phalloidine and Colonic-Neoplasms

The herbal compound 4-hydroxybenzyl alcohol (HBA) is a pleiotropic agent, which has been shown to effectively inhibit the development of new blood vessels by targeting multiple mechanisms of the angiogenic process. Because angiogenesis is a major prerequisite for tumor growth, the aim of this study was to analyze for the first time, whether HBA may be used for anti-cancer therapy.. CT26.WT colon carcinoma cells were exposed to different HBA doses to study their viability, migration, invasiveness and protein expression compared to vehicle-treated controls. Moreover, CT26.WT cell spheroids were transplanted into the dorsal skinfold chamber of HBA-treated and vehicle-treated BALB/c mice for the analysis of tumor vascularization and growth by means of repetitive intravital fluorescence microscopy, histology and immunohistochemistry.. As shown by water-soluble tetrazolium (WST)-1 and lactate dehydrogenase (LDH) assays, HBA treatment dose-dependently reduced the viability and integrity of the tumor cells. Moreover, phalloidin staining of HBA-treated cells revealed a disorganized cytoskeleton, which was associated with a decreased cellular migratory and invasive activity. In addition, the cells presented with a significantly increased expression of the apoptosis marker cleaved caspase-3 and a decreased expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinase (MMP)-9 when compared to controls. Finally, HBA treatment inhibited the vascularization and growth of newly developing CT26.WT tumors in the mouse dorsal skinfold chamber model without affecting the normal behavior of the animals.. These novel findings indicate that HBA represents a promising candidate for the establishment of anti-angiogenic treatment strategies in cancer therapy.

Topics: Analysis of Variance; Animals; Benzyl Alcohols; Blotting, Western; Caspase 3; Cell Line, Tumor; Cell Movement; Colonic Neoplasms; Dose-Response Relationship, Drug; Histological Techniques; Immunohistochemistry; L-Lactate Dehydrogenase; Matrix Metalloproteinase 9; Mice; Mice, Inbred BALB C; Microscopy, Fluorescence; Neoplasm Invasiveness; Neovascularization, Pathologic; Phalloidine; Vascular Endothelial Growth Factor A

Cripto-1 (CR-1) is a glycosylphosphatidylinositol (GPI)-anchored membrane glycoprotein that has been shown to play an important role in embryogenesis and cellular transformation. CR-1 is reported to function as a membrane-bound co-receptor and as a soluble ligand. Although a number of studies implicate the role of CR-1 as a soluble ligand in tumor progression, it is unclear how transition from the membrane-bound to the soluble form is physiologically regulated and whether differences in biological activity exist between these forms. Here, we demonstrate that CR-1 protein is secreted from tumor cells into the conditioned medium after treatment with serum, epidermal growth factor, or lysophosphatidic acid, and this soluble form of CR-1 exhibits the ability to promote endothelial cell migration as a paracrine chemoattractant. On the other hand, membrane-bound CR-1 can stimulate endothelial cell sprouting through direct cell-cell interaction. Shedding of CR-1 occurs at the GPI-anchorage site by the activity of GPI-phospholipase D (GPI-PLD), because CR-1 shedding was suppressed by siRNA knockdown of GPI-PLD and enhanced by overexpression of GPI-PLD. These findings describe a novel molecular mechanism of CR-1 shedding, which may contribute to endothelial cell migration and possibly tumor angiogenesis.

Topics: Adenocarcinoma; Animals; Cell Line; Cell Line, Tumor; Cell Movement; Chlorocebus aethiops; Coculture Techniques; Colonic Neoplasms; COS Cells; Dogs; Endothelial Cells; Endothelium, Vascular; Epidermal Growth Factor; Fluorescent Dyes; Glycosylphosphatidylinositols; GPI-Linked Proteins; Growth Substances; Humans; Indoles; Intercellular Signaling Peptides and Proteins; Kidney; Mass Spectrometry; Membrane Glycoproteins; Neoplasm Proteins; Phalloidine; Phospholipase D; Rhodamines; RNA, Small Interfering; Umbilical Veins

