Caffeic Acid-Grafted PLGA as a Novel Material for the Design of Fluvastatin-Eluting Nanoparticles for the Prevention of Neointimal Hyperplasia.

Drug-eluting nanoparticles (NPs) administered by an eluting balloon represent a novel tool to prevent restenosis after angioplasty, even if the selection of the suitable drug and biodegradable material is still a matter of debate. Herein, we provide the proof of concept of the use of a novel material obtained by combining the grafting of caffeic acid or resveratrol on a poly(lactide-co-glycolide) backbone (g-CA-PLGA or g-RV-PLGA) and the pleiotropic effects of fluvastatin chosen because of its low lipophilic profile which is challenging for the encapsulation in NPs and delivery to the artery wall cells. NPs made of such materials are biocompatible with macrophages, human smooth muscle cells (SMCs), and endothelial cells (ECs). Their cellular uptake is demonstrated and quantified by confocal microscopy using fluorescent NPs, while their distribution in the cytoplasm is verified by TEM images using NPs stained with an Ag-PVP probe appositely synthetized. g-CA-PLGA assures the best control of the FLV release from NP sizing around 180 nm and the faster SMC uptake, as demonstrated by confocal analyses. Interestingly and surprisingly, g-CA-PLGA improves the FLV efficacy to inhibit the SMC migration, without altering its effects on EC proliferation and migration. The improved trophism of NPs toward SMCs, combined with the excellent biocompatibility and low modification of the microenvironment pH upon polymer degradation, makes g-CA-PLGA a suitable material for the design of drug-eluting balloons.

Adipocyte-Derived Extracellular Vesicles Promote Prostate Cancer Cell Aggressiveness by Enabling Multiple Phenotypic and Metabolic Changes.

BACKGROUND: In recent decades, obesity has widely emerged as an important risk factor for prostate cancer (PCa). Adipose tissue and PCa cells have been shown to orchestrate a complex interaction network to support tumor growth and evolution; nonetheless, the study of this communication has only been focused on soluble factors, although increasing evidence highlights the key role of extracellular vesicles (EVs) in the modulation of tumor progression. METHODS AND RESULTS: In the present study, we found that EVs derived from 3T3-L1 adipocytes could affect PC3 and DU145 PCa cell traits, inducing increased proliferation, migration and invasion. Furthermore, conditioning of both PCa cell lines with adipocyte-released EVs resulted in lower sensitivity to docetaxel, with reduced phosphatidylserine externalization and decreased caspase 3 and PARP cleavage. In particular, these alterations were paralleled by an Akt/HIF-1α axis-related Warburg effect, characterized by enhanced glucose consumption, lactate release and ATP production. CONCLUSIONS: Collectively, these findings demonstrate that EV-mediated crosstalk exists between adipocytes and PCa, driving tumor aggressiveness.

Simvastatin Prevents Liver Microthrombosis and Sepsis Induced Coagulopathy in a Rat Model of Endotoxemia.

Background: Endotoxemia causes endothelial dysfunction and microthrombosis, which are pathogenic mechanisms of coagulopathy and organ failure during sepsis. Simvastatin has potential anti-thrombotic effects on liver endothelial cells. We investigated the hemostatic changes induced by lipopolysaccharide (LPS) and explored the protective effects of simvastatin against liver vascular microthrombosis. Methods and results: We compared male Wistar rats exposed to LPS (5 mg/kg one i.p. dose) or saline in two experimental protocols­placebo (vehicle) and simvastatin (25 mg/kg die, orally, for 3 days before LPS). Morphological studies were performed by light- and electron-microscopy analyses to show intravascular fibrin deposition, vascular endothelial structure and liver damage. Peripheral- and organ-hemostatic profiles were analyzed using whole blood viscoelastometry by ROTEM, liver biopsy and western-blot/immunohistochemistry of thrombomodulin (TM), as well as immunohistochemistry of the von Willebrand factor (VWF). LPS-induced fibrin deposition and liver vascular microthrombosis were combined with a loss of sinusoidal endothelial TM expression and VWF-release. These changes were associated with parenchymal eosinophilia and necrosis. ROTEM analyses displayed hypo-coagulability in the peripheral blood that correlated with the degree of intrahepatic fibrin deposition (p < 0.05). Simvastatin prevented LPS-induced fibrin deposition by preserving TM expression in sinusoidal cells and completely reverted the peripheral hypo-coagulability caused by endotoxemia. These changes were associated with a significant reduction of liver cell necrosis without any effect on eosinophilia. Conclusions: Simvastatin preserves the antithrombotic properties of sinusoidal endothelial cells disrupted by LPS, deserving pharmacological properties to contrast sepsis-associated coagulopathy and hepatic failure elicited by endotoxemia

