Enhanced Delivery of 5-Aminolevulinic Acid by Lecithin Invasomes in 3D Melanoma Cancer Model.

Photodynamic therapy (PDT) is a noninvasive therapeutic approach for the treatment of skin cancer and diseases. 5-Aminolevulinic acid is a prodrug clinically approved for PDT. Once internalized by cancer cells, it is rapidly metabolized to the photosensitizer protoporphyrin IX, which under the proper light irradiation, stimulates the deleterious reactive oxygen species (ROS) production and leads to cell death. The high hydrophilicity of 5-aminolevulinic acid limits its capability to cross the epidermis. Lipophilic derivatives of 5-aminolevulinic acid only partly improved skin penetration, thus making its incorporation into nanocarriers necessary. Here we have developed and characterized 5-aminolevulinic acid loaded invasomes made of egg lecithin, either 1,2-dilauroyl-sn-glycero-3-phosphocholine or 1,2-dioleoyl-sn-glycero-3-phosphocholine, and the terpene limonene. The obtained invasomes are highly thermostable and display a spherical morphology with an average size of 150 nm and an encapsulation efficiency of 80%; moreover, the ex vivo epidermis diffusion tests established that nanovesicles containing the terpene led to a much higher skin penetration (up to 80% in 3 h) compared to those without limonene and to the free fluorescent tracer (less than 50%). Finally, in vitro studies with 2D and 3D human cell models of melanoma proved the biocompatibility of invasomes, the enhanced intracellular transport of 5-aminolevulinic acid, its ability to generate ROS upon irradiation, and consequently, its antiproliferative effect. A simplified scaffold-based 3D skin model containing melanoma spheroids was also prepared. Considering the results obtained, we conclude that the lecithin invasomes loaded with 5-aminolevulinic acid have a good therapeutic potential and may represent an efficient tool that can be considered a valid alternative in the topical treatment of melanoma and other skin diseases.

Aquaponics-Derived Tilapia Skin Collagen for Biomaterials Development.

Collagen is one of the most widely used biomaterials in health-related sectors. The industrial production of collagen mostly relies on its extraction from mammals, but several issues limited its use. In the last two decades, marine organisms attracted interest as safe, abundant, and alternative source for collagen extraction. In particular, the possibility to valorize the huge quantity of fish industry waste and byproducts as collagen source reinforced perception of fish collagen as eco-friendlier and particularly attractive in terms of profitability and cost-effectiveness. Especially fish byproducts from eco-sustainable aquaponics production allow for fish biomass with additional added value and controlled properties over time. Among fish species, Oreochromis niloticus is one of the most widely bred fish in large-scale aquaculture and aquaponics systems. In this work, type I collagen was extracted from aquaponics-raised Tilapia skin and characterized from a chemical, physical, mechanical, and biological point of view in comparison with a commercially available analog. Performed analysis confirmed that the proprietary process optimized for type I collagen extraction allowed to isolate pure native collagen and to preserve its native conformational structure. Preliminary cellular studies performed with mouse fibroblasts indicated its optimal biocompatibility. All data confirmed the eligibility of the extracted Tilapia-derived native type I collagen as a biomaterial for healthcare applications.

Poly(l-lactide-co-caprolactone-co-glycolide)-Based Nanoparticles as Delivery Platform: Effect of the Surfactants on Characteristics and Delivery Efficiency.

Polymeric nanoparticles made of the copolymer Poly(L-lactide-co-caprolactone-co-glycolide) were prepared using the solvent evaporation method. Two different surfactants, polyvinyl alcohol and dextran, and a mixture of the two were employed. The three types of nanoparticles were used as hosting carriers of two chemotherapeutic drugs, the hydrophilic doxorubicin and the hydrophobic SN-38. The morphostructural characterization showed similar features for the three types of nanoparticles, while the drug encapsulation efficiency indicated that the dextran-based systems are the most effective with both drugs. Cellular studies with breast cancer cells were performed to compare the delivery capability and the cytotoxicity profile of the three nanosystems. The results show that the unloaded nanoparticles are highly biocompatible at the administered concentrations and confirmed that dextran-coated nanoparticles are the most efficient vectors to release the two drugs, exerting cytotoxic activity. PVA, on the other hand, shows limited drug release in vitro, probably due to strong interactions with both drugs. Data also show the release is more efficient for doxorubicin than for SN-38; indeed, the doxorubicin IC50 value for the dextran-coated nanoparticles was about 35% lower than the free drug. This indicates that these nanocarriers are suitable candidates to deliver hydrophilic drugs while needing further modification to host hydrophobic molecules.

