Examination of the dielectrophoretic spectra of MCF7 breast cancer cells and leukocytes

The detection of circulating tumor cells (CTCs) in blood is crucial to assess metastatic progression and to guide therapy. Dielectrophoresis (DEP) is a powerful cell surface marker-free method that allows intrinsic dielectric properties of suspended cells to be exploited for CTC enrichment/isolation from blood. Design of a successful DEP-based CTC enrichment/isolation system requires that the DEP response of the targeted particles should accurately be known. This paper presents a DEP spectrum method to investigate the DEP spectra of cells without directly analyzing their membrane and cytoplasmic properties in contrast to the methods in literature, which employ theoretical assumptions and complex modeling. Integrating electric field simulations based on DEP theory with the experimental data enables determination of the DEP spectra of leukocyte subpopulations, polymorphonuclear and mononuclear leukocytes, and MCF7 breast cancer cells as a model of CTC due to their metastatic origin over the frequency range 100 kHz–50 MHz at 10 V_pp. In agreement with earlier findings, differential DEP responses were detected for mononuclear and polymorphonuclear leukocytes due to the richness of the cell surface features and morphologies of the different leukocyte types. The data reveal that the strength of the DEP force exerted on MCF7 cells was particularly high between 850 kHz and 20 MHz. These results illustrate that the proposed technique has the potential to provide a generic platform to identify DEP responses of different biological particles.     

Novel microfluidic device for the continuous separation of cancer cells using dielectrophoresis

We describe the design, microfabrication, and testing of a microfluidic device for the separation of cancer cells based on dielectrophoresis. Cancer cells, specifically green fluorescent protein‐labeled MDA‐MB‐231, are successfully separated from a heterogeneous mixture of the same and normal blood cells. MDA‐MB‐231 cancer cells are separated with an accuracy that enables precise detection and counting of circulating tumor cells present among normal blood cells. The separation is performed using a set of planar interdigitated transducer electrodes that are deposited on the surface of a glass wafer and slightly protrude into the separation microchannel at one side. The device includes two parts, namely, a glass wafer and polydimethylsiloxane element. The device is fabricated using standard microfabrication techniques. All experiments are conducted with low conductivity sucrose‐dextrose isotonic medium. The variation in response between MDA‐MB‐231 cancer cells and normal cells to a certain band of alternating‐current frequencies is used for continuous separation of cells. The fabrication of the microfluidic device, preparation of cells and medium, and flow conditions are detailed. The proposed microdevice can be used to detect and separate malignant cells from heterogeneous mixture of cells for the purpose of early screening for cancer.     

Resveratrol-induced cell inhibition of growth and apoptosis in MCF7 human breast cancer cells are associated with modulation of phosphorylated akt and caspase-9

Resveratrol (trans-3,4N,-5-trihydroxystilbene), a phytoalexin present in grapes and red wine, is emerging as a natural compound with potential anticancer properties. Here we show that resveratrol affects the growth of human breast cancer cell lines MCF7, MDA-MB-231, SK-BR-3, and Bcap-37 in a dose-dependent manner and that MCF7 is the most sensitive among the four cell lines. MCF7 cells treated with resveratrol showed typical characteristics of apoptosis including the poly (ADP-ribose) polymerase cleavage, TdT-mediated dUTP nick end labeling-positive staining, and morphologic changes. Phosphorylation of the oncogene product Akt was significantly reduced followed by decreased phosphorylation and increased processing of pro-caspase-9 on resveratrol treatment. These results indicate that resveratrol seems to exert its growth-inhibitory/apoptotic effect on the breast cancer cell line MCF7 via the Akt-caspase-9 pathway.     

PEGylated anti-MUC1 aptamer-doxorubicin complex for targeted drug delivery to MCF7 breast cancer cells

Targeted drug delivery is especially important in cancer treatment as many anti-cancer drugs are non-specific and highly toxic to both cancer and normal cells. The targeted drug delivery of DOX to the MUC1-expressing breast cancer cell line (MCF7) was obtained using APT as a carrier. Modification of the APT-DOX complex by PEG increases the survivability of the macrophage control (RAW 264.7) by about six-fold as compared to free DOX treatment without significantly affecting the cytotoxicity toward the target cell line. Thus, PEG-APT-DOX is potentially a new therapeutic agent for targeted drug delivery to MUC1-expressing cell lines.     

