cytochalasin-d and Lung-Neoplasms

Background Despite inherent differences between the cytoskeletal networks of malignant and normal cells, and the clinical antineoplastic activity of microtubule-directed agents, there has yet to be a microfilament-directed agent approved for clinical use. One of the most studied microfilament-directed agents has been cytochalasin B, a mycogenic toxin known to disrupt the formation of actin polymers. Therefore, this study sought to expand on our previous work with the microfilament-directed agent, along with other less studied cytochalasin congeners. Materials and Methods We determined whether cytochalasin B exerted significant cytotoxic effects in vitro on adherent M109 lung carcinoma and B16BL6 and B16F10 murine melanomas, or on suspension P388/ADR murine leukemia cells. We also examined whether cytochalasin B, its reduced congener 21, 22-dihydrocytochalasin B (DiHCB), or cytochalasin D could synergize with doxorubicin (ADR) against ADR-resistant P388/ADR leukemia cells, and produce significant cytotoxicity in vitro. For in vivo characterization, cytochalasins B and D were administered intraperitoneally (i.p.) to Balb/c mice challenged with drug sensitive P388-S or multidrug resistant P388/ADR leukemias. Results Cytochalasin B demonstrated higher cytotoxicity against adherent lung carcinoma and melanoma cells than against suspension P388/ADR leukemia cells, as assessed by comparative effects on cell growth, and IC₅₀ and IC₈₀ values. Isobolographic analysis indicated that both cytochalasin B and DiHCB demonstrate considerable drug synergy with ADR against ADR-resistant P388/ADR leukemia, while cytochalasin D exhibits only additivity with ADR against the same cell line. In vivo, cytochalasins B and D substantially increased the life expectancy of mice challenged with P388/S and P388/ADR leukemias, and in some cases, produced long-term survival. Conclusion Taken together, it appears that cytochalasins have unique antineoplastic activity that could potentiate a novel class of chemotherapeutic agents.

Topics: Animals; Antineoplastic Agents; Cell Survival; Cytochalasin B; Cytochalasin D; Cytochalasins; Doxorubicin; Drug Synergism; Leukemia P388; Lung Neoplasms; Melanoma; Mice; Mice, Inbred BALB C; Neoplasms; Tumor Cells, Cultured

Therapeutic delivery of small interfering RNA (siRNA) is a major challenge that limits its potential clinical application. Four fatty acids derivatives of octa-arginine (R8) were synthesized and evaluated for the delivery of siRNA into hepatocellular carcinoma Hep G2 and human lung adenocarcinoma A549 cells. The results showed that the long chain acid oleic acid or stearic acid derivatives of R8, OA-R8 and StA-R8, were more efficient in siRNA complexation and form nanoparticles with greater stability compared to the native R8. Cellular uptake of fluorescence-labeled siRNA delivered by OA-R8 and StA-R8 in Hep G2 and A549 cells was substantially 40-50 times higher than unmodified R8. A significant reduction in siRNA cellular uptake was observed in the presence of sucrose and cytochalasin D, indicating endocytosis as a primary mechanism of cellular entry. A survivin siRNA was used to prepare nanoparticles with OA-R8 or StA-R8 and evaluated for silencing of survivin mRNA and protein in A549 cells, and the inhibition efficiencies of survivin protein reached to 50.3% and 54.6%, respectively. The results showed greater effectiveness with the derivatized R8. Taken together, these findings showed that long chain fatty acid derivatives of R8 are efficient delivery agents for siRNA and may facilitate its therapeutic application.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Arginine; Cell Line, Tumor; Cell-Penetrating Peptides; Cytochalasin D; Dose-Response Relationship, Drug; Endocytosis; Fatty Acids; Hep G2 Cells; Humans; Lung Neoplasms; Nanoparticles; RNA, Small Interfering; Sucrose