The Antagonism of the Prokineticin System Counteracts Bortezomib Induced Side Effects: Focus on Mood Alterations.

The development of neuropathy and of mood alterations is frequent after chemotherapy. These complications, independent from the antitumoral mechanism, are interconnected due to an overlapping in their processing pathways and a common neuroinflammatory condition. This study aims to verify whether in mice the treatment with the proteasome inhibitor bortezomib (BTZ), at a protocol capable of inducing painful neuropathy, is associated with anxiety, depression and supraspinal neuroinflammation. We also verify if the therapeutic treatment with the antagonist of the prokineticin (PK) system PC1, which is known to contrast pain and neuroinflammation, can prevent mood alterations. Mice were treated with BTZ (0.4 mg/kg three times/week for 4 weeks); mechanical allodynia and locomotor activity were evaluated over time while anxiety (dark light and marble burying test), depression (sucrose preference and swimming test) and supraspinal neuroinflammation were checked at the end of the protocol. BTZ treated neuropathic mice develop anxiety and depression. The presence of mood alterations is related to the presence of neuroinflammation and PK system activation in prefrontal cortex, hippocampus and hypothalamus with high levels of PK2 and PKR2 receptor, IL-6 and TNF-α, TLR4 and an upregulation of glial markers. PC1 treatment, counteracting pain, prevented the development of supraspinal inflammation and depression-like behavior in BTZ mice.

Pre-Drawn Syringes of Comirnaty for an Efficient COVID-19 Mass Vaccination: Demonstration of Stability.

Moving towards a real mass vaccination in the context of COVID-19, healthcare professionals are required to face some criticisms due to limited data on the stability of a mRNA-based vaccine (Pfizer-BioNTech COVID-19 Vaccine in the US or Comirnaty in EU) as a dose in a 1 mL-syringe. The stability of the lipid nanoparticles and the encapsulated mRNA was evaluated in a "real-life" scenario. Specifically, we investigated the effects of different storing materials (e.g., syringes vs. glass vials), as well as of temperature and mechanical stress on nucleic acid integrity, number, and particle size distribution of lipid nanoparticles. After 5 h in the syringe, lipid nanoparticles maintained the regular round shape, and the hydrodynamic diameter ranged between 80 and 100 nm with a relatively narrow polydispersity (<0.2). Samples were stable independently of syringe materials and storage conditions. Only strong mechanical stress (e.g., shaking) caused massive aggregation of lipid nanoparticles and mRNA degradation. These proof-of-concept experiments support the hypothesis that vaccine doses can be safely prepared in a dedicated area using an aseptic technique and transferred without affecting their stability.

Activity of Experimental Mouthwashes and Gels Containing DNA-RNA and Bioactive Molecules against the Oxidative Stress of Oral Soft Tissues: The Importance of Formulations. A Bioreactor-Based Reconstituted Human Oral Epithelium Model.

