Down-regulation of 21A Alu RNA as a tool to boost proliferation maintaining the tissue regeneration potential of progenitor cells

21A is an Alu non-coding (nc) RNA transcribed by RNA polymerase (pol) III. While investigating the biological role of 21A ncRNA we documented an inverse correlation between its expression level and the rate of cell proliferation. The downregulation of this ncRNA not only caused a boost in cell proliferation, but was also associated to a transient cell dedifferentiation, suggesting a possible involvement of this RNA in cell dedifferentiation/reprogramming. In this study, we explored the possibility to enhance proliferation and dedifferentiation of cells of interest, by 21A down-regulation, using a mixture of chemically modified Anti-21A RNAs. Our results confirmed the validity of this approach that allows the amplification of specific cell populations, in a controlled manner and without inducing permanent effects. In addition to induce cell proliferation, the procedure did not decrease the tissue regeneration potential of progenitor cells in two different cell systems.</p

Minimalist Hybrid Ligand/Receptor-Based Pharmacophore Model for CXCR4 Applied to a Small-Library of Marine Natural Products Led to the Identification of Phidianidine A as a New CXCR4 Ligand Exhibiting Antagonist Activity

Here, we present a minimal hybrid ligand/receptor-based pharmacophore model (PM) for CXCR4, a chemokine receptor deeply involved in several pathologies, such as HIV infection, rheumatoid arthritis, cancer development/progression, and metastasization. This model, considerably simpler than those thus far proposed for this receptor, has been used to search for new CXCR4 inhibitors in a small marine natural product library available at ICB-CNR Institute (Pozzuoli, NA, Italy), since natural products, with their naturally selected chemical and functional diversity, represent a rich source of bioactive scaffolds; computational approaches allow searching for new scaffolds with a minimal waste of possibly precious natural product samples; and our “stripped-down” model substantially increases the probabilities of identifying potential hits even in small-sized libraries. This search, also validated by a systematic virtual screening of the same library, has led to the identification of a new CXCR4 ligand, phidianidine A (PHIA). Docking studies supported PHIA activity and suggested its possible binding modes to CXCR4. Using the CXCR4-expressing/CXCR7-negative GH4C1 cell line we show that PHIA inhibits CXCL12-induced DNA synthesis, cell migration, and ERK1/2 activation. The specificity of these effects was confirmed by the lack of PHIA activity in GH4C1 cells, in which siRNA highly reduces CXCR4 expression and the lack of cytoxicity of PHIA was also verified. Thus, PHIA represents a promising lead for a new family of CXCR4 modulators with wide margins of improvement in potency and specificity offered by the small and very simple underlying PM

<i>R</i>. <i>graveolens</i> a.e. induces apoptosis in A1 cells.

<p>(A) Cell cycle was analyzed by means of Tali image-based cytometry on proliferating A1 cells in control conditions (dark grey) and 48h after 1mg/ml <i>R</i>. <i>graveolens</i> a.e. treatment (light grey) *p<0.01 <i>vs</i> controls (B) Number of apoptotic nuclei/100 cells treated (R48) or not (CTRL) with 1 mg/ml <i>R</i>. <i>graveolens</i> a.e. for 48 hours. *p<0.01 <i>vs</i> controls. (C-E) Caspase 3 activity expressed as absorbance at 400 nm in A1 cells (C), U87MG cells (D) and C6 cells (E) treated with vehicle (CTRL), 1mg/ml <i>R</i>. <i>graveolens</i> a.e. for 24 (R24) or 48 (R48) hours, 10μM PD98059 in combination with ml <i>R</i>. <i>graveolens</i> a.e. for 48 hours (PD+R48), 1μM wortmannin in combination with ml <i>R</i>. <i>graveolens</i> a.e. for 48 hours (W+R48) or the combination of the two inhibitors (PD+W+R48) for 48 hours. *p<0.01 <i>vs</i> control conditions.</p

Temozolomide and cisplatin affect viability of proliferating and differentiated A1.

<p>(A-B) MTT assay on proliferating A1 cells treated with increasing concentrations of temozolomide (A) and cisplatin (B) for 48 (♦) and 72 (■) hours; *p<0.01 <i>vs</i> controls; (C-D) MTT assay on differentiated A1 cells treated with increasing concentrations of temozolomide (C) and cisplatin (D) for 48 (♦) and 72 (■) hours; *p<0.01 <i>vs</i> controls.</p

Rutin does not influence proliferating A1 cells viability.

