探究分析乌司他丁和多西他赛对体外培养的人乳腺癌细胞MDA-MB-231增殖、侵袭和迁移能力的影响。方法将人乳腺癌细胞MDA-MB-231随机分为乌司他丁组、多西他赛组、乌司他丁联合多西他赛组以及对照组四个组,分别予以800 U/ml乌司他丁、3. 7μg/ml多西他赛、800 U/ml乌司他丁+3. 7μg/ml多西他赛以及等量生理盐水干预。分别在干预后0 h、24 h、48 h、72 h时点采用CCK8法检测MDA-MB-231的增殖能力,在干预后48 h采用Transwell法检测MDA-MB-231的侵袭、迁移能力。结果与对照组相比,乌司他丁组、多西他赛组、乌司他丁联合多西他赛组24 h、48 h、72 h等各时点MDA-MB-231的增殖能力均明显降低(P 0. 05),且多西他赛组的增殖能力较同时点乌司他丁组较低(P 0. 05),乌司他丁联合多西他赛组各时点增殖能力则明显降低(P 0. 05),组间比较差异明显,具有统计学意义。与对照组相比,乌司他丁组、多西他赛组、乌司他丁联合多西他赛组各组MDA-MB-231细胞侵袭、迁移能力明显降低(P 0. 05),且乌司他丁联合多西他赛组细胞侵袭、迁移能力明显低于多西他赛组及乌司他丁组水平(P 0. 05),多西他赛组细胞侵袭、迁移能力明显低于乌司他丁组(P 0. 05),组间差异具有显著统计学意义。结论乌司他丁与多西他赛均可在一定程度上抑制人乳腺癌细胞MDA-MB-231的增殖、侵袭和迁移,且乌司他丁和多西他赛联合应用抑制效果优势明显。     

沉默SETD8基因表达对骨肉瘤MG-63细胞增殖、侵袭和迁移的抑制作用

目的探究siRNA下调含SET结构域(赖氨酸甲基转移酶) 8(SETD8)基因表达对骨肉瘤MG-63细胞增殖、侵袭和迁移的影响及其可能的机制。方法采用脂质体法将特异性针对SETD8基因的siRNA转入骨肉瘤MG-63细胞中,得到关于SETD8基因沉默表达的MG-63稳定的细胞系即实验组,将杂序siRNA用脂质体法同时转染骨肉瘤MG-63细胞,得到关于Control siRNA的MG-63细胞系即对照组,未经转染的骨肉瘤MG-63的稳定的细胞系即空白组。以上三组细胞系通过应用细胞毒性活性检测法(CCK-8法)、平板克隆法同时检测被SETD8基因沉默后骨肉瘤MG-63细胞的增殖能力,侵袭迁移小室即(Transwell小室)法检测骨肉瘤MG-63细胞的侵袭和迁移能力,实时荧光定量法即(PCR法)以及蛋白质印迹法即(Western Blot法)检测SETD8基因沉默表达效果及细胞内SETD8,β-catenin,cyclin D1,vimentin、MMP3的mRNA和相应蛋白的表达水平。结果 SETD8 siRNA转染后,骨肉瘤MG-63细胞的增殖能力显著低于对照组(control siRNA转染MG-63细胞)和空白组(未经转染的MG-63细胞)(P 0. 05); Transwell小室结果显示,沉默SETD8基因组的侵袭和迁移能力明显下降;实验组(SETD8 siRNA转染MG-63细胞)中SETD8、β-catenin、cyclin D1、vimentin、MMP3的mRNA及蛋白表达水平显著低于阴性对照组和空白对照组(P 0. 05)。结论 siRNA下调SETD8基因表达可以抑制骨肉瘤MG-63细胞的增殖、侵袭和迁移能力,Wnt/β-catenin信号通路可能参与并调控这一过程。     

