RGD Cyclopeptide Equipped with a Lysine-Engaging Salicylaldehyde Showing Enhanced Integrin Affinity and Cell Detachment Potency.

Salicylaldehyde (SA) derivatives are emerging as useful fragments to obtain reversible-covalent inhibitors interacting with the lysine residues of the target protein. Here the SA installation at the C terminus of an integrin-binding cyclopeptide, leading to enhanced ligand affinity for the receptor as well as stronger biological activity in cultured glioblastoma cells is reported.

The Importance of Detail: How Differences in Ligand Structures Determine Distinct Functional Responses in Integrin αv ß3.

Ligand-based control of protein functional motions can provide novel opportunities in the study of fundamental biological mechanisms and in the development of novel therapeutics. In this work we addressed the ligand-based modulation of integrin functions. Inhibitors of integrin αv ß3 are interesting anticancer agents but their molecular mechanisms are still unclear: Peptides and peptidomimetics characterized by the Arg-Gly-Asp (RGD) or isoAsp-Gly-Arg (isoDGR) binding motifs have shown controversial agonist/antagonist effects. We have investigated the differential mechanisms of integrin activation/deactivation by three distinct ligands (cyclo-RGDf(NMe)V (Cilengitide), cyclo[DKP3-RGD], cyclo[DKP3-isoDGR]; DKP=diketopiperazine) through a comparative analysis of ligand-controlled protein internal dynamics: Although RGD facilitates the onset of dynamic states leading to activation, isoDGR induces a diffuse rigidification of the complex consistent with antagonist activities. Computational predictions have been experimentally probed by showing that the antibody AP5, which is capable of recognizing the active form of integrin, binds specifically to the RGD complexes and not to the isoDGR complex, which supports opposite functional roles of the two motifs targeting the same binding site.

A dimeric bicyclic RGD ligand displays enhanced integrin binding affinity and strong biological effects on U-373 MG glioblastoma cells.

A C2-symmetric bicyclic peptide bearing two RGD motifs was developed as a dimeric ligand, and it displayed enhanced inhibition of ECM protein binding to purified integrin receptors as compared to monomeric RGD analogues. Moreover, the dimeric bicyclic ligand induced cell detachment and inhibited FAK phosphorylation in U-373 MG glioblastoma cells.

Extracellular Matrix Alterations in Metastatic Processes.

The extracellular matrix (ECM) is a complex network of extracellular-secreted macromolecules, such as collagen, enzymes and glycoproteins, whose main functions deal with structural scaffolding and biochemical support of cells and tissues. ECM homeostasis is essential for organ development and functioning under physiological conditions, while its sustained modification or dysregulation can result in pathological conditions. During cancer progression, epithelial tumor cells may undergo epithelial-to-mesenchymal transition (EMT), a morphological and functional remodeling, that deeply alters tumor cell features, leading to loss of epithelial markers (i.e., E-cadherin), changes in cell polarity and intercellular junctions and increase of mesenchymal markers (i.e., N-cadherin, fibronectin and vimentin). This process enhances cancer cell detachment from the original tumor mass and invasiveness, which are necessary for metastasis onset, thus allowing cancer cells to enter the bloodstream or lymphatic flow and colonize distant sites. The mechanisms that lead to development of metastases in specific sites are still largely obscure but modifications occurring in target tissue ECM are being intensively studied. Matrix metalloproteases and several adhesion receptors, among which integrins play a key role, are involved in metastasis-linked ECM modifications. In addition, cells involved in the metastatic niche formation, like cancer associated fibroblasts (CAF) and tumor associated macrophages (TAM), have been found to play crucial roles in ECM alterations aimed at promoting cancer cells adhesion and growth. In this review we focus on molecular mechanisms of ECM modifications occurring during cancer progression and metastatic dissemination to distant sites, with special attention to lung, liver and bone. Moreover, the functional role of cells forming the tumor niche will also be reviewed in light of the most recent findings.

Integrins in glioblastoma: Still an attractive target?