Green-synthesized Zinc oxide nanoparticle,an efficient safe anticancer compound for human breast MCF7 cancer cells

There are many types of researches investigating anticancer therapeutics for breast cancer therapy. Zinc oxide nanoparticles (ZnONPs) as an efficient drug delivery system, has been widely being used in various biomedical applications. In the current study, we synthesized ZnONP applying Rheum rhaponticum Waste (RRW) as a novel bio-platform to investigate its anticancer impacts on MCF7 breast cancer cells compared with normal Human HFF and HDF cells. In this regard, RRW was triggered to synthesize the ZnONPs. Then, they were characterized by XRD, FTIR, TEM, and SEM analysis. Next, the MCF-7, HFF, and HDF cell lines were cultured and treated as the following plane: Incubation of all cell lines for 72, 48, and 24 hours at the presence of different ZnONPs doses. Finally, the cell morphology, BCL2- BAX genes expression profile and AO/PI-fluorescent cell staining on the 48-hour incubated cells were analyzed to check the ZnONP apoptotic activity. Moreover, the ZnONP antioxidant activity was analyzed by a DPPH antioxidant test. We produced the 30 nm ZnONPs which significantly increased the BAX and decreased the BCL-2 gene expression. According to the results including the Sub G1 enhancement peaks, apoptotic hallmarks, MTT assay, and the AO/PI-fluorescent stained cells, ZnONPs can specifically induce apoptotic death in MCF7 breast cancer cells compared with normal HFF and HDF cells. The IC₅₀ values of MCF-7 in 72, 48, and 24 hr were measured at 8, 11, and 12 μg/ml in 72, 48, and 24 hr, respectively. This is while the mentioned values in the normal cells (HFF, HDF) were estimated at higher treatment doses. In conclusion, we suggest that the ZnONPs have the potential to be applied as a safe cell-specific apoptosis inducer in breast cancer treatment. However, there are many challenges that need to be clarified for applying them as an efficient anticancer agent.     

Graphene-Based Biosensors with High Sensitivity for Detection of Ovarian Cancer Cells

Ovarian cancer has the highest mortality rate in the world. Therefore, it is urgent but still challenging to develop an efficient circulating tumor cell (CTC) detection method to sensitively detect ovarian cancer. To address such issues, herein, for the first time, we present a novel CTC detection method for ovarian cancer cells by designing sensitive and rapid graphene-based biosensors. This graphene-based sensor, consisting of a cell pool and two electrodes, can be prepared by a conventional chip fabrication process. It demonstrates high-sensitivity detection even for several ovarian cancer cells by comparing the electrical signal before and after adding cell solution. Moreover, the graphene-based biosensors can perform rapid detection with good repeatability. This suggests that this novel method is possible to use for the early detection of ovarian cancer with very low CTC cell concentration. This work provides a novel and quick strategy to detect ovarian cancer and further judge or predict the risk of the transfer of ovarian cancer.     

On the design of deterministic dielectrophoresis for continuous separation of circulating tumor cells from peripheral blood cells

Detection and analysis of circulating tumor cells (CTCs) have emerged as a promising way to diagnose cancer, study its cellular mechanism, and test or develop potential treatments. However, the rarity of CTCs among peripheral blood cells is a big challenge toward CTC detection. In addition, in cases where there is similar size range between certain types of CTCs (e.g. breast cancer cells) and white blood cells (WBCs), high‐resolution techniques are needed. In the present work, we propose a deterministic dielectrophoresis (DEP) method that combines the concept of deterministic lateral displacement (DLD) and insulator‐based dielectrophoresis (iDEP) techniques that rely on physical markers such as size and dielectric properties to differentiate different type of cells. The proposed deterministic DEP technology takes advantage of frequency‐controlled AC electric field for continuous separation of CTCs from peripheral blood cells. Utilizing numerical modeling, different aspects of coupled DLD‐DEP design such as the required applied voltages, velocities, and geometrical parameters of DLD arrays of microposts are investigated. Regarding the inevitable difference and uncertainty ranges for the reported crossover frequencies of cells, a comprehensive analysis is conducted on applied electric field frequency as design's determinant factor. Deterministic DEP design provides continuous sorting of CTCs from WBCs even with similar size and has the future potential for high throughput and efficiency.     

