cytochalasin-d and Prostatic-Neoplasms

Aiming to establish the noninvasive discrimination of cancer cells from normal cells in adherent culture, we examined in this study the reason why the laser phase shift, which is the product of refractive index and height of malignant cancer cells, was markedly smaller than that of normal cells. Both of the phase shift measured by phase-shifting laser microscopy (PLM) and the relative actin content of cells of the adhesive human prostatic carcinoma epithelial cell line (PC-3) were markedly lower than those of adhesive human prostate epithelial cells (PRECs), while there was almost no difference in morphology observed under a conventional inverted microscope, between them. The decrease in relative actin content by the addition of cytochalasin D resulted in the decrease in phase shift in both cell lines, and these cell lines showed a marked positive correlation between phase shift and relative actin content (r = 0.84). The height and refractive index of adhesive cells were determined using PLM and the height of PC-3 cells were apparently lower than those of PRECs, while there was no difference in refractive indices between PC-3 cells and PRECs. Consequently, the smaller height of PC-3 cells caused by lower actin content than of PRECs might be the reason for the lower phase shift of PC-3 cells.

Topics: Actins; Cell Adhesion; Cell Line; Cell Line, Tumor; Cytochalasin D; Epithelial Cells; Humans; Lasers; Male; Microscopy, Confocal; Prostate; Prostatic Neoplasms; Refractometry

Neuroendocrine differentiation (NED) is a hallmark of advanced androgen-independent prostate cancer, for which no successful therapy exists. NED tumour cells escape apoptotic cell death by alterations of Ca(2+) homeostasis where the store-operated Ca(2+) entry (SOCE) is known to be a key event. We have previously shown that the downregulation of Orai1 protein representing the major molecular component of endogenous SOCE in human prostate cancer cells, and constituting the principal source of Ca(2+) influx used by the cell to trigger apoptosis, contributes to the establishment of an apoptosis-resistant phenotype (Cell Death Dis. 2010 Sep 16;1:e75.). Here, we report for the first time that the decrease of SOCE during NED may be caused by alternative NED-induced mechanism involving cytoskeleton reorganisation. NED induced by androgen deprivation resulted in a decrease of SOCE due to cortical F-actin over-polymerization which inhibits thapsigargin-induced SOCE. The disruption of F-actin polymerization by Cytochalasin D in NED cells restored SOCE, while the induction of F-actin polymerization by jasplakinolide or calyculin A diminished SOCE without changing the expression of key SOCE players: Orai1, STIM1, and TRPC1. Our data suggest that targeting cytoskeleton-induced pathways of malignant cells together with SOCE-involved channels may prove a useful strategy in the treatment of advanced prostate cancer.

Topics: Actins; Androgens; Apoptosis; Calcium; Calcium Channels; Cell Differentiation; Cell Line, Tumor; Cytochalasin D; Cytoskeleton; DNA Primers; Electrophysiology; Humans; Male; Marine Toxins; Membrane Proteins; Neoplasm Proteins; Neuroendocrine Cells; ORAI1 Protein; Oxazoles; Phenotype; Prostatic Neoplasms; Stromal Interaction Molecule 1; TRPC Cation Channels

In this study, we identify the extent of deformation that causes cell lysis using a simple technique where a drop of cell suspension is compressed by two flat plates. The viability of human prostatic adenocarcinoma PC-3 cells in solutions of various concentrations of NaCl is determined as a function of the gap size between the plates. The viability declines with decreasing gap size in the following order: 700 mM >150 mM >75 mM NaCl. This is considered to be due to the difference in cell size, which is caused by the osmotic volume change before deformation; cell diameter becomes smaller in a solution of higher NaCl concentration, which appears to increase the survival ratio in a given gap size. The deformation-induced decrease in cell viability is correlated with the cell surface strain, which is dependent on the increase in surface area, irrespective of NaCl concentration. In addition, the treatment of cells with cytochalasin D results in the disappearance of cortical actin filaments and a marked drop in the viability, indicating that cell lysis is closely related to the deformation of the cytoskeleton.

