融合蛋白1hFVII-LDM的制备及其对乳腺癌细胞的抑制作用

该文主要研究了强化融合蛋白lhFⅦ-LDM对乳腺癌MDA-MB-231细胞的抑制作用。通过PCR和重叠PCR的方法构建了pET19b-lhFⅦ-LDP表达载体,重组质粒转化BL21,经IPTG诱导后表达并用Co~(2+)亲和层析纯化lhFⅦ-LDP融合蛋白,Western blot检测融合蛋白的正确性,再通过分子重组的方法将lhFⅦ-LDP与力达霉素(LDM)的活性发色团(AE)组装成为强化融合蛋白lhFⅦ-LDM。通过免疫共沉淀实验鉴定lhFⅦ-LDP与组织因子(TF)的特异性结合作用,利用平板克隆形成实验观察lhFⅦ-LDM对细胞增殖的影响,采用Hoechst33342染色检测lhFⅦ-LDM诱导MDA-MB-231细胞凋亡情况,建立了人乳腺癌裸鼠肿瘤模型,研究lhFⅦ-LDM对MDA-MB-231肿瘤生长的抑制作用。结果显示,lhFⅦ-LDM强化融合蛋白在体外能很好的诱导MDA-MB-231细胞凋亡,动物实验结果表明,强化融合蛋白对MDA-MB-231肿瘤的生长具有显著的抑制作用。     

小檗碱对乳腺癌MDA-MB-231细胞增殖抑制作用及其分子机制研究

本研究的目的是为了探索小檗碱对乳腺癌MDA-MB-231细胞增殖的影响以及阐明小檗碱促乳腺癌细胞凋亡的分子机制。在实验过程中,我们通过MTT检测小檗碱对乳腺癌MDA-MB-231细胞增殖的抑制作用,采用Annexin-V/PI染色定量考察小檗碱对肿瘤细胞凋亡的影响,运用Western Blot实验检测肿瘤相关通路蛋白表达来进行研究。实验表明小檗碱对乳腺癌MDA-MB-231细胞增殖具有抑制作用,使细胞中自噬因子Beclin 1表达增加,诱导细胞自噬泡的形成,导致肿瘤细胞发生凋亡。综上说明小檗碱是通过抑制AKT-mTOR通路,诱导MDA-MB-231细胞的自噬以及凋亡,从而发挥抗肿瘤作用。     

分泌蛋白YKL-40增强肿瘤细胞的上皮间质样转化

目的:分泌糖蛋白YKL-40在多种晚期肿瘤病人的血液中显著升高,提示YKL-40蛋白的血浓度是肿瘤恶变的生物标志物。本课题研究YKL-40重组蛋白和过表达YKL-40肿瘤细胞对肿瘤细胞的上皮间质样转化的作用。方法:构建YKL-40过表达的纤维状乳腺癌细胞系MDA-MB-231和非纤维状结肠癌细胞系HCT-116,观察细胞形态学变化,收集细胞和细胞培养液用于Western Blot(WB)检测YKL-40和上皮间质转化标记蛋白Vimentin和N-cadherin。观察重组蛋白YKL-40对原代MDA-MB-231细胞在无血清条件下的细胞存活影响;另外,用细胞存活试剂盒检测YKL-40过表达HCT-116细胞在无血清的培养液中细胞存活情况。最后,用细胞侵袭试验检测YKL-40过表达MDA-MB-231细胞的侵袭力,并用WB和Zymography来测定细胞分泌MMP9蛋白的表达和酶活性。结果:YKL-40过表达增强MDA-MB-231细胞的形态向上皮间质样转化,并显著提高Vimentin、N-cadherin蛋白的表达,但对HCT-116细胞无法诱导上皮间质样转化。在无血清培养基培养条件下,YKL-40可以增强两种细胞的存活能力,并且YKL-40过表达的MDA-MB-231细胞增强了细胞的侵袭能力,促进了MMP9蛋白表达和蛋白活性。结论:YKL-40可以增加肿瘤细胞的存活力,增强纤维状细胞向上皮间质样转化;并且,YKL-40增加MMP9蛋白表达和活性,增强细胞侵袭力。YKL-40是间质样肿瘤细胞EMT的增强子,此发现为抑制肿瘤恶变提供新靶点。     

