ABSTRACT
The transfer of redox-labelled bioelectrochemical sensors from proteins to cells is not straightforward because of the cell downward force issue on the surface of the sensors. In this paper, 20-nm-thick nanopillars are introduced to overcome this issue, in a well-controlled manner. We show on both molecular dynamics simulations and experiments that suspending cells a few nanometers above an electrode surface enables redox-labelled tethered DNA aptamer probes to move freely, while remaining at an interaction distance from a target membrane protein, i. e. epithelial cell adhesion molecule (EpCAM), which is typically overexpressed in cancer cells. By this nanopillar configuration, the interaction of aptamer with cancer cells is clearly observable, with 13 cells as the lower limit of detection. Nanoconfinement induced by the gap between the electrode surface and the cell membrane appears to improve the limit of detection and to lower the melting temperature of DNA aptamer hairpins, offering an additional degree of freedom to optimize molecular recognition mechanisms. This novel nanosupported electrochemical DNA cell sensor scheme including Brownian-fluctuating redox species opens new opportunities for the design of all-electrical sensors using redox-labelled probes.
Subject(s)
Aptamers, Nucleotide , Biosensing Techniques , Neoplasms , Aptamers, Nucleotide/chemistry , DNA/chemistry , Electrochemical Techniques , Epithelial Cell Adhesion Molecule , Oxidation-ReductionABSTRACT
The Trk family of neurotrophin tyrosine kinase receptors is emerging as an important player in carcinogenic progression in non-neuronal tissues. Here, we show that breast tumors present high levels of TrkA and phospho-TrkA compared to normal breast tissues. To further evaluate the precise functions of TrkA overexpression in breast cancer development, we have performed a series of biological tests using breast cancer cells that stably overexpress TrkA. We show that (1) TrkA overexpression promoted cell growth, migration and invasion in vitro; (2) overexpression of TrkA per se conferred constitutive activation of its tyrosine kinase activity; (3) signal pathways including PI3K-Akt and ERK/p38 MAP kinases were activated by TrkA overexpression and were required for the maintenance of a more aggressive cellular phenotype; and (4) TrkA overexpression enhanced tumor growth, angiogenesis and metastasis of xenografted breast cancer cells in immunodeficient mice. Moreover, recovered metastatic cells from the lungs exhibited enhanced anoikis resistance that was abolished by the pharmacological inhibitor K252a, suggesting that TrkA-promoted breast tumor metastasis could be mediated at least in part by enhancing anoikis resistance. Together, these results provide the first direct evidence that TrkA overexpression enhances the tumorigenic properties of breast cancer cells and point to TrkA as a potential target in breast cancer therapy.
Subject(s)
Breast Neoplasms/genetics , Cell Proliferation , Receptor, trkA/genetics , Animals , Anoikis/physiology , Biopsy , Breast Neoplasms/enzymology , Breast Neoplasms/pathology , Breast Neoplasms/surgery , Cell Line, Tumor , Cell Movement , Extracellular Signal-Regulated MAP Kinases/physiology , Female , Green Fluorescent Proteins/metabolism , Humans , Immunohistochemistry , In Situ Nick-End Labeling , Mice , Mice, SCID , Neoplasm Invasiveness , Neoplasm Metastasis , Neovascularization, Pathologic , Phosphatidylinositol 3-Kinases/physiology , Proto-Oncogene Proteins c-akt/physiology , RNA, Messenger/metabolism , Signal Transduction/physiology , Xenograft Model Antitumor Assays/methods , p38 Mitogen-Activated Protein Kinases/physiologyABSTRACT
Tamoxifen (TAM), is widely used as a single agent in adjuvant treatment of breast cancer. Here, we investigated the effects of TAM in combination with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in estrogen receptor-alpha (ER-alpha)-positive and -negative breast cancer cells. We showed that cotreatment with TAM and TRAIL synergistically induced apoptosis regardless of ER-alpha status. By contrast, cotreatment did not affect the viability of normal breast epithelial cells. Cotreatment with TAM and TRAIL in breast cancer cells decreased the levels of antiapoptotic proteins including FLIPs and Bcl-2, and enhanced the levels of proapoptotic proteins such as FADD, caspase 8, tBid, Bax and caspase 9. Furthermore, cotreatment-induced apoptosis was efficiently reduced by FADD- or Bid-siRNA, indicating the implication of both extrinsic and intrinsic pathways in synergistic apoptosis induction. Importantly, cotreatment totally arrested tumor growth in an ER-alpha-negative MDA-MB-231 tumor xenograft model. The abrogation of tumor growth correlated with enhanced apoptosis in tumor tissues. Our findings raise the possibility to use TAM in combination with TRAIL for breast cancers, regardless of ER-alpha status.
