Myo-Inositol Reverses TGF-ß1-Induced EMT in MCF-10A Non-Tumorigenic Breast Cells.

Epithelial-Mesenchymal Transition (EMT), triggered by external and internal cues in several physiological and pathological conditions, elicits the transformation of epithelial cells into a mesenchymal-like phenotype. During EMT, epithelial cells lose cell-to-cell contact and acquire unusual motility/invasive capabilities. The associated architectural and functional changes destabilize the epithelial layer consistency, allowing cells to migrate and invade the surrounding tissues. EMT is a critical step in the progression of inflammation and cancer, often sustained by a main driving factor as the transforming growth factor-ß1 (TGF-ß1). Antagonizing EMT has recently gained momentum as an attractive issue in cancer treatment and metastasis prevention. Herein, we demonstrate the capability of myo-inositol (myo-Ins) to revert the EMT process induced by TGF-ß1 on MCF-10A breast cells. Upon TGF-ß1 addition, cells underwent a dramatic phenotypic transformation, as witnessed by structural (disappearance of the E-cadherin-ß-catenin complexes and the emergence of a mesenchymal shape) and molecular modifications (increase in N-cadherin, Snai1, and vimentin), including the release of increased collagen and fibronectin. However, following myo-Ins, those changes were almost completely reverted. Inositol promotes the reconstitution of E-cadherin-ß-catenin complexes, decreasing the expression of genes involved in EMT, while promoting the re-expression of epithelial genes (keratin-18 and E-cadherin). Noticeably, myo-Ins efficiently inhibits the invasiveness and migrating capability of TGF-ß1 treated cells, also reducing the release of metalloproteinase (MMP-9) altogether with collagen synthesis, allowing for the re-establishment of appropriate cell-to-cell junctions, ultimately leading the cell layer back towards a more compact state. Inositol effects were nullified by previous treatment with an siRNA construct to inhibit CDH1 transcripts and, hence, E-cadherin synthesis. This finding suggests that the reconstitution of E-cadherin complexes is an irreplaceable step in the inositol-induced reversion of EMT. Overall, such a result advocates for the useful role of myo-Ins in cancer treatment.

Microgravity Modifies the Phenotype of Fibroblast and Promotes Remodeling of the Fibroblast-Keratinocyte Interaction in a 3D Co-Culture Model.

Microgravity impairs tissue organization and critical pathways involved in the cell-microenvironment interplay, where fibroblasts have a critical role. We exposed dermal fibroblasts to simulated microgravity by means of a Random Positioning Machine (RPM), a device that reproduces conditions of weightlessness. Molecular and structural changes were analyzed and compared to control samples growing in a normal gravity field. Simulated microgravity impairs fibroblast conversion into myofibroblast and inhibits their migratory properties. Consequently, the normal interplay between fibroblasts and keratinocytes were remarkably altered in 3D co-culture experiments, giving rise to several ultra-structural abnormalities. Such phenotypic changes are associated with down-regulation of α-SMA that translocate in the nucleoplasm, altogether with the concomitant modification of the actin-vinculin apparatus. Noticeably, the stress associated with weightlessness induced oxidative damage, which seemed to concur with such modifications. These findings disclose new opportunities to establish antioxidant strategies that counteract the microgravity-induced disruptive effects on fibroblasts and tissue organization.

Increase in motility and invasiveness of MCF7 cancer cells induced by nicotine is abolished by melatonin through inhibition of ERK phosphorylation.

Through activation of the ERK pathway, nicotine, in both normal MCF-10A and low-malignant breast cancer cells (MCF7), promotes increased motility and invasiveness. Melatonin antagonizes both these effects by inhibiting almost completely ERK phosphorylation. As melatonin has no effect on nonstimulated cells, it is likely that melatonin can counteract ERK activation only downstream of nicotine-induced activation. This finding suggests that melatonin hampers ERK phosphorylation presumably by targeting a still unknown intermediate factor that connects nicotine stimulation to ERK phosphorylation. Furthermore, downstream of ERK activation, melatonin significantly reduces fascin and calpain activation while restoring normal vinculin levels. Melatonin also counteracts nicotine effects by reshaping the overall cytoskeleton architecture and abolishing invasive membrane protrusion. In addition, melatonin decreases nicotine-dependent ROCK1/ROCK2 activation, thus further inhibiting cell contractility and motility. Melatonin actions are most likely attributable to ERK inhibition, although melatonin could display other ERK-independent effects, namely through a direct modulation of additional molecular and structural factors, including coronin, cofilin, and cytoskeleton components.