Integrin-mediated signaling pathways have been found to promote the invasiveness and survival of glioma cells by modifying the brain microenvironment to support the formation of the tumoral niche. A variety of cells in the niche express integrin receptors, including tumor-associated macrophages, fibroblasts, endothelial cells and pericytes. In particular, RGD-binding integrins have been demonstrated to have an important role in the epithelial-mesenchymal transition process, considered the first step in the infiltration of tissue by cancer cells and molecular markers of which have been found in glioma cells. In simultaneous research, Small Molecule Integrin Antagonists (SMIA) yielded initially promising results in in vitro and in vivo studies, leading to clinical trials to test their safety and efficacy in combination with other anticancer drugs in the treatment of several tumor types. The initially high expectations, especially because of their antiangiogenic activity, which appeared to be a winning strategy against GBM, were not confirmed and this cast serious doubts on the real benefits to be gained from the use of SMIA for the treatment of cancer in humans. In this review, we provide an overview of recent findings concerning the functional roles of integrins, especially RGD-binding integrins, in the processes related to glioma cells survival and brain tissue infiltration. These findings disclose a new scenario in which recently developed SMIA might become useful tools to hinder glioblastoma cell dissemination.

Cyclic isoDGR and RGD peptidomimetics containing bifunctional diketopiperazine scaffolds are integrin antagonists.

The cyclo[DKP-isoDGR] peptidomimetics 2-5, containing bifunctional diketopiperazine (DKP) scaffolds that differ in the configuration of the two DKP stereocenters and in the substitution at the DKP nitrogen atoms, were prepared and examined in vitro in competitive binding assays with purified αv ß3 and αv ß5 integrin receptors. IC50 values ranged from low nanomolar (ligand 3) to submicromolar with αv ß3 integrin. The biological activities of ligands cyclo[DKP3-RGD] 1 and cyclo[DKP3-isoDGR] 3, bearing the same bifunctional DKP scaffold and showing similar αV ß3 integrin binding values, were compared in terms of their cellular effects in human U373 glioblastoma cells. Compounds 1 and 3 displayed overlapping inhibitory effects on the FAK/Akt integrin activated transduction pathway and on integrin-mediated cell infiltration processes, and qualify therefore, despite the different RGD and isoDGR sequences, as integrin antagonists. Both compounds induced apoptosis in glioma cells after 72 hour treatment.

Small Molecules against Metastatic Tumors: Concrete Perspectives and Shattered Dreams.

Metastasis is the main cause of anti-cancer therapy failure, leading to unfavorable prognosis for patients. The true challenge to increase cancer patient life expectancy by making cancer a chronic disease with periodic but manageable relapses relies on the development of efficient therapeutic strategies specifically directed against key targets in the metastatic process. Traditional chemotherapy with classical alkylating agents, microtubule inhibitors, and antimetabolites has demonstrated its limited efficacy against metastatic cells due to their capacity to select chemo-resistant cell populations that undergo epithelial-to-mesenchymal transition (EMT), thus promoting the colonization of distant sites that, in turn, sustain the initial metastatic process. This scenario has prompted efforts aimed at discovering a wide variety of small molecules and biologics as potential anti-metastatic drugs directed against more specific targets known to be involved in the various stages of metastasis. In this short review, we give an overview of the most recent advances related to important families of antimetastatic small molecules: intracellular tyrosine kinase inhibitors, cyclin-dependent kinase inhibitors, KRAS inhibitors, and integrin antagonists. Although the majority of these small molecules are not yet approved and not available in the drug market, any information related to their stage of development could represent a precious and valuable tool to identify new targets in the endless fight against metastasis.

Effect of the simulated digestion process on the chlorogenic acid trapping activity against methylglyoxal.

