phalloidine and Adenocarcinoma

ErbB family receptor tyrosine kinases (ErbBs) play a role in cell adhesion and migration and are frequently overexpressed in esophageal squamous cell carcinomas (ESCCs) or esophageal adenocarcinomas (EACs). Targeting ErbBs by tyrosine kinase inhibitors (TKIs) may therefore limit esophageal cancer cell migration. Here, we studied the impact of TKIs on ErbB dimerization, cell signaling pathways, and cell migration in three esophageal cell lines: OE21 (ESCC), OE33 (EAC), and Het-1A (non-neoplastic esophageal epithelium). In OE21 cells, the TKIs erlotinib, gefitinib, and lapatinib slightly affected epidermal growth factor receptor EGFR/EGFR, but not EGFR/HER2 dimerization as detected by in situ proximity ligation assay (in situ PLA). Still, TKIs inhibited ERK1/2, Akt, STAT3, and RhoA activity in OE21 cells, as assessed by Western blot, antibody arrays, and Rho GTPase effector pull-down assays. This was accompanied by reduced OE21 cell migration, induction of focal adhesions, and actin cytoskeleton reorganization, as shown by Oris™ migration assay and focal adhesion kinase (FAK)/phalloidin staining. In contrast, in OE33 cells, only lapatinib decreased STAT5, Src family kinase (SFK), and FAK activity as well as β-catenin expression. This impeded cell migration and induced morphological changes in OE33 cells. No alterations were seen for the non-neoplastic Het-1A cells. Thus, we identified the ErbB signaling network as regulator of esophageal cancer cell's actin cytoskeleton, focal adhesions, and cell migration. ErbB targeted TKIs therefore also limit ESCC and EAC cell motility and migration.. Clinical tyrosine kinase inhibitors (TKIs) reduce esophageal cancer cell migration. Loss of cell migration is linked to reduced Akt, ERK1/2, STAT (3 or 5), FAK, SFKs, and RhoA activity. Clinical TKIs act via distinct signaling in the two main histotypes of esophageal cancer.

Topics: Actin Cytoskeleton; Adenocarcinoma; Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; ErbB Receptors; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Fluorescent Antibody Technique, Indirect; Gene Expression Regulation; Humans; Neoplasms; Phalloidine; Phosphorylation; Protein Multimerization; Signal Transduction

Several studies have shown that curcumin can induce apoptosis and inhibit growth in human A549 lung adenocarcinoma cells. However, the mechanism is not completely understood yet. In the present study, we investigated the in vitro effect of curcumin on cell viability, apoptosis and disorganization of the actin cytoskeleton in A549 cells. Our results showed that curcumin significantly inhibited the viability of A549 cells in a dose- and time-dependent manner by induced apoptosis. The apoptotic process was associated with a disorganization of the architecture of actin microfilaments and a decrease in the levels of F-actin. DMSO-treated control cells exhibited a well-defined F-actin network that was mainly organized into stress fibers. The actin fibers in cells treated with curcumin or the positive control drug cytochalasin B were disorganized, disassembled, or disrupted, however, the disorganization of actin fibers and apoptosis could be prevented by phalloidin, an F-actin stabilizing compound. Thus, these results demonstrated that actin filament disorganization might play a central role in the curcumin-induced apoptosis of A549 cells.

Topics: Actin Cytoskeleton; Actins; Adenocarcinoma; Antineoplastic Agents, Phytogenic; Apoptosis; Cell Line, Tumor; Cell Survival; Curcuma; Curcumin; Cytochalasin B; Cytoskeleton; Dose-Response Relationship, Drug; Humans; Lung Neoplasms; Phalloidine; Phytotherapy; Plant Extracts; Poisons