Cytochalasin D (CytD) targets actin, a ubiquitous protein in eukaryotic cells. Previous studies have focused mainly on the antitumor effects of CytD. We previously found CytD to promote lung metastasis in B16 melanoma cells, which we had not anticipated, and, therefore, in the present study we investigated the possible underlying mechanisms. B16 melanoma cells were co-cultured with CytD and other agents and used to establish a lung metastatic model. In this B16 melanoma metastatic model, significantly increased lung metastasis and lung weight were found in CytD-treated mice, which was almost completely suppressed by tissue factor (TF) RNA interference expressed via lentivirus. The results of northern and western blot, and real-time RT-PCR analysis showed that the expression of TF was significantly upregulated in B16 cells treated with CytD but was significantly inhibited by TF RNA interference. In addition, upregulation and phosphorylation of mitogen-activated protein kinase p38 were also found in the metastatic lung tissues treated with CytD and in the B16 cells co-cultured with CytD and factor VIIa (FVIIa), but not in cells cultured with CytD, dimethyl sulfoxide or FVIIa alone. These results indicate that CytD stimulates the expression of TF in B16 melanoma cells, activating both coagulation-dependent and -independent pathways via binding to FVIIa, eventually promoting lung metastasis. TF interference is a potential approach to the prevention of B16 melanoma metastasis.

Topics: Animals; Cell Line, Tumor; Cytochalasin D; Factor VIIa; Female; Gene Expression Regulation, Neoplastic; Lung Neoplasms; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Nucleic Acid Synthesis Inhibitors; p38 Mitogen-Activated Protein Kinases; Phosphorylation; RNA Interference; RNA, Small Interfering; Thromboplastin; Up-Regulation

It is generally accepted that compounds of nanomolecular size penetrate into cells by different endocytic processes. The vehiculization strategy of a compound is a factor that could determine its uptake mechanism. Understanding the influence of the vehicle in the precise mechanism of drug penetration into cells makes possible to improve or modify the therapeutic effects. In this study, using human A-549 cells, we have characterized the possible internalization mechanism of the photosensitizer Zn(II)-phthalocyanine (ZnPc), either dissolved in dimethylformamide (ZnPc-DMF) or included in liposomes of dipalmitoyl-phosphatidyl-choline. Specific inhibitors involved in the main endocytic pathways were used. Co-incubation of cells with ZnPc-liposomes and dynasore (dinamin-mediated endocytosis inhibitor) resulted in a significant decrease of photodamage, whereas other inhibitors did not alter the photodynamic effect of ZnPc. On the contrary, cells treated with ZnPc-DMF in the presence of dynasore, genistein (caveolin-mediated endocytosis inhibitor) or cytochalasin D (macropinocytosis and caveolin-mediated endocytosis inhibitor) showed a significant decrease in ZnPc uptake and photodynamic damage. These results suggest that ZnPc-DMF penetrates into cells mainly by caveolin-mediated endocytosis, whereas ZnPc-liposomes are internalized into cells preferentially by clathrin-mediated endocytosis. We conclude that using different drug vehiculization systems, it is possible to modify the internalization mechanism of a therapeutic compound, which could be of great interest in clinical research.

Topics: Caveolins; Cell Line, Tumor; Cell Membrane; Cell Shape; Cell Survival; Clathrin; Cytochalasin D; Endocytosis; Genistein; Humans; Hydrazones; Indoles; Isoindoles; Liposomes; Lung Neoplasms; Organometallic Compounds; Photochemotherapy; Photosensitizing Agents; Time Factors; Zinc Compounds