Chlorogenic acids are hydroxycinnamic derivatives widespread in food or food by-products, known for their antioxidant effects and ability to interfere with the formation of advanced glycation end products (AGEs). AGEs are potential glycotoxins involved in age-related disorders, such as diabetes, cardiovascular diseases, and neurological disorders. The ability of chlorogenic acids to inhibit AGE formation under physiological conditions needs further investigation other than the in vitro assays. Therefore, in this study, the capacity of 5-caffeoylquinic acid (5-CQA) to effectively trap methylglyoxal (MGO), an AGE precursor compound also present in daily consumed food, was investigated by evaluating 5-CQA and MGO metabolic fate when subjected to digestion. Two different in vitro digestion approaches (static based on the Infogest protocol and dynamic based on a novel millifluidic gastrointestinal model) were set up and the samples collected at different steps of the static and dynamic processes were analyzed by a validated RP-HPLC-DAD method. The obtained results indicated that the gastrointestinal process strongly affected the 5-CQA capacity to trap MGO and its resulting antiglycation activity. Therefore, preliminary investigation using advanced in vitro tests, particularly dynamic approaches, should always be performed to predict the effect of the digestion process on the potential bioactives present in food, food by-products, or plant extracts.

cRGD-Functionalized Silk Fibroin Nanoparticles: A Strategy for Cancer Treatment with a Potent Unselective Naphthalene Diimide Derivative.

Developing drug delivery systems to target cytotoxic drugs directly into tumor cells is still a compelling need with regard to reducing side effects and improving the efficacy of cancer chemotherapy. In this work, silk fibroin nanoparticles (SFNs) have been designed to load a previously described cytotoxic compound (NDI-1) that disrupts the cell cycle by specifically interacting with non-canonical secondary structures of DNA. SFNs were then functionalized on their surface with cyclic pentapeptides incorporating the Arg-Gly-Asp sequence (cRGDs) to provide active targeting toward glioma cell lines that abundantly express ανß3 and ανß5 integrin receptors. Cytotoxicity and selective targeting were assessed by in vitro tests on human glioma cell lines U373 (highly-expressing integrin subunits) and D384 cell lines (low-expressing integrin subunits in comparison to U373). SFNs were of nanometric size (d50 less than 100 nm), round shaped with a smooth surface, and with a negative surface charge; overall, these characteristics made them very likely to be taken up by cells. The active NDI-1 was loaded into SFNs with high encapsulation efficiency and was not released before the internalization and degradation by cells. Functionalization with cRGDs provided selectivity in cell uptake and thus cytotoxicity, with a significantly higher cytotoxic effect of NDI-1 delivered by cRGD-SFNs on U373 cells than on D384 cells. This manuscript provides an in vitro proof-of-concept of cRGD-silk fibroin nanoparticles' active site-specific targeting of tumors, paving the way for further in vivo efficacy tests.

In Vitro Glioblastoma Models: A Journey into the Third Dimension.

Glioblastoma multiforme (GBM) is the most lethal primary brain tumor in adults, with an average survival time of about one year from initial diagnosis. In the attempt to overcome the complexity and drawbacks associated with in vivo GBM models, together with the need of developing systems dedicated to screen new potential drugs, considerable efforts have been devoted to the implementation of reliable and affordable in vitro GBM models. Recent findings on GBM molecular features, revealing a high heterogeneity between GBM cells and also between other non-tumor cells belonging to the tumoral niche, have stressed the limitations of the classical 2D cell culture systems. Recently, several novel and innovative 3D cell cultures models for GBM have been proposed and implemented. In this review, we first describe the different populations and their functional role of GBM and niche non-tumor cells that could be used in 3D models. An overview of the current available 3D in vitro systems for modeling GBM, together with their major weaknesses and strengths, is presented. Lastly, we discuss the impact of groundbreaking technologies, such as bioprinting and multi-omics single cell analysis, on the future implementation of 3D in vitro GBM models.

Fostering "Education": Do Extracellular Vesicles Exploit Their Own Delivery Code?

