Changes in Electric Properties of Human Breast Cancer Cells

Studies of the electrical surface properties of biological cells have provided fundamental knowledge about the cell surface. The change in biological functions of cells may affect the surface properties and can be detected by electrokinetic measurements. The surface density of fibroblasts and breast cancer cells (MDA-MB-231 and MCF-7) as a function of pH was measured by electrophoresis. The interaction between solution ions and the breast cancer cell or fibroblast surface was described by a four-component equilibrium model. The agreement between the experimental and theoretical charge variation curves of the breast cancer cells and fibroblasts was good at pH 2.5–9. The extent of fibroblast and breast cancer cell lipid peroxidation was estimated by HPLC measurement of the malondialdehyde level. The acid (C _TA) and basic (C _TB) functional group concentrations and the average association constant with hydroxyl (K _BOH) ions values of the breast cancer cell membranes were higher than in normal cells, while the average association constant with hydrogen (K _AH) value was smaller. The level of lipid peroxidation products was higher in breast cancer cells than in normal cells.     

Enhanced Gene Delivery and Expression in Human Hepatocellular Carcinoma Cells by Cationic Immunoliposomes

Abstract

A targeted vector allowing enhanced gene transfer to human hepatocellular carcinoma (HCC¹) cells in vitro was developed using cationic liposomes covalently conjugated with the mAb AF-20. This high affinity antibody recognizes a rapidly internalized 180 kDa cell surface glycoprotein which is abundantly expressed on the surface of human HCC and other cancer cells. Quantitative binding analysis of liposomes with target cells by flow cytometry showed specific association of mAb-targeted liposomes with human HCC cells. Using mAb-targeted cationic liposomes containing 20% DOTAP, in the presence or absence of serum, gene expression in HuH-7 cells was enhanced up to 40-fold as compared to liposomes conjugated with an isotype-matched non-relevant control antibody. Transfection specificity was not observed in a control cell line that does not express the antigen recognized by mAb AF-20. This study demonstrates that cationic liposome formulations can be targeted with monoclonal antibodies (mAbs) to enhance specific in vitro gene delivery and expression in the presence or absence of serum.     

Breast Carcinoma With Osteoclast-Like Giant Cells

The cytological and histological findings of a case of breast carcinoma with osteoclast-like giant cells are presented. A fine needle aspiration specimen demonstrated the characteristic combination of malignant epithelium and reactive multinucleated cells and enabled identification of this rare variant of breast cancer. Immunocytochemical studies using the monoclonal antibody KP1[CD68] support a histiocytic origin for the osteoclast-like cells.     

Human Breast Tumor Cells Are More Resistant to Cardiac Glycoside Toxicity Than Non-Tumorigenic Breast Cells

Cardiotonic steroids (CTS), specific inhibitors of Na,K-ATPase activity, have been widely used for treating cardiac insufficiency. Recent studies suggest that low levels of endogenous CTS do not inhibit Na,K-ATPase activity but play a role in regulating blood pressure, inducing cellular kinase activity, and promoting cell viability. Higher CTS concentrations inhibit Na,K-ATPase activity and can induce reactive oxygen species, growth arrest, and cell death. CTS are being considered as potential novel therapies in cancer treatment, as they have been shown to limit tumor cell growth. However, there is a lack of information on the relative toxicity of tumor cells and comparable non-tumor cells. We have investigated the effects of CTS compounds, ouabain, digitoxin, and bufalin, on cell growth and survival in cell lines exhibiting the full spectrum of non-cancerous to malignant phenotypes. We show that CTS inhibit membrane Na,K-ATPase activity equally well in all cell lines tested regardless of metastatic potential. In contrast, the cellular responses to the drugs are different in non-tumor and tumor cells. Ouabain causes greater inhibition of proliferation and more extensive apoptosis in non-tumor breast cells compared to malignant or oncogene-transfected cells. In tumor cells, the effects of ouabain are accompanied by activation of anti-apoptotic ERK1/2. However, ERK1/2 or Src inhibition does not sensitize tumor cells to CTS cytotoxicity, suggesting that other mechanisms provide protection to the tumor cells. Reduced CTS-sensitivity in breast tumor cells compared to non-tumor cells indicates that CTS are not good candidates as cancer therapies.     

