Naturally-derived phenethyl isothiocyanate modulates apoptotic induction through regulation of the intrinsic cascade and resulting apoptosome formation in human malignant melanoma cells.

Malignant melanoma is the most aggressive type of skin cancer with increasing incidence rates worldwide. On the other hand, watercress is a rich source of phenethyl isothiocyanate (PEITC), among others, which has been widely investigated for its anticancer properties against various cancers. In the present study, we evaluated the role of a watercress extract in modulating apoptotic induction in an in vitro model of human malignant melanoma consisting of melanoma (A375, COLO-679, COLO-800), non-melanoma epidermoid carcinoma (A431) and immortalized, non-tumorigenic keratinocyte (HaCaT) cells. Moreover, the chemical composition of the watercress extract was characterized through UPLC MS/MS and other analytical methodologies. In addition, cytotoxicity was assessed by the alamar blue assay whereas apoptosis was determined, initially, by a multiplex activity assay kit (measuring levels of activated caspases -3, -8 and -9) as well as by qRT-PCR for the identification of major genes regulating apoptosis. In addition, protein expression levels were evaluated by western immunoblotting. Our data indicate that the extract contains various phytochemicals (e.g. phenolics, flavonoids, pigments, etc.) while isothiocyanates (ITCs; especially PEITC) were the most abundant. In addition, the extract was shown to exert a significant time- and dose-dependent cytotoxicity against all malignant melanoma cell lines while non-melanoma and non-tumorigenic cells exhibited significant resistance. Finally, expression profiling revealed a number of genes (and corresponding proteins) being implicated in regulating apoptotic induction through activation of the intrinsic apoptotic cascade. Overall, our data indicate the potential of PEITC as a promising anti-cancer agent in the clinical management of human malignant melanoma.

Nanomechanical and Microstructural Characterization of Biocompatible Ti3Au Thin Films Grown on Glass and Ti6Al4V Substrates.

Ti-Au intermetallic-based material systems are being extensively studied as next-generation thin film coatings to extend the lifetime of implant devices. These coatings are being developed for application to the articulating surfaces of total joint implants and, therefore, must have excellent biocompatibility combined with superior mechanical hardness and wear resistance. However, these key characteristics of Ti-Au coatings are heavily dependent upon factors such as the surface properties and temperature of the underlying substrate during thin film deposition. In this work, Ti3Au thin films were deposited by magnetron sputtering on both glass and Ti6Al4V substrates at an ambient and elevated substrate temperature of 275 °C. These films were studied for their mechanical properties by the nanoindentation technique in both variable load and fixed load mode using a Berkovich tip. XRD patterns and cross-sectional SEM images detail the microstructure, while AFM images present the surface morphologies of these Ti3Au thin films. The biocompatibility potential of the films is assessed by cytotoxicity tests in L929 mouse fibroblast cells using Alamar blue assay, while leached ion concentrations in the film extracts are quantified using ICPOEMS. The standard deviation for hardness of films deposited on glass substrates is ∼4 times lower than that on Ti6Al4V substrates and is correlated with a corresponding increase in surface roughness from 2 nm for glass to 40 nm for Ti6Al4V substrates. Elevating substrate temperature leads to an increase in film hardness from 5.1 to 8.9 GPa and is related to the development of a superhard ß phase of the Ti3Au intermetallic. The standard deviation of this peak mechanical hardness value is reduced by ∼3 times when measured in fixed load mode compared to the variable load mode due to the effect of nanoindentation tip penetration depth. All tested Ti-Au thin films also exhibit excellent biocompatibility against L929 fibroblast cells, as viability levels are above 95% and leached Ti, Al, V, and Au ion concentrations are below 0.1 ppm. Overall, this work demonstrates a novel Ti3Au thin film system with a unique combination of high hardness and excellent biocompatibility with potential to be developed into a new wear-resistant coating to extend the lifetime of articulating total joint implants.

Isothiocyanates Potentiate Tazemetostat-Induced Apoptosis by Modulating the Expression of Apoptotic Genes, Members of Polycomb Repressive Complex 2, and Levels of Tri-Methylating Lysine 27 at Histone 3 in Human Malignant Melanoma Cells.

In this study, we utilized an in vitro model consisting of human malignant melanoma as well as non-tumorigenic immortalized keratinocyte cells with the aim of characterizing the therapeutic effectiveness of the clinical epigenetic drug Tazemetostat alone or in combination with various isothiocyanates. In doing so, we assessed markers of cell viability, apoptotic induction, and expression levels of key proteins capable of mediating the therapeutic response. Our data indicated, for the first time, that Tazemetostat caused a significant decrease in viability levels of malignant melanoma cells in a dose- and time-dependent manner via the induction of apoptosis, while non-malignant keratinocytes were more resistant. Moreover, combinatorial treatment protocols caused a further decrease in cell viability, together with higher apoptotic rates. In addition, a significant reduction in the Polycomb Repressive Complex 2 (PRC2) members [e.g., Enhancer of Zeste Homologue 2 (EZH2), Embryonic Ectoderm Development (EED), and suppressor of zeste 12 (SUZ12)] and tri-methylating lysine 27 at Histone 3 (H3K27me3) protein expression levels was observed, at least partially, under specific combinatorial exposure conditions. Reactivation of major apoptotic gene targets was determined at much higher levels in combinatorial treatment protocols than Tazemetostat alone, known to be involved in the induction of intrinsic and extrinsic apoptosis. Overall, we developed an optimized experimental therapeutic platform aiming to ensure the therapeutic effectiveness of Tazemetostat in malignant melanoma while at the same time minimizing toxicity against neighboring non-tumorigenic keratinocyte cells.

