Maria Malicka-Błaszkiewicz

Cofilin overexpression affects actin cytoskeleton organization and migration of human colon adenocarcinoma cells

The dynamic reorganization of actin cytoskeleton is regulated by a large number of actin-binding proteins. Among them, the interaction of ADF/cofilin with monomeric and filamentous actin is very important, since it severs actin filaments. It also positively influences actin treadmilling. The activity of ADF/cofilin is reversibly regulated by phosphorylation and dephosphorylation at Ser-3, with the phosphorylated form (P-cofilin) being inactive. Here, we studied the effects of overexpression of cofilin and two cofilin variants in the human colon adenocarcinoma LS180 cell line. We have generated the LS180 cells expressing three different cofilin variants: WT (wild type), Ser 3 Ala (S3A) (constitutively active) or Ser 3 Asp (S3D) (constitutively inactive cofilin). The cells expressing WT cofilin were characterized by abundant cell spreading and colocalization of cofilin with the submembranous F-actin. Similar effects were observed in cells expressing S3A cofilin. In contrast, LS180 cells expressing S3D cofilin remained longitudinal in morphology and cofilin was equally distributed within the cell body. Furthermore, the migration ability of LS180 cells expressing different cofilin mutants was analyzed. In comparison to control cells, we have noticed a significant, approximately fourfold increase in the migration factor value of cells overexpressing WT type cofilin. The overexpression of S3D cofilin resulted in an almost complete inhibition of cell motility. The estimation of actin pool in the cytosol of LS180 cells expressing S3A cofilin has shown a significantly lower level of total actin in reference to control cells. The opposite effect was observed in LS180 cells overexpressing S3D cofilin. In summary, the results of our experiments indicate that phosphorylation “status” of cofilin is a factor affecting the actin cytoskeleton organization and migration abilities of colon adenocarcinoma LS180 cells.

Overexpression of lumican affects the migration of human colon cancer cells through up-regulation of gelsolin and filamentous actin reorganization.

Cell migration is a multistep process initiated by extracellular matrix components that leads to cytoskeletal changes and formation of different protrusive structures at the cell periphery. Lumican, a small extracellular matrix leucine-rich proteoglycan, has been shown to inhibit human melanoma cell migration by binding to α2β1 integrin and affecting actin cytoskeleton organization. The aim of this study was to determine the effect of lumican overexpression on the migration ability of human colon adenocarcinoma LS180 cells. The cells stably transfected with plasmid containing lumican cDNA were characterized by the increased chemotactic migration measured on Transwell filters. Lumican-overexpressing cells presented the elevated filamentous to monomeric actin ratio and gelsolin up-regulation. This was accompanied by a distinct cytoskeletal actin rearrangement and gelsolin subcellular relocation, as observed under laser scaning confocal microscope. Moreover, LS180 cells overexpressing lumican tend to form podosome-like structures as indicated by vinculin redistribution and its colocalization with gelsolin and actin at the submembrane region of the cells. In conclusion, the elevated level of lumican secretion to extracellular space leads to actin cytoskeletal remodeling followed by an increase in migration capacity of human colon LS180 cells. These data suggest that lumican expression and its presence in ECM has an impact on colon cancer cells motility and may modulate invasiveness of colon cancer.

Lumican affects actin cytoskeletal organization in human melanoma A375 cells

AIMS Lumican, a small leucine-rich proteoglycan (SLRP), has attracted attention as a molecule of the extracellular matrix possibly involved in signalling pathways affecting cancer cell behaviour. The remodelling of the actin cytoskeleton, induced in response to external stimuli, is crucial for cell motility and intracellular signal transduction. The main goal of this study was to examine the effects of recombinant lumican on actin organization, the state of actin polymerization, actin isoform expression, and their sub-cellular distribution in the A375 human melanoma cell line. MAIN METHODS Fluorescence and confocal microscopy were used to observe actin cytoskeletal organization and the sub-cellular distribution of cytoplasmic beta- and gamma-actins. The ability of actin to inhibit DNaseI activity was used to quantify actin. Western blotting and real-time PCR were used to determine the expression levels of the actin isoforms. KEY FINDINGS A375 cells grown on lumican coatings changed in morphology and presented rearranged actin filament organization: from filaments evenly spread throughout the whole cell body to their condensed sub-membrane localization. In the presence of lumican, both actin isoforms were concentrated under the cellular membrane. A statistically significant increase in the total, filamentous, and monomeric actin pools was observed in A375 cells grown on lumican. SIGNIFICANCE Novel biological effects of lumican, an extracellular matrix SLRP, on the actin pool and organization are identified, which may extend our understanding of the mechanism underlying the inhibitory effect of lumican on the migration of melanoma cells.