AFM对人乳腺癌细胞外纤连蛋白原纤维的形态学观察

探讨原子力显微镜在研究细胞和细胞外基质间的相互作用及细胞外基质的功能等方面的应用前景。应用原子力显微镜观察培养的人乳腺癌MCF 7 R细胞分泌的纤连蛋白原纤维的分布和排列规律 ,并与其他常规观察技术进行比较。应用原子力显微镜获得了多个乳腺癌细胞和细胞外纤连蛋白原纤维的整体和局部形貌图像 ,发现这些原纤维的分布和排列方式非常有规律 ,而且这些规律与其功能相适应。由于样品制备简单和分辨率较高等优点 ,原子力显微镜较适合于细胞外基质的原位观察     

Investigation of Gallic Acid Induced Anticancer Effect in Human Breast Carcinoma MCF‐7 Cells

Gallic acid (GA), a polyhydroxylphenolic compound abundantly distributed in plants, fruits, and foods, has been reported to have various biological activities including an anticancer effect. In this study, we extensively investigated the anticancer effect of GA in human breast carcinoma MCF‐7 cells. Our study indicated that treatment with GA resulted in inhibition of proliferation and induction of apoptosis in MCF‐7 cells. Then, the molecular mechanism of GA's apoptotic action in MCF‐7 cells was further investigated. The results revealed that GA induced apoptosis by triggering the extrinsic or Fas/FasL pathway as well as the intrinsic or mitochondrial pathway. Furthermore, the apoptotic signaling induced by GA was amplified by cross‐link between the two pathways. Taken together, our findings may be useful for understanding the mechanism of action of GA on breast cancer cells and provide new insights into the possible application of such compound and its derivatives in breast cancer therapy.     