Extracellular vesicles (EVs), comprising large microvesicles (MVs) and exosomes (EXs), play a key role in intercellular communication, both in physiological and in a wide variety of pathological conditions. However, the education of EV target cells has so far mainly been investigated as a function of EX cargo, while few studies have focused on the characterization of EV surface membrane molecules and the mechanisms that mediate the addressability of specific EVs to different cell types and tissues. Identifying these mechanisms will help fulfill the diagnostic, prognostic, and therapeutic promises fueled by our growing knowledge of EVs. In this review, we first discuss published studies on the presumed EV "delivery code" and on the combinations of the hypothesized EV surface membrane "sender" and "recipient" molecules that may mediate EV targeting in intercellular communication. Then we briefly review the main experimental approaches and techniques, and the bioinformatic tools that can be used to identify and characterize the structure and functional role of EV surface membrane molecules. In the final part, we present innovative techniques and directions for future research that would improve and deepen our understandings of EV-cell targeting.

Berberine Photo-Activation Potentiates Cytotoxicity in Human Astrocytoma Cells through Apoptosis Induction.

Photodynamic therapy (PDT) has recently attracted interest as an innovative and adjuvant treatment for different cancers including malignant gliomas. Among these, Glioblastoma (GBM) is the most prevalent neoplasm in the central nervous system. Despite conventional therapeutic approaches that include surgical removal, radiation, and chemotherapy, GBM is characterized by an extremely poor prognosis and a high rate of recurrence. PDT is a physical process that induces tumor cell death through the genesis and accumulation of reactive oxygen species (ROS) produced by light energy interaction with a photosensitizing agent. In this contribution, we explored the potentiality of the plant alkaloid berberine (BBR) as a photosensitizing and cytotoxic agent coupled with a PDT scheme using a blue light source in human established astrocytoma cell lines. Our data mainly indicated for the combined BBR-PDT scheme a potent activation of the apoptosis pathway, through a massive ROS production, a great extent of mitochondria depolarization, and the sub-sequent activation of caspases. Altogether, these results demonstrated that BBR is an efficient photosensitizer agent and that its association with PDT may be a potential anticancer strategy for high malignant gliomas.

Silk Fibroin Nanoparticle Functionalization with Arg-Gly-Asp Cyclopentapeptide Promotes Active Targeting for Tumor Site-Specific Delivery.

Arg-Gly-Asp (RGD)-based cyclopentapeptides (cRGDs) have a high affinity towards integrin αvß3 and αvß5, which are overexpressed by many tumor cells. Here, curcumin-loaded silk fibroin nanoparticles (SFNs) have been functionalized on the surface with cRGD to provide active targeting towards tumor cells; a "click reaction" between the RGD-based cyclopentapeptide carrying an azide group and triple-bond-functionalized nanoparticles has been exploited. Both naked and functionalized SFNs were less than 200 nm in diameter and showed a round-shaped morphology but, after functionalization, SFNs increased in size and protein molecular weight. The functionalization of SFNs' surfaces with cRGD provided active internalization by cells overexpressing integrin receptors. At the lowest concentration tested (0.01 mg/mL), functionalized SFNs showed more effective uptake with respect to the naked by tumor cells that overexpress integrin receptors (but not for non-overexpressing ones). In contrast, at higher concentrations, the non-specific cell membrane protein-particle interactions are promoted and coupled to specific and target mediated uptake. Visual observations by fluorescence microscopy suggested that SFNs bind to integrin receptors on the cell surface and are then internalized by endocytosis. Overall, SFN functionalization provided in vitro active targeting for site-specific delivery of anticancer drugs, boosting activity and sparing healthy organs.

Endothelin B receptor antagonists block proliferation and induce apoptosis in glioma cells.

The proliferative and antiapoptotic actions of endothelin (ET)-1 in cancer cells have been documented and ET receptor antagonists have been exploited as potential anticancer drugs. Glioblastoma cell lines express both ETA and ETB receptors and previous works have shown that ETB receptors are involved in the proliferation of different cancer cell types. In this study we have investigated the effects of two structurally unrelated ETB receptor antagonists, BQ788 and A192621, on cell survival, proliferation and apoptosis in 1321-N1, U87 and IPDDCA2 glioma cell lines. BQ788 and A192621 reduced glioma cells viability and proliferation assessed by BrdU incorporation and cell cycle analysis by flow cytometry, while in contrast the ETA receptor antagonist BQ123 had no effect on cell survival. TUNEL assay and immunocytochemical experiments showed that BQ788 and A192621 trigger apoptotic processes mainly via activation of the intrinsic mitochondrial pathway involving caspase-9 activation, AIF release and cytochrome c translocation. Furthermore, treatment with ETB antagonists downregulates ERK- and p38MAPK-dependent pathways but does not affect VEGF mRNA levels. Our findings support the hypothesis that ETB antagonists represent a new promising therapeutic strategy for the treatment of high grade gliomas.