Methotrexate induces apoptosis in CaSki and NRK cells and influences the organization of their actin cytoskeleton

Methotrexate is a widely used drug in treatments of various types of malignancies and in the therapy of rheumatoid arthritis. The goal of our study was to look at the effect of this dihydrofolate reductase inhibitor on the actin cytoskeleton, since actin plays an important role in cancer transformation and metastasis. For this reason we compared results obtained from experiments on CaSki (human uterine cervix cancer) and NRK (normal fibroblastic rat kidney) cells treated with methotrexate. It has been shown previously that methotrexate can induce apoptosis. Therefore we first examined whether methotrexate induces apoptosis in our model cells. For this aim we applied several assays like Caspase Glo 3/7, DNA fragmentation and binding of phosphatidylserine by annexin V-fluorescein. The data obtained indicated that methotrexate induces programmed cell death in CaSki and NRK cells. However, differences between CaSki and NRK cells were observed in the morphological alterations and dynamics of apoptosis induced by methotrexate. It seemed that cancer cells were more sensitive towards the cell death inducing activity at lower concentrations of methotrexate. Analysis by confocal microscopy of methotrexate-treated cells demonstrated that treatment with this folate antagonist affected the actin cytoskeleton, although the dis-organization of the actin cytoskeleton after treatment with methotrexate differed between cancer and normal cells.

Liposomal Formulation of DIMIQ, Potential Antitumor Indolo(2,3-b)Quinoline Agent and Its Cytotoxicity on Hepatoma Morris 5123 Cells

The cytotoxic and antitumor activity of DIMIQ (5,11-dimethyl-5H-indolo[2,3-b]quinoline), synthetic analog of neocryptolepine, makes this compound a potential antitumor agent. An attempt to obtain liposomal form of DIMIQ·HCl was undertaken in the present study. Standard experimental conditions were chosen and information on the physicochemical parameters of the liposome dispersion containing studied indoloquinoline agent was collected. The effective and efficient encapsulation of DIMIQ·HCl (66.6%) in conventional liposomes (FAT-MLV, DMPC:DMPG 7:3 w/w at pH 7.0), uniformity of the size of liposomal vesicles, and high stability at pH 6.5 were demonstrated. Hemolysis of sheep erythrocytes induced by free form of DIMIQ·HCl was dramatically decreased after addition of liposome-entrapped DIMIQ·HCl. Treatment of hepatoma Morris 5123 cells for 24 hr with different concentrations of both free and its liposomal formulation of DIMIQ·HCl resulted in significant changes in cell morphology accompanied by reduction of cell viability.

Lumican - derived peptides inhibit melanoma cell growth and migration.

Lumican, a small leucine-rich proteoglycan of the extracellular matrix, presents potent anti-tumor properties. Previous works from our group showed that lumican inhibited melanoma cell migration and tumor growth in vitro and in vivo. Melanoma cells adhered to lumican, resulting in a remodeling of their actin cytoskeleton and preventing their migration. In addition, we identified a sequence of 17 amino acids within the lumican core protein, named lumcorin, which was able to inhibit cell chemotaxis and reproduce anti-migratory effect of lumican in vitro. The aim of the present study was to characterize the anti-tumor mechanism of action of lumcorin. Lumcorin significantly decreased the growth in monolayer and in soft agar of two melanoma cell lines – mice B16F1 and human SK-MEL-28 cells – in comparison to controls. Addition of lumcorin to serum free medium significantly inhibited spontaneous motility of these two melanoma cell lines. To characterize the mechanisms involved in the inhibition of cell migration by lumcorin, the status of the phosphorylation/dephosphorylation of proteins was examined. Inhibition of focal adhesion kinase phosphorylation was observed in presence of lumcorin. Since cancer cells have been shown to migrate and to invade by mechanisms that involve matrix metalloproteinases (MMPs), the expression and activity of MMPs were analyzed. Lumcorin induced an accumulation of an intermediate form of MMP-14 (~59kDa), and inhibited MMP-14 activity. Additionally, we identified a short, 10 amino acids peptide within lumcorin sequence, which was able to reproduce its anti-tumor effect on melanoma cells. This peptide may have potential pharmacological applications.