A thermo-sensitive chitosan/pectin hydrogel for long-term tumor spheroid culture.

Hydrogels represent a key element in the development of in vitro tumor models, by mimicking the typical 3D tumor architecture in a physicochemical manner and allowing the study of tumor mechanisms. Here we developed a thermo-sensitive, natural polymer-based hydrogel, where chitosan and pectin were mixed and, after a weak base-induced chitosan gelation, a stable semi-Interpenetrating Polymer Network formed. This resulted thermo-responsive at 37 °C, injectable at room temperature, stable up to 6 weeks in vitro, permeable to small/medium-sized molecules (3 to 70 kDa) and suitable for cell-encapsulation. Tunable mechanical and permeability properties were obtained by varying the polymer content. Optimized formulations successfully supported the formation and growth of human colorectal cancer spheroids up to 44 days of culture. The spheroid dimension and density were influenced by the semi-IPN stiffness and permeability. These encouraging results would allow the implementation of faithful tumor models for the study and development of personalized oncological treatments.

Oxidative Stress and Multi-Organel Damage Induced by Two Novel Phytocannabinoids, CBDB and CBDP, in Breast Cancer Cells.

Over the last few years, much attention has been paid to phytocannabinoids derived from Cannabis for their therapeutic potential. Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD) are the most abundant compounds of the Cannabis sativa L. plant. Recently, novel phytocannabinoids, such as cannabidibutol (CBDB) and cannabidiphorol (CBDP), have been discovered. These new molecules exhibit the same terpenophenolic core of CBD and differ only for the length of the alkyl side chain. Roles of CBD homologs in physiological and pathological processes are emerging but the exact molecular mechanisms remain to be fully elucidated. Here, we investigated the biological effects of the newly discovered CBDB or CBDP, compared to the well-known natural and synthetic CBD (nat CBD and syn CBD) in human breast carcinoma cells that express CB receptors. In detail, our data demonstrated that the treatment of cells with the novel phytocannabinoids affects cell viability, increases the production of reactive oxygen species (ROS) and activates cellular pathways related to ROS signaling, as already demonstrated for natural CBD. Moreover, we observed that the biological activity is significantly increased upon combining CBD homologs with drugs that inhibit the activity of enzymes involved in the metabolism of endocannabinoids, such as the monoacylglycerol lipase (MAGL) inhibitor, or with drugs that induces the activation of cellular stress pathways, such as the phorbol ester 12-myristate 13-acetate (PMA).

Investigation on the Composition of Agarose-Collagen I Blended Hydrogels as Matrices for the Growth of Spheroids from Breast Cancer Cell Lines.

Three-dimensional (3D) cell culture systems mimic the structural complexity of the tissue microenvironment and are gaining increasing importance as they resemble the extracellular matrix (ECM)-cell and cell-cell physical interactions occurring in vivo. Several scaffold-based culture systems have been already proposed as valuable tools for large-scale production of spheroids, but they often suffer of poor reproducibility or high costs of production. In this work, we present a reliable 3D culture system based on collagen I-blended agarose hydrogels and show how the variation in the agarose percentage affects the physical and mechanical properties of the resulting hydrogel. The influence of the different physical and mechanical properties of the blended hydrogels on the growth, size, morphology, and cell motility of the spheroids obtained by culturing three different breast cancer cell lines (MCF-7, MDA-MB-361, and MDA-MB-231) was also evaluated. As proof of concept, the cisplatin penetration and its cytotoxic effect on the tumor spheroids as function of the hydrogel stiffness were also investigated. Noteworthily, the possibility to recover the spheroids from the hydrogels for further processing and other biological studies has been considered. This feature, in addition to the ease of preparation, the lack of cross-linking chemistry and the high reproducibility, makes this hydrogel a reliable biomimetic matrix for the growth of 3D cell structures.