Integrin-Targeting Dye-Doped PEG-Shell/Silica-Core Nanoparticles Mimicking the Proapoptotic Smac/DIABLO Protein.

Cancer cells demonstrate elevated expression levels of the inhibitor of apoptosis proteins (IAPs), contributing to tumor cell survival, disease progression, chemo-resistance, and poor prognosis. Smac/DIABLO is a mitochondrial protein that promotes apoptosis by neutralizing members of the IAP family. Herein, we describe the preparation and in vitro validation of a synthetic mimic of Smac/DIABLO, based on fluorescent polyethylene glycol (PEG)-coated silica-core nanoparticles (NPs) carrying a Smac/DIABLO-derived pro-apoptotic peptide and a tumor-homing integrin peptide ligand. At low µM concentration, the NPs showed significant toxicity towards A549, U373, and HeLa cancer cells and modest toxicity towards other integrin-expressing cells, correlated with integrin-mediated cell uptake and consequent highly increased levels of apoptotic activity, without perturbing cells not expressing the α5 integrin subunit.

Development of Artificial Plasma Membranes Derived Nanovesicles Suitable for Drugs Encapsulation.

Extracellular vesicles (EVs) are considered as promising nanoparticle theranostic tools in many pathological contexts. The increasing clinical employment of therapeutic nanoparticles is contributing to the development of a new research area related to the design of artificial EVs. To this aim, different approaches have been described to develop mimetic biologically functional nanovescicles. In this paper, we suggest a simplified procedure to generate plasma membrane-derived nanovesicles with the possibility to efficiently encapsulate different drugs during their spontaneously assembly. After physical and molecular characterization by Tunable Resistive Pulse Sensing (TRPS) technology, transmission electron microscopy, and flow cytometry, as a proof of principle, we have loaded into mimetic EVs the isoquinoline alkaloid Berberine chloride and the chemotherapy compounds Temozolomide or Givinostat. We demonstrated the fully functionality of these nanoparticles in drug encapsulation and cell delivery, showing, in particular, a similar cytotoxic effect of direct cell culture administration of the anticancer drugs. In conclusion, we have documented the possibility to easily generate scalable nanovesicles with specific therapeutic cargo modifications useful in different drug delivery contexts.

Gene expression analysis of an EGFR indirectly related pathway identified PTEN and MMP9 as reliable diagnostic markers for human glial tumor specimens.

In this study the mRNA levels of five EGFR indirectly related genes, EGFR, HB-EGF, ADAM17, PTEN, and MMP9, have been assessed by Real-time PCR in a panel of 37 glioblastoma multiforme specimens and in 5 normal brain samples; as a result, in glioblastoma, ADAM17 and PTEN expression was significantly lower than in normal brain samples, and, in particular, a statistically significant inverse correlation was found between PTEN and MMP9 mRNA levels. To verify if this correlation was conserved in gliomas, PTEN and MMP9 expression was further investigated in an additional panel of 16 anaplastic astrocytoma specimens and, in parallel, in different human normal and astrocytic tumor cell lines. In anaplastic astrocytomas PTEN expression was significantly higher than in glioblastoma multiforme, but no significant correlation was found between PTEN and MMP9 expression. PTEN and MMP9 mRNA levels were also employed to identify subgroups of specimens within the different glioma malignancy grades and to define a gene expression-based diagnostic classification scheme. In conclusion, this gene expression survey highlighted that the combined measurement of PTEN and MMP9 transcripts might represent a novel reliable tool for the differential diagnosis of high-grade gliomas, and it also suggested a functional link involving these genes in glial tumors.