Passivated‐electrode insulator‐based dielectrophoretic separation of heterogeneous cell mixtures

Rapid and accurate purification of various heterogeneous mixtures is a critical step for a multitude of molecular, chemical, and biological applications. Dielectrophoresis has shown to be a promising technique for particle separation due to its exploitation of the intrinsic electrical properties, simple fabrication, and low cost. Here, we present a geometrically novel dielectrophoretic channel design which utilizes an array of localized electric fields to separate a variety of unique particle mixtures into distinct populations. This label‐free device incorporates multiple winding rows with several nonuniform structures on to sidewalls to produce high electric field gradients, enabling high locally generated dielectrophoretic forces. A balance between dielectrophoretic forces and Stokes’ drag is used to effectively isolate each particle population. Mixtures of polystyrene beads (500 nm and 2 μm), breast cancer cells spiked in whole blood, and for the first time, neuron and satellite glial cells were used to study the separation capabilities of the design. We found that our device was able to rapidly separate unique particle populations with over 90% separation yields for each investigated mixture. The unique architecture of the device uses passivated‐electrode insulator‐based dielectrophoresis in an innovative microfluidic device to separate a variety of heterogeneous mixture without particle saturation in the channel.     

微流控芯片分选富集循环肿瘤细胞的研究进展

近年来,循环肿瘤细胞(CTCs)研究得到了越来越多的关注,许多研究报告已经证实其在肿瘤转移的早期诊断、治疗方案选择、个体化治疗及探索肿瘤转移机制等方面具有潜在的价值,然而CTCs在循环系统中的含量极低,这成为限制其临床相关应用的主要难点。微流控芯片技术具有低成本、快速、高通量及操作简单等优势,利用微流控芯片可实现CTCs的高速、高回收率、高纯度的分选富集,近年来得到广泛的关注。本文综述了近年来在微流控芯片内进行CTCs分选富集的研究并探讨了各种方法的优缺点,并在本研究团队的研究基础上进行了展望。     

Anticancer effects of brucine and gemcitabine combination in MCF-7 human breast cancer cells

This study was designed to investigate the combination effects of brucine and gemcitabine, each with anticancer properties, in MCF-7 human breast cancer cells in culture. With regard to cell viability, effects of both the drugs and their combinations were inversely proportional to dose and time. For various proportional drug combinations studied, combination effects were analysed using CompuSyn software. The analyses revealed synergistic and/or additive effects regarding cell viability, anchorage-independent growth and cell migration. Combination analyses exhibited diversified impacts of the type of combination treatment, namely pretreatment with either drug followed by exposure to the other, or treatment with both drugs at the same time. Compared with untreated cells, combination treatment of asynchronised MCF-7 cells resulted in 17.2 × decrease in G2 phase, increasing G1 (2.1 × ) and S (1.5 × ) phase cells in cell cycle analysis. Brucine, either individually or in combination, but not gemcitabine, inhibited NF-kB subunit (p65) expression in MCF-7 cells.     

Anti-proliferative and pro-apoptotic effects of cinobufagin on human breast cancer MCF-7 cells and its molecular mechanism

Cinobufagin (CBF) is an active ingredient isolated from Venenum Bufonis extracted and dried from the secretory glands of Bufo gargarizans Cantor. The purpose of the study was to investigate the effects and underlying mechanisms of CBF on human breast cancer MCF-7 cells in vitro. Our results showed that CBF exhibited obvious cytotoxicity on MCF-7 cells in a dose- and time-dependent manner, as indicated by CCK-8 assays. Also, Hoechst 33258 staining and flow cytometry assays showed that CBF strongly induced MCF-7 cell apoptosis and G1 phase arrest. In addition, further molecular mechanistic investigation demonstrated that cinobufagin significantly increased Bax expression, decreased Bcl-2 expression level and up-regulated the ratio of the pro-apoptosis/anti-apoptosis protein Bax/Bcl-2, which were demonstrated by RT-qPCR and western blot assays. Taken together, our data confirm that CBF inhibits growth and triggers apoptosis of MCF-7 cells by affecting the expression of Bax and Bcl-2 in vitro.     