Topics: Actin Cytoskeleton; Biomechanical Phenomena; Carcinoma; Cell Line, Tumor; Cell Membrane; Cell Shape; Cell Size; Cell Survival; Cytochalasin D; Cytoskeleton; Dose-Response Relationship, Drug; Humans; Male; Osmolar Concentration; Osmosis; Pressure; Prostatic Neoplasms; Sodium Chloride; Stress, Mechanical

Bombesin-like peptides, including the mammalian homologue gastrin-releasing peptide, are highly expressed and secreted by neuroendocrine cells in prostate carcinoma tissues and are likely to be related to the progression of this neoplastic disease. Previously, we demonstrated that bombesin increased migration and protease expression in androgen-independent cells. In this work we show that bombesin is able to activate pro-MMP-9 through a mechanism involving the beta1 integrin subunit. In fact, MMP-9 processing was evident only when beta1 integrin was engaged with specific adhesive substrates, such as type I collagen, or when cells were seeded on dishes coated with antibodies against beta1 integrin, resulting in activation of the surface ligand. When exogenous pro-MMP-9 was added to PC3 cells, MMP-9 active forms were produced within 30 min by bombesin-treated cultures while control cultures expressed activated forms only after a longer time and at lower levels. MMP-9 activation required cytoskeleton integrity since this effect was abolished by cytochalasin D. Engagement of beta1 integrin caused an increased membrane-linked uPA activity which was required for MMP-9 activation. The cross talk between bombesin- and beta1-integrin-engaged signals seems to be crucial for the modulation of both membrane-linked uPA activity and MMP-9 activation and triggers complex intracellular signaling pathways requiring activation of tyrosine kinase activity, including that of src and PI3K. The beta1 integrin may be considered an important mechanism by which bombesin induces MMP-9 activation. This finding supports the idea that cellular responses to growth factors may be driven by cell-matrix interactions and stresses the role of neuroendocrine factors in prostate carcinoma progression.

Topics: Bombesin; Cell Adhesion; Cell Line; Chemotaxis; Collagen Type I; Collagenases; Cytochalasin D; Cytoskeleton; Enzyme Activation; Enzyme Inhibitors; Enzyme Precursors; Extracellular Matrix; Fibrinolytic Agents; Gelatinases; Humans; Integrin beta1; Male; Matrix Metalloproteinase 9; Nucleic Acid Synthesis Inhibitors; Plasminogen; Prostatic Neoplasms; Signal Transduction; src-Family Kinases; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator

As transmembrane heterodimers, integrins bind to both extracellular ligands and intracellular proteins. We are currently investigating the interaction between integrins and the intracellular protein calreticulin. A prostatic carcinoma cell line (PC-3) was used to demonstrate that calreticulin can be found in the alpha3 immunoprecipitates of cells plated on collagen type IV, but not when plated on vitronectin. Conversely, alphav immunoprecipitates contained calreticulin only when cells were plated on vitronectin, i. e. not when plated on collagen IV. The interactions between these integrins and calreticulin were independent of actin cytoskeleton assembly and were transient, being maximal approx. 10-30 min after the cells came into contact with the substrates prior to complete cell spreading and formation of firm adhesive contacts. We demonstrate that okadaic acid, an inhibitor of intracellular serine/threonine protein phosphatases, inhibited the alpha3beta1-mediated adhesion of PC-3 cells to collagen IV and the alpha2beta1-mediated attachment of Jurkat cells to collagen I. This inhibition by okadaic acid was accompanied by inhibition of the ligand-specific interaction of calreticulin with the respective integrins in the two cell types. Additionally, we found that pharmacological inhibition of mitogen-activated protein kinase kinase (MEK) resulted in prolongation of the calreticulin-integrin interaction, and enhancement of PC-3 cell attachment to collagen IV. We conclude that calreticulin interacts transiently with integrins during cell attachment and spreading. This interaction depends on receptor occupation, is ligand-specific, and can be modulated by protein phosphatase and MEK activity.

Topics: Actins; Calcium-Binding Proteins; Calreticulin; Cell Adhesion; Cell Size; Collagen; Cytochalasin D; Cytoskeleton; Extracellular Matrix Proteins; Humans; Integrin alpha3beta1; Integrins; Jurkat Cells; Ligands; Male; MAP Kinase Kinase 1; Mitogen-Activated Protein Kinase Kinases; Okadaic Acid; Phosphoprotein Phosphatases; Phosphorylation; Prostatic Neoplasms; Protein Binding; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Receptors, Collagen; Ribonucleoproteins; Tumor Cells, Cultured; Vitronectin