Carbonic Anhydrase XII Expression Is Modulated during Epithelial Mesenchymal Transition and Regulated through Protein Kinase C Signaling.

Members of the carbonic anhydrase family are functionally involved in the regulation of intracellular and extracellular pH in physiological and pathological conditions. Their expression is finely regulated to maintain a strict control on cellular homeostasis, and it is dependent on the activation of extracellular and intracellular signaling pathways. Combining RNA sequencing (RNA-seq), NanoString, and bioinformatics data, we demonstrated that the expression of carbonic anhydrase 12 (CAXII) is significantly different in luminal and triple negative breast cancer (BC) models and patients, and is associated with the activation of an epithelial mesenchymal transition (EMT) program. In BC models, the phorbol ester 12-myristate 13-acetate (PMA)-mediated activation of protein kinase C (PKC) induced a down-regulation of CAXII with a concomitant modulation of other members of the transport metabolon, including CAIX and the sodium bicarbonate cotransporter 3 (NBCn1). This is associated with a remodeling of tumor glycolytic metabolism induced after PKC activation. Overall, this analysis highlights the dynamic nature of transport metabolom and identifies signaling pathways finely regulating this plasticity.

A specific lipid metabolic profile is associated with the epithelial mesenchymal transition program.

Several studies have identified a specific metabolic program that is associated with the process of epithelial-mesenchymal transition (EMT). Whereas much is known about the association between glucose metabolism and EMT, the contribution of lipid metabolism is not still completely understood. Here, we studied epithelial and mesenchymal breast cancer cells by proteomic and lipidomic approaches and identified significant differences that characterised these models concerning specific metabolic enzymes and metabolites including fatty acids and phospholipids. Higher levels of monounsaturated fatty acids together with increased expression of enzymes of de novo fatty acid synthesis is the distinct signature of epithelial with respect to mesenchymal cells that, on the contrary, show reduced lipogenesis, higher polyunsaturated fatty acids level and increased expression of genes involved in the triacylglycerol (TAG) synthesis and lipid droplets formation. In the mesenchymal model, the diacylglycerol acyltransferase (DGAT)-1 appears to be the major enzyme involved in TAG synthesis and inhibition of DGAT1, but not DGAT2, drastically reduces the incorporation of labeled palmitate into TAG. Moreover, knockdown of ß-catenin demonstrated that this metabolic phenotype in under the control of a network of transcriptional factors and that ß-catenin has a specific role in the regulation of lipid metabolism in mesenchymal cells.

An SPR based immunoassay for the sensitive detection of the soluble epithelial marker E-cadherin.

Protein biomarkers are important diagnostic tools for cancer and several other diseases. To be validated in a clinical context, a biomarker should satisfy some requirements including the ability to provide reliable information on a pathological state by measuring its expression levels. In parallel, the development of an approach capable of detecting biomarkers with high sensitivity and specificity would be ideally suited for clinical applications. Here, we performed an immune-based label free assay using Surface Plasmon Resonance (SPR)-based detection of the soluble form of E-cadherin, a cell-cell contact protein that is involved in the maintaining of tissue integrity. With this approach, we obtained a specific and quantitative detection of E-cadherin from a few hundred microliters of serum of breast cancer patients by obtaining a 10-fold enhancement in the detection limit over a traditional colorimetric ELISA.

Quartz Crystal Microbalance as Cell-Based Biosensor to Detect and Study Cytoskeletal Alterations and Dynamics.

Several techniques can be used to monitor cell dynamism after a perturbation. Among these, Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D) offers the great advantage to study the mechanical properties of cells in real-time and with a great sensitivity. Here, we used QCM-D to investigate the effects of two cytoskeleton-targeting agents, cytochalasin D (CytoD) and Y27632, on human MCF-7 cells. Cell adhesion on the sensor surface, crucial for in-flow experiments, was obtained by covalent adsorption of a fibronectin (FN) film, an extracellular matrix (ECM) protein. Direct analysis of MCF-7 cells on FN-coated sensor, shows a specific cellular response that was revealed and quantified by QCM-D after drugs exposure. Notably, upon treatment with Y27632, we observed a two-regime dissipation behavior that we associated with specific modifications of actin filaments and signaling proteins providing a link between biophysical and molecular mechanisms. Overall, this approach opens new opportunities for studying cellular response to mechanical cues in different biological conditions.

