RESUMEN
The nanostructures and hydrophobic properties of cancer cell membranes are important for membrane fusion and cell adhesion. They are directly related to cancer cell biophysical properties, including aggressive growth and migration. Additionally, chemical component analysis of the cancer cell membrane could potentially be applied in clinical diagnosis of cancer by identification of specific biomarker receptors expressed on cancer cell surfaces. In the present work, a combined Raman microspectroscopy (RM) and atomic force microscopy (AFM) technique was applied to detect the difference in membrane chemical components and nanomechanics of three cancer cell lines: human lung adenocarcinoma epithelial cells (A549), and human breast cancer cells (MDA-MB-435 with and without BRMS1 metastasis suppressor). Raman spectral analysis indicated similar bands between the A549, 435 and 435/BRMS1 including ~720 cm(-1) (guanine band of DNA), 940 cm(-1) (skeletal mode polysaccharide), 1006 cm(-1) (symmetric ring breathing phenylalanine), and 1451 cm(-1) (CH deformation). The membrane surface adhesion forces for these cancer cells were measured by AFM in culture medium: 0.478 ± 0.091 nN for A549 cells, 0.253 ± 0.070 nN for 435 cells, and 1.114 ± 0.281 nN for 435/BRMS1 cells, and the cell spring constant was measured at 2.62 ± 0.682 mN m(-1) for A549 cells, 2.105 ± 0.691 mN m(-1) for 435 cells, and 5.448 ± 1.081 mN m(-1) for 435/BRMS1 cells.
Asunto(s)
Microscopía de Fuerza Atómica , Nanoestructuras/química , Espectrometría Raman , Biomarcadores/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Adhesión Celular/fisiología , Línea Celular Tumoral , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Módulo de Elasticidad , Femenino , Humanos , Cinética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Microscopía Confocal , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Análisis de Componente Principal , Proteínas RepresorasRESUMEN
Following the discovery of the human histamine H4 receptor, a high throughput screen of our corporate compound collection identified compound 6 as a potential lead. Investigation of the SAR resulted in the discovery of novel compounds 10e and 10l, which are the first potent and selective histamine H4 receptor antagonists to be described.
Asunto(s)
Antagonistas de los Receptores Histamínicos/química , Antagonistas de los Receptores Histamínicos/farmacología , Piperazinas/química , Piperazinas/farmacología , Receptores de Superficie Celular/efectos de los fármacos , Receptores Acoplados a Proteínas G , Receptores Histamínicos , Animales , Línea Celular , Relación Dosis-Respuesta a Droga , Antagonistas de los Receptores Histamínicos/metabolismo , Humanos , Indoles/química , Indoles/metabolismo , Indoles/farmacología , Cinética , Ligandos , Neuronas/citología , Piperazinas/metabolismo , Ensayo de Unión Radioligante , Ratas , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Receptores Histamínicos H4 , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relación Estructura-Actividad , TransfecciónRESUMEN
Restoring BReast cancer Metastasis Suppressor 1 (BRMS1) expression suppresses metastasis in MDA-MB-435 human breast carcinoma cells at ectopic sites without affecting tumor formation at orthotopic site in the body. BRMS1 expression induces many phenotypic alterations in 435 cells such as cell adhesion, cytoskeleton rearrangement, and the down regulation of epidermal growth factor receptor (EGFR) expression. In order to better understand the role of cellular biomechanics in breast cancer metastasis, the qualitative and quantitative detection of cellular biomechanics and biochemical composition is urgently needed. In the present work, using atomic force microscopy (AFM) and fluorescent microscopy we revealed that BRMS1 expression in 435 cells induced reorganization of F-actin and caused alteration in cytoarchitectures (cell topography and ultrastructure). Results from AFM observed increase in biomechanical properties which include cell adhesion, cellular spring constant, and Young's modulus in 435/BRMS1 cells. Raman microspectroscopy showed weaker vibrational spectroscopic bands in 435/BRMS1 cells, implying decrease in concentration of cellular biochemical components in these cells. This was despite the similar spectral patterns observed between 435 and 435/BRMS1 cells. This work demonstrated the feasibility of applying AFM and Raman techniques for in situ measurements of the cellular biomechanics and biochemical components of breast carcinoma cells. It provides vital clues in understanding of the role of cellular biomechanics in cancer metastasis, and further the development of new techniques for early diagnosis of breast cancer.