二甲双胍联合阿霉素应用对人乳腺癌细胞增殖和凋亡的影响

目的：观察二甲双胍联合阿霉素应用对人乳腺癌细胞MDA-MB-231增殖和凋亡的影响。方法：MTT法分别检测二甲双胍、阿霉素和二甲双胍联合阿霉素对MDA-MB-231细胞生长的抑制作用;平板克隆实验检测二甲双胍联合阿霉素对MDA-MB-231细胞克隆形成能力的影响;流式细胞仪检测二甲双胍联合阿霉素对MDA-MB-231细胞凋亡的影响。结果：二甲双胍和阿霉素分别对MDA-MB-231细胞生长有抑制作用,二甲双胍联合阿霉素应用对MDA-MB-231细胞生长的抑制作用更加显著,并且随着药物浓度的增加而增加;二甲双胍联合阿霉素应用与单药相比能够明显降低MDA-MB-231细胞克隆形成率,并且促进细胞凋亡。结论：二甲双胍联合阿霉素应用与单药相比能够显著抑制人乳腺癌细胞MDA-MB-231细胞的增值,促进其凋亡,可见两药联用对肿瘤细胞的杀伤具有协同性。     

人乳腺癌MDA-MB-231细胞裸鼠移植瘤模型的建立及生物学特性研究

目的-建立人乳腺癌MDA-MB-231细胞株裸小鼠模型,研究其生物学特性,观察MDA-MB-231乳腺癌细胞在移植前后的形态学变化。方法-将人乳腺癌细胞MDA-MB-231接种于裸鼠腋窝处皮下,每3天测量肿瘤大小,第30天处死小鼠。肿瘤组织及相关脏器送病理切片。皮下肿瘤组织细胞及细胞株培养HE染色。结果-肿瘤生长较快,成功率为72%,病理检查符合人乳腺癌细胞特征。肿瘤组织细胞及培养细胞形态学未见显著差异。结论-人乳腺癌细胞株MDA-MB-231裸小鼠模型建立方法较简便,细胞形态无明显差异,且保持了人乳腺癌的生物学特性。     

亲骨转移乳腺癌细胞MDA-MB-231BO成瘤性的初步研究

目的通过比较亲骨转移乳腺癌细胞（MDA-MB-231BO）和亲代乳腺癌细胞（MDA-MB-231）的生长曲线和致瘤性,初步探讨MDA-MB-231BO细胞的生物学特性。方法MTT法测定两种细胞的生长曲线,并将两种乳腺癌细胞接种于裸鼠腋窝处皮下,建立乳腺癌细胞异种移植瘤动物模型,30 d后处死裸鼠,肿瘤组织及相关脏器官做病理检查。结果MTT法测得MDA-MB-231BO细胞生长速率高于MDA-MB-231细胞。接种两种乳腺癌细胞的裸鼠均长出肿瘤,成瘤率为100%。病理检查符合人乳腺癌细胞特征,MDA-MB-231BO组瘤体体积明显大于MDA-MB-231组（P〈0.05）。结论MDA-MB-231BO细胞生长速率高于MDA-MB-231细胞,而且MDA-MB-231BO在裸鼠体内的致瘤性强于MDA-MB-231。     

山柰酚通过线粒体凋亡通路诱导三阴性乳腺癌细胞凋亡的机制研究

本文旨在探讨山柰酚对三阴性乳腺癌细胞MDA-MB-231的抑制作用及其可能的分子机制。用不同浓度的山柰酚处理细胞,CCK-8法检测山柰酚对MDA-MB-231和人正常乳腺上皮细胞MCF-10A活力的影响,倒置显微镜观察各组细胞形态变化,克隆形成法检测细胞增殖,流式细胞仪检测细胞凋亡,JC-1染色法检测细胞线粒体膜电位的改变,Western blot检测B细胞淋巴瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、细胞色素C(Cyt C)和细胞周期蛋白D1(CyclinD1)的蛋白表达,比色法检测半胱氨酸天冬氨酸蛋白水解酶-3(Caspase-3)以及半胱氨酸天冬氨酸蛋白水解酶-9(Caspase-9)的活性。结果显示,山柰酚体外可显著抑制MDA-MB-231细胞的增殖,诱导细胞凋亡,且对正常乳腺上皮细胞活力无影响,降低细胞线粒体膜电位,上调Bax、Cyt C的表达,下降Bcl-2和Cyclin D1的表达,增强Caspase-3、Caspase-9活性。结果表明,线粒体凋亡信号通路的激活是山柰酚诱导MDA-MB-231细胞凋亡的途径之一。     