The tumor suppressor p21WAF/CIP1 mediates the proliferation arrest via p53-dependent or -independent gene transactivation following distinct environmental stresses. In this study, we show that direct destabilization of the actin cytoskeleton by actin-targeting reagents leads to a p53-independent up-regulation of p21WAF/CIP1. The actin-targeting agent cytochalasin B (10 microM) quickly disrupted the actin cytoskeleton of p53 wild-type and p53-null cells accompanied by up-regulation of p21WAF/CIP1. Nevertheless, both total p53 and ser-15 phosphorylated p53 were not accumulated concomitantly, compared to the effect caused by ionizing irradiation. P53-independent up-regulation of p21WAF/CIP1 was also observed by two other actin-targeting agents cytochalasin D and latrunculin B, but not by the microtubule inhibitor colcemid. Furthermore, we showed that p21WAF/CIP1 mRNA level was not increased, whereas the protein degradation was delayed. A reduction of ubiquitination for p21WAF/CIP1 protein was detected using immunoprecipitation/immunoblot analysis. Up-regulation of p21WAF/CIP1 was not associated with cytotoxicity induced by cytochalasin B that influenced DNA integrity and plating efficiency only after 24 h of treatment. In addition, up-regulated p21WAF/CIP1 was accompanied by reduction of phosphorylation on retinoblastoma (Rb) protein in p53-null cells, implying that p21WAF/CIP1 might in part account for the molecular regulation of cytochalasin B induced G1 phase arrest. Together, current results suggest that p21WAF/CIP1 level can be mediated by actin organization in the absence of p53 via a post-transcriptional machinery, and it may contribute to the growth ablation by agents targeting the actin cytoskeleton.

Topics: Actins; Adenocarcinoma; Bone Neoplasms; Bridged Bicyclo Compounds, Heterocyclic; Carcinoma, Non-Small-Cell Lung; Cell Cycle; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p21; Cytochalasin D; Cytoskeleton; DNA, Neoplasm; Genes, p53; Humans; Lung Neoplasms; Osteosarcoma; RNA Processing, Post-Transcriptional; RNA, Messenger; RNA, Neoplasm; Thiazolidines; Ubiquitin

Metastases kill 90% of cancer patients. It is thus a major challenge in cancer therapy to inhibit the spreading of tumor cells from primary tumor sites to those particular organs where metastases are likely to occur. Whereas the actin cytoskeleton is a key component involved in cell migration, agents targeting actin dynamics have been relatively poorly investigated. Consequently, valuable in vitro pharmacological tools are needed to selectively identify this type of agent. In response to the absence of any standardized process, the present work aims to develop a multi-assay strategy for screening actin-affecting drugs with anti-migratory potentials. To validate our approach, we used two cancer cell lines (MCF7 and A549) and three actin-affecting drugs (cytochalasin D, latrunculin A, and jasplakinolide). We quantified the effects of these drugs on the kinetics of actin polymerization in tubes (by means of spectrofluorimetry) and on the dynamics of actin cytoskeletons within whole cells (by means of fluorescence microscopy). Using quantitative videomicroscopy, we investigated the actual effects of the drugs on cell motility. Finally, the combined drug effects on cell motility and cell growth were evaluated by means of a scratch-wound assay. While our results showed concordant drug-induced effects on actin polymerization occurring in vitro in test tubes and within whole cells, the whole cell assay appeared more sensitive than the tube assay. The inhibition of actin polymerization induced by cytochalasin D was paralleled by a decrease in cell motility for both cell types. In the case of jasplakinolide, which induces actin polymerization, while it significantly enhanced the locomotion of the A549 cells, it significantly inhibited that of the MCF-7 ones. All these effects were confirmed by means of the scratch-wound assay except of the jasplakinolide-induced effects on MCF-7 cell motility. These later seemed compensated by an additional effect occurring during wound recolonization (possibly acting on the cell growth features). In conclusion, the use of multi-assays with different levels of sophistication and biological relevance is recommended in the screening of new actin-affecting drugs with potentially anti-migratory effects.