细胞代谢组学用于羽扇豆醇干预人乳腺癌细胞MCF-7的机理探究

应用基于气相色谱-质谱联用（GC-MS）的代谢组学方法结合细胞周期实验,研究羽扇豆醇体外抑制人乳腺癌细胞MCF-7增殖的作用机理。代谢组学的研究结果表明：通过正交偏最小方差判别分析（OPLS-DA）可以很好地区分羽扇豆醇作用的MCF-7细胞代谢谱与对照组细胞代谢谱,模型参数为：R²Ycum=0.988,Q²Ycum=0.964。VIP（variable importance in the projection）值大于1的差异代谢物进一步用t检验进行单位分析,选择t<0.05（VIP>1）的代谢物作为羽扇豆醇作用组的生物标志物,得到琥珀酸、磷酸、亮氨酸、异亮氨酸等11种代谢差异物。结合羽扇豆醇将细胞周期抑制在G1期这一现象,推测羽扇豆醇可能是主要抑制了三羧酸循环中的琥珀酰辅酶A的生成和底物磷酸化生成ATP的反应来抑制MCF-7细胞的增殖。本实验从代谢组学角度为乳腺癌抗肿瘤机制提供新的线索。     

Discovery of cancer cell proliferation inhibitors from Salviae miltiorrhizae radix et rhizoma by a trace peak enrichment approach

Salviae miltiorrhizae radix et rhizoma is a traditional herbal medicine with anti‐cancer activities. In this work, a trace peak enrichment approach combined with a cell proliferation assay was applied for screening cancer cell proliferation inhibitors from the extract of S. miltiorrhiza. A set of 123 peak fractions were prepared, and by comprehensive screening, 21 tanshinones were screened out as cancer cell proliferation inhibitors and their structures were tentatively identified by liquid chromatography coupled with quadrupole time‐of‐flight tandem mass spectrometry analysis. The inhibitory activities of nine available screened tanshinones were validated, with their IC₅₀ values ranging from 0.63 to 28.40 μM, indicating their activities strongly inhibit the proliferation of cancer cells. This study presents tanshinones that are potential cancer cell proliferation inhibitors and may explain the anti‐cancer activity of S. miltiorrhiza.     

Characterization of a hybrid dielectrophoresis and immunocapture microfluidic system for cancer cell capture

The capture of circulating tumor cells (CTCs) from cancer patient blood enables early clinical assessment as well as genetic and pharmacological evaluation of cancer and metastasis. Although there have been many microfluidic immunocapture and electrokinetic techniques developed for isolating rare cancer cells, these techniques are often limited by a capture performance tradeoff between high efficiency and high purity. We present the characterization of shear‐dependent cancer cell capture in a novel hybrid DEP–immunocapture system consisting of interdigitated electrodes fabricated in a Hele‐Shaw flow cell that was functionalized with a monoclonal antibody, J591, which is highly specific to prostate‐specific membrane antigen expressing prostate cancer cells. We measured the positive and negative DEP response of a prostate cancer cell line, LNCaP, as a function of applied electric field frequency, and showed that DEP can control capture performance by promoting or preventing cell interactions with immunocapture surfaces, depending on the sign and magnitude of the applied DEP force, as well as on the local shear stress experienced by cells flowing in the device. This work demonstrates that DEP and immunocapture techniques can work synergistically to improve cell capture performance, and it will aid in the design of future hybrid DEP–immunocapture systems for high‐efficiency CTC capture with enhanced purity.     