肿瘤坏死因子-α在乳腺肿瘤中的表达及其与影像学特征的关系

目的:检测乳腺肿瘤中肿瘤坏死因子α(Tumor necrosis factor alpha,TNF-α)表达及其与影像学特征的关系。方法:利用免疫荧光和流式细胞术检测MDA-MB-231及MCF-7乳腺癌细胞中TNF-α的表达;收集82例经病理证实的乳腺疾病患者组织及其病理资料、影像学资料,通过免疫组化检测乳腺组织TNF-α的表达,并分析其表达量与病理特征及影像学特征之间的关系。结果:TNF-α在MDA-MB-231细胞中呈高表达,恶性乳腺肿瘤组织中TNF-α的表达显著高于乳腺良性肿瘤,其表达量与淋巴结转移、TNM分期相关、核磁共振(MRI)强化是否均匀、钼靶X射线的边界的平滑度形状是否规则以及B超中彩色血流信号强弱均显著相关(P0.05)。结论:乳腺肿瘤组织中TNF-α呈异常高表达,且与乳腺肿瘤的某些影像学特征密切相关。     

辣椒碱对乳腺癌MDA-MB-231细胞迁移和侵袭的抑制作用及其机制

本研究旨在观察辣椒碱对人乳腺癌MDA-MB-231细胞迁移和侵袭的影响,并探讨其分子机制。乳腺癌MDA-MB-231细胞经不同浓度辣椒碱作用24 h后进行实验,采用CCK-8法检测细胞活性,采用细胞划痕实验测量细胞迁移率,采用Transwell侵袭实验检测细胞侵袭率,采用Western blot检测乳腺癌MDA-MB-231细胞中c-Src、p-c-Src(Tyr416)、黏着斑激酶(focal adhesion kinase,FAK)、p-FAK(Tyr576/577)、桩蛋白(Paxillin)、p-Paxillin(Tyr118)、基质金属蛋白酶2(matrix metalloproteinase 2,MMP2)和MMP9蛋白表达水平,采用RT-PCR法检测乳腺癌MDA-MB-231细胞中MMP2和MMP9的mRNA水平。结果显示,10~50μmol/L辣椒碱对MDA-MB-231细胞存活率无显著影响,100~500μmol/L辣椒碱显著降低MDA-MB-231细胞存活率(P0.05);与正常对照组比较,25和50μmol/L辣椒碱组MDA-MB-231细胞体外迁移率和侵袭率均显著降低(P0.05),c-Src、FAK和Paxillin蛋白的磷酸化水平均显著下降(P0.05),MMP2和MMP9的mRNA和蛋白表达水平均下降(P0.05);并且辣椒碱对MDA-MB-231细胞的上述作用具有剂量依赖性。以上结果提示,低浓度辣椒碱可剂量依赖性地抑制人乳腺癌MDA-MB-231细胞迁移和侵袭,其机制可能涉及辣椒碱对c-Src/FAK/Paxillin信号通路以及MMP2和MMP9表达的抑制。     

迷迭香酸对乳腺癌MDA-MB-231细胞增殖、凋亡和迁移能力的影响

研究迷迭香酸对乳腺癌MDA-MB-231细胞增殖、凋亡和迁移能力的影响。采用磺酰罗丹明B(SRB)法测定迷迭香酸对乳腺癌MDA-MB-231细胞增殖的影响,Hoechst 33258荧光染色法观察细胞凋亡形态,Annexin VFITC/PI检测细胞凋亡率;同时,细胞划痕实验检测迷迭香酸对MDA-MB-231细胞体外迁移能力的影响,实时荧光PCR(qPCR)法检测Bax、Bcl-2、Caspase-3、MMP-2和MMP-9基因的表达水平。研究结果显示迷迭香酸能抑制乳腺癌MDA-MB-231细胞的增殖,且呈时间剂量依赖性;迷迭香酸处理后的MDA-MB-231细胞出现明显的凋亡形态,且细胞凋亡率明显增加,Bax和caspase-3 mRNA表达水平增加,而Bcl-2 mRNA表达水平降低。另外,迷迭香酸作用后可剂量依赖性地降低MDA-MB-231细胞的体外迁移能力;同时降低MMP-2和MMP-9 mRNA的表达。因此,迷迭香酸能有效的抑制MDA-MB-231细胞的增殖,诱导细胞凋亡,降低细胞迁移能力。     