On Stem Cells in the Human Breast

In the human breast, stem cells give rise to myoepithelial and luminal epithelial cells that are needed during monthly estrous cycles and lactation. The mechanisms that govern mammary stem cells are hotly debated.Petersen and Polyak (2011) elegantly explain the developmental hierarchy of the human mammary gland as it is currently understood. It is amazing that the small pool of stem cells can be cyclically called on to give rise to the progenitors and more differentiated myoepithelial and luminal epithelial cells that are needed for expansion during monthly estrous cycles and in preparation for lactation. How do the stem cells respond so perfectly and repetitively throughout a woman’s childbearing years? Do stem cells harbor internal circadian clocks that work in time with similar clocks in the endocrine system and ovaries? Or are mammary stem cells able to respond to shifting physiological needs of the organism because their functions are controlled by their microenvironment, which changes with the ebb and flow of the physiological tide? That estrous cycles can vary according to life’s circumstances (e.g., changes in nutrition, exercise, or stress) and because lengths of pregnancies and lactation periods differ for each pregnancy, it seems likely that mammary stem cells are governed by an external control mechanism that interacts with systemic changes in physiology.That stem cells and all of their progeny share identical genomes, and that stem cells reside inside niche microenvironments that are completely unique as compared to those of the surrounding tissue, suggests that microenvironments exert a tremendous influence over stem cell behavior. Even mathematical models of hematopoiesis suggested that stem cell-extrinsic regulation was the best way to explain why stem cells responded to a wide variety of physiological needs (Loeffler and Roeder 2002; Roeder and Lorenz 2006). Experimentally, microenvironmental control of mammary progenitors, hematopoietic stem cells, embryonic stem cells, and neural progenitors has been shown by use of functional assays on arrayed combinatorial microenvironments (Flaim et al. 2005; Soen et al. 2006; LaBarge et al. 2009; Lutolf et al. 2009). And perhaps the grandest demonstration of the power of the microenvironment over stem cell function was shown repeatedly when adult stem cells from one tissue were shown to give rise to other tissues after they were placed in tissue-specific microenvironments different from their native ones (Blau et al. 2001; Boulanger et al. 2007; Booth et al. 2008). An important likelihood that arises from those experiments is that the phenotype of a stem cell is probably affected by its microenvironment (LaBarge et al. 2007). To completely understand the identity and control of mammary stem cells, we must meticulously define the microenvironment(s) they inhabit.As Petersen and Polyak point out, the methodology used to describe the stem cell hierarchy in adult hematopoietic systems has guided many subsequent studies aimed at identifying hierarchies in epithelial tissues. Accordingly, tissues are dissociated into suspensions of single cells, fractionated with a cell sorting technology, and the fractions are assayed for stem cell activity in a number of culture assays and in vivo when possible. Demonstration that a single cell can give rise to the tissue in question is often referred to as the “gold standard” experiment, and it is an unfortunate burden of proof held over from the hematopoietic field. Blood is an interesting tissue in that centers for hematopoiesis shift throughout development among locations that are anatomically close to the circulation, and it is thought that in adults, blood is produced from niches in marrow and vasculature (Sacchetti et al. 2007; Hooper et al. 2009; Butler et al. 2010). Thus, it makes sense that a single hematopoietic stem cell should regenerate the blood as nothing more is being asked of it than to do exactly what it was meant to, and to do it in the perfect microenvironment. Moreover, in the hematopoiesis model, where the microenvironment was essentially perfect, it stands to reason that one would only observe different activities according to the fraction from which single cells were derived. These facts may help explain why it has taken so long for acceptance of the idea that microenvironments can control stem cell activity. By comparison, adult mammary epithelial stem cells are distinct from the neonatal stem cells that made the initial mammary rudiment, and the adult stem cells evolved within an intricate epithelial architecture that was within a tissue stroma. That a single mouse mammary stem cell could give rise to an outgrowth was shown to occur at very low frequency (Shackleton et al. 2006; Stingl et al. 2006), and it has yet to be shown that a single normal human mammary stem cell can generate an outgrowth in a murine fat pad. Given the biological differences between blood and mammary epithelium, it is no surprise that the identity of human mammary stem cells and the mechanisms that govern them are still hotly debated.     

Time-Dependent Micromechanical Responses of Breast Cancer Cells and Adjacent Fibroblasts to Electric Treatment

Direct-current, low-intensity, electric fields were suggested as a minimally invasive treatment for various cancers. The tumor microenvironment may affect treatment efficacy, albeit it has not generally been considered when evaluating novel anti-cancer treatments. We evaluate the effects of electric treatment on epithelial, breast-cancer cells, co-cultured with non-cancerous fibroblasts, a simplified model for the tumor-microenvironment. We evaluate changes in morphology, cytoskeleton, and focus on dynamic intracellular structure and mechanics. Multiple-particle tracking was used within living cells to quantify time-dependent structural and mechanical changes. Cancer cells suffer severe cell death and exhibit transient rounding and changes in internal structural and mechanics. Interestingly, treating cancer cells in co-culture with fibroblasts delays and reduces their responses to treatment. Our particle-tracking data indicates a mechanism relating the observed changes in intracellular transport to transient changes in the microtubule network and its motors. In contrast, fibroblasts are only minimally affected by treatment, separately or in co-culture. To conclude, intracellular mechanics reveal time-dependent responses after treatment, unavailable by bulk measurements. This time-dependence could provide a window of opportunity for continued treatment. We demonstrate the importance of evaluating anti-cancer treatments within their microenvironment, which can affect response magnitude and time-course.     

