jasplakinolide and Breast-Neoplasms

Alpha-1-syntrophin (SNTA1) is emerging as a novel modulator of the actin cytoskeleton. SNTA1 binds to F-actin and regulates intracellular localization and activity of various actin organizing signaling molecules. Aberration in syntrophin signaling has been closely linked with deregulated growth connected to tumor development/metastasis and its abnormal over expression has been observed in breast cancer. In the present work the effect of jasplakinolide, an actin-binding cyclodepsipeptide, on the SNTA1 protein activity and SNTA1 mediated downstream cellular events was studied in MDA-MB-231 breast cancer cell line.. SNTA1 protein levels and phosphorylation status were determined in MDA-MB-231 cells post jasplakinolide exposure using western blotting and immunoprecipitation techniques respectively. MDA-MB-231 cells were transfected with WT SNTA1 and DM SNTA1 (Y. Jasplakinolide treatment decreases proliferation of MDA-MB-231 cells in both dose and time dependent manner. Results suggest that subtoxic doses of jasplakinolide induce morphological changes in MDA-MB-231 cells from flat spindle shape adherent cells to round weakly adherent forms. Mechanistically, jasplakinolide treatment was found to decrease SNTA1 protein levels and its tyrosine phosphorylation status. Moreover, migratory potential of jasplakinolide treated cells was significantly inhibited in comparison to control cells.. Our results demonstrate that jasplakinolide inhibits cell migration by impairing SNTA1 functioning in breast cancer cells.

Topics: Breast Neoplasms; Calcium-Binding Proteins; Cell Line, Tumor; Cell Movement; Cell Survival; Depsipeptides; Female; Humans; Membrane Proteins; Muscle Proteins; Phosphorylation

In the clinical treatment of breast cancer, antimitotic cytotoxic agents are one of the most commonly employed chemotherapies, owing largely to their antiproliferative effects on the growth and survival of adherent cells in studies that model primary tumor growth. Importantly, the manner in which these chemotherapeutics impact the metastatic process remains unclear. Furthermore, since dissemination of tumor cells through the systemic circulation and lymphatics necessitates periods of detached survival, it is equally important to consider how circulating tumor cells respond to such compounds. To address this question, we exposed both nontumorigenic and tumor-derived epithelial cell lines to two antitumor compounds, jasplakinolide and paclitaxel (Taxol), in a series of attached and detached states. We report here that jasplakinolide promoted the extension of microtubule-based projections and microtentacle protrusions in adherent and suspended cells, respectively. These protrusions were specifically enriched by upregulation of a stable post-translationally modified form of alpha-tubulin, and this occurred prior to, and independently of any reductions in cellular viability. Microtubule stabilization with Taxol significantly enhanced these effects. Additionally, Taxol promoted the attachment and spreading of suspended tumor cell populations on extracellular matrix. While the antiproliferative effects of these compounds are well recognized and clinically valuable, our findings that microfilament and microtubule binding chemotherapeutics rapidly increase the mechanisms that promote endothelial adhesion of circulating tumor cells warrant caution to avoid inadvertently enhancing metastatic potential, while targeting cell division.

Topics: Antimitotic Agents; Breast Neoplasms; Cell Adhesion; Cell Line, Tumor; Depsipeptides; Female; Fluorescent Antibody Technique; Humans; Immunoblotting; Microscopy, Confocal; Microtubules; Neoplastic Cells, Circulating; Paclitaxel

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

One of the intriguing questions regarding cell motility concerns the mechanism that makes stationary cells move. Here, we provide the first physical evidence that the onset of breast cancer cell motility in response to insulin-like growth factor I (IGF-I) correlates with lowering of adhesion strength from 2.52 +/- 0.20 to 1.52 +/- 0.13 microdynes/microm2 in cells attached to fibronectin via alpha5beta1 integrin. The adhesion strength depends on the dose of IGF-I and time of IGF-I treatment. Weakening of cell-matrix adhesion is blocked significantly (p < 0.01) by the catalytically inactive IGF-I receptor (IGF-IR) and the phosphoinositide 3-kinase (PI-3 kinase) inhibitor LY-294002, but it is unaffected by mitogen-activated protein kinase kinase inhibitor UO-126 and Src kinase inhibitor PP2. Sustained blockade of Rho-associated kinase (ROCK) with Y-27632 down-regulates adhesion strength in stationary, but not in IGF-I-treated, cells. Jasplakinolide, a drug that prevents actin filament disassembly, counteracts the effect of IGF-I on integrin-mediated cell adhesion. In the absence of growth factor signaling, ROCK supports a strong adhesion via alpha5beta1 integrin, whereas activation of the IGF-IR kinase reduces cell-matrix adhesion through a PI-3K-dependent, but ROCK-independent, mechanism. We propose that disassembly of the actin filaments via PI-3 kinase pathway contributes to weakening of adhesion strength and induction of cell movement. Understanding how cell adhesion and migration are coordinated has an important application in cancer research, developmental biology, and tissue bioengineering.

Topics: Actins; Amides; Blotting, Western; Breast Neoplasms; Butadienes; Carcinoma; Catalysis; Cell Adhesion; Cell Line, Tumor; Cell Movement; Chromones; CSK Tyrosine-Protein Kinase; Culture Media, Serum-Free; Depsipeptides; Down-Regulation; Fibronectins; Flow Cytometry; Humans; Immunoprecipitation; Insulin-Like Growth Factor I; Integrin alpha5beta1; Intracellular Signaling Peptides and Proteins; MAP Kinase Signaling System; Microscopy, Phase-Contrast; Models, Biological; Morpholines; Nitriles; Phosphatidylinositol 3-Kinases; Phosphorylation; Protein Serine-Threonine Kinases; Protein Structure, Tertiary; Protein-Tyrosine Kinases; Pyridines; Pyrimidines; rho GTP-Binding Proteins; rho-Associated Kinases; Signal Transduction; src-Family Kinases; Time Factors