肿瘤抑制基因WWOX转染MDA-MB-231细胞系的实验研究

目的探讨肿瘤抑制基因WWOX与乳腺癌疾病相关性,为进一步研究WWOX基因的作用机制及功能奠定基础。方法构建真核表达载体pcDNA4．0／Myc—His-WWOX,并转染至MDA．MB．231细胞中,采用RT-PCR及Westernblot方法,检测WWOXmRNA及融合蛋白在MDA-MB-231细胞中的表达。结果测序结果显示,RT-PCR法获得的WWOX序列与GenBank中报道的一致。真核表达载体pcDNA4．0／Myc．His—WWOX转染MDA—MB-231细胞48h后,从RT-PCR及Westernblot结果中观察到WWOX基因的有效转录。结论成功构建了肿瘤抑制基因WWOX真核表达载体pcDNA4．0／Myc．His—WWOX,为进一步研究WWOX在乳腺癌发生和进展中的作用及其靶向基因治疗奠定了实验基础。     

TRAIL联合Res对乳腺癌MDA-MB-231细胞DR和Caspase表达的影响

目的研究TRAIL联合白藜芦醇（Res）对乳腺癌MDA-MB-231细胞DR和caspase表达的影响。方法MTT法检测细胞增殖;流式细胞术检测细胞凋亡及细胞表面DR4、DR5蛋白的表达;分光光度法检测caspase-3、caspase-8相对活性;荧光定量PCR检测DR4、DR5及caspase-3、caspase-8 mRNA的表达。结果50μmol／L和100μmo/L,Res分别与50ng／mL TRAIL联合作用后,对MDA-MB-231细胞增殖的抑制率及细胞凋亡率分别与单独应用相应浓度的Res和TRAIL比较,均存在显著差异（P〈0．01）。50μmol／L和100μmol／LRes分别与50ng／mLT RAIL联合作用后,caspase-3、caspase-8的相对活性与对照组及单用TRAIL、Res比较均明显增强,差异显著（P〈0．01）。Res作用后,细胞表面DR4、DR5荧光指数与对照组比较明显增强。荧光定量PCR结果显示与对照组比较．Res作用后DR4、DR5mRNA表达上调;与单独用药比较,TRAIL联合Resetcagpase-3、easpase-8 mRNA表达上调。结论TRAIL和Res联合应用对诱导乳腺癌MDA-MB-231细胞凋亡具有明显的协同效果,其作用机制可能与增加DR和caspase活性有关。     

miR-203调控NEDD9抑制乳腺癌细胞MDA-MB-231侵袭及迁移作用机制研究

目的 :研究mi R-203抑制乳腺癌MDA-MB-231细胞NEDD9蛋白的作用对细胞侵袭和迁移的影响。方法:mi R-203脂质体转染MDA-MB-231细胞,通过细胞划痕实验和Transwell实验观察细胞侵袭和迁移能力的变化;通过蛋白印迹检测mi R-203对NEDD9表达的调控作用,并用sensor reporter确定mi R-203的靶标位点;最后,通过细胞"拯救"实验研究相关蛋白表达量的变化,用免疫荧光-激光共聚焦显微镜观察细胞片状伪足和黏着斑的改变,探究mi R-203对MDA-MB-231细胞侵袭和迁移能力的调节机制。结果:细胞划痕实验和Transwell实验显示,mi R-203抑制了MDA-MB-231细胞的侵袭和迁移能力;通过生物信息学网站预测mi R-203的靶基因是NEDD9;蛋白印迹和sensor reporter检测结果显示,mi R-203通过与NEDD9 3′-UTR结合,下调NEDD9;共转染si NEDD9和mi R-203的"拯救"实验后的细胞划痕实验、蛋白印迹和免疫荧光-激光共聚焦结果显示,mi R-203抑制NEDD9表达导致激活态Rac1-GTP减少,致使细胞运动状态改变,侵袭迁移能力减弱,而si NEDD9干涉作用能"拯救"mi R-203对MDA-MB-231细胞迁移能力的抑制。结论 :mi R-203通过降解NEDD9,下调激活的Rac1水平,导致乳腺癌细胞MDA-MB-231片状伪足和黏着斑的减少或消失,从而抑制细胞的侵袭及迁移。     