Increased extracellular pressure stimulates colon cancer cell adhesion by activating focal adhesion kinase (FAK) and Src. We investigated the role of the cytoskeleton in pressure-induced inside-out FAK and Src phosphorylation and pressure-stimulated adhesion. We perturbed actin polymerization with phalloidin, cytochalasin D and latrunculin B, and microtubule organization with colchicine and paclitaxol. We compared the effects of these agents on pressure-induced SW620 and human primary colon cancer cell adhesion and inside-out FAK/Src activation with outside-in adhesion-dependent FAK/Src activation. Cells pretreated with cytoskeletal inhibitors were subjected to 15 mmHg increased pressure and allowed to adhere to collagen I coated plates or prevented from adhesion to pacificated plates for 30 min. Phalloidin, cytochalasin D, latrunculin B and colchicine pretreatment completely prevented pressure-stimulated and significantly inhibited basal SW620 cell adhesion. Taxol did not inhibit pressure-induced colon cancer cell adhesion, but significantly lowered basal adhesion. Cytochalasin D and colchicine had similar effects in pressure-stimulated primary human malignant colonocytes. Phalloidin, cytochalasin D, latrunculin B and colchicine prevented pressure-induced SW620 FAK phosphorylation but not Src phosphorylation. FAK phosphorylation in response to collagen I adhesion was significantly attenuated but not completely prevented by these inhibitors. Although Src phosphorylation was not increased on adhesion, the cytoskeleton disrupting agents significantly lowered basal Src phosphorylation in adherent cells. These results suggest that both cytoskeleton-dependent FAK activation and cytoskeleton-independent Src activation may be required for extracellular pressure to stimulate colon cancer cell adhesion. Furthermore, the cytoskeleton plays a different role in pressure-activated FAK and Src signaling than in FAK and Src activation in adherent cells. We, therefore, hypothesize that cytoskeletal interactions with focal adhesion signals mediate the effects of extracellular pressure on colon cancer cell adhesion.

Topics: Cell Adhesion; Cell Line, Tumor; Cells, Cultured; Colchicine; Colon; Colonic Neoplasms; Cytochalasin D; Cytoskeleton; Dimethyl Sulfoxide; Humans; Integrins; Models, Biological; Paclitaxel; Phalloidine; Pressure; Signal Transduction

Few circulating tumor cells implant or cause metastasis. We hypothesized that venous or lymphatic pressure or iatrogenic pressure during resection activates signals governing malignant colonocyte adhesion.. We studied the effect of 15 mm Hg increased pressure for 30 minutes on adhesion of primary human colon cancer cells and SW620 colonocytes to collagen and endothelial cells. We modulated integrin affinity with extracellular cations. We assessed binding affinity by detachment assay; integrin surface expression by flow cytometry; and focal adhesion kinase (FAK), Src, and extracellular signal-regulated kinase (ERK) activation by Western analysis and Src in vitro kinase assay. We inhibited Src (PP2), FAK (small RNA interference, SiRNA, or FRNK transfection), MEK (PD98059), PKC (calphostin C), and actin destabilization (phalloidin).. Pressure and manganese stimulated primary and SW620 colonocyte adhesion to collagen. Pressure also stimulated SW620 adhesion to endothelial monolayers. Pressure strengthened SW620 binding force to matrix without changing integrin surface expression. Pressure activated SW620 FAK and Src, but not ERK. Manganese did not. Calcium-inhibited adhesion but stimulated FAK (but not Src). PP2 prevented pressure activation of Src, Src phosphorylation of FAK576, and pressure-stimulated adhesion but not FAK397 autophosphorylation. FRNK transfection or FAK SiRNA also prevented pressure-stimulated adhesion. FAK SiRNA ablated pressure-activated FAK397, Src, and FAK576 phosphorylation. Neither Src nor FAK inhibition blocked cation effects. Phalloidin prevented pressure-stimulated adhesion. PD98059 or calphostin C did not.. In contrast to divalent cations, extracellular pressure may increase integrin affinity and promote colon cancer adhesion via actin dependent inside-out FAK and Src signals. This mechanotransduced pathway may regulate metastasizing tumor cell adhesion.