Gelsolin affects the migratory ability of human colon adenocarcinoma and melanoma cells

AIMS Formation of different protrusive structures by migrating cells is driven by actin polymerization at the plasma membrane region. Gelsolin is an actin binding protein controlling the length of actin filaments by its severing and capping activity. The main goal of this study was to determine the effect of gelsolin expression on the migration of human colon adenocarcinoma LS180 and melanoma A375 cells. MAIN METHODS Colon adenocarcinoma cell line LS180 was stably transfected with plasmid containing human cytoplasmic gelsolin cDNA tagged to enhanced green fluorescence protein (EGFP). Melanoma A375 cells were transfected with siRNAs directed against gelsolin. Real-time PCR and Western blotting were used to determine the level of gelsolin. The ability of actin to inhibit DNase I activity was used to quantify monomeric and total actin level and calculate the state of actin polymerization. Fluorescence confocal microscopy was applied to observe gelsolin and vinculin distribution along with actin cytoskeleton organization. KEY FINDINGS Increased level of gelsolin expression leads to its accumulation at the submembranous region of the cell accompanied by distinct changes in the state of actin polymerization and an increase in the migration of LS180 cells. In addition, LS180 cells overexpressing gelsolin form podosome-like structures as indicated by vinculin redistribution and its colocalization with gelsolin and actin. Downregulation of gelsolin expression in melanoma A375 cells significantly reduces their migratory potential. SIGNIFICANCE Our experimental data indicate that alterations in the expression level of gelsolin and its subcellular distribution may be directly responsible for determining migration capacity of human cancer cells.

Effect of overexpression of β- and γ-actin isoforms on actin cytoskeleton organization and migration of human colon cancer cells

Abstract Actins are eukaryotic proteins, which are involved in diverse cellular functions including muscle contraction, cell motility, adhesion and maintenance of cell shape. Cytoplasmic actin isoforms β and γ are ubiquitously expressed and essential for cell functioning. However, their unique contributions are not very well understood. The aim of this study was to determine the effect of β- and γ-actin overexpression on the migration capacity and actin cytoskeleton organization of human colon adenocarcinoma BE cells. In cells overexpressing β- or γ-actin, distinct cytoskeletal actin rearrangements were observed under the laser scanning confocal microscope. Overexpressed actins localized at the submembranous region of the cell body, especially near to the leading edge and on the tips of pseudopodia. The cells transfected with plasmids containing cDNA for β- or γ-actin were characterized by increased migration and invasion capacities. However, the migration velocity was statistically significantly higher only in the case of γ-actin overexpressing cells. In conclusion, the increased level of β- or γ-actin leads to actin cytoskeletal remodeling followed by an increase in migration and invasion capacities of human colon BE cells. These data suggest that expression of both actin isoforms has an impact on cancer cell motility, with the subtle predominance of γ-actin, and may influence invasiveness of human colon cancer.

DNase I-like activity and actin content in the liver of some vertebrates

Total actin content and F:G actin ratio were determined in the liver cytosol of fish, frogs and mouse by measurements of inhibition of exogenous crystalline bovine pancreatic DNase I. Endogenous DNase I-like activity, was found in all examined livers after electrophoresis in the presence of sodium dodecyl sulfate and subsequent enzyme renaturation. It is concluded that DNase I-like enzymes occur in the liver cytosol in a latent form, probably bound to cytoplasmic actin.

Apoptotic effect of imatinib on human colon adenocarcinoma cells: Influence on actin cytoskeleton organization and cell migration

Imatinib mesylate (STI571) is the first member of a new class of agents that act by inhibiting specific tyrosine kinases, rather than killing all rapidly dividing cells. This drug is usually used in the treatment of chronic myelogenous leukemia and gastrointestinal stromal tumors. It was recognized to inhibit activity of kinases such as Bcr/Abl, platelet-derived growth factor receptor, and c-kit. These proteins play important roles in cell growth, motility, and survival. Therefore, studies on the biological effects of imatinib on different cellular models are very important. Human colon adenocarcinoma LS180 cell line was used in the studies presented. Cells were exposed to 0.1-100 μM imatinib for 24 and 48 h. Dose-dependent decreases in cell viability and morphological changes were observed. Moreover, the apoptotic effect of imatinib (10 μM, 50 μM) after 24 h of exposure was demonstrated as evaluated by translocation of phosphatidylserine to external membrane leaflet and by increased activity of caspase-3. Special attention was focused on imatinib influence on actin cytoskeleton organization and migration ability of LS180 cells. Distinct alterations in actin cytoskeleton architecture occurred in response to drug treatment, accompanied by appearance of filamentous actin aggregates and decrease in actin polymerization state. These changes were correlated with remarkable decrease in cell migration capacity. In summary, our data clearly demonstrate that imatinib induces apoptosis and inhibits human colon adenocarcinoma cell migration. Therefore, this drug may have potential in colon cancer therapy in the future.