A new millifluidic-based gastrointestinal platform to evaluate the effect of simulated dietary methylglyoxal intakes.

The search for new in vitro modular bioreactors to simulate flow-mediated transport and absorption of chemical substances is a very important issue in toxicology and in drug and bioactive delivery research. The possibility of setting up a dynamic microenvironment leads to experimental conditions that may more closely resemble the in vivo model, especially to measure acute or chronic intake of compounds. We propose a novel millifluidic-based gastrointestinal model as an evolution of the common in vitro methods, to evaluate the exposure to exogenous methylglyoxal (MGO), a highly reactive α-oxoaldehyde responsible for the formation of advanced glycation end products involved in a number of chronic diseases. Gastric and intestinal cells were seeded into two different chambers, creating a multi-compartmental system where fluids dynamically interact with human gastric stromal and intestinal cells. MGO was tested at concentrations simulating different MGO food intakes (meal, daily, and hypothetically weekly). Cell viability was measured over time, and simultaneously, extracellular MGO was quantified by a validated RP-HPLC-DAD method to evaluate its absorption/metabolization. This new platform gives the opportunity to connect different compartments, allowing studying kinetic and metabolic profiles of different substances and representing a very promising alternative to animal models, at least in preliminary investigations.

Stem-Like Cancer Cells in a Dynamic 3D Culture System: A Model to Study Metastatic Cell Adhesion and Anti-Cancer Drugs.

Metastatic spread is mainly sustained by cancer stem cells (CSC), a subpopulation of cancer cells that displays stemness features. CSC are thought to be derived from cancer cells that undergo epithelial to mesenchymal transition (EMT), thus acquiring resistance to anoikis and anti-cancer drugs. After detachment from the primary tumor mass, CSC reach the blood and lymphatic flow, and disseminate to the target tissue. This process is by nature dynamic and in vitro models are quite far from the in vivo situation. In this study, we have tried to reproduce the adhesion process of CSC to a target tissue by using a 3D dynamic cell culture system. We isolated two populations of 3D tumor spheroids displaying CSC-like features from breast carcinoma (MCF-7) and lung carcinoma (A549) cell lines. Human fibroblasts were layered on a polystyrene scaffold placed in a dynamically perfused millifluidic system and then the adhesion of tumor cell derived from spheroids to fibroblasts was investigated under continuous perfusion. After 24 h of perfusion, we found that spheroid cells tightly adhered to fibroblasts layered on the scaffold, as assessed by a scanning electron microscope (SEM). To further investigate mechanisms involved in spheroid cell adhesion to fibroblasts, we tested the effect of three RGD integrin antagonists with different molecular structures on cell adhesion; when injected into the circuit, only cilengitide was able to inhibit cell adhesion to fibroblasts. Although our model needs further refinements and improvements, we do believe this study could represent a promising approach in improving current models to study metastatic infiltration in vitro and a new tool to screen new potential anti-metastatic molecules.

Glioblastoma under Siege: An Overview of Current Therapeutic Strategies.

Glioblastoma is known to be one of the most lethal and untreatable human tumors. Surgery and radiotherapy in combination with classical alkylating agents such as temozolomide offer little hope to escape a poor prognosis. For these reasons, enormous efforts are currently devoted to refine in vivo and in vitro models with the specific goal of finding new molecular aberrant pathways, suitable to be targeted by a variety of therapeutic approaches, including novel pharmaceutical formulations and immunotherapy strategies. In this review, we will first discuss current molecular classification based on genomic and transcriptomic criteria. Also, the state of the art in current clinical practice for glioblastoma therapy in the light of the recent molecular classification, together with ongoing phases II and III clinical trials, will be described. Finally, new pharmaceutical formulations such as nanoparticles and viral vectors, together with new strategies entailing the use of monoclonal antibodies, vaccines and immunotherapy agents, such as checkpoint inhibitors, will also be discussed.