Polymeric Nano-Micelles as Novel Cargo-Carriers for LY2157299 Liver Cancer Cells Delivery.

LY2157299 (LY), which is very small molecule bringing high cancer diffusion, is a pathway antagonist against TGFß. LY dosage can be diluted by blood plasma, can be captured by immune system or it might be dissolved during digestion in gastrointestinal tract. The aim of our study is to optimize a "nano-elastic" carrier to avoid acidic pH of gastrointestinal tract, colon alkaline pH, and anti-immune recognition. Polygalacturonic acid (PgA) is not degradable in the gastrointestinal tract due to its insolubility at acidic pH. To avoid PgA solubility in the colon, we have designed its conjugation with Polyacrylic acid (PAA). PgA-PAA conjugation has enhanced their potential use for oral and injected dosage. Following these pre-requisites, novel polymeric nano-micelles derived from PgA-PAA conjugation and loading LY2157299 are developed and characterized. Efficacy, uptake and targeting against a hepatocellular carcinoma cell line (HLF) have also been demonstrated.

Resveratrol Modulation of Protein Expression in parkin-Mutant Human Skin Fibroblasts: A Proteomic Approach.

In this study, we investigated by two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) analysis the effects of resveratrol treatment on skin primary fibroblasts from a healthy subject and from a parkin-mutant early onset Parkinson's disease patient. Parkin, an E3 ubiquitin ligase, is the most frequently mutated gene in hereditary Parkinson's disease. Functional alteration of parkin leads to impairment of the ubiquitin-proteasome system, resulting in the accumulation of misfolded or aggregated proteins accountable for the neurodegenerative process. The identification of proteins differentially expressed revealed that resveratrol treatment can act on deregulated specific biological process and molecular function such as cellular redox balance and protein homeostasis. In particular, resveratrol was highly effective at restoring the heat-shock protein network and the protein degradation systems. Moreover, resveratrol treatment led to a significant increase in GSH level, reduction of GSSG/GSH ratio, and decrease of reduced free thiol content in patient cells compared to normal fibroblasts. Thus, our findings provide an experimental evidence of the beneficial effects by which resveratrol could contribute to preserve the cellular homeostasis in parkin-mutant fibroblasts.

ß-Catenin Knockdown Affects Mitochondrial Biogenesis and Lipid Metabolism in Breast Cancer Cells.

ß-catenin plays an important role as regulatory hub in several cellular processes including cell adhesion, metabolism, and epithelial mesenchymal transition. This is mainly achieved by its dual role as structural component of cadherin-based adherens junctions, and as a key nuclear effector of the Wnt pathway. For this dual role, different classes of proteins are differentially regulated via ß-catenin dependent mechanisms. Here, we applied a liquid chromatography-mass spectrometry (LC-MS/MS) approach to identify proteins modulated after ß-catenin knockdown in the breast cancer cell line MCF-7. We used a label free analysis to compare trypsin-digested proteins from CTR (shCTR) and ß-catenin knockout cells (shßcat). This led to the identification of 98 differentially expressed proteins, 53 of them were up-regulated and 45 down-regulated. Loss of ß-catenin induced morphological changes and a significant modulation of the expression levels of proteins associated with primary metabolic processes. In detail, proteins involved in carbohydrate metabolism and tricarboxylic acid cycle were found to be down-regulated, whereas proteins associated to lipid metabolism were found up-regulated in shßcat compared to shCTR. A loss of mitochondrial mass and membrane potential was also assessed by fluorescent probes in shßcat cells with respect to the controls. These data are consistent with the reduced expression of transcriptional factors regulating mitochondrial biogenesis detected in shßcat cells. ß-catenin driven metabolic reprogramming resulted also in a significant modulation of lipogenic enzyme expression and activity. Compared to controls, ß-catenin knockout cells showed increased incorporation of [1-14C]acetate and decreased utilization of [U-14C]glucose for fatty acid synthesis. Our data highlight a role of ß-catenin in the regulation of metabolism and energy homeostasis in breast cancer cells.

Translating epithelial mesenchymal transition markers into the clinic: Novel insights from proteomics.

