Monastrol suppresses invasion and metastasis in human colorectal cancer cells by targeting fascin independent of kinesin-Eg5 pathway.

Rearrangement of the actin cytoskeleton is a prerequisite for carcinoma cells to develop cellular protrusions, which are required for migration, invasion, and metastasis. Fascin is a key protein involved in actin bundling and is expressed in aggressive and invasive carcinomas. Additionally, fascin appears to be involved in tubulin-binding and microtubule rearrangement. Pharmacophoric-based in silico screening was performed to identify compounds with better fascin inhibitory properties than migrastatin, a gold-standard fascin inhibitor. We hypothesized that monastrol displays anti-migratory and anti-invasive properties via fascin blocking in colorectal cancer cell lines. Biophysical (thermofluor and ligand titration followed by fluorescence spectroscopy), biochemical (NMR), and cellular assays (MTT, invasion of human tissue), as well as animal model studies (zebrafish invasion) were performed to characterize the inhibitory effect of monastrol on fascin activity. In silico analysis revealed that monastrol is a potential fascin-binding compound. Biophysical and biochemical assays demonstrated that monastrol binds to fascin and interferes with its actin-bundling activity. Cell culture studies, including a 3D human myoma disc model, showed that monastrol inhibited fascin-driven cytoplasmic protrusions as well as invasion. In silico, confocal microscopy, and immunoprecipitation assays demonstrated that monastrol disrupted fascin-tubulin interactions. These anti-invasive effects were confirmed in vivo. In silico confocal microscopy and immunoprecipitation assays were carried out to test whether monastrol disrupted the fascin-tubulin interaction. This study reports, for the first time, the in vitro and in vivo anti-invasive properties of monastrol in colorectal tumor cells. The number and types of interactions suggest potential binding of monastrol across actin and tubulin sites on fascin, which could be valuable for the development of antitumor therapies.

Pdx1 Is Transcriptionally Regulated by EGR-1 during Nitric Oxide-Induced Endoderm Differentiation of Mouse Embryonic Stem Cells.

The transcription factor, early growth response-1 (EGR-1), is involved in the regulation of cell differentiation, proliferation, and apoptosis in response to different stimuli. EGR-1 is described to be involved in pancreatic endoderm differentiation, but the regulatory mechanisms controlling its action are not fully elucidated. Our previous investigation reported that exposure of mouse embryonic stem cells (mESCs) to the chemical nitric oxide (NO) donor diethylenetriamine nitric oxide adduct (DETA-NO) induces the expression of early differentiation genes such as pancreatic and duodenal homeobox 1 (Pdx1). We have also evidenced that Pdx1 expression is associated with the release of polycomb repressive complex 2 (PRC2) and P300 from the Pdx1 promoter; these events were accompanied by epigenetic changes to histones and site-specific changes in the DNA methylation. Here, we investigate the role of EGR-1 on Pdx1 regulation in mESCs. This study reveals that EGR-1 plays a negative role in Pdx1 expression and shows that the binding capacity of EGR-1 to the Pdx1 promoter depends on the methylation level of its DNA binding site and its acetylation state. These results suggest that targeting EGR-1 at early differentiation stages might be relevant for directing pluripotent cells into Pdx1-dependent cell lineages.

AICAR Stimulates the Pluripotency Transcriptional Complex in Embryonic Stem Cells Mediated by PI3K, GSK3ß, and ß-Catenin.

Pluripotent stem cells maintain the property of self-renewal and differentiate into all cell types under clear environments. Though the gene regulatory mechanism for pluripotency has been investigated in recent years, it is still not completely understood. Here, we show several signaling pathways involved in the maintenance of pluripotency. To investigate whether AMPK is involved in maintaining the pluripotency in mouse embryonic stem cells (mESCs) and elucidating the possible molecular mechanisms, implicated D3 and R1/E mESC lines were used in this study. Cells were cultured in the absence or presence of LIF and treated with 1 mM and 0.5 mM 5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside (AICAR), 2 mM metformin, compound C, and the PI3K inhibitor LY294002 for 24, 72, and 120 h. The levels of Nanog, Oct3/4, and REX1 and Brachyury, Notch2, and Gata4 mRNAs and Nanog or OCT3/4 protein levels were analyzed. Alkaline phosphatase and the cellular cycle were determined. The pGSK3ß, GSK3ß, p-ß-catenin, and ß-catenin protein levels were also investigated. We found that AMPK activators such as AICAR and metformin increase mRNA expression of pluripotency markers and decrease mRNA expression of differentiation markers in R1/E and D3 ES cells. AICAR increases phosphatase activity and arrests the cellular cycle in the G1 phase in these cells. We describe that AICAR effects were mediated by AMPK activation using a chemical inhibitor or by silencing this gene. AICAR effects were also mediated by PI3K, GSK3ß, and ß-catenin in R1/E ES cells. According to our findings, we provide a mechanism by which AICAR increases and maintains a pluripotency state through enhanced Nanog expression, involving AMPK/PI3K and p-GSK3ß Ser21/9 pathways backing up the AICAR function as a potential target for this drug controlling pluripotency. The highlights of this study are that AICAR (5-aminoimidazole-4-carboxamied-1-b-riboside), an AMP protein kinase (AMPK) activator, blocks the ESC differentiation and AMPK is a key enzyme for pluripotency and shows valuable data to clarify the molecular pluripotency mechanism.