blister and Neoplasm-Metastasis

Blebbistatin is a widely used inhibitor of myosin 2 that enables the study of a broad range of cytoskeleton-related processes. However, blebbistatin has several limitations hindering its applicability: it is fluorescent, poorly water soluble, cytotoxic, and prone to (photo)degradation. Despite these adverse effects, being the only available myosin 2-specific inhibitor, blebbistatin is rather a choice of necessity. Blebbistatin has been modified to improve its properties and some of the new compounds have proven to be useful replacements of the original molecule. This review summarizes recent results on blebbistatin development. We also discuss the pharmacological perspectives of these efforts, as myosins are becoming promising drug target candidates for a variety of conditions ranging from neurodegeneration to muscle disease, wound healing, and cancer metastasis.

Topics: Animals; Drug Delivery Systems; Heterocyclic Compounds, 4 or More Rings; Humans; Muscular Diseases; Myosins; Neoplasm Metastasis; Neoplasms; Neurodegenerative Diseases; Wound Healing

Cortical actin is a thin layer of filamentous (F-)actin that lies beneath the plasma membrane, and its role in pathophysiology remains unclear. We investigated the subcellular localization of cortical actin by the histopathological and experimental studies of lung adenocarcinomas.. The subcellular localization of cortical actin was studied in surgically resected lung adenocarcinomas tissues and in 3-dimensionally cultured lung adenocarcinoma A549 cells.. In normal type II alveolar cells and the bronchiolar epithelium, cortical actin was localized to the apical-side cytoplasm. In invasive adenocarcinoma cells, cortical actin was frequently localized to the matrix side. The degree of cortical actin localized to the matrix side was associated with the loss of basement membrane and a poor prognosis. In A549 cell spheroids cultured in a type I collagen and basement membrane extract Matrigel™ mixed gel, cortical F-actin was localized to the matrix side with phosphorylated myosin light chain. Super-resolution and electron microscopy results suggest that compact wrinkling of the plasma membrane by myosin-mediated F-actin contraction is an explanation for cortical actin accumulation at the matrix side. The myosin II inhibitor blebbistatin suppressed the 3-dimensional collective migration of A549 cells induced by constitutively active Cdc42 and MT1-MMP.. Cortical actin accumulation at the matrix-side cytoplasm of cancer cells occurs in invasive lung adenocarcinomas and it possibly participates in the migration of cancer cells through myosin-mediated contraction.

Topics: A549 Cells; Actins; Adenocarcinoma; Adenocarcinoma of Lung; Cell Membrane; Cell Movement; Cytoplasm; Heterocyclic Compounds, 4 or More Rings; Humans; Immunohistochemistry; Lung Neoplasms; Myosins; Neoplasm Invasiveness; Neoplasm Metastasis; Prognosis

The chemokine receptor cysteine (C)-X-C receptor (CXCR4) is a G-protein-coupled receptor that exerts a vital role in distant metastasis of renal cell carcinoma (RCC). Emerging evidence demonstrates that CXCR4 as the cytomembrane receptor translocated into the nucleus to facilitate cell migration and, therefore, determine the prognosis of several types of malignancies. However, the biological mechanism of nuclear location of CXCR4 remains unclear. In the present study, we confirmed the significant implications of the putative nuclear localization sequence (NLS) '146RPRK149̓ on CXCR4 subcellular localization and metastatic potential by point-mutation assay in RCC cell lines. Importantly, mass spectrum followed by immunoprecipitation identified non-muscle myosin heavy chain-IIA (NMMHC-IIA) as the CXCR4-interacting protein. Furthermore, pharmaceutical inhibition of NMMHC-IIA by blebbistatin dampened the nuclear translocation of CXCR4 as well as the metastatic capacity of RCC cells. In conclusion, the present study may drive the comprehensive progress toward elucidating the mechanism responsible for CXCR4 nuclear function and metastasis in tumors.