The growing understanding of the molecular mechanisms underlying epithelial-to-mesenchymal transition (EMT) may represent a potential source of clinical markers. Despite EMT drivers have not yet emerged as candidate markers in the clinical setting, their association with established clinical markers may improve their specificity and sensitivity. Mass spectrometry-based platforms allow analyzing multiple samples for the expression of EMT candidate markers, and may help to diagnose diseases or monitor treatment efficiently. This review highlights proteomic approaches applied to elucidate the differences between epithelial and mesenchymal tumors and describes how these can be used for target discovery and validation.

The complete 12 Mb genome and transcriptome of Nonomuraea gerenzanensis with new insights into its duplicated "magic" RNA polymerase.

In contrast to the widely accepted consensus of the existence of a single RNA polymerase in bacteria, several actinomycetes have been recently shown to possess two forms of RNA polymerases due the to co-existence of two rpoB paralogs in their genome. However, the biological significance of the rpoB duplication is obscure. In this study we have determined the genome sequence of the lipoglycopeptide antibiotic A40926 producer Nonomuraea gerenzanensis ATCC 39727, an actinomycete with a large genome and two rpoB genes, i.e. rpoB(S) (the wild-type gene) and rpoB(R) (the mutant-type gene). We next analyzed the transcriptional and metabolite profiles in the wild-type gene and in two derivative strains over-expressing either rpoB(R) or a mutated form of this gene to explore the physiological role and biotechnological potential of the "mutant-type" RNA polymerase. We show that rpoB(R) controls antibiotic production and a wide range of metabolic adaptive behaviors in response to environmental pH. This may give interesting perspectives also with regard to biotechnological applications.

Altered protein expression pattern in skin fibroblasts from parkin-mutant early-onset Parkinson's disease patients.

Parkinson's disease (PD) is the most common neurodegenerative movement disorder caused primarily by selective degeneration of the dopaminergic neurons in substantia nigra. In this work the proteomes extracted from primary fibroblasts of two unrelated, hereditary cases of PD patients, with different parkin mutations, were compared with the proteomes extracted from commercial adult normal human dermal fibroblasts (NHDF) and primary fibroblasts from the healthy mother of one of the two patients. The results show that the fibroblasts from the two different cases of parkin-mutant patients display analogous alterations in the expression level of proteins involved in different cellular functions, like cytoskeleton structure-dynamics, calcium homeostasis, oxidative stress response, protein and RNA processing.

Cytoskeletal Alterations and Biomechanical Properties of parkin-Mutant Human Primary Fibroblasts.

Parkinson's disease (PD) is one of the most common neurodegenerative diseases. Genes which have been implicated in autosomal-recessive PD include PARK2 which codes for parkin, an E3 ubiquitin ligase that participates in a variety of cellular activities. In this study, we compared parkin-mutant primary fibroblasts, from a patient with parkin compound heterozygous mutations, to healthy control cells. Western blot analysis of proteins obtained from patient's fibroblasts showed quantitative differences of many proteins involved in the cytoskeleton organization with respect to control cells. These molecular alterations are accompanied by changes in the organization of actin stress fibers and biomechanical properties, as revealed by confocal laser scanning microscopy and atomic force microscopy. In particular, parkin deficiency is associated with a significant increase of Young's modulus of null-cells in comparison to normal fibroblasts. The current study proposes that parkin influences the spatial organization of actin filaments, the shape of human fibroblasts, and their elastic response to an external applied force.

Effect of resveratrol on mitochondrial function: implications in parkin-associated familiar Parkinson's disease.