丙泊酚对人乳腺癌MCF-7细胞增殖、侵袭、迁移及相关标志物表达的影响

目的 探讨不同浓度丙泊酚对人乳腺癌MCF-7细胞增殖、侵袭、迁移能力的影响及对程序性死亡配体1(PD-L1)、细胞增殖核抗原(Ki-67)等相关标志物的影响。方法 体外培养人乳腺癌雌激素受体阳性细胞系MCF-7,随机将MCF-7分为3组,对每一组加入不同浓度的丙泊酚,即P₀组(空白对照组)、P₂₅组(25μg/ml丙泊酚组)和P₅₀组(50μg/ml丙泊酚组)。对三组细胞分别进行克隆形成实验、Transwell细胞迁移实验、侵袭实验、划痕愈合实验以研究各组细胞的增殖、以及细胞的迁移、侵袭变化差异。使用Western blot检测不同浓度丙泊酚做用下对人乳腺癌相关蛋白表达改变情况。结果 与P₀组及P₂₅组相比,P₅₀组乳腺癌MCF-7细胞增殖及细胞迁移、侵袭能力显著升高(P<0.05)。通过Western blot实验发现：P₅₀组的PD-L1、Ki-67蛋白表达显著高于P₀组(P<0.05),其中PD...     

青蒿琥酯对生存素在乳腺癌细胞株MDA-MB-231表达的影响

目的 探讨青蒿琥酯对生存素(survivin)在乳腺癌细胞株MDA-MB-231中表达的影响.方法 以不同浓度(0、25、50 μg/mL)的青蒿琥酯处理MDA-MB-231细胞,分别于药物处理后24、48和72 h收集细胞及培养上清液,用RT-PCR检测不同组别、不同时间点的细胞中survivin的基因表达情况,酶联免疫吸附试验(ELISA)检测各组、各时间点培养上清液中survivin的蛋白质表达水平.结果 PCR结果和ELISA结果均显示,随着药物浓度的增加和处理时间的延长,survivin基因水平和蛋白质水平的表达也逐渐降低,同一时间点各组间survivin的表达量进行单因素方差分析,差异有统计学意义(P＜0.05);对照组(0 μg/mL)在24 h和48 h survivin的表达量变化不大,经t检验分析,差异无统计学意义(P＞0.05),而各浓度经药物处理48 h后survivin的表达量明显低于24 h的表达,差异有统计学意义(P＜0.05).结论 青蒿琥酯可通过抑制survivin的表达促进乳腺癌MDA-MB-231细胞凋亡.     

Apoptotic effect of tolfenamic acid on MDA-MB-231 breast cancer cells and xenograft tumors

Recent studies have indicated that non-steroidal anti-inflammatory drug (NSAID), particularly tolfenamic acid, can inhibit proliferation and induce apoptosis invarious cancer cells. Breast cancer represents one-third of all cancers diagnosed in women and is the second leading cause of cancer death in Western European and North American women. In the present study, we investigated the apoptotic effect of tolfenamic acid in MDA-MB-231 estrogen receptor-negative human breast carcinoma cells and in a xenograft tumor model. Treatment of cells with tolfenamic acid significantly decreased cell viability in a concentration-dependent manner. Notably, tolfenamic acid increased apoptosis-related proteins, such as p53 and p21, within 48 h. Furthermore, in vivo experiments showed that tolfenamic acid treatment resulted in a significant reduction in tumor volume over 5 weeks. Immunohistochemistry results showed that apoptosis-related protein induction by tolfenamic acid was significantly higher in the 50 mg/kg-treated group compared to the control group. Together, these results indicate that tolfenamic acid induces apoptosis in MDA-MB-231 breast cancer cells and tumor xenograft model and it may be a potential chemotherapeutic agent against breast cancer.     