Asunto(s)
Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/ultraestructura , Proteínas de Neoplasias/biosíntesis , Actinas/metabolismo , Fenómenos Biomecánicos , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Adhesión Celular/fisiología , Línea Celular Tumoral , Membrana Celular/metabolismo , Membrana Celular/fisiología , Elasticidad , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Microscopía de Fuerza Atómica/métodos , Metástasis de la Neoplasia , Proteínas de Neoplasias/genética , Proteínas Represoras , Espectrometría Raman/métodos , TransfecciónRESUMEN
Cathepsin S is the key protease responsible for the removal of the invariant chain from MHC class II molecules and, as such, has attracted much attention as a target for developing immunosuppressive drugs. To help in testing candidate compounds, the monkey (Saimiri boliviensis) and dog (Canis familiaris) cathepsin S cDNAs have been cloned. The monkey cDNA sequence encodes a 330 amino acid protein with 95% homology to human cathepsin S. The dog cDNA sequence encodes a 331 amino acid protein with 91% homology to human cathepsin S. The amino acid differences do not have a major effect on the hydrolysis of the substrate Z-VVR-AMC, but may affect the substrate specificity. As for human and bovine cathepsin S, activity against Z-VVR-AMC extends into the neutral pH range. These parameters are important for understanding the role of cathepsin S in different species and for testing inhibitors in animal models of autoimmunity.
Asunto(s)
Catepsinas/aislamiento & purificación , Catepsinas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Catepsinas/biosíntesis , Catepsinas/genética , Clonación Molecular , ADN Complementario , Perros , Expresión Génica , Humanos , Concentración de Iones de Hidrógeno , Cinética , Ratones , Datos de Secuencia Molecular , Saimiri/genética , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
Cathepsin S is considered crucial for normal presentation of major histocompatibility complex (MHC) class II-restricted antigens by antigen presenting cells to CD4+ T cells. It is a key enzyme for the degradation of the class II-associated invariant chain, a process that is required for effective antigen loading of class II molecules. Here, we report a selective, orally available, high-affinity cathepsin S inhibitor, 1-[3-[4-(6-Chloro-2,3-dihydro-3-methyl-2-oxo-1H-benzimidazol-1-yl)-1-piperidinyl]propyl]-4,5,6,7-tetrahydro-5-(methylsulfonyl)-3-[4-(trifluoromethyl)phenyl]-1H-pyrazolo[4,3-c]pyridine. (JNJ 10329670), that represents a novel class of immunosuppressive compounds. JNJ 10329670 is a highly potent (Ki of approximately 30 nM), nonpeptidic, noncovalent inhibitor of human cathepsin S, but it is much less active against the mouse, dog, monkey, and bovine enzymes. The compound is inactive against other proteases, including the closely related cathepsins L, F, and K. This selectivity makes JNJ 10329670 an excellent tool for exploring the role of cathepsin S in human systems. Treatment of human B cell lines and primary human dendritic cells with JNJ 10329670 resulted in the accumulation of the p10 fragment of the invariant chain (IC50 of approximately 1 microM). In contrast, inhibition of invariant chain proteolysis was much less effective in a human monocytic cell line, suggesting that other enzymes may degrade the invariant chain in this cell type. JNJ 10329670 was shown to block the proteolysis of the invariant chain in vivo by using immunocompromised mice injected with human peripheral blood mononuclear cells (PBMCs). Furthermore, this inhibitor blocks the presentation of tetanus toxoid and giant ragweed by human PBMCs. The properties of JNJ 10329670 make it a candidate for immunosuppressive therapy of allergies and autoimmune diseases.