BACKGROUND: DNA-RNA compounds have shown promising protection against cell oxidative stress. This study aimed to assess the cytotoxicity, protective, or preventive effect of different experimental formulations on oral epithelia's oxidative stress in vitro. METHODS: Reconstituted human oral epithelia (RHOE) were grown air-lifted in a continuous-flow bioreactor. Mouthwashes and gels containing DNA-RNA compounds and other bioactive molecules were tested on a model of oxidative stress generated by hydrogen peroxide treatment. Epithelia viability was evaluated using a biochemical MTT-based assay and confocal microscopy; structural and ultrastructural morphology was evaluated by light microscopy and TEM. RESULTS: DNA-RNA showed non-cytotoxic activity and effectively protected against oxidative stress, but did not help in its prevention. Gel formulations did not express adequate activity compared to the mouthwashes. Excipients played a fundamental role in enhancing or even decreasing the bioactive molecules' effect. CONCLUSION: A mouthwash formulation with hydrolyzed DNA-RNA effectively protected against oxidative stress without additional enhancement by other bioactive molecules. Active compounds, such as hyaluronic acid, ß-Glucan, allantoin, bisabolol, ruscogenin, and essential oils, showed a protective effect against oxidative stress, which was not synergistic with the one of DNA-RNA. Incorporation of surfactant agents showed a reduced, yet significant, cytotoxic effect.

Ca2+ overload- and ROS-associated mitochondrial dysfunction contributes to δ-tocotrienol-mediated paraptosis in melanoma cells.

Melanoma is an aggressive tumor with still poor therapy outcomes. δ-tocotrienol (δ-TT) is a vitamin E derivative displaying potent anti-cancer properties. Previously, we demonstrated that δ-TT triggers apoptosis in human melanoma cells. Here, we investigated whether it might also activate paraptosis, a non-canonical programmed cell death. In accordance with the main paraptotic features, δ-TT was shown to promote cytoplasmic vacuolization, associated with endoplasmic reticulum/mitochondrial dilation and protein synthesis, as well as MAPK activation in A375 and BLM cell lines. Moreover, treated cells exhibited a significant reduced expression of OXPHOS complex I and a marked decrease in oxygen consumption and mitochondrial membrane potential, culminating in decreased ATP synthesis and AMPK phosphorylation. This mitochondrial dysfunction resulted in ROS overproduction, found to be responsible for paraptosis induction. Additionally, δ-TT caused Ca2+ homeostasis disruption, with endoplasmic reticulum-derived ions accumulating in mitochondria and activating the paraptotic signaling. Interestingly, by using both IP3R and VDAC inhibitors, a close cause-effect relationship between mitochondrial Ca2+ overload and ROS generation was evidenced. Collectively, these results provide novel insights into δ-TT anti-melanoma activity, highlighting its ability to induce mitochondrial dysfunction-mediated paraptosis. δ-tocotrienol induces paraptotic cell death in human melanoma cells, causing endoplasmic reticulum dilation and mitochondrial swelling. These alterations induce an impairment of mitochondrial function, ROS production and calcium overload.

Effects of Na-DNA mouthwash solutions on oral soft tissues. A bioreactor-based reconstituted human oral epithelium model.

PURPOSE: To investigate whether the addition of sodium-DNA (Na-DNA) to chlorhexidine (CHX)-containing mouthwash influenced morphology and viability of a reconstituted human oral epithelium (ROE), and protects ROE against oxidative stress. METHODS: Multi-layered 0.5 cm² ROE specimens were positioned inside a continuous flow bioreactor and grown air-lifted for 24 hours. They were treated with phosphate-buffered saline (PBS) (n= 16) or 1 vol% H2O2 for 1 minute (n= 16). Then, they were treated for 5 (n= 8) or 30 minutes (n= 8) with the experimental mouthwash solutions containing: 0.2 wt% CHX, 0.2 wt% CHX + 0.2 wt% Na-DNA, 0.2 wt% Na-DNA, PBS. After 60 minutes washout specimens were subjected to tetrazolium-based viability assay (MTT) confocal laser-scanning microscopy (CLSM), and histological evaluation using optical microscopy and transmission electron microscopy (TEM). RESULTS: ROE treated with Na-DNA for 30 minutes revealed significantly higher viability than PBS, and CHX + Na-DNA showed higher viability after 30-minute treatment than after 5 minutes, suggesting a significant protective activity of Na-DNA. Moreover, the protective effect of Na-DNA on cell viability was higher after the induction of oxidative stress. After treatment with CHX, CLSM revealed cell stress, leading to cell death in the outer layer. On the contrary, specimens treated with Na-DNA showed a much lower number of dead cells compared to PBS, both in the absence or presence of oxidative stress. Histological examination showed that the protective action of Na-DNA formulations reached more in-depth into the epithelium exposed to oxidative stress, due to intercellular spaces opening in the outer epithelium layers, giving way to Na-DNA to the inner parts of the epithelium. It can be concluded that Na-DNA had a topical protective activity when applied for 30 minutes unless the epithelium barrier is damaged, allowing it to act more in-depth. CLINICAL SIGNIFICANCE: Na-DNA showed a clear and protective action against cellular degeneration due to oxidative stress and, partly, to the exposure to CHX. Its addition to chlorhexidine mouthwash or gels could be clinically helpful in contrasting the detrimental activity of CHX on oral tissues, and in the preservation of cell viability, control of inflammation and wound healing.