Topics: Actins; Adenocarcinoma; Antineoplastic Agents; Breast Neoplasms; Bridged Bicyclo Compounds, Heterocyclic; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Cytochalasin D; Cytoskeleton; Depsipeptides; Humans; Inhibitory Concentration 50; Lung Neoplasms; Neoplasm Invasiveness; Statistics, Nonparametric; Thiazoles; Thiazolidines

Recent studies have identified novel actions for 2-aminoethoxydiphenyl borate (2-APB) in triggering calcium release and enhancing calcium influx induced by the depletion of intracellular calcium stores. In this study, we have examined the effects of 2-APB on the human lung adenocarcinoma A549 cell line, which we have previously shown displays a unique calcium influx response, when ER calcium stores are depleted by thapsigargin (TG) treatment. Here, we show that low concentrations of 2-APB failed to induce the rapid augmentation of TG-activated calcium influx previously reported for other cell types. We observed that store-operated calcium (SOC) channels in the A549 cell line exhibited short-term sensitivity to low doses of 2-APB, perhaps reflecting a delayed augmentation of SOC channel activity or the recruitment of 2-APB-insensitive SOC channels. In both intact and permeabilized cells, 2-APB effectively discharged a subset of A549 calcium pools corresponding to the hormone-sensitive intracellular calcium stores. The 2-APB-induced calcium release produced a long-lasting perturbation of the adenosine triphosphate (ATP)-releasable calcium pools, effectively uncoupling ATP-activated calcium release even, when stores are replenished with calcium. In contrast to previous reports, we found that disruption of either the actin or microtubule-based cytoskeleton failed to block the 2-APB-induced effects on calcium signaling in A549 cells. Our study describes novel cytoskeletal-independent effects of 2-APB on Ca2+-signaling pathways, revealing differentially sensitive Ca2+-influx pathways and long-term perturbation of hormone-sensitive Ca2+ stores.

Topics: Actins; Adenocarcinoma; Adenosine Triphosphate; Antineoplastic Agents, Phytogenic; Boron Compounds; Calcium; Calcium Channels; Calcium Signaling; Cell Line, Tumor; Cytochalasin D; Cytoskeleton; Digitonin; Dose-Response Relationship, Drug; Enzyme Inhibitors; Green Fluorescent Proteins; Humans; Indicators and Reagents; Inositol 1,4,5-Trisphosphate; Ionomycin; Lung Neoplasms; Microscopy, Confocal; Nucleic Acid Synthesis Inhibitors; Paclitaxel; Thapsigargin; Time Factors

The possible functional role of voltage-gated Na(+) channel (VGSC) expression in controlling endocytic membrane activity in human small-cell lung cancer (SCLC) cell lines (H69, H209, H510) was studied using uptake of horseradish peroxidase (HRP). The normal human airway epithelial (16HBE14o) cell line was used in a comparative approach. Uptake of HRP was vesicular, strongly temperature-sensitive and suppressed by cytoskeletal poisons (cytochalasin D and colchicine), consistent with endocytosis. Compared with the normal cells, HRP uptake into SCLC cells was kinetically more efficient, resulting in more than four-fold higher uptake under optimized conditions. Importantly, HRP uptake into SCLC cells was inhibited significantly by the specific VGSC blocker tetrodotoxin, as well as lidocaine and phenytoin. These effects were dose-dependent. None of these drugs had any effect on the uptake into the 16HBE14o cells. Uptake of HRP into SCLC cells was reduced by approximately 66% in Na(+)-free medium and was partially ( approximately 30%) dependent on extracellular Ca(2+). The possibility that the endocytic activity in the H510 SCLC cells involved an endogenous cholinergic system was investigated by testing the effects of carbachol (a cholinergic receptor agonist) and eserine (an inhibitor of acetylcholinesterase). Both drugs inhibited HRP uptake, thereby suggesting that basal cholinergic activity occurred. It is concluded that VGSC upregulation could enhance metastatic cell behavior in SCLC by enhancing endocytic membrane activity.