Topics: Actins; Animals; Aorta; Cell Adhesion; Cell Membrane; Cells, Cultured; Collagen Type I; Colonic Neoplasms; Cytoskeleton; Endothelium, Vascular; Enzyme Activation; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Humans; Integrins; Mitogen-Activated Protein Kinases; Phalloidine; Pressure; Protein-Tyrosine Kinases; Pyrimidines; Rats; RNA, Small Interfering; Signal Transduction; src-Family Kinases

Malignant cells shed from tumors during surgical resection or spontaneous metastasis experience physical forces such as shear stress and turbulence within the peritoneal cavity during irrigation, laparoscopic air insufflation, or surgical manipulation, and within the venous or lymphatic system. Since physical forces can activate intracellular signals that modulate the biology of various cell types in vitro, we hypothesized that shear stress and turbulence might increase colon cancer cell adhesion to extracellular matrix, potentiating metastatic implantation. Primary human malignant colon cancer cells isolated from resected tumors and SW620 were subjected to shear stress and turbulence by stirring cells in suspension at 600 rpm for 10 min. Shear stress for 10 min increased subsequent SW620 colon cancer cell adhesion by 40.0 +/- 3.0% (n = 3; P < 0.001) and primary cancer cells by 41.0 +/- 3.0% to collagen I when compared to control cells. In vitro kinase assay (1.5 +/- 0.13 fold) and Western analysis (1.34 +/- 0.04 fold) demonstrated a significant increase in Src kinase activity in cells exposed shear stress. Src kinase inhibitors PP1 (0.1 microM), PP2 (20 microM), and actin-cytoskeleton stabilizer phalloidin (10 microM) prevented the shear stress stimulated cell adhesion to collagen I. Furthermore, PP2 inhibited basal (50.0 +/- 2.8%) and prevented shear stress induced src activation but phalloidin pretreatment did not. These results raise the possibility that shear stress and turbulence may stimulate the adhesion of malignant cells shed from colon cancers by a mechanism that requires both actin-cytoskeletal reorganization an independent physical force activation of Src kinase. Blocking this pathway might reduce tumor metastasis during surgical resection.

Topics: Actins; Cell Adhesion; Collagen; Colonic Neoplasms; Cytoskeleton; Enzyme Activation; Enzyme Inhibitors; Humans; Phalloidine; Pyrazoles; Pyrimidines; Reproducibility of Results; src-Family Kinases; Stress, Mechanical; Tumor Cells, Cultured

The role of the polymorphonuclear leukocyte (PMN) cytoskeleton during the transmigration across colonic epithelial cells is not very well understood. In order to study the role of different components of the PMN cytoskeleton during transepithelial migration across a colonic epithelial cell monolayer (T84), PMN were preincubated with drugs affecting either the actin cytoskeleton (cytochalasin B, iota toxin of Clostridium perfringens, and phalloidin) or the microtubules (colchicine and taxol). The role of PMN myosin during transepithelial migration was investigated using the inhibitor 2,3-butanedione monoxime (BDM) and DC3B toxin. PMN intracellular Ca2+, during neutrophil adhesion and translocation across the epithelium, was assessed by the Ca2+ chelator 1, 2bis-(2-aminophenoxy)-ethane-N,N,N', N'-tetra-acetic acid tetrakis (acetoxymethyl) ester (BAPTA-AM). Transmigration of PMN was initiated by applying either interleukin-8 or formyl-met-leu-phe (fMLP). While colchicine and taxol preexposure did not influence PMN transepithelial migration, treatment with cytochalasin B, iota toxin, phalloidin, BDM, DC3B toxin and BAPTA-AM greatly diminished migration of PMN across T84 monolayers. Similarly, cell-cell contacts established between PMN and epithelial cells during the transmigration were diminished after treatment of PMN with iota toxin or cytochalasin B. These data show that the neutrophil actin cytokeleton and myosin, but not the microtubules, evoke a Ca2+ -dependent motility that facilitates migration across the colonic epithelial barrier.

Topics: Actins; ADP Ribose Transferases; Bacterial Toxins; Calcium; Cell Adhesion; Cells, Cultured; Chelating Agents; Chemotaxis, Leukocyte; Colchicine; Colonic Neoplasms; Cytochalasin B; Cytoskeleton; Diacetyl; Egtazic Acid; Epithelial Cells; Humans; Interleukin-8; Intestinal Mucosa; Microtubules; Myosins; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Paclitaxel; Phalloidine; Tumor Cells, Cultured