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

[14C]Glucosamine metabolic labeling and concanavalin A blots were used to identify four major glycoprotein species associated with ascites tumor cell microvillar microfilament cores and with a transmembrane complex containing actin. Phalloidin shift analysis of glucosamine-labeled microvilli showed that glycoproteins of 110-120, 80, 65, and 55 kDa are stably associated with the microfilament cores. Analysis of large (greater than 10(6) kDa) transmembrane complexes from microvillar membranes made under microfilament-depolymerizing conditions (Carraway, C. A. C., Jung, G., and Carraway, K. L. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 430-434) revealed glycoproteins of the same Mr values, showing the same relative staining or labeling patterns as those observed with the microfilament cores. Gel filtration of high salt, high pH extracts of intact microvilli, microfilament cores, or transmembrane complexes showed that in all of these fractions the glycoproteins are associated in a very large, stable complex. The glycoprotein multimer was isolated essentially free of actin and other components by Sephacryl S-1000 chromatography of microvilli, microvillar membranes prepared at pH 11, microfilament cores, or transmembrane complex fractions in Triton X-100, 1 M KCl, glycine, pH 9.5. Purified glycoprotein complex bound actin when incubated under polymerizing conditions. The presence of the glycoprotein heteromultimer in both microfilament cores and transmembrane complex from isolated membranes and the association of the purified glycoprotein complex with actin are consistent with our hypothesis that the glycoprotein-containing transmembrane complex is an association site for microfilaments at the plasma membrane.

Topics: Actins; Adenocarcinoma; Animals; Ascites; Blotting, Western; Chromatography, Gel; Concanavalin A; Electrophoresis, Polyacrylamide Gel; Glucosamine; Membrane Glycoproteins; Microfilament Proteins; Microvilli; Phalloidine; Rats; Tumor Cells, Cultured

Microfilament-associated proteins and membrane-microfilament interactions are being investigated in microvilli isolated from 13,762 rat mammary ascites tumor cells. "Phalloidin shift" analyses on velocity sedimentation gradients of Triton X-100 extracts of [3H]-glucosamine-labeled microvilli identified a 120-kDa cell-surface glycoprotein associated with the microvillar microfilament core. The identification was verified by concanavalin A (Con A) blots of one- and two-dimensional (2D) electrophoresis gels of sedimented microfilament cores. By 2D-electrophoresis and lectin analyses the 120-kDa protein appeared to be a fraction of ASGP-2, the major Con A-binding glycoprotein of the sialomucin complex of the 13,762 cells. This identity was confirmed by immunoblot analyses using immunoblot-purified anti-ASGP-2 from anti-membrane serum prepared against microvillar membranes. Proteolysis of the microvilli with subtilisin or trypsin resulted in an increase in the amount of ASGP-2 associated with the microfilament cores. An increase was also observed with sialidase treatment of the microvilli, suggesting that negative charges, probably present on the highly sialated sialomucin ASGP-1 of the ASGP-1/ASGP-2 sialomucin complex, reduce ASGP-2 association with the microfilament core. Proteolysis of isolated microvillar membranes, which contain actin but not microfilaments, also increased the association of ASGP-2 with a Triton-insoluble, actin-containing membrane fraction. Purified ASGP-2 does not bind to microfilaments in sedimentation assays. Since the Triton-insoluble membrane residue is enriched in an actin-containing transmembrane complex, which contains a different glycoprotein, we suggest that the ASGP-2 is binding indirectly via this complex to the microfilament core in the intact microvilli.

