The molecular basis of the anticancer effect of statins.

Statins, widely used cardiovascular drugs that lower cholesterol by inhibiting HMG-CoA reductase, have been increasingly recognized for their potential anticancer properties. This study elucidates the underlying mechanism, revealing that statins exploit Synthetic Lethality, a principle where the co-occurrence of two non-lethal events leads to cell death. Our computational analysis of approximately 37,000 SL pairs identified statins as potential drugs targeting genes involved in SL pairs with metastatic genes. In vitro validation on various cancer cell lines confirmed the anticancer efficacy of statins. This data-driven drug repurposing strategy provides a molecular basis for the anticancer effects of statins, offering translational opportunities in oncology.

Zwitterionic nanoparticles for thermally activated drug delivery in hyperthermia cancer treatment.

Hyperthermia is considered a promising strategy to boost the curative outcome of traditional chemotherapeutic treatments. However, this thermally mediated drug delivery is still affected by important limitations. First, the poor accumulation of the conventional anticancer formulations in the target site limits the bioavailability of the active ingredient and induces off-site effects. In addition, some tumoral scenarios, such as ovarian carcinoma, are characterized by cell thermotolerance, which induces tumoral cells to activate self-protecting mechanisms against high temperatures. To overcome these constraints, we developed thermoresponsive nanoparticles (NPs) with an upper critical solution temperature (UCST) to intracellularly deliver a therapeutic payload and release it on demand through hyperthermia stimulation. These NPs were synthesized via reversible addition-fragmentation chain transfer (RAFT) emulsion polymerization and combine polyzwitterionic stabilizing segments and an oligoester-based biodegradable core. By leveraging the pseudo-living nature of RAFT polymerization, important physicochemical properties of the NPs were controlled and optimized, including their cloud point (Tcp) and size. We have tuned the Tcp of NPs to match the therapeutic needs of hyperthermia treatments at 43 °C and tested the nanocarriers in the controlled delivery of paclitaxel, a common anticancer drug. The NPs released almost entirely the encapsulated drug only following 1 h incubation at 43 °C, whereas they retained more than 95% of the payload in the physiological environment (37 °C), thus demonstrating their efficacy as on-demand drug delivery systems. The administration of drug-loaded NPs to ovarian cancer cells led to therapeutic effects outperforming the conventional administration of non-encapsulated paclitaxel, which highlights the potential of the zwitterionic UCST-type NPs as an innovative hyperthermia-responsive drug delivery system.

Bone Marrow Mesenchymal Stem Cells Expanded Inside the Nichoid Micro-Scaffold: a Focus on Anti-Inflammatory Response.

Purpose: Mesenchymal stem cells (MSCs) represent a promising source for stem cell therapies in numerous diseases, including pediatric respiratory system diseases. Characterized by low immunogenicity, high anti-inflammatory, and immunoregulatory features, MSCs demonstrated an excellent therapeutic profile in numerous in vitro and preclinical models. MSCs reside in a specialized physiologic microenvironment, characterized by a unique combination of biophysical, biochemical, and cellular properties. The exploitation of the 3D micro-scaffold Nichoid, which simulates the native niche, enhanced the anti-inflammatory potential of stem cells through mechanical stimulation only, overcoming the limitation of biochemical and xenogenic growth factors application. Materials and Methods: In this work, we expanded pediatric bone marrow MSCs (BM-MSCs) inside the Nichoid and performed a complete cellular characterization with different approaches including viability assays, immunofluorescence analyses, RNA sequencing, and gene expression analysis. Results: We demonstrated that BM-MSCs inside the scaffold remain in a stem cell quiescent state mimicking the condition of the in vivo environment. Moreover, the gene expression profile of these cells shows a significant up-regulation of genes involved in immune response when compared with the flat control. Conclusion: The significant changes in the expression profile of anti-inflammatory genes could potentiate the therapeutic effect of BM-MSCs, encouraging the possible clinical translation for the treatment of pediatric congenital and acquired pulmonary disorders, including post-COVID lung manifestations. Lay Summary: Regenerative medicine is the research field integrating medicine, biology, and biomedical engineering. In this context, stem cells, which are a fundamental cell source able to regenerate tissues and restore damage in the body, are the key component for a regenerative therapeutic approach. When expanded outside the body, stem cells tend to differentiate spontaneously and lose regenerative potential due to external stimuli. For this reason, we exploit the scaffold named Nichoid, which mimics the in vivo cell niche architecture. In this scaffold, mesenchymal stem cells "feel at home" due to the three-dimensional mechanical stimuli, and our findings could be considered as an innovative culture system for the in vitro expansion of stem cells for clinical translation. Future Perspective: The increasing demand of safe and effective cell therapies projects our findings toward the possibility of improving cell therapies based on the use of BM-MSCs, particularly for their clinical translation in lung diseases.