Anti-RhoA and anti-RhoC siRNAs inhibit the proliferation and invasiveness of MDA-MB-231 breast cancer cells in vitro and in vivo.

Overexpression of RhoA or RhoC in breast cancer indicates a poor prognosis, due to increased tumor cell proliferation and invasion and tumor-dependent angiogenesis. Until now, the strategy of blockage of the Rho-signaling pathway has used either GGTI or HMG-CoA reductase inhibitors, but they are not specific to RhoA or RhoC inhibition. In this study, a new approach with anti-RhoA and anti-RhoC siRNAs was used to inhibit specifically RhoA or RhoC synthesis. Two transfections of either RhoA or RhoC siRNA (8.5 nM) into MDA-MB-231 human breast cancer cells or HMEC-1 endothelial cells induced extensive degradation of the target mRNA and led to a dramatic decrease in synthesis of the corresponding protein. In vitro, these siRNAs inhibited cell proliferation and invasion more effectively than conventional blockers of Rho cell signaling. Finally, in a nude mouse model, intratumoral injections of anti-RhoA siRNA (100 microl at 85 nM) every 3 days for 20 days almost totally inhibited the growth and angiogenesis of xenografted MDA-MB-231 tumors. One may infer from these observations that specific inhibition of the Rho-signaling pathway with siRNAs represents a promising approach for the treatment of aggressive breast cancers.     

Genes commonly upregulated by hypoxia in human breast cancer cells MCF-7 and MDA-MB-231.

Hypoxia is a stress that causes alterations in signal transduction and gene instability. In the cancer microenvironment, hypoxia plays a significant role in forming a tumor phenotype and tumor progression. We aimed to identify the genes upregulated by hypoxia in human breast cancer cell lines, a hormone-dependent MCF-7 and a hormone-independent MDA-MB-231, using microarray analysis. These cells were exposed to two oxygen concentrations such as 21% and 1% in a time-course. Out of 12625 genes, 26 genes were identified as commonly upregulated in both MCF-7 and MDA-MB-231 cells. Some of these genes were already reported as hypoxia-related, but some of those were identified newly. These commonly upregulated genes between hormone-dependent and hormone-independent cells would be a clue to study hypoxia-related events and to explore the novel therapeutic targets in human breast cancer.     

硫酸乙酰肝素在MDA-MB-231细胞增殖抑制中的作用

目的:探讨硫酸乙酰肝素(heparan sufate,HS)在抑制人乳腺癌MDA-MB-231细胞增殖过程中对葡萄糖调节蛋白78(glucose-regulated pr otein,GRP78)及相关凋亡蛋白表达的影响.方法:培养人乳腺癌上皮MCF-7细胞株,生长达汇合期及汇合后期时收集培养液,提取其中的HS链.采用噻唑蓝比色法检测HS对MDA-MB-231细胞增殖的抑制作用.采用流式细胞仪检测细胞凋亡情况.应用Westem blot法分别检测GRP78蛋白和凋亡相关蛋白Bcl-2、Bax的表达.结果:HS对MDA-MB-231细胞增殖有明显的抑制作用,且具有剂量和时间依赖性(P<0.05).HS可诱导MDA-MB-231细胞凋亡,并随着剂量的增大,细胞的凋亡率升高(P<0.05).HS抑制MDA-MB-231细胞GRP78蛋白的表达(P<0.05).HS可促进Bax蛋白表达(P<0.05),但抑制Bcl-2蛋白的表达(P<0.05).结论:HS通过减少MDA-MB-231细胞的GRF78、Bcl-2的表达,增加Bax表达来促进肿瘤细胞的凋亡,发挥其抗肿瘤作用.     