Delivery of Human Adipose Stem Cells Spheroids into Lockyballs

Adipose stem cells (ASCs) spheroids show enhanced regenerative effects compared to single cells. Also, spheroids have been recently introduced as building blocks in directed self-assembly strategy. Recent efforts aim to improve long-term cell retention and integration by the use of microencapsulation delivery systems that can rapidly integrate in the implantation site. Interlockable solid synthetic microscaffolds, so called lockyballs, were recently designed with hooks and loops to enhance cell retention and integration at the implantation site as well as to support spheroids aggregation after transplantation. Here we present an efficient methodology for human ASCs spheroids biofabrication and lockyballs cellularization using micro-molded non-adhesive agarose hydrogel. Lockyballs were produced using two-photon polymerization with an estimated mechanical strength. The Young’s modulus was calculated at level 0.1362 +/-0.009 MPa. Interlocking in vitro test demonstrates high level of loading induced interlockability of fabricated lockyballs. Diameter measurements and elongation coefficient calculation revealed that human ASCs spheroids biofabricated in resections of micro-molded non-adhesive hydrogel had a more regular size distribution and shape than spheroids biofabricated in hanging drops. Cellularization of lockyballs using human ASCs spheroids did not alter the level of cells viability (p › 0,999) and gene fold expression for SOX-9 and RUNX2 (p › 0,195). The biofabrication of ASCs spheroids into lockyballs represents an innovative strategy in regenerative medicine, which combines solid scaffold-based and directed self-assembly approaches, fostering opportunities for rapid in situ biofabrication of 3D building-blocks.     

Selective Lentiviral Gene Delivery to CD133-Expressing Human Glioblastoma Stem Cells

Glioblastoma multiforme (GBM) is a deadly primary brain malignancy. Glioblastoma stem cells (GSC), which have the ability to self-renew and differentiate into tumor lineages, are believed to cause tumor recurrence due to their resistance to current therapies. A subset of GSCs is marked by cell surface expression of CD133, a glycosylated pentaspan transmembrane protein. The study of CD133-expressing GSCs has been limited by the relative paucity of genetic tools that specifically target them. Here, we present CD133-LV, a lentiviral vector presenting a single chain antibody against CD133 on its envelope, as a vehicle for the selective transduction of CD133-expressing GSCs. We show that CD133-LV selectively transduces CD133+ human GSCs in dose-dependent manner and that transduced cells maintain their stem-like properties. The transduction efficiency of CD133-LV is reduced by an antibody that recognizes the same epitope on CD133 as the viral envelope and by shRNA-mediated knockdown of CD133. Conversely, the rate of transduction by CD133-LV is augmented by overexpression of CD133 in primary human GBM cultures. CD133-LV selectively transduces CD133-expressing cells in intracranial human GBM xenografts in NOD.SCID mice, but spares normal mouse brain tissue, neurons derived from human embryonic stem cells and primary human astrocytes. Our findings indicate that CD133-LV represents a novel tool for the selective genetic manipulation of CD133-expressing GSCs, and can be used to answer important questions about how these cells contribute to tumor biology and therapy resistance.     

Transpapillary Drug Delivery to the Breast

The study was aimed at investigating localized topical drug delivery to the breast via mammary papilla (nipple). 5-fluorouracil (5-FU) and estradiol (EST) were used as model hydrophilic and hydrophobic compounds respectively. Porcine and human nipple were used for in-vitro penetration studies. The removal of keratin plug enhanced the drug transport through the nipple. The drug penetration was significantly higher through the nipple compared to breast skin. The drug’s lipophilicity had a significant influence on drug penetration through nipple. The ducts in the nipple served as a major transport pathway to the underlying breast tissue. Results showed that porcine nipple could be a potential model for human nipple. The topical application of 5-FU on the rat nipple resulted in high drug concentration in the breast and minimal drug levels in plasma and other organs. Overall, the findings from this study demonstrate the feasibility of localized drug delivery to the breast through nipple.     