The nuclear import of the transcription factor MyoD is reduced in mesenchymal stem cells grown in a 3D micro-engineered niche.

Smart biomaterials are increasingly being used to control stem cell fate in vitro by the recapitulation of the native niche microenvironment. By integrating experimental measurements with numerical models, we show that in mesenchymal stem cells grown inside a 3D synthetic niche both nuclear transport of a myogenic factor and the passive nuclear diffusion of a smaller inert protein are reduced. Our results also suggest that cell morphology modulates nuclear proteins import through a partition of the nuclear envelope surface, which is a thin but extremely permeable annular portion in cells cultured on 2D substrates. Therefore, our results support the hypothesis that in stem cell differentiation, the nuclear import of gene-regulating transcription factors is controlled by a strain-dependent nuclear envelope permeability, probably related to the reorganization of stretch-activated nuclear pore complexes.

Characterization of Mesenchymal Stem Cell Differentiation within Miniaturized 3D Scaffolds through Advanced Microscopy Techniques.

Three-dimensional culture systems and suitable substrates topographies demonstrated to drive stem cell fate in vitro by mechanical conditioning. For example, the Nichoid 3D scaffold remodels stem cells and shapes nuclei, thus promoting stem cell expansion and stemness maintenance. However, the mechanisms involved in force transmission and in biochemical signaling at the basis of fate determination are not yet clear. Among the available investigation systems, confocal fluorescence microscopy using fluorescent dyes enables the observation of cell function and shape at the subcellular scale in vital and fixed conditions. Contrarily, nonlinear optical microscopy techniques, which exploit multi-photon processes, allow to study cell behavior in vital and unlabeled conditions. We apply confocal fluorescence microscopy, coherent anti-Stokes Raman scattering (CARS), and second harmonic generation (SHG) microscopy to characterize the phenotypic expression of mesenchymal stem cells (MSCs) towards adipogenic and chondrogenic differentiation inside Nichoid scaffolds, in terms of nuclear morphology and specific phenotypic products, by comparing these techniques. We demonstrate that the Nichoid maintains a rounded nuclei during expansion and differentiation, promoting MSCs adipogenic differentiation while inhibiting chondrogenesis. We show that CARS and SHG techniques are suitable for specific estimation of the lipid and collagenous content, thus overcoming the limitations of using unspecific fluorescent probes.

Live-cell p53 single-molecule binding is modulated by C-terminal acetylation and correlates with transcriptional activity.

Live-cell microscopy has highlighted that transcription factors bind transiently to chromatin but it is not clear if the duration of these binding interactions can be modulated in response to an activation stimulus, and if such modulation can be controlled by post-translational modifications of the transcription factor. We address this question for the tumor suppressor p53 by combining live-cell single-molecule microscopy and single cell in situ measurements of transcription and we show that p53-binding kinetics are modulated following genotoxic stress. The modulation of p53 residence times on chromatin requires C-terminal acetylation-a classical mark for transcriptionally active p53-and correlates with the induction of transcription of target genes such as CDKN1a. We propose a model in which the modification state of the transcription factor determines the coupling between transcription factor abundance and transcriptional activity by tuning the transcription factor residence time on target sites.Both transcription binding kinetics and post-translational modifications of transcription factors are thought to play a role in the modulation of transcription. Here the authors use single-molecule tracking to directly demonstrate that p53 acetylation modulates promoter residence time and transcriptional activity.