Topics: Carbachol; Carcinoma, Small Cell; Cell Membrane; Cells, Cultured; Cholinergic Agonists; Cholinesterase Inhibitors; Colchicine; Cytochalasin D; Endocytosis; Epithelial Cells; Gout Suppressants; Horseradish Peroxidase; Humans; In Vitro Techniques; Ion Channel Gating; Lung Neoplasms; Membrane Potentials; Nucleic Acid Synthesis Inhibitors; Physostigmine; Respiratory System; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sodium Channel Blockers; Sodium Channels

The tumor suppressor gene TSLC1, which we recently identified in human non-small cell lung cancer, encodes a membrane glycoprotein of the immunoglobulin superfamily. Here, we report that TSLC1 directly associates with DAL-1, a gene product of another lung tumor suppressor belonging to the protein 4.1 family. TSLC1 additionally interacts with the actin filament through DAL-1 at the cell-cell attached site where the complex formation of TSLC1 and DAL-1 is dependent on the integrity of actin cytoskeleton. Redistribution of both TSLC1 and DAL-1 to the newly generated membrane ruffling areas suggests that these proteins are also involved in cell motility accompanying the actin rearrangement. Furthermore, restoration of TSLC1 expression strongly suppressed the metastasis of a human non-small cell lung cancer cell line, A549, from the spleen to the liver in nude mice. These findings, together with frequent loss of their expression in lung cancers, suggest that TSLC1 and DAL-1 play a critical role in the same pathway involved in the suppression of lung tumor formation and metastasis.

Topics: Actins; Amino Acid Sequence; Animals; Carcinoma, Non-Small-Cell Lung; Cell Adhesion; Cell Adhesion Molecule-1; Cell Adhesion Molecules; COS Cells; Cytochalasin D; Cytoskeleton; Humans; Immunoglobulins; Lung Neoplasms; Membrane Proteins; Microfilament Proteins; Microscopy, Fluorescence; Molecular Sequence Data; Protein Structure, Tertiary; Proteins; Sequence Homology, Amino Acid; Tumor Cells, Cultured; Tumor Suppressor Proteins

The requirement of an intact cytoskeleton organization for G1/S cell cycle progression has been demonstrated in cultured cells. In the non-small-cell lung carcinoma cell line A549, the kinase inhibitor staurosporine induced G1 cell cycle arrest with an accumulation of the cyclin-dependent kinase inhibitor p27kip1. Staurosporine induced also a drastic change in cell shape that was accompanied by changes in the actin cytoskeleton. The cytoskeleton disruption agents, cytochalasin D (cyto D) and 2,3-butanedione 2-monoxime (BDM), also induced G1 cell cycle arrest in A549 cells but without an accumulation of p27kip1. A comparison of the cell shape changes caused by these agents revealed that a conversion from an epithelial polygonal shape to an elongated fibroblast-like shape was specific for staurosporine. The shape change induced by staurosporine preceded the accumulation of p27kip1 by about 4 h. The accumulation of p27kip1 was not due to enhanced transcription but to stabilization of the protein resulting from the inhibition of proteolytic degradation. Staurosporine, however, did not inhibit directly the proteasome that was involved in the cell-cycle-dependent p27kip1 degradation. The results indicate that the cell shape change caused by staurosporine correlates with the accumulation of p27kip1 and that staurosporine interferes with the p27kip1-specific proteolysis activity.