Effects of Novel Dinuclear Cisplatinum(II) Complexes on the Electric Properties of Human Breast Cancer Cells

The aim of this study was to determine the influence of cisplatin and novel dinuclear platinum(II) complexes on the electrical properties of the membrane and the level of lipid peroxidation in the human breast cancer cell lines MDA-MB-231 and MCF-7. The basal electrical surface properties of cells are known. Changes in cell function may affect these surface properties, and those changes can be detected by electrokinetic measurements. The surface charge density of the breast cancer cell lines MDA-MB-231 and MCF-7 were measured as a function of pH. A four-component equilibrium model was used to describe the interaction between the solution ions and the breast cancer cell surface. The experimental and the theoretical charge variation curves of the breast cancer cells at pH 2.5–9 were in agreement. Measurements of the cellular malondialdehyde levels with high performance liquid chromatography were used to determine the extent of lipid peroxidation. The acid and base functional group concentrations and average association constants with hydroxyl ions were smaller in breast cancer cell membranes treated with cisplatin or novel dinuclear platinum(II) complexes compared with untreated cancer cells, and the average association constants with hydrogen ions were higher. The levels of lipid peroxidation products in breast cancer cells treated with cisplatin or novel dinuclear platinum(II) complexes were also higher than in untreated cancer cells.     

RNA干扰敲减FucT VII表达抑制人结肠癌细胞HT-29和HUVECs的粘附能力

 探讨利用RNA干扰（RNAi）技术抑制岩藻糖基转移酶Ⅶ（FucT Ⅶ）表达对人结肠癌细胞HT-29与人脐静脉内皮细胞（HUVEC）粘附能力的影响及其机制.本课题构建3对针对FucT Ⅶ基因的RNAi表达载体,并将其转染入人结肠癌细胞HT-29,Western 印迹检测FucT Ⅶ及其下游产物sLeX蛋白的变化;实时PCR 检测FucT Ⅶ mRNA表达的变化;玫瑰红染色法检测RNAi 对HT-29与HUVECs细胞粘附能力的影响.结果显示,3对FucT Ⅶ siRNA表达载体均可有效抑制HT-29细胞FucT Ⅶ mRNA和蛋白表达,以pSilencer 2.0 FucT Ⅶ 2最为有效;与空白细胞组比较,转染pSilencer 2.0-FucT Ⅶ的HT-29细胞表面sLeX表达水平明显下降,以pSilencer 2.0-FucT Ⅶ 2最为显著;RNA干扰FucT Ⅶ表达后HT 29细胞和HUVEC之间的粘附能力明显受到抑制.研究表明,RNAi靶向沉默HT-29细胞中FucT Ⅶ基因表达可显著降低其下游产物sLeX的合成,进而抑制HT-29细胞与HUVECs的粘附能力.     

Anti-Proliferative Effects of Evodiamine on Human Breast Cancer Cells

Endocrine sensitivity, assessed by the expression of estrogen receptor (ER), has long been the predict factor to guide therapeutic decisions. Tamoxifen has been the most successful hormonal treatment in endocrine-sensitive breast cancer. However, in estrogen-insensitive cancer tamoxifen showed less effectiveness than in estrogen-sensitive cancer. It is interesting to develop new drugs against both hormone-sensitive and insensitive tumor. In this present study we examined anticancer effects of evodiamine extracted from the Chinese herb, Evodiae fructus, in estrogen-dependent and –independent human breast cancer cells, MCF-7 and MDA-MB-231 cells, respectively. Evodiamine inhibited the proliferation of MCF-7 and MDA-MB-231 cells in a concentration-dependent manner with concentration of 1×10⁻⁶ and 1×10⁻⁵ M. Evodiamine also induced apoptosis via up-regulation of caspase 7 activation, PARP cleavage (Bik and Bax expression). The expression of ER α and β in protein and mRNA levels was down-regulated by evodiamine according to data from immunoblotting and RT-PCR analysis. Overall, our results indicate that evodiamine mediates degradation of ER and induces caspase-dependent pathway leading to inhibit proliferation of breast cancer cell lines. It suggests that evodiamine may in part mediate through ER-inhibitory pathway to inhibit breast cancer cell proliferation.     