MICAL2 is a novel human cancer gene controlling mesenchymal to epithelial transition involved in cancer growth and invasion.

The MICAL (Molecules Interacting with CasL) proteins catalyze actin oxidation-reduction reactions destabilizing F-actin in cytoskeletal dynamics. Here we show for the first time that MICAL2 mRNA is significantly over-expressed in aggressive, poorly differentiated/undifferentiated, primary human epithelial cancers (gastric and renal). Immunohistochemistry showed MICAL2-positive cells on the cancer invasive front and in metastasizing cancer cells inside emboli, but not at sites of metastasis, suggesting MICAL2 expression was 'on' in a subpopulation of primary cancer cells seemingly detaching from the tissue of origin, enter emboli and travel to distant sites, and was turned 'off' upon homing at metastatic sites. In vitro, MICAL2 knock-down resulted in mesenchymal to epithelial transition, reduction of viability, and loss of motility and invasion properties of human cancer cells. Moreover, expression of MICAL2 cDNA in MICAL2-depleted cells induced epithelial to mesenchymal transition. Altogether our data indicate that MICAL2 over-expression is associated with cancer progression and metastatic disease. MICAL2 might be an important regulator of epithelial to mesenchymal transition and therefore a promising target for anti-metastatic therapy.

Visualization of single proteins from stripped native cell membranes: a protocol for high-resolution atomic force microscopy.

Atomic force microscopy (AFM) proved to be able to obtain high-resolution three-dimensional images of single-membrane proteins, isolated, crystallized, or included in reconstructed model membranes. The extension of this technique to native systems, such as the protein immersed in a cell membrane, needs a careful manipulation of the biological sample to meet the experimental constraints for high-resolution AFM imaging. In this article, a general protocol for sample preparation is presented, based on the mechanical stretch of the cell membrane. The effectiveness for AFM imaging has been tested on the basis of an integrated optical and AFM approach and the proposed method has been applied to cells expressing cystic fibrosis transmembrane conductance regulator, a channel involved in cystic fibrosis, showing the possibility to identify and analyze single proteins in the plasma membrane.

Endothelial differentiation of mesenchymal stromal cells: when traditional biology meets mechanotransduction.

In the last few years, scientists have focused their attention on adult stem cells. It has long been known that stem cells are capable of renewing themselves and can generate multiple cell types. Today, it is demonstrated that stem cells are present in far more tissues and organs than once thought and that they are capable of developing into more kinds of cells than previously imagined. Adult human mesenchymal stromal cells (h-MSC) are prime candidates for many cell-based tissue-engineering applications thanks to their plasticity and easy accessibility. Despite such potential, their use in therapeutic application is still restricted because there are some open issues, which need to be deeply investigated. In this review, we focus on endothelial differentiation of h-MSCs. We comment on some controversies and show how these might arise from h-MSCs heterogeneity and morpho-functional variability. Finally, we highlight the role of extracellular mechanical cues and report on the mechanotransduction pathways activated by micro-/nanotopographies as promising alternative differentiation inducers.

Dextran-catechin conjugate: a potential treatment against the pancreatic ductal adenocarcinoma.

PURPOSE: A polysaccharide-flavonoid conjugate was developend and proposed for the treatment of pancreatic ductal adenocarcinoma (PDAC). METHODS: The conjugate was synthesized by free radical grafting reaction between catechin and dextran. The chemical characterization of the conjugate was obtained by UV-Vis, 1H-NMR, FT-IR and GPC analyses, while the functionalization degree was determined by the Folin-Ciocalteu assay. The biological activity of the catechin-dextran conjugate was tested on two different cell lines derived from human pancreatic cancer (MIA PaCa-2 and PL45 cells), and the toxicity towards human pancreatic nestin-expressing cells evaluated. RESULTS: Both the cancer cell lines are killed when exposed to the conjugate, and undergo apoptosis after the incubation with catechin-dextran which resulted more effective in killing pancreatic tumor cells compared to the catechin alone. Moreover, our experimental data indicate that the conjugate was less cytotoxic to human pancreatic nestin-expressing cells which are considered a good model of non-neoplastic pancreatic cells. CONCLUSION: The suitability of newly synthesized Dextran-Catechin conjugate in the treatment of PDAC was proved confirming the high potential application of the proposed macromolecula system in the cancer therapy.