Mitochondrial functional and structural impairment is involved in the antitumor activity of δ-tocotrienol in prostate cancer cells.

The therapeutic options for castration-resistant prostate cancer (CRPC) are still limited. Natural bioactive compounds were shown to possess pro-death properties in different tumors. We previously reported that δ-tocotrienol (δ-TT) induces apoptosis, paraptosis and autophagy in CRPC cells. Here, we investigated whether δ-TT might exert its activity by impairing mitochondrial functions. We demonstrated that, in PC3 and DU145 cells, δ-TT impairs mitochondrial respiration and structural dynamics. In both cell lines, δ-TT triggers mitochondrial Ca2+ and ROS overload. In PC3 cells, both Ca2+ and ROS mediate the δ-TT-related anticancer activities (decrease of cell viability, apoptosis, paraptosis, autophagy and mitophagy). As expected, in autophagy-defective DU145 cells, Ca2+ overload was involved in δ-TT-induced pro-death effects but not in autophagy and mitophagy. In this cell line, we also demonstrated that ROS overload is not involved in the anticancer activities of δ-TT, supporting a low susceptibility of these cells to ROS-related oxidative stress. Taken together, these data demonstrate that, in CRPC cells, δ-TT triggers cell death by inducing mitochondrial functional and structural impairments, providing novel mechanistic insights in its antitumor activity.

Acid Sphingomyelinase Downregulation Enhances Mitochondrial Fusion and Promotes Oxidative Metabolism in a Mouse Model of Melanoma.

Melanoma is the most severe type of skin cancer. Its unique and heterogeneous metabolism, relying on both glycolysis and oxidative phosphorylation, allows it to adapt to disparate conditions. Mitochondrial function is strictly interconnected with mitochondrial dynamics and both are fundamental in tumour progression and metastasis. The malignant phenotype of melanoma is also regulated by the expression levels of the enzyme acid sphingomyelinase (A-SMase). By modulating at transcriptional level A-SMase in the melanoma cell line B16-F1 cells, we assessed the effect of enzyme downregulation on mitochondrial dynamics and function. Our results demonstrate that A-SMase influences mitochondrial morphology by affecting the expression of mitofusin 1 and OPA1. The enhanced expression of the two mitochondrial fusion proteins, observed when A-SMase is expressed at low levels, correlates with the increase of mitochondrial function via the stimulation of the genes PGC-1alpha and TFAM, two genes that preside over mitochondrial biogenesis. Thus, the reduction of A-SMase expression, observed in malignant melanomas, may determine their metastatic behaviour through the stimulation of mitochondrial fusion, activity and biogenesis, conferring a metabolic advantage to melanoma cells.

Targeting prokineticin system counteracts hypersensitivity, neuroinflammation, and tissue damage in a mouse model of bortezomib-induced peripheral neuropathy.