Continuous separation of breast cancer cells from blood samples using multi-orifice flow fractionation (MOFF) and dielectrophoresis (DEP)

Circulating tumor cells (CTCs) are highly correlated with the invasive behavior of cancer, so their isolations and quantifications are important for biomedical applications such as cancer prognosis and measuring the responses to drug treatments. In this paper, we present the development of a microfluidic device for the separation of CTCs from blood cells based on the physical properties of cells. For use as a CTC model, we successfully separated human breast cancer cells (MCF-7) from a spiked blood cell sample by combining multi-orifice flow fractionation (MOFF) and dielectrophoretic (DEP) cell separation technique. Hydrodynamic separation takes advantage of the massive and high-throughput filtration of blood cells as it can accommodate a very high flow rate. DEP separation plays a role in precise post-processing to enhance the efficiency of the separation. The serial combination of these two different sorting techniques enabled high-speed continuous flow-through separation without labeling. We observed up to a 162-fold increase in MCF-7 cells at a 126 μL min(-1) flow rate. Red and white blood cells were efficiently removed with separation efficiencies of 99.24% and 94.23% respectively. Therefore, we suggest that our system could be used for separation and detection of CTCs from blood cells for biomedical applications.     

Interdigitated comb-like electrodes for continuous separation of malignant cells from blood using dielectrophoresis

In this paper, a method for continuous flow separation of circulating malignant cells from blood in a microfluidic device using dielectrophoresis is discussed. Separation of MDA231 breast cancer cells after mixing with normal blood cells was achieved with a level of accuracy that enabled precise counting of the malignant cells, separation and eventually, sub-culturing. MDA231 cells were separated from the blood to a daughter channel using two pairs of interdigitated activated comb-like electrode structures. All experiments are performed with conductivity adjusted medium samples. The electrode pairs were positioned divergent and convergent with respect to the flow. The AC signals used in the separation are 20 V peak-to-peak with frequencies of 10-50 kHz. The separation is based on balance of magnitude of the dielectrophoretic force and hydrodynamic force. The difference in response between circulating malignant cells and normal cells at a certain band of alternating current frequencies was used for rapid separation of cancer cells from blood. The significance of these experimental results is discussed in this paper, with detailed reporting on the suspension medium, preparation of cells, flow condition and the fabrication process of the microfluidic chip. The present technique could potentially be applied to identify incident cancer at a stage and size that is not yet detectable by standard diagnostic techniques (imaging and biochemical testing). Alternatively, it may also be used to detect cancer recurrences.     

Aspirin-induced Bcl-2 translocation and its phosphorylation in the nucleus trigger apoptosis in breast cancer cells

Here, we report that B-cell lymphoma 2 (Bcl-2) is a novel target molecule of aspirin in breast cancer cells. Aspirin influenced the formation of a complex by Bcl-2 and FKBP38 and induced the nuclear translocation of Bcl-2 and its phosphorylation. These events inhibited cancer cell proliferation and subsequently enhanced MCF-7 breast cancer cell apoptosis. Bcl-2 knockdown using small interfering RNA (siRNA) delayed apoptotic cell death, which correlated with increased proliferation following aspirin exposure. In contrast, Bcl-2 overexpression enhanced the onset of aspirin-induced apoptosis, which was also associated with a significant increase in Bcl-2 phosphorylation in the nucleus. Therefore, this study may provide novel insight into the molecular mechanism of aspirin, particularly its anticancer effects in Bcl-2- and estrogen receptor-positive breast cancer cells.     

Valtrate from Valeriana jatamansi Jones induces apoptosis and inhibits migration of human breast cancer cells in vitro

Abstract

Valtrate is a principle compound isolated from Valeriana jatamansi Jones, a traditional Chinese folk medicine originally used to treat various nervous disorders. Here, we found that valtrate exhibited significant anti-cancer activity in vitro, especially in human breast cancer cells, while displayed relatively low cytotoxicity to normal human breast epithelial cells (MCF 10A). Valtrate induced cell cycle arrest at G2/M stage and apoptosis in MDA-MB-231 and MCF-7 cells, with reduced expression of p-Akt (Ser 473), cyclin B1 and caspase 8, and increased expression of p21, p-cdc2, cleaved-caspase 3, cleaved-caspase 7 and poly (ADP-ribose) polymerase (PARP). In addition, valtrate inhibited cell migration through down-regulation of MMP-9 and MMP-2 expression. These results demonstrate that valtrate possesses anti-breast cancer activities via cell cycle arrest, apoptosis, and inhibition of cell migration, thus supporting valtrate as a potential antitumor agent.