Topics: Cell Cycle; Cell Cycle Proteins; Cell Size; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Cycloheximide; Cysteine Endopeptidases; Cytochalasin D; Cytoskeleton; Diacetyl; Enzyme Inhibitors; Female; Flow Cytometry; Humans; Lung Neoplasms; Microscopy, Confocal; Multienzyme Complexes; Nucleic Acid Synthesis Inhibitors; Proteasome Endopeptidase Complex; Protein Synthesis Inhibitors; Staurosporine; Tumor Cells, Cultured; Tumor Suppressor Proteins; Uterine Cervical Neoplasms

The effects of neurotensin on focal adhesion kinase were investigated using lung cancer cells. Neurotensin bound with high affinity to large cell carcinoma cell line NCI-H1299 as did neurotensin-(8-13), but not neurotensin-(1-7) or levocabastine. Addition of 100 nM neurotensin to NCI-H1299 cells caused transient tyrosine phosphorylation of focal adhesion kinase which was maximal after 1-2.5 min. Also, neurotensin-(8-13), but not neurotensin-(1-8) or levocabastine, caused tyrosine phosphorylation of focal adhesion kinase after addition to NCI-H1299 cells. Focal adhesion kinase tyrosine phosphorylation caused by neurotensin was inhibited by the nonpeptide neurotensin receptor antagonist (2-(1-(7-chloroquinolin-4-yl)-5-(2,6-dimethoxyphenyl)-1H-pyrazole-3-carbonyl)amino)-adamantane-2-carboxylic acid) (SR48692). SR48692 inhibited the clonal growth of NCI-H1299 cells, whereas neurotensin-stimulated proliferation and levocabastine had no effect. These results indicate that lung cancer cells have functional neurotensin receptors which regulate focal adhesion kinase tyrosine phosphorylation. It remains to be determined if neurotensin receptors and focal adhesion kinase plays a role in lung cancer cellular adhesion and migration.

Topics: Binding, Competitive; Cell Division; Cytochalasin D; Dose-Response Relationship, Drug; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Humans; Lung Neoplasms; Neurotensin; Nucleic Acid Synthesis Inhibitors; Peptide Fragments; Phosphorylation; Piperidines; Protein-Tyrosine Kinases; Pyrazoles; Quinolines; Receptors, Neurotensin; Tumor Cells, Cultured; Tyrosine

Lipocortin 1 (annexin I) is a calcium- and phospholipid-binding annexin protein which can be externalised from cells despite the lack of a signal sequence. To determine its cellular distribution lipocortin 1 in A549 human lung adenocarcinoma cells was localised by light- and electron-microscopic immunocytochemistry and by cell fractionation and western blotting. Lipocortin 1 immunoreactivity is concentrated in prominent patches associated with the plasma membrane. The intensity of these patches varied with the confluence and duration of the culture and was not detectably diminished by an EDTA wash before fixation. Tubulin and cytokeratin 8 were colocalized with lipocortin 1 in the patches. Within the cells lipocortin 1 was distributed throughout the cytoplasm. Electron microscopy revealed prominent immunoreactivity along the plasma membrane with occasional large clusters of gold particles in contact with the membrane surface of the cells; within the cytoplasm the membrane of some vesicle/vacuole structures and some small electron-dense bodies was immunoreactive, but no immunogold particles were associated with the multilamellar bodies. Subcellular fractionation, extraction and western blotting showed that lipocortin 1 in the membrane pellet was present as two distinct fractions; one, intimately associated with the lipid bilayer, which behaved like an integral membrane protein and one loosely attached which behaved like a peripheral membrane protein. The results show that a substantial amounts of lipocortin 1 is concentrated in focal structures associated with and immediately beneath the plasma membrane. These might form part of the mechanism by which lipocortin 1 is released from the cells.

Topics: Adenocarcinoma; Annexin A1; Biological Transport; Blotting, Western; Cell Compartmentation; Cell Membrane; Cytochalasin D; Cytoplasm; Cytoskeleton; Fluorescent Antibody Technique; Humans; Keratins; Lung Neoplasms; Membrane Proteins; Microscopy, Immunoelectron; Microtubules; Nucleic Acid Synthesis Inhibitors; Tubulin; Tumor Cells, Cultured