BACKGROUND: Neuropathy is a dose-limiting side effect of many chemotherapeutics, including bortezomib. The mechanisms underlying this condition are not fully elucidated even if a contribution of neuroinflammation was suggested. Here, we investigated the role of a chemokine family, the prokineticins (PKs), in the development of bortezomib-induced peripheral neuropathy (BIPN), and we used a PK receptor antagonist to counteract the development and progression of the pathology. METHODS: Neuropathy was induced in male C57BL/6J mice by using a protocol capable to induce a detectable neuropathic phenotype limiting systemic side effects. The presence of allodynia (both mechanical and thermal) and thermal hyperalgesia was monitored over time. Mice were sacrificed at two different time points: 14 and 28 days after the first bortezomib (BTZ) injection. At these times, PK system activation (PK2 and PK-Rs), macrophage and glial activation markers, and cytokine production were evaluated in the main station involved in pain transmission (sciatic nerve, DRG, and spinal cord), and the effect of a PK receptors antagonist (PC1) on the same behavioral and biochemical parameters was assessed. Structural damage of DRG during BTZ treatment and an eventual protective effect of PC1 were also evaluated. RESULTS: BTZ induces in mice a dose-related allodynia and hyperalgesia and a progressive structural damage to the DRG. We observed a precocious increase of macrophage activation markers and unbalance of pro- and anti-inflammatory cytokines in sciatic nerve and DRG together with an upregulation of GFAP in the spinal cord. At higher BTZ cumulative dose PK2 and PK receptors are upregulated in the PNS and in the spinal cord. The therapeutic treatment with the PK-R antagonist PC1 counteracts the development of allodynia and hyperalgesia, ameliorates the structural damage in the PNS, decreases the levels of activated macrophage markers, and prevents full neuroimmune activation in the spinal cord. CONCLUSIONS: PK system may be a strategical pharmacological target to counteract BTZ-induced peripheral neuropathy. Blocking PK2 activity reduces progressive BTZ toxicity in the DRG, reducing neuroinflammation and structural damage to DRG, and it may prevent spinal cord sensitization.

δ-Tocotrienol induces apoptosis, involving endoplasmic reticulum stress and autophagy, and paraptosis in prostate cancer cells.

OBJECTIVES: Prostate cancer, after the phase of androgen dependence, may progress to the castration-resistant prostate cancer (CRPC) stage, with resistance to standard therapies. Vitamin E-derived tocotrienols (TTs) possess a significant antitumour activity. Here, we evaluated the anti-cancer properties of δ-TT in CRPC cells (PC3 and DU145) and the related mechanisms of action. MATERIALS AND METHODS: MTT, Trypan blue and colony formation assays were used to assess cell viability/cell death/cytotoxicity. Western blot, immunofluorescence and MTT analyses were utilized to investigate apoptosis, ER stress and autophagy. Morphological changes were investigated by light and transmission electron microscopy. RESULTS: We demonstrated that δ-TT exerts a cytotoxic/proapoptotic activity in CRPC cells. We found that in PC3 cells: (a) δ-TT triggers both the endoplasmic reticulum (ER) stress and autophagy pathways; (b) autophagy induction is related to the ER stress, and this ER stress/autophagy axis is involved in the antitumour activity of δ-TT; in autophagy-defective DU145 cells, only the ER stress pathway is involved in the proapoptotic effects of δ-TT; (c) in both CRPC cell lines, δ-TT also induces an intense vacuolation prevented by the ER stress inhibitor salubrinal and the protein synthesis inhibitor cycloheximide, together with increased levels of phosphorylated JNK and p38, supporting the induction of paraptosis by δ-TT. CONCLUSIONS: These data demonstrate that apoptosis, involving ER stress and autophagy (in autophagy positive PC3 cells), and paraptosis are involved in the anti-cancer activity of δ-TT in CRPC cells.

Characterization of an in vitro model to study the possible role of polyomavirus BK in prostate cancer.