In vitro lung cancer multicellular tumor spheroid formation using a microfluidic device

The purpose of this study was to demonstrate self-organizing in vitro multicellular tumor spheroid (MCTS) formation in a microfluidic system and to observe the behavior of MCTSs under controlled microenvironment. The employed microfluidic system was designed for simple and effective formation of MCTSs by generating nutrient and oxygen gradients. The MCTSs were composed of cancer cells, vascular endothelial cells, and type I collagen matrix to mimic the in vivo tumor microenvironment (TME). Cell culture medium was perfused to the microfluidic device loaded with MCTSs by a passive fluidic pump at a constant flow rate. The dose response to an MMPs inhibitor was investigated to demonstrate the effects of biochemical substances. The result of long-term stability of MCTSs revealed that continuous perfusion of cell culture medium is one of the major factors for the successful MCTS formation. A continuous flow of cell culture medium in the in vitro TME greatly affected both the proliferation of cancer cells in the micro-wells and the sustainability of the endothelial cell-layer integrity in the lumen of microfluidic channels. Addition of MMP inhibitor to the cell culture medium improved the stability of the collagen matrix by preventing the detachment and shrinkage of the collagen matrix surrounding the MCTSs. In summary, the present constant flow assisted microfluidic system is highly advantageous for long-term observation of the MCTS generation, tumorous tissue formation process and drug responses. MCTS formation in a microfluidic system may serve as a potent tool for studying drug screening, tumorigenesis and metastasis.     

Snail1 induced in breast cancer cells in 3D collagen I gel environment suppresses cortactin and impairs effective invadopodia formation

Although an in vitro 3D environment cannot completely mimic the in vivo tumor site, embedding tumor cells in a 3D extracellular matrix (ECM) allows for the study of cancer cell behaviors and the screening of anti-metastatic reagents with a more in vivo-like context. Here we explored the behaviors of MDA-MB-231 breast cancer cells embedded in 3D collagen I. Diverse tumor environmental conditions (including cell density, extracellular acidity, or hypoxia as mimics for a continuous tumor growth) reduced JNKs, enhanced TGFβ1/Smad signaling activity, induced Snail1, and reduced cortactin expression. The reduced JNKs activity blocked efficient formation of invadopodia labeled with actin, cortactin, or MT1-MMP. JNKs inactivation activated Smad2 and Smad4, which were required for Snail1 expression. Snail1 then repressed cortactin expression, causing reduced invadopodia formation and prominent localization of MT1-MMP at perinuclear regions. MDA-MB-231 cells thus exhibited less efficient collagen I degradation and invasion in 3D collagen I upon JNKs inhibition. These observations support a signaling network among JNKs, Smads, Snail1, and cortactin to regulate the invasion of MDA-MB-231 cells embedded in 3D collagen I, which may be targeted during screening of anti-invasion reagents.     

Oxygen-mediated regulation of gelatinase and tissue inhibitor of metalloproteinases-1 expression by invasive cells.

The relative expression of matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) is an important determinant in trophoblast invasion of the uterus and tumor invasion and metastasis. Our previous studies have shown that low oxygen levels increase the in vitro invasiveness of trophoblast and tumor cells. The present study examined whether changes in oxygen levels affect TIMP and MMP expression by cultured trophoblast and breast cancer cells. Reverse zymographic analysis demonstrated reduced TIMP-1 protein secretion by HTR-8/SVneo trophoblast cells as well as MDA-MB-231 and MCF-7 breast carcinoma cells cultured in 1% vs 20% oxygen for 24 h. While gelatin zymography revealed no changes in the levels of MMP-9 secreted by HTR-8/SVneo trophoblasts cultured under various oxygen concentrations for 24 h, human MDA-MB-231 breast carcinoma cells displayed increased MMP-9 secretion and human MCF-7 breast cancer cells exhibited reduced secretion of this enzyme when cultured under similar conditions. In contrast, MMP-2 levels remained unchanged in all cultures incubated under similar conditions. Western blot analysis of MMP-9 protein in cell extracts confirmed the results of zymography. To assess the contribution of enhanced MMP activity to hypoxia-induced invasion, the effect of an MMP inhibitor (llomastat) on the ability of MDA-MB-231 cells to penetrate reconstituted extracellular matrix (Matrigel) was examined. Results showed that MMP inhibition significantly decreased the hypoxic upregulation of invasion by these cells. These findings indicate that the increased cellular invasiveness observed under reduced oxygen conditions may be due in part to a shift in the balance between MMPs and their inhibitors favoring increased MMP activity.     