Modulation of Progestin Binding Activity in Cultured Human Breast Carcinoma Cells: The Effect of Serum Type and Concentration

Abstract

Progesterone receptor levels in MCF-7 human breast cancer cells increase as a specific response to estrogen and to some nonsteroidal antiestrogens. In the present study we demonstrate that the type and quantity of serum present during culture of these cells modifies the level of progestin binding activity, but not the level of estradiol binding activity.

MCF-7 cells maintained in media supplemented with 5% charcoal-dextran treated calf serum (CDCS) contain 0.3 - 0.4 pmol of cytosol progesterone receptor (PR_c) per mg DNA. When cells previously maintained in 5% CDCS-media are shifted to media containing 5% charcoal-dextran treated fetal calf serum (CDFCS), the level of progestin binding increases after day 16, and stabilizes at 2 - 3 pmol/mg DNA at days 30 to 40. Shifting these cells back to 5% CDCS-media, reduces PR_c to 0.2 - 0.4 pmol/mg DNA within 3 days. This reduction is dose dependent with a half-optimal decrease at 1% CDCS, and a full decrease at 2% CDCS (4d incubation). Nuclear progestin binding was uniformly low (0.2 - 0.4 pmol/mg DNA) and unaffected by type or concentration of serum, and no consistent change in cytosol or nuclear estrogen receptor levels was observed. These cytoplasmic progestin binding sites are translocated to the nucleus by progesterone, and are similar to estradiol (E₂) induced sites by Scatchard binding and sucrose gradient analysis. Similar serum-dependent changes are also observed in the T₄₇D human breast cancer cell line where growth in CDFCS-media results in 4-fold higher progestin binidng levels than observed in CDCS-media. Our findings suggest the presence of non-dialyzable stimulatory factor(s) in CDFCS that influence the progestin receptor level the highlight the fact that serum components can alter dramatically the cellular progestin binding activity.     

Tumor Specific Peptides Selected for Targeted Delivery of Therapeutic Agents to Glioma Human Cells

Russian Journal of Bioorganic Chemistry - Brain tumors are among the most intractable types of malignant neoplasms. Despite advances in the treatment of cancer, in particular, the development of...     

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。     

Soluble T-Cell Receptors Produced in Human Cells for Targeted Delivery

Recently, technology has become available to generate soluble T-cell receptors (sTCRs) that contain the antigen recognition part. In contrast to antibodies, sTCRs recognize intracellular in addition to extracellular epitopes, potentially increasing the number of applications as reagents for target detection and immunotherapy. Moreover, recent data show that they can be used for identification of their natural peptide ligands in disease. Here we describe a new and simplified expression method for sTCRs in human cells and show that these sTCRs can be used for antigen-specific labeling and elimination of human target cells. Four different TCRs were solubilized by expression of constructs encoding the TCR alpha (α) and beta (β) chains lacking the transmembrane and intracellular domains, linked by a ribosomal skipping 2A sequence that facilitates equimolar production of the chains. Cell supernatants containing sTCRs labeled target cells directly in a peptide (p)-human leukocyte antigen (HLA)-specific manner. We demonstrated that a MART-1p/HLA-A*02:01-specific sTCR fused to a fluorescent protein, or multimerized onto magnetic nanoparticles, could be internalized. Moreover, we showed that this sTCR and two sTCRs recognizing CD20p/HLA-A*02:01 could mediate selective elimination of target cells expressing the relevant pHLA complex when tetramerized to streptavidin-conjugated toxin, demonstrating the potential for specific delivery of cargo. This simple and efficient method can be utilized to generate a wide range of minimally modified sTCRs from the naturally occurring TCR repertoire for antigen-specific detection and targeting.