Topics: Carcinoma, Renal Cell; Cell Line, Tumor; Cell Movement; Cell Nucleus; Heterocyclic Compounds, 4 or More Rings; Humans; Molecular Motor Proteins; Myosin Heavy Chains; Neoplasm Invasiveness; Neoplasm Metastasis; Point Mutation; Receptors, CXCR4; Signal Transduction

Cancer cells are defined by their ability to invade through the basement membrane, a critical step during metastasis. While increased secretion of proteases, which facilitates degradation of the basement membrane, and alterations in the cytoskeletal architecture of cancer cells have been previously studied, the contribution of the mechanical properties of cells in invasion is unclear. Here, we applied a magnetic tweezer system to establish that stiffness of patient tumor cells and cancer cell lines inversely correlates with migration and invasion through three-dimensional basement membranes, a correlation known as a power law. We found that cancer cells with the highest migratory and invasive potential are five times less stiff than cells with the lowest migration and invasion potential. Moreover, decreasing cell stiffness by pharmacologic inhibition of myosin II increases invasiveness, whereas increasing cell stiffness by restoring expression of the metastasis suppressor TβRIII/betaglycan decreases invasiveness. These findings are the first demonstration of the power-law relation between the stiffness and the invasiveness of cancer cells and show that mechanical phenotypes can be used to grade the metastatic potential of cell populations with the potential for single cell grading. The measurement of a mechanical phenotype, taking minutes rather than hours needed for invasion assays, is promising as a quantitative diagnostic method and as a discovery tool for therapeutics. By showing that altering stiffness predictably alters invasiveness, our results indicate that pathways regulating these mechanical phenotypes are novel targets for molecular therapy of cancer.

Topics: Actomyosin; Ascites; Cell Line, Tumor; Cell Movement; Cell Shape; Collagen; Compliance; Drug Combinations; Drug Design; Female; Heterocyclic Compounds, 4 or More Rings; Humans; Laminin; Magnetics; Micromanipulation; Microscopy, Atomic Force; Microspheres; Molecular Targeted Therapy; Myosin Type II; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Proteins; Ovarian Neoplasms; Proteoglycans; Receptors, Transforming Growth Factor beta; Tumor Cells, Cultured

Cancer deaths are primarily caused by metastases, not by the parent tumor. During metastasis, malignant cells detach from the parent tumor, and spread through the circulatory system to invade new tissues and organs. The physical-chemical mechanisms and parameters within the cellular microenvironment that initiate the onset of metastasis, however, are not understood. Here we show that human colon carcinoma (HCT-8) cells can exhibit a dissociative, metastasis-like phenotype (MLP) in vitro when cultured on substrates with appropriate mechanical stiffness. This rather remarkable phenotype is observed when HCT-8 cells are cultured on gels with intermediate-stiffness (physiologically relevant 21-47 kPa), but not on very soft (1 kPa) and very stiff (3.6 GPa) substrates. The cell-cell adhesion molecule E-Cadherin, a metastasis hallmark, decreases 4.73 ± 1.43 times on cell membranes in concert with disassociation. Both specific and nonspecific cell adhesion decrease once the cells have disassociated. After reculturing the disassociated cells on fresh substrates, they retain the disassociated phenotype regardless of substrate stiffness. Inducing E-Cadherin overexpression in MLP cells only partially reverses the MLP phenotype in a minority population of the dissociated cells. This important experiment reveals that E-Cadherin does not play a significant role in the upstream regulation of the mechanosensing cascade. Our results indicate, during culture on the appropriate mechanical microenvironment, HCT-8 cells undergo a stable cell-state transition with increased in vitro metastasis-like characteristics as compared to parent cells grown on standard, very stiff tissue culture dishes. Nuclear staining reveals that a large nuclear deformation (major/minor axis ratio, 2:5) occurs in HCT-8 cells when cells are cultured on polystyrene substrates, but it is markedly reduced (ratio, 1:3) in cells grown on 21 kPa substrates, suggesting the cells are experiencing different intracellular forces when grown on stiff as compared to soft substrates. Furthermore, MLP can be inhibited by blebbistatin, which inactivates myosin II activity and relaxes intracellular forces. This novel finding suggests that the onset of metastasis may, in part, be linked to the intracellular forces and the mechanical microenvironment of the tumor.

Topics: Actins; Biomechanical Phenomena; Cadherins; Cell Adhesion; Cell Line, Tumor; Cell Nucleus; Colonic Neoplasms; Gene Expression Regulation, Neoplastic; Heterocyclic Compounds, 4 or More Rings; Humans; Mechanical Phenomena; Neoplasm Metastasis; Phenotype