Mitochondrial dysfunction and oxidative stress occur in Parkinson's disease (PD), but the molecular mechanisms controlling these events are not completely understood. Peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α) is a transcriptional coactivator known as master regulator of mitochondrial functions and oxidative metabolism. Recent studies, including one from our group, have highlighted altered PGC-1α activity and transcriptional deregulation of its target genes in PD pathogenesis suggesting it as a new potential therapeutic target. Resveratrol, a natural polyphenolic compound proved to improve mitochondrial activity through the activation of several metabolic sensors resulting in PGC-1α activation. Here we have tested in vitro the effect of resveratrol treatment on primary fibroblast cultures from two patients with early-onset PD linked to different Park2 mutations. We show that resveratrol regulates energy homeostasis through activation of AMP-activated protein kinase (AMPK) and sirtuin 1 (SIRT1) and raise of mRNA expression of a number of PGC-1α's target genes resulting in enhanced mitochondrial oxidative function, likely related to a decrease of oxidative stress and to an increase of mitochondrial biogenesis. The functional impact of resveratrol treatment encompassed an increase of complex I and citrate synthase activities, basal oxygen consumption, and mitochondrial ATP production and a decrease in lactate content, thus supporting a switch from glycolytic to oxidative metabolism. Moreover, resveratrol treatment caused an enhanced macro-autophagic flux through activation of an LC3-independent pathway. Our results, obtained in early-onset PD fibroblasts, suggest that resveratrol may have potential clinical application in selected cases of PD-affected patients.

Sphingomonas cynarae sp. nov., a proteobacterium that produces an unusual type of sphingan.

Strain SPC-1(T) was isolated from the phyllosphere of Cynara cardunculus L. var. sylvestris (Lamk) Fiori (wild cardoon), a Mediterranean native plant considered to be the wild ancestor of the globe artichoke and cultivated cardoon. This Gram-stain-negative, catalase-positive, oxidase-negative, non-spore-forming, rod-shaped and non-motile strain secreted copious amounts of an exopolysaccharide, formed slimy, viscous, orange-pigmented colonies and grew optimally at around pH 6.0-6.5 and 26-30 °C in the presence of 0-0.5 % NaCl. Phylogenetic analysis based on comparisons of 16S rRNA gene sequences demonstrated that SPC-1(T) clustered together with species of the genus Sphingomonas sensu stricto. The G+C content of the DNA (66.1 mol%), the presence of Q-10 as the predominant ubiquinone, sym-homospermidine as the predominant polyamine, 2-hydroxymyristic acid (C(14 : 0) 2-OH) as the major hydroxylated fatty acid, the absence of 3-hydroxy fatty acids and the presence of sphingoglycolipid supported this taxonomic position. 16S rRNA gene sequence analysis showed that SPC-1(T) was most closely related to Sphingomonas hankookensis ODN7(T), Sphingomonas insulae DS-28(T) and Sphingomonas panni C52(T) (98.19, 97.91 and 97.11 % sequence similarities, respectively). However, DNA-DNA hybridization analysis did not reveal any relatedness at the species level. Further differences were apparent in biochemical traits, and fatty acid, quinone and polyamine profiles leading us to conclude that strain SPC-1(T) represents a novel species of the genus Sphingomonas, for which the name Sphingomonas cynarae sp. nov. is proposed; the type strain is SPC-1(T) ( = JCM 17498(T) = ITEM 13494(T)). A component analysis of the exopolysaccharide suggested that it represents a novel type of sphingan containing glucose, rhamnose, mannose and galactose, while glucuronic acid, which is commonly found in sphingans, was not detected.

An active mitochondrial biogenesis occurs during dendritic cell differentiation.

Dendritic cells (DC) are sentinels of the immune system deriving from circulating monocyte precursors recruited to sites of inflammation. In a previous report (Del Prete et al., 2008) we showed that, after differentiation, DC exhibited increased number of condensed mitochondria and dynamic changes in their energy metabolism. A study is presented here showing that the DC differentiation process is characterized by increased expression level and activity of mitochondrial respiratory complexes, as well as by an increased mitochondrial DNA (mtDNA) copy number. Moreover, DC are equipped with more efficient antioxidant protection systems, over expressed most likely to detoxify increased ROS production, as a consequence of the much higher mitochondrial activity. Kinetic analysis of the three main mitochondrial biogenesis-associated genes revealed that the peak in PPARγ coactivator-1alpha (PGC-1α) gene expression was suddenly reached few hours after the onset of the differentiation. While PGC-1α expression rapidly declines, the mitochondrial transcription factor A (TFAM) and nuclear respiratory factor-1 (NRF-1) expression gradually increased. These findings demonstrate that an active mitochondrial biogenesis occurs during DC differentiation and further suggest that an early input by the master regulator of mitochondrial biogenesis PGC-1α is needed to trigger the subsequent activation of the downstream transcription factors, NRF-1 and TFAM in this process.