Breast cancer cell behaviors on staged tumorigenesis-mimicking matrices derived from tumor cells at various malignant stages

Extracellular matrix (ECM) has been focused to understand tumor progression in addition to the genetic mutation of cancer cells. Here, we prepared “staged tumorigenesis-mimicking matrices” which mimic in vivo ECM in tumor tissue at each malignant stage to understand the roles of ECM in tumor progression. Breast tumor cells, MDA-MB-231 (invasive), MCF-7 (non-invasive), and MCF-10A (benign) cells, were cultured to form their own ECM beneath the cells and formed ECM was prepared as staged tumorigenesis-mimicking matrices by decellularization treatment. Cells showed weak attachment on the matrices derived from MDA-MB-231 cancer cells. The proliferations of MDA-MB-231 and MCF-7 was promoted on the matrices derived from MDA-MB-231 cancer cells whereas MCF-10A cell proliferation was not promoted. MCF-10A cell proliferation was promoted on the matrices derived from MCF-10A cells. Chemoresistance of MDA-MB-231 cells against 5-fluorouracil increased on only matrices derived from MDA-MB-231 cells. Our results showed that the cells showed different behaviors on staged tumorigenesis-mimicking matrices according to the malignancy of cell sources for ECM preparation. Therefore, staged tumorigenesis-mimicking matrices might be a useful in vitro ECM models to investigate the roles of ECM in tumor progression.     

HDAC6 is required for invadopodia activity and invasion by breast tumor cells

Invasion across tissue boundaries by metastatic tumor cells depends on the proteolytic degradation of the extracellular matrix, initiated by the formation of invadopodia, actin-driven membrane protrusions with matrix-degradative activity. Yet, mechanisms underlying invadopodia formation remain largely unknown. In this report, we examined the role of the histone deacetylase HDAC6 in invadopodia formation and invasion by breast cancer cells. Using small interfering RNA silencing of protein expression in highly invasive MDA-MB-231 breast adenocarcinoma cells, we show that HDAC6 is required for two-dimensional matrix proteolysis. In addition, we demonstrate that HDAC6 acts as a tubulin and cortactin deacetylase. We also report that the inhibition of HDAC6 by siRNA or treatment with HDAC inhibitor TSA results in a decreased invasion capacity of a three-dimensional type I collagen matrix by MDA-MB-231 cells. These data identify HDAC6 as a critical component of the invasive apparatus of tumor cells, in both two- and three-dimensional matrices.     

Engineering tumor constructs to study matrix-dependent angiogenic signaling of breast cancer cells

Breast cancer is the leading cause of cancer deaths among females globally. The crosstalk between tumor microenvironment and neoplastic cells is the key for promoting tumor growth, stimulating tumor angiogenesis, and metastasis to distant organs. Thus, it is highly important to investigate tumor cell–matrix interactions to facilitate screening of different anti-cancer agents, individually or in combination. We, herein report, the development of an in vitro three-dimensional (3D) breast cancer model to investigate the effect of stromal crosslinking and consequent, stiffening on the angiogenic activity of cancer cells. Crosslinking of collagen gels was altered via non-enzymatic glycation and highly aggressive breast cancer cells, MDA-MB-231, were encapsulated in these gels. Cells encapsulated in glycated/stiffer matrices displayed an increased expression of pro-angiogenesis-related signals. Inhibition of mechanotransduction pathways on the angiogenic activity of aggressive tumor cells in stiff matrices was investigated using Y-27632, blebbistatin, and cytochalasin D. Rho-associated kinase (ROCK) inhibitor, Y-27632, diminished the pro-angiogenic signaling, thereby suggesting the potential dependence of breast cancer cells on the Rho/ROCK pathway in regulating tumor angiogenesis. Our findings highlight the potential of the developed model to be used as a tool to investigate matrix-associated tumor angiogenesis and screen different therapeutic agents towards inhibiting it.