STAT3 is an oncoprotein overexpressed in different types of tumors, including prostate cancer (PCa), and its activity is modulated by a variety of post-translational modifications (PTMs). Prostate cancer represents the most common cancer diagnosed in men, and each phase of tumor progression displays specific cellular conditions: inflammation is predominant in tumor’s early stage, whereas oxidative stress is typical of clinically advanced PCa. The aim of this research is to assess the correspondence between the stimulus-specificity of STAT3 PTMs and definite STAT3-mediated transcriptional programs, in order to identify new suitable pharmacological targets for PCa treatment. Experiments were performed on less-aggressive LNCaP and more aggressive DU-145 cell lines, simulating inflammatory and oxidative-stress conditions. Cellular studies confirmed pY705-STAT3 as common denominator of all STAT3-mediated signaling. In addition, acK685-STAT3 was found in response to IL-6, whereas glutC328/542-STAT3 and pS727-STAT3 occurred upon tert-butyl hydroperoxyde (tBHP) treatment. Obtained results also provided evidence of an interplay between STAT3 PTMs and specific protein interactors such as P300 and APE1/Ref-1. In accordance with these outcomes, mRNA levels of STAT3-target genes seemed to follow the differing STAT3 PTMs. These results highlighted the role of STAT3 and its PTMs as drivers in the progression of PCa.

Cell culture is usually performed in 2D polymer surfaces; however, several studies are conducted with the aim to screen functional coating molecules to find substrates more suitable for cell adhesion and proliferation. The aim of this manuscript is to compare the cell adhesion and cytoskeleton organization of different cell types on different surfaces. Human primary fibroblasts, chondrocytes and osteoblasts isolated from patients undergoing surgery were seeded on polystyrene, poly-d-lysine-coated glass and titanium carbide slides and left to grow for several days. Then their cytoskeleton was analyzed, both by staining cells with phalloidin, which highlights actin fibers, and using Atomic Force Microscopy. We also monitored the production of Fibroblast Growth Factor-2, Bone Morphogenetic Protein-2 and Osteocalcin, using ELISA, and we highlighted production of Collagen type I in fibroblasts and osteoblasts and Collagen type II in chondrocytes by immunofluorescences. Fibroblasts, chondrocytes and osteoblasts showed both an improved proliferative activity and a good adhesion ability when cultured on titanium carbide slides, compared to polystyrene and poly-d-lysine-coated glass. In conclusion, we propose titanium carbide as a suitable surface to cultivate cells such as fibroblasts, chondrocytes and osteoblasts, allowing the preservation of their differentiated state and good adhesion properties.

The glucosamine derivative 2-(N-Acetyl)-L-phenylalanylamido-2-deoxy-$\beta$-D-glucose (NAPA), was shown to inhibit the kinase activity of IKK$\alpha$, one of the two catalytic subunits of IKK complex, decreasing the inflammatory status in osteoarthritis chondrocytes. In the present work we have investigated the inhibition mechanism of IKK$\alpha$ by NAPA by combining computational simulations, in vitro assays and Mass Spectrometry (MS) technique. The kinase in vitro assay was conducted using a recombinant IKK$\alpha$ and IKKtide, a 20 amino acid peptide substrate derived from IkB$\alpha$ kinase protein and containing the serine residues Ser32 and Ser36. Phosphorylated peptide production was measured by Ultra Performance Liquid Chromatography coupled with Mass Spectrometry (UPLC-MS), and the atomic interaction between IKK$\alpha$ and NAPA has been studied by molecular docking and Molecular Dynamics (MD) approaches. Here we report that NAPA was able to inhibit the IKK$\alpha$ kinase activity with an IC₅₀ of 0.5 mM, to decrease the K_m value from 0.337 mM to 0.402 mM and the V_max from 0.0257 mM·min${}^{-1}$ to 0.0076 mM·min${}^{-1}$. The computational analyses indicate the region between the KD, ULD and SDD domains of IKK$\alpha$ as the optimal binding site explored by NAPA. Biochemical data indicate that there is a non-significant difference between K_m and K_i whereas there is a statistically significant difference between the two V_max values. This evidence, combined with computational results, consistently indicates that the inhibition is non-competitive, and that the NAPA binding site is different than that of ATP or IKKtide.

TPX2, a protein involved in mitosis, is considered a good marker for actively proliferating tissues, highly expressed in a number of cancer cells. We show the presence of high-affinity binding site for STAT3 in the 5′-flanking region of the Tpx2 gene, which is in vivo bound by activated STAT3. A specific STAT3 peptide inhibitor represses the expression of the Tpx2 gene and inhibits the binding of STAT3 to its consensus sequence in human cell lines where STAT3 is activated. These results indicate that activated STAT3 contributes to the over-expression of Tpx2 through the binding to an enhancer site.