Nicotine increases colon cancer cell migration and invasion through epithelial to mesenchymal transition (EMT): COX-2 involvement.

Cigarette smoking is a recognized risk factor for colon cancer and nicotine, the principal active component of tobacco, plays a pivotal role in increasing colon cancer cell growth and survival. The aim of this study was to determine the effect of nicotine on cellular Caco-2 and HCT-8 migration and invasion, focusing on epithelial to mesenchymal transition (EMT) induction, and COX-2 pathway involvement. In both these cell lines, treatment with nicotine increased COX-2 expression and the release of its enzymatic product PGE2 . Moreover, nicotine-stimulated cells showed increased migratory and invasive behavior, mesenchymal markers up-regulation and epithelial markers down-regulation, nuclear translocation of the ß-catenin, increase of MMP-2 and MMP-9 activity, and enhanced NF-κB expression. Noticeably, all these effects are largely mediated by COX-2 activity, as simultaneous treatment of both cell lines with nicotine and NS-398, a selective COX-2 inhibitor, greatly reduced the number of migrating and invading cells and reverted nicotine-induced EMT. These findings emphasize that nicotine triggers EMT, leading hence to increased migration and invasiveness of colon cancer cells. Thereby, the use of COX-2 inhibitor drugs might likely counteract nicotine-mediated EMT effects on colon cancer development and progression.

Inositol induces mesenchymal-epithelial reversion in breast cancer cells through cytoskeleton rearrangement.

Inositol displays multi-targeted effects on many biochemical pathways involved in epithelial-mesenchymal transition (EMT). As Akt activation is inhibited by inositol, we investigated if such effect could hamper EMT in MDA-MB-231 breast cancer cells. In cancer cells treated with pharmacological doses of inositol E-cadherin was increased, ß-catenin was redistributed behind cell membrane, and metalloproteinase-9 was significantly reduced, while motility and invading capacity were severely inhibited. Those changes were associated with a significant down-regulation of PI3K/Akt activity, leading to a decrease in downstream signaling effectors: NF-kB, COX-2, and SNAI1. Inositol-mediated inhibition of PS1 leads to lowered Notch 1 release, thus contributing in decreasing SNAI1 levels. Overall, these data indicated that inositol inhibits the principal molecular pathway supporting EMT. Similar results were obtained in ZR-75, a highly metastatic breast cancer line. These findings are coupled with significant changes on cytoskeleton. Inositol slowed-down vimentin expression in cells placed behind the wound-healing edge and stabilized cortical F-actin. Moreover, lamellipodia and filopodia, two specific membrane extensions enabling cell migration and invasiveness, were no longer detectable after inositol addiction. Additionally, fascin and cofilin, two mandatory required components for F-actin assembling within cell protrusions, were highly reduced. These data suggest that inositol may induce an EMT reversion in breast cancer cells, suppressing motility and invasiveness through cytoskeleton modifications.

Multiwalled carbon nanotube buckypaper induces cell cycle arrest and apoptosis in human leukemia cell lines through modulation of AKT and MAPK signaling pathways.

MWCNT buckypaper (BP) shows physico-chemical and mechanical properties that make it potentially useful as a substrate in nano-bio interface research including in tissue engineering. When used as a scaffold material, BP comes into contact with host cells and surrounding tissues; therefore it is critical to determine its biocompatibility and interaction with living systems. The aim of this study was to investigate BP effects on cell growth, apoptosis and reactive oxygen species (ROS) production in three human leukemia cell lines HL-60, U-937 and K-562. BP was able to induce both the reduction of cell proliferation, associated with an arrest in G0/G1 phase of cell cycle and the increase of apoptosis in leukemic cell lines, thus exerting both cytostatic and cytotoxic effects. The growth inhibitory effect was likely mediated by the decrease of cyclins D, E, A, B1 levels and CDK4 expression; meanwhile, the apoptotic effect, not mediated by ROS production, was presumably due to the combined action of the survival and pro-apoptotic AKT and MAPK signal transduction pathways. These results raised the issue of biocompatibility of MWCNT BP for the creation of carbon nanotubes based scaffolds to utilize as prostheses in tissue engineering.