We have previously established a vincristine resistant human lung cancer cell line (PC-9/VCR) by a stepwise exposure of parental line PC-9 to vincristine. In this study the resistant cells showed enhanced vincristine cytotoxicity in the presence of cytochalasin B and D. The increase in cytotoxicity was associated with an enhanced accumulation and a reduced efflux of vincristine. Colchicine and taxol had no effects on vincristine accumulation. Several cytoplasmic proteins were overexpressed in the resistant cells. The two major ones, with molecular weights of 58.8 kDa and 83.2 kDa, were shown by western blotting to be beta-tubulin and actin, respectively. The polymerized tubulin level in the resistant cells was significantly (p < 0.05) higher than that in the parental cells. These results suggest that the cellular cytoskeletons might play an important role in VCR resistance in the PC-9/VCR human lung cancer cell line.

Topics: Adenocarcinoma; Cell Survival; Colchicine; Cyclosporine; Cytochalasin B; Cytochalasin D; Cytoskeleton; Drug Resistance, Neoplasm; Drug Synergism; Humans; Lung Neoplasms; Neoplasm Proteins; Paclitaxel; Tubulin; Tumor Cells, Cultured; Verapamil; Vincristine

Metastasis is one of the most important factors responsible for the pathogenesis of small cell lung carcinoma (SCLC). SCLC cells express cadherins, which are homophilic cell-cell adhesion molecules that play an important role in the regulation of metastasis. We present the first evidence that altering the activity of the small GTP-binding protein Rho induces cadherin-mediated adhesion. ADP-ribosylation of Rho upon incubation or electroporation with recombinant C3 exoenzyme induces rapid aggregation and compaction of SCLC cells. Aggregation and compaction induced by C3 exoenzyme are diminished by removal of extracellular Ca2+ and by the HECD blocking antibody to E-cadherin but not by antibodies to other adhesion molecules. Altering the activity of Rho by ADP-ribosylation does not alter surface expression of E-cadherin, but it alters G actin content, as indicated by the binding of DNase I. Treatment with cytochalasin D also alters G actin content and increases aggregation and compaction of SCLC cells. These findings implicate Rho in the regulation of cadherin-mediated adhesion and identify Rho as a potential therapeutic target for the control of SCLC metastasis.

Topics: Actin Cytoskeleton; Actins; Adenosine Diphosphate Ribose; ADP Ribose Transferases; Botulinum Toxins; Cadherins; Carcinoma, Small Cell; Cell Adhesion; Cell Aggregation; Cytochalasin D; Cytoskeleton; GTP-Binding Proteins; Humans; Lung Neoplasms; rho GTP-Binding Proteins; Time Factors; Tumor Cells, Cultured

During cancer progression, tumor cells interact with stromal cells. As a consequence, matrix metalloproteinases are produced that contribute to the degradation of the extracellular matrix. This study used coculture systems to investigate fibroblast interaction with three colon cancer cell lines isolated from a single patient. Cells from primary colorectal carcinoma, but not from corresponding liver or lymph node metastases, induced gelatinase B expression by fibroblasts of different tissue origin. Remarkably, direct cell-cell contact was required for this induction, which occurred at the pretranslational level (as revealed by Northern blot analysis) and was completely blocked by anti-beta1 integrin monoclonal antibody, but only partially blocked by anti-alpha5 or anti-alpha(v). Induction was also inhibited by cytochalasin D, staurosporine, or dexamethasone, suggesting the need, respectively, for an organized actin cytoskeleton, protein kinase C, and AP-1-driven gene transcription. Our data suggest that direct tumor-stromal cell contact is one inductive event involved in matrix metalloproteinase expression by stromal cells.