Topics: Actin Cytoskeleton; Adenocarcinoma; Animals; Centrifugation, Density Gradient; Concanavalin A; Cytoskeleton; Electrophoresis, Polyacrylamide Gel; Immunoblotting; Isoelectric Focusing; Mammary Neoplasms, Experimental; Microvilli; Mucin-4; Phalloidine; Rats; Sialoglycoproteins; Subtilisins; Tumor Cells, Cultured

The cell-surface enzyme 5'-nucleotidase in microvilli from 13762 rat mammary adenocarcinoma cells remains largely associated with microfilament-containing high-speed pellets from Triton X-100 extracts of the microvilli. The fraction remaining with the insoluble portion is higher under ionic conditions which enhance microfilament stability. To minimize trapping and cosedimentation we have analyzed the distribution of microfilaments and 5'-nucleotidase activity on velocity sedimentation sucrose gradients of the microvillar extracts. A large fraction of the total enzyme activity is found in the filament fractions in the middle of the gradient. When phalloidin is included in the extraction buffer to stabilize the microfilaments, both the microfilaments and the bulk of the nucleotidase activity are shifted further into the gradients. Both the position of the filament fraction and the percentage of the total nucleotidase activity remaining with the filament fraction varies with extraction buffer composition and conditions. Nonetheless, under all conditions tested, a large percentage of the activity was shifted, along with the microfilaments, in the presence of phalloidin. These results are consistent with a specific association of 5'-nucleotidase with microfilaments in the ascites tumor cell microvilli.

Topics: 5'-Nucleotidase; Actin Cytoskeleton; Adenocarcinoma; Animals; Cell Compartmentation; Cell Line; Centrifugation, Density Gradient; Cytoskeleton; Mammary Neoplasms, Experimental; Microvilli; Nucleotidases; Phalloidine; Rats

The association of microvillar microfilaments with the microvillar membrane actin-containing transmembrane complex of MAT-C1 13762 ascites tumor cell microvilli has been investigated by differential centrifugation, gel electrophoresis and electron microscopy of detergent extracts of the isolated microvilli. Several methods have been used to reduce breakdown and solubilization of the microfilament core actin during the detergent extractions for preparation of microvillar core microfilaments. Gel electrophoresis of differential centrifugation fractions demonstrated that over 70% of the total microvillus actin could be pelleted with microfilament cores at 10 000 g under extraction conditions which reduce filament breakdown. Transmission electron microscopy (TEM) of all of the core preparations showed arrays of microfilaments and small microfilament bundles. The major protein components of the microfilament cores, observed by sodium dodecyl sulfate (SDS) electrophoresis, were actin and alpha-actinin. Among the less prominent polypeptide components was a 58 000 Dalton polypeptide (58 K), previously identified as a member of the MAT-Cl transmembrane complex. This three-component complex contains, in addition to 58 K, actin associated directly and stably with a cell surface glycoprotein (Carraway, CAC, Jung, G & Carraway, K L, Proc. natl acad. sci. US 80 (1983) 430). Evidence that the apparent association of complex with the microfilament core was not due simply to co-sedimentation was provided by myosin affinity precipitation. These results provide further evidence that the transmembrane complex is a site for the interaction of microfilaments with the microvillar plasma membrane.

Topics: Actins; Adenocarcinoma; Animals; Ascites; Buffers; Cell Fractionation; Cell Line; Centrifugation; Cross-Linking Reagents; Cytoskeleton; Electrophoresis, Polyacrylamide Gel; Isoelectric Focusing; Mammary Neoplasms, Experimental; Microfilament Proteins; Microscopy, Electron; Microvilli; Phalloidine; Rats

A fluorescent derivative of phalloidin with a high affinity for F-actin was microinjected into tissue culture cells and its intracellular reorganization was followed by TV image intensification and video recording. When the F-actin stabilizing drug is used at concentrations, which do not inhibit cellular movement, rearrangement of fluorescently labelled microfilament bundles can be followed directly. We discuss the possibility that active ruffles are governed by structural rules different from those applying to stress fibers and raise the possibility that actin may be released from microfilaments in a form different from G-actin.

Topics: Actins; Adenocarcinoma; Animals; Cells, Cultured; Cytoskeleton; Dipodomys; Female; Fluorescent Dyes; Mammary Glands, Animal; Mammary Neoplasms, Experimental; Microscopy, Fluorescence; Oligopeptides; Phalloidine; Rats