Prostate cancer (PCa) is the most common male neoplasms in the Western world. Various risk factors may lead to carcinogenesis, including infectious agents such as polyomavirus BK (BKPyV), which infects the human renourinary tract, establishes latency, and encodes oncoproteins. Previous studies suggested that BKPyV plays a role in PCa pathogenesis. However, the unspecific tropism of BKPyV and the lack of in vitro models of BKPyV-infected prostate cells cast doubt on this hypothesis. The aim of the present study was to determine whether BKPyV could (a) infect normal and/or tumoral epithelial prostate cells and (b) affect their phenotype. Normal epithelial prostate RWPE-1 cells and PCa PC-3 cells were infected with BKPyV for 21 days. Cell proliferation, cytokine production, adhesion, invasion ability, and epithelial-to-mesenchymal transition (EMT) markers were analyzed. Our results show that (a) RWPE-1 and PC-3 cells are both infectable with BKPyV, but the outcome of the infection varies, (b) cell proliferation and TNF-α production were increased in BKPyV-infected RWPE-1, but not in PC-3 cells, (c) adhesion to matrigel and invasion abilities were elevated in BKPyV-infected RWPE-1 cells, and (d) loss of E-cadherin and expression of vimentin occurred in both uninfected and infected RWPE-1 cells. In conclusion, BKPyV may change some features of the normal prostate cells but is not needed for maintaining the transformed phenotype in the PCa cells The fact that RWPE-1 cells exhibit some phenotype modifications related to EMT represents a limit of this in vitro model.

Effects of caloric restriction on neuropathic pain, peripheral nerve degeneration and inflammation in normometabolic and autophagy defective prediabetic Ambra1 mice.

There is a growing interest on the role of autophagy in diabetes pathophysiology, where development of neuropathy is one of the most frequent comorbidities. We have previously demonstrated that neuropathic pain after nerve damage is exacerbated in autophagy-defective heterozygous Ambra1 mice. Here, we show the existence of a prediabetic state in Ambra1 mice, characterized by hyperglycemia, intolerance to glucose and insulin resistance. Thus, we further investigate the hypothesis that prediabetes may account for the exacerbation of allodynia and chronic pain and that counteracting the autophagy deficit may relieve the neuropathic condition. We took advantage from caloric restriction (CR) able to exert a double action: a powerful increase of autophagy and a control on the metabolic status. We found that CR ameliorates neuropathy throughout anti-inflammatory and metabolic mechanisms both in Ambra1 and in WT animals subjected to nerve injury. Moreover, we discovered that nerve lesion represents, per se, a metabolic stressor and CR reinstates glucose homeostasis, insulin resistance, incomplete fatty acid oxidation and energy metabolism. As autophagy inducer, CR promotes and anticipates Schwann cell autophagy via AMP-activated protein kinase (AMPK) that facilitates remyelination in peripheral nerve. In summary, we provide new evidence for the role of autophagy in glucose metabolism and identify in energy depletion by dietary restriction a therapeutic approach in the fight against neuropathic pain.

Tumor⁻Stroma Cross-Talk in Human Pancreatic Ductal Adenocarcinoma: A Focus on the Effect of the Extracellular Matrix on Tumor Cell Phenotype and Invasive Potential.

The extracellular matrix (ECM) in the tumor microenvironment modulates the cancer cell phenotype, especially in pancreatic ductal adenocarcinoma (PDAC), a tumor characterized by an intense desmoplastic reaction. Because the epithelial-to-mesenchymal transition (EMT), a process that provides cancer cells with a metastatic phenotype, plays an important role in PDAC progression, the authors aimed to explore in vitro the interactions between human PDAC cells and ECM components of the PDAC microenvironment, focusing on the expression of EMT markers and matrix metalloproteinases (MMPs) that are able to digest the basement membrane during tumor invasion. EMT markers and the invasive potential of HPAF-II, HPAC, and PL45 cells grown on different ECM substrates (fibronectin, laminin, and collagen) were analyzed. While N-cadherin, αSMA, and type I collagen were not significantly affected by ECM components, the E-cadherin/ß-catenin complex was highly expressed in all the experimental conditions, and E-cadherin was upregulated by collagen in PL45 cells. Cell migration was unaffected by fibronectin and delayed by laminin. In contrast, collagen significantly stimulated cell migration and the secretion of MMPs. This study's results showed that ECM components impacted cell migration and invasive potential differently. Collagen exerted a more evident effect, providing new insights into the understanding of the intricate interplay between ECM molecules and cancer cells, in order to find novel therapeutic targets for PDAC treatment.