Topics: Animals; Antibodies, Monoclonal; Carcinoma; Cell Communication; Cells, Cultured; Coculture Techniques; Collagenases; Colonic Neoplasms; Connective Tissue; Connective Tissue Cells; Cytochalasin D; Cytoskeleton; Dexamethasone; Enzyme Induction; Enzyme Inhibitors; Extracellular Matrix Proteins; Fibroblasts; Genistein; Humans; Integrin beta1; Isoflavones; Keratinocytes; Liver Neoplasms; Lung Neoplasms; Matrix Metalloproteinase 9; Mice; Mice, Nude; Neoplasm Metastasis; Protein Kinase C; Staurosporine; Transcription Factor AP-1; Tumor Cells, Cultured

Although infection by group A streptococci is a model of extracellular mucosal pathogenesis, these organisms can be associated with highly invasive infections resulting in sepsis and shock. Over the last 6 yr this species has renewed its reputation as a significant cause of sepsis and has piqued interest in the mechanism by which some strains are better able to breach mucosal barriers to gain access to the bloodstream than are others. An internalization assay was developed on the basis of resistance of intracellular streptococci to penicillin and gentamicin. Experiments showed that stationary-phase, as opposed to logarithmic-phase, bacteria are efficiently internalized and can persist in cultured human cells. Electron microscopy confirmed that streptococci were contained within intracellular vacuoles. Various strains of streptococci revealed significant differences in their capacity to be internalized. Two type M1 streptococci isolated from blood infections were internalized at frequencies equal to those reported for Salmonella and Listeria monocytogenes and greater than the frequency of a clonal variant from a case of pharyngitis.

Topics: Cell Line; Colchicine; Cytochalasin D; Endocytosis; Epithelium; Humans; Kinetics; Listeria monocytogenes; Lung Neoplasms; Microscopy, Electron; Respiratory System; Salmonella; Streptococcus pyogenes; Time Factors; Tumor Cells, Cultured

We recently reported that disruption of tumor cell microfilaments or intermediate filaments resulted in an inhibition of the ability of tumor cells to induce the aggregation of homologous platelets in vitro (H. Chopra et al., Cancer Res., 48: 3787-3800, 1988). Previous investigators demonstrated that disruption of the tumor cell cytoskeleton decreases the ability of these cells to form lung colonies. We proposed that this latter effect is due, in part, to decreased interaction of tumor cells with platelets, following their arrest in the microvasculature. To test this hypothesis, B16 amelanotic melanoma cell microtubules, microfilaments, or vimentin intermediate filaments were disrupted with colchicine (50 microns), cytochalasin D (50 microns), or cycloheximide (50 microns), respectively, and then cells were tail vein injected into syngeneic mice. Both cytochalasin D- and cycloheximide-treated cells formed fewer lung colonies than did control cells. Colchicine, however, failed to inhibit lung colony formation. Neither colchicine nor cycloheximide treatment altered initial pulmonary arrest; however, fewer cycloheximide-treated cells remained in the lungs 8 h postinjection. Greater than 90% of control or colchicine-treated cells were found to be associated with activated platelets, and they also demonstrated typical cell membrane process formation 10 min and 8 h post-tumor cell injection. In contrast, less than 10% of cycloheximide-treated cells were in contact with activated platelets 10 min postinjection. However, by 8 h approximately 90% of cycloheximide-treated cells were in contact with activated platelets. This recovery coincided with the reformation of the B16 amelanotic melanoma vimentin intermediate filament network and the reacquisition of the ability to induce platelet aggregation in vitro. Neither colchicine nor cycloheximide treatment altered initial B16 amelanotic melanoma cell adhesion to murine microvessel-derived endothelial cells. This study provides in vivo evidence in support of our previous findings that disruption of certain cytoskeletal elements (i.e., vimentin intermediate filaments) inhibits the tumor cell ability to activate platelets. This study also suggests that platelet activation may stabilize the initial tumor cell arrest in the microvasculature.

Topics: Animals; Blood Platelets; Cell Communication; Colchicine; Cycloheximide; Cytochalasin D; Cytoskeleton; Endothelium; Lung Neoplasms; Male; Melanoma; Mice; Mice, Inbred C57BL; Microscopy, Electron; Neoplasm Metastasis; Platelet Aggregation