Autocrine abscisic acid plays a key role in quartz-induced macrophage activation.

Inhalation of quartz induces silicosis, a lung disease where alveolar macrophages release inflammatory mediators, including prostaglandin-E(2) (PGE(2)) and tumor necrosis factor α (TNF-α). Here we report the pivotal role of abscisic acid (ABA), a recently discovered human inflammatory hormone, in silica-induced activation of murine RAW264.7 macrophages and of rat alveolar macrophages (AMs). Stimulation of both RAW264.7 cells and AMs with quartz induced a significant increase of ABA release (5- and 10-fold, respectively), compared to untreated cells. In RAW264.7 cells, autocrine ABA released after quartz stimulation sequentially activates the plasma membrane receptor LANCL2 and NADPH oxidase, generating a Ca(2+) influx resulting in NFκ B nuclear translocation and PGE(2) and TNF-α release (3-, 2-, and 3.5-fold increase, respectively, compared to control, unstimulated cells). Quartz-stimulated RAW264.7 cells silenced for LANCL2 or preincubated with a monoclonal antibody against ABA show an almost complete inhibition of NFκ B nuclear translocation and PGE(2) and TNF-α release compared to controls electroporated with a scramble oligonucleotide or preincubated with an unrelated antibody. AMs showed similar early and late ABA-induced responses as RAW264.7 cells. These findings identify ABA and LANCL2 as key mediators in quartz-induced inflammation, providing possible new targets for antisilicotic therapy.

Regulation of human mesenchymal stem cell functions by an autocrine loop involving NAD+ release and P2Y11-mediated signaling.

In several cell types, a regulated efflux of NAD(+) across Connexin 43 hemichannels (Cx43 HC) can occur, and extracellular NAD(+) (NAD(+)(e)) affects cell-specific functions. We studied the capability of bone marrow-derived human mesenchymal stem cells (MSC) to release intracellular NAD(+) through Cx43 HC. NAD(+) efflux, quantified by a sensitive enzymatic cycling assay, was significantly upregulated by low extracellular Ca(2+) (5-6-fold), by shear stress (13-fold), and by inflammatory conditions (3.1- and 2.5-fold in cells incubated with lipopolysaccharide (LPS) or at 39°C, respectively), as compared with untreated cells, whereas it was downregulated in Cx43-siRNA-transfected MSC (by 53%) and by cell-to-cell contact (by 45%). Further, we show that NAD(+)(e) activates the purinergic receptor P2Y(11) and a cyclic adenosin monophosphate (cAMP)/cyclic ADP-ribose/[Ca(2+)](i) signaling cascade, involving the opening, unique to MSC, of L-type Ca(2+) channels. Extracellular NAD(+) enhanced nuclear translocation of cAMP/Ca(2+)-dependent transcription factors. Moreover, NAD(+), either extracellularly added or autocrinally released, resulted in stimulation of MSC functions, including proliferation, migration, release of prostaglandin E(2) and cytokines, and downregulation of T lymphocyte proliferation compared with controls. No detectable modifications of MSC markers and of adipocyte or osteocyte differentiation were induced by NAD(+)(e). Controls included Cx43-siRNA transfected and/or NAD(+)-glycohydrolase-treated MSC (autocrine effects), and NAD(+)-untreated or P2Y(11)-siRNA-transfected MSC (exogenous NAD(+)). These findings suggest a potential beneficial role of NAD(+)(e) in modulating MSC functions relevant to MSC-based cell therapies.

Diadenosine homodinucleotide products of ADP-ribosyl cyclases behave as modulators of the purinergic receptor P2X7.