Gellan Nanohydrogels: Novel Nanodelivery Systems for Cutaneous Administration of Piroxicam.

The feasibility to use gellan nanohydrogels (Ge-NHs) as delivery system for the cutaneous administration of piroxicam (PRX) was investigated using gellan conjugated with cholesterol or riboflavin. The in vitro skin penetration studies through human epidermis were performed using a saturated aqueous drug solution, a 50% w/v Transcutol aqueous solution, and a commercially available PRX plaster as controls. Confocal microscopy, ATR-FTIR spectroscopy, circular dichroism, and a dynamometer assisted extrusion assay were performed to clarify the permeation mechanism of Ge-NHs. The skin permeation studies evidenced that Ge-NHs enhance the PRX retention in the epidermis and, at the same time, slow down the permeation process with respect to the controls. NHs can penetrate the stratum corneum, and then gradually disassemble thus diffusing in the viable epidermis reaching the spinosum layer. In conclusion, NHs represent a novel strategy to target poorly permeable compounds in the epidermis, thus improving the management of cutaneous pathologies.

3D-spheroids: What can they tell us about pancreatic ductal adenocarcinoma cell phenotype?

We aimed at analyzing the effect of the 3D-arrangement on the expression of some genes and proteins which play a key role in pancreatic adenocarcinoma (PDAC) progression in HPAF-II, HPAC and PL45 PDAC cells cultured in either 2D-monolayers or 3D-spheroids. Cytokeratins 7, 8, 18, 19 were differently expressed in 3D-spheroids compared to 2D-monolayers. Syndecan 1 was upregulated in HPAF-II and PL45 3D-spheroids, and downregulated in HPAC. Heparanase mRNA levels were almost unchanged in HPAF-II, and increased in HPAC and PL45 3D-spheroids. Hyaluronan synthase (HAS) 2 and 3 mRNA increased in all 3D-spheroids compared to 2D-monolayers. CD44 and CD44s were expressed to a lower extent in HPAF-II and HPAC 3D-spheroids. By contrast, the CD44s/v3 and the CD44s/v6 ratio increased in HPAC and PL45 3D-spheroids, compared to 2D-monolayers. The expression of MMP-7 was strongly upregulated in 3D-spheroids. STAT3 was similarly expressed 3D-spheroids or 2D-monolayers, while pSTAT3 was almost undetectable in 2D-monolayers and strongly upregulated in 3D-spheroids. These results suggest that 3D-spheroids represent a cell culture model that allows the characterization of PDAC cell phenotype, adding new information that contributes to a better understanding of the biology and behavior of PDAC cells.

Tuning the Extent and Depth of Penetration of Flexible Liposomes in Human Skin.

In this work we made an attempt to assess the effect of drug-induced changes of flexibility on the penetration of deformable vesicles into the human skin. Eight cationic liposomes with different degrees of flexibility were obtained by entrapping unfractionated heparin, enoxaparin, and nadroparin. The deformability was studied by a novel, facile, and reliable extrusion assay appositely developed and validated by means of quantitative nanoscale mechanical AFM measurements of vesicle elastic modulus (log10(YM)). The proposed extrusion assay, determining the forces involved in vesicles deformation, resulted very sensitive to evidence of minimal changes in bilayer rigidity (σ) and vesicle deformation (K). The drug loading caused a reduction of liposome flexibility with respect to the reference plain liposomes and in accordance to the heparin type, drug to cationic lipid (DOTAP) ratio, and drug distribution within the vesicles. Interestingly, the σ and log10(YM) values perfectly correlated (R2 = 0.935), demonstrating the reliability of the deformability data obtained with both approaches. The combination of TEM and LC-MS/MS spectrometry allowed the pattern of the penetration of the entire vesicles into the skin to be followed. In all cases, intact liposomes in the epidermis layers were observed and a relationship between the depth of penetration and the liposome flexibility was found, supporting the hypothesis of the whole vesicle penetration mechanism. Moreover, the results of the extent (R24) of vesicle penetration in the human skin samples showed a direct relation to the flexibility values (σ1 = 0.65 ± 0.10 MPa → R24 = 3.33 ± 0.02 µg/mg; σ2 = 0.95 ± 0.04 MPa → R24 = 1.18 ± 0.26 µg/mg; σ3 = 1.89 ± 0.30 MPa → R24 = 0.53 ± 0.33 µg/mg).