<p>Trypan blue exclusion test on proliferating A1 cells treated with or without (control) increasing concentrations of rutin (3μg/ml, 30μg/ml and 300μg/ml) for 24, 48 and 72h.</p

<i>R</i>. <i>graveolens</i> a.e. induces cell death in proliferating but not in differentiated A1 cells.

<p>(A) Microphotographs of the mouse mesencephalic embryonic cell line A1 mes c-myc (A1). They are proliferating/undifferentiated in the presence of serum (left panel) but acquire a neuronal phenotype upon serum withdrawal and stimulation with cAMP (right panel). (B) MTT assay on proliferating A1 cells in control conditions (♦) or treated with 1mg/ml <i>R</i>. <i>graveolens</i> a.e. (■) for 24, 48, 72 and 96 hours. *p<0.01 <i>vs</i> controls. (C) MTT assay on differentiated A1 cells in control conditions (♦) or treated with 1mg/ml <i>R</i>. <i>graveolens</i> a.e. (■) for 48 and 72 hours *p<0.01 <i>vs</i> controls. (D) Trypan blue exclusion test on proliferating A1 cells treated (light grey) or not (dark grey) with 1mg/ml <i>R</i>. <i>graveolens</i> a.e. for 24, 48 and 72h; *p<0.01 <i>vs</i> controls. (E) Trypan blue exclusion test on differentiated A1 cells treated (light gray) or not (dark gray) with 1mg/ml <i>R</i>. <i>graveolens</i> a.e. for 48 and 72h; *p<0.01 <i>vs</i> controls.</p

<i>R</i>. <i>graveolens</i> extract induces cell death of U87MG human glioma cells.

<p>(A) MTT assay on proliferating U87MG human glioma cells treated with vehicle (♦) or with 1mg/ml <i>R</i>. <i>graveolens</i> a.e. (■) for 24, 48, 72, 96h, *p<0,01 vs control conditions. (B) Trypan blue exclusion test on U87MG glioma cells treated (light gray) or not (dark gray) with 1mg/ml <i>R</i>. <i>graveolens</i> a.e. for 24, 48 and 72h; *<0,01 <i>vs</i> control conditions.</p

<i>R</i>. <i>graveolens</i> a.e. is able to induce ERK1/2 phosphorylation in glioma and in A1 proliferating neural cells.

<p>Western blotting detection of p-ERK1/2 and ERK1/2 proteins in C6 glioma cells (A), in U138 glioma cells (B), in A1 cells (C) and in U87MG (D) treated with 1mg/ml <i>R</i>. <i>graveolens</i> a.e. for 5, 10, 30 and 60 minutes. Two specific bands are observed respectively at 44 and 42 kDa. Each blot is representative of three separate experiments. The graphs show the relative quantitation of p-ERK1/2 and ERK1/2 in the different cell lines. Data are expressed as ratios of p-ERK/ERK. Asterisks represent p<0.05 <i>vs</i> controls. (G-H) MTT assay in U87MG cells (E), A1 cells (F), C6 glioma cells (G) and U138 cells (H) treated for 48 hours with 1mg/ml <i>R</i>. <i>graveolens</i> a.e. (R48), 10μM PD98059 (PD) or in combination (PD+R48); *p<0.01 <i>vs</i> control conditions.°p<0.05 <i>vs</i> R48.</p

Supplementary Data from Overexpression of Stromal Cell–Derived Factor 1 and Its Receptor CXCR4 Induces Autocrine/Paracrine Cell Proliferation in Human Pituitary Adenomas

Supplementary Data from Overexpression of Stromal Cell–Derived Factor 1 and Its Receptor CXCR4 Induces Autocrine/Paracrine Cell Proliferation in Human Pituitary Adenoma

Figure S1 from Mutual Influence of ROS, pH, and CLIC1 Membrane Protein in the Regulation of G₁–S Phase Progression in Human Glioblastoma Stem Cells

IAA-94 blockade induces G1 cell cycle arrest in GBM cancer stem cells.</p