Silencing TRIM37 inhibits the proliferation and migration of non-small cell lung cancer cells

Tripartite motif containing 37 (TRIM37), a member of the tripartite motif (TRIM) family, has been involved in the development and progression of several tumors. However, its role in non-small cell lung cancer (NSCLC) is still unclear. Therefore, the aim of this study was to investigate the expression pattern and role of TRIM37 in NSCLC. Our results showed that TRIM37 was highly expressed in human NSCLC cell lines. Knockdown of TRIM37 obviously inhibited the proliferation in vitro and xenografted tumor growth in vivo. Furthermore, knockdown of TRIM37 suppressed NSCLC cell migration and invasion by inhibiting the epithelial–mesenchymal transition (EMT) phenotype. Lastly, knockdown of TRIM37 greatly down-regulated the protein expression levels of β-catenin, cyclinD1 and c-myc in A549 cells. In conclusion, the present study revealed that TRIM37 plays an important role in the development and progression of NSCLC. Thus, TRIM37 may act a potential therapeutic target for treating NSCLC.

Tripartite motif containing 37 (TRIM37), a member of the tripartite motif (TRIM) family, has been involved in the development and progression of several tumors.     

脂肪组织释放细胞因子脂联素的球状结构域对人乳腺癌细胞MDA-MB-231生长抑制及诱导凋亡的效应

目的：脂联素作为一种脂肪组织释放的细胞因子,具有抗糖尿病、抗动脉粥样硬化等生物学功能,脂联素球状结构域（gapM1）为其重要的功能结构域。最近研究发现,脂联素对恶性肿瘤细胞有一定的抑制作用。实验拟进一步讨论gapM1对人乳腺癌细胞MDA-MB-231的生长抑制作用及诱导凋亡作用。方法：实验于2005-03/2006-03在复旦大学上海医学院病理实验室完成。①实验材料：gapM1购自R＆D公司。②实验过程：选用人乳腺癌MDA-MB-231细胞进行体外培养,不同剂量的gapM1（0,0.5,2,8mg/L）处理细胞48h后,显微镜进行形态学观察。③评估：采用BrdU掺入法检测细胞增殖情况;采用TdT介导的dUTP末端标记法原位观察癌细胞凋亡,并计算平均凋亡指数;流式细胞仪测定细胞周期和凋亡率。结果：①倒置显微镜观察结果：不同剂量的gapM1作用MDA-MB-231细胞后,未见明显的细胞形态学改变,但细胞收缩、脱落,细胞增殖明显受到抑制,且随浓度的增大,细胞数量减少。②BrdU掺入法检测结果：不同剂量的gapM1对细胞生长均有一定的抑制作用,且同一处理时相点各组间比较差异均有显著性（P〈0.05）。③TUNEL法检测结果：不同剂量的gapM1作用于MDA-MB-231细胞48h,0.5,2,8mg/L组细胞的凋亡率高于0mg/L组（P〈0.05）。④流式细胞仪分析结果：gapM1作用MDA-MB-231细胞48h,G0～G1期细胞增多,S期细胞显著减少,细胞周期分布特点与药物浓度有关,随浓度增大,变化更加明显。同时可观察到细胞凋亡峰,细胞凋亡率也呈剂量依赖趋势。结论：gapM1对人乳腺癌MDA-MB-231细胞有明显的抑制增殖和诱导凋亡作用。     

Paeoniflorin inhibits proliferation and invasion of breast cancer cells through suppressing Notch-1 signaling pathway

Paeoniflorin (PF), one of the major active ingredients of Chinese peony, was reported to possess anti-tumor effect. However, the role of PF in breast cancer remains to be clarified. Therefore, in this context, the present study investigated the effects of PF on breast cancer cell proliferation and invasion, as well as the underlying mechanism. Our results found that PF suppressed the proliferation and invasion of breast cancer cells. We further demonstrated that PF down-regulated the expression of Notch-1; in addition, overexpression of Notch-1 reversed PF-inhibited proliferation and invasion, and knockdown of Notch-1 enhanced PF-inhibited proliferation and invasion in breast cancer cells. In conclusion, the present study suggests that PF inhibits proliferation and invasion of breast cancer cells through suppressing Notch-1 signaling pathway. Therefore, PF may represent a chemopreventive and/or therapeutic agent in the prevention of breast cancer.