Epithelial-to-mesenchymal transition in pancreatic ductal adenocarcinoma: Characterization in a 3D-cell culture model.

AIM: To analyze the effect of three-dimensional (3D)-arrangement on the expression of epithelial-to-mesenchymal transition markers in pancreatic adenocarcinoma (PDAC) cells. METHODS: HPAF-II, HPAC, and PL45 PDAC cells were cultured in either 2D-monolayers or 3D-spheroids. Ultrastructure was analyzed by transmission electron microscopy. The expression of E-cadherin, ß-catenin, N-cadherin, collagen type I (COL-I), vimentin, α-smooth muscle actin (αSMA), and podoplanin was assayed by confocal microscopy in cells cultured on 12-mm diameter round coverslips and in 3D-spheroids. Gene expression for E-cadherin, Snail, Slug, Twist, Zeb1, and Zeb2 was quantified by real-time PCR. E-cadherin protein level and its electrophoretic pattern were studied by Western blot in cell lysates obtained from cells grown in 2D-monolayers and 3D-spheroids. RESULTS: The E-cadherin/ß-catenin complex was expressed in a similar way in plasma membrane cell boundaries in both 2D-monolayers and 3D-spheroids. E-cadherin increased in lysates obtained from 3D-spheroids, while cleavage fragments were more evident in 2D-monolayers. N-cadherin expression was observed in very few PDAC cells grown in 2D-monolayers, but was more evident in 3D-spheroids. Some cells expressing COL-I were observed in 3D-spheroids. Podoplanin, expressed in collectively migrating cells, and αSMA were similarly expressed in both experimental conditions. The concomitant maintenance of the E-cadherin/ß-catenin complex at cell boundaries supports the hypothesis of a collective migration for these cells, which is consistent with podoplanin expression. CONCLUSION: We show that a 3D-cell culture model could provide deeper insight into understanding the biology of PDAC and allow for the detection of marked differences in the phenotype of PDAC cells grown in 3D-spheroids.

Essential role for acid sphingomyelinase-inhibited autophagy in melanoma response to cisplatin.

The sphingolipid metabolising enzyme Acid Sphingomyelinase (A-SMase) has been recently shown to inhibit melanoma progression and correlate inversely to tumour grade. In this study we have investigated the role of A-SMase in the chemo-resistance to anticancer treatmentusing mice with melanoma allografts and melanoma cells differing in terms of expression/activity of A-SMase. Since autophagy is emerging as a key mechanism in tumour growth and chemo-resistance, we have also investigated whether an action of A-SMase in autophagy can explain its role. Melanoma sensitivity to chemotherapeutic agent cisplatin in terms of cell viability/apoptosis, tumour growth, and animal survival depended directly on the A-SMase levels in tumoural cells. A-SMase action was due to inhibition of autophagy through activation of Akt/mammalian target of rapamycin (mTOR) pathway. Treatment of melanoma-bearing mice with the autophagy inhibitor chloroquine restored sensitivity to cisplatin of tumours expressing low levels of A-SMase while no additive effects were observed in tumours characterised by sustained A-SMase levels. The fact that A-SMase in melanomas affects mTOR-regulated autophagy and plays a central role in cisplatin efficacy encourages pre-clinical testing on the modulation of A-SMase levels/activity as possible novel anti-neoplastic strategy.