ADP-ribosyl cyclases from both vertebrates and invertebrates were previously shown to produce two isomers of P1,P2 diadenosine 5',5'"-P1, P2-diphosphate, P18 and P24, from cyclic ADP-ribose (cADPR) and adenine. P18 and P24 are characterized by an unusual N-glycosidic linkage in one of the adenylic mononucleotides (Basile, G., Taglialatela-Scafati, O., Damonte, G., Armirotti, A., Bruzzone, S., Guida, L., Franco, L., Usai, C., Fattorusso, E., De Flora, A., and Zocchi, E. (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 14509-14514). P24, but not P18, proved to increase the intracellular Ca(2+) concentration ([Ca(2+)](i)) in HeLa cells and to negatively affect mitochondrial function. Here we show that micromolar P24, but not P18, triggers a slow and sustained influx of extracellular Ca(2+) through the opening of the purinergic receptor/channel P2X7. On the other hand, P18 inhibits the Ca(2+) influx induced by 0.6 mm ATP in HEK293 cells stably transfected with P2X7, with an IC(50) of approximately 1 mum. Thus, P18 is devoid of intrinsic P2X7 stimulatory activity and behaves as an ATP antagonist. A P2X7-mediated increase of the basal [Ca(2+)](i) has been demonstrated to negatively affect Schwann cell (SC) function in rats with the inherited, peripheral neuropathy Charcot-Marie-Tooth 1A (CMT1A) (Nobbio, L., Sturla, L., Fiorese, F., Usai, C., Basile, G., Moreschi, I., Benvenuto, F., Zocchi, E., De Flora, A., Schenone, A., and Bruzzone S. (2009) J. Biol. Chem. 284, 23146-23158). Preincubation of CMT1A SC with 200 nm P18 restored the basal [Ca(2+)](i) to values similar to those recorded in wild-type SC. These results identify P18 as a new P2X7 antagonist, potentially useful in the treatment of CMT1A.

Ex vivo expansion of human circulating myogenic progenitors on cluster-assembled nanostructured TiO2.

Ex vivo expansion of hematopoietic stem cells has been explored in the fields of stem cell biology, gene therapy and clinical transplantation. Recently, we demonstrated the existence of a circulating myogenic progenitor expressing the CD133 antigen. The relative inability of circulating CD133+ stem cells to reproduce themselves ex vivo imposes substantial limitations on their use for clinical applications in muscular dystrophies. Here we report that the use of cluster-assembled nanostructured titanium dioxide (ns-TiO(2)) substrates, in combination with cytokine enriched medium, enables high-level expansion of circulating CD133+ stem cells in vitro. Furthermore, we demonstrate that expanded circulating CD133+ stem cells retain their in vitro capacity to differentiate into myogenic cells. The exploitation of cluster-assembled ns-TiO(2) substrates for the expansion of CD133+ stem cells in vitro could therefore make the clinical application of these stem cells for the treatment of muscle diseases practical.

LANCL2 is necessary for abscisic acid binding and signaling in human granulocytes and in rat insulinoma cells.

Abscisic acid (ABA) is a plant hormone regulating fundamental physiological functions in plants, such as response to abiotic stress. Recently, ABA was shown to be produced and released by human granulocytes, by insulin-producing rat insulinoma cells, and by human and murine pancreatic beta cells. ABA autocrinally stimulates the functional activities specific for each cell type through a receptor-operated signal transduction pathway, sequentially involving a pertussis toxin-sensitive receptor/G-protein complex, cAMP, CD38-produced cADP-ribose and intracellular calcium. Here we show that the lanthionine synthetase C-like protein LANCL2 is required for ABA binding on the membrane of human granulocytes and that LANCL2 is necessary for transduction of the ABA signal into the cell-specific functional responses in granulocytes and in rat insulinoma cells. Co-expression of LANCL2 and CD38 in the human HeLa cell line reproduces the ABA-signaling pathway. Results obtained with granulocytes and CD38(+)/LANCL2(+) HeLa transfected with a chimeric G-protein (G alpha(q/i)) suggest that the pertussis toxin-sensitive G-protein coupled to LANCL2 is a G(i). Identification of LANCL2 as a critical component of the ABA-sensing protein complex will enable the screening of synthetic ABA antagonists as prospective new anti-inflammatory and anti-diabetic agents.