Biocompatible Ti3Au-Ag/Cu thin film coatings with enhanced mechanical and antimicrobial functionality.

BACKGROUND: Biofilm formation on medical device surfaces is a persistent problem that shelters bacteria and encourages infections and implant rejection. One promising approach to tackle this problem is to coat the medical device with an antimicrobial material. In this work, for the first time, we impart antimicrobial functionality to Ti3Au intermetallic alloy thin film coatings, while maintaining their superior mechanical hardness and biocompatibility. METHODS: A mosaic Ti sputtering target is developed to dope controlled amounts of antimicrobial elements of Ag and Cu into a Ti3Au coating matrix by precise control of individual target power levels. The resulting Ti3Au-Ag/Cu thin film coatings are then systematically characterised for their structural, chemical, morphological, mechanical, corrosion, biocompatibility-cytotoxicity and antimicrobial properties. RESULTS: X-ray diffraction patterns reveal the formation of a super hard ß-Ti3Au phase, but the thin films undergo a transition in crystal orientation from (200) to (211) with increasing Ag concentration, whereas introduction of Cu brings no observable changes in crystal orientation. Scanning and transmission electron microscopy analysis show the polyhedral shape of the Ti3Au crystal but agglomeration of Ag particles between crystal grains begins at 1.2 at% Ag and develops into large granules with increasing Ag concentration up to 4.1 at%. The smallest doping concentration of 0.2 at% Ag raises the hardness of the thin film to 14.7 GPa, a 360% improvement compared to the ∼4 GPa hardness of the standard Ti6Al4V base alloy. On the other hand, addition of Cu brings a 315-330% improvement in mechanical hardness of films throughout the entire concentration range of 0.5-7.1 at%. The thin films also show good electrochemical corrosion resistance and a > tenfold reduction in wear rate compared to Ti6Al4V alloy. All thin film samples exhibit very safe cytotoxic profiles towards L929 mouse fibroblast cells when analysed with Alamar blue assay, with ion leaching concentrations lower than 0.2 ppm for Ag and 0.08 ppm for Cu and conductivity tests reveal the positive effect of increased conductivity on myogenic differentiation. Antimicrobial tests show a drastic reduction in microbial survival over a short test period of < 20 min for Ti3Au films doped with Ag or Cu concentrations as low as 0.2-0.5 at%. CONCLUSION: Therefore, according to these results, this work presents a new antimicrobial Ti3Au-Ag/Cu coating material with excellent mechanical performance with the potential to develop wear resistant medical implant devices with resistance to biofilm formation and bacterial infection.

Expression of CD47 and SIRPα Macrophage Immune-Checkpoint Pathway in Non-Small-Cell Lung Cancer.

BACKGROUND: Cancer cells escape macrophage phagocytosis by expressing the CD47 integrin-associated protein that binds to the SIRPα ligand (signal regulatory protein alpha) expressed by macrophages. Immunotherapy targeting this pathway is under clinical development. METHODS: We investigated the expression of CD47/SIRPα molecules in a series of 98 NSCLCs, in parallel with the infiltration of tumor stroma by CD68+ macrophages, tumor-infiltrating lymphocytes (TILs), and PD-L1/PD-1 molecules. RESULTS: Extensive membranous CD47 expression by cancer cells characterized 29/98 cases. SIRPα and CD68 were expressed, to a varying extent, by tumor-associated macrophages (Μφ, TAMs). A high CD68Mφ-score in inner tumor areas was linked with improved overall survival (p = 0.005); and this was independent of the stage (p = 0.02, hazard ratio 0.4). In contrast, high SIRPα expression by CD68+ TAMs (SIRPα/CD68-ratio) was linked with CD47 expression by cancer cells, low TIL-score, and poor prognosis (p = 0.02). A direct association of CD47 expression by cancer cells and the % FOXP3+ TILs (p = 0.01, r = 0.25) was also noted. CONCLUSIONS: TAMs play an important role in the prognosis of operable NSCLC. As SIRPα+ macrophages adversely affect prognosis, it is suggested that the CD47/SIRPα axis is a sound target for adjuvant immunotherapy policies, aiming to improve the cure rates in operable NSCLC.

Thermal activation of Ti(1-x)Au(x) thin films with enhanced hardness and biocompatibility.

The lifetime of orthopaedic implants can be extended by coating the softer Ti6Al4V alloy with harder biocompatible thin films. In this work, thin films of Ti(1-x)Au(x) are grown on Ti6Al4V and glass substrates by magnetron sputtering in the entire x = 0-1 range, before their key biomechanical properties are performance tuned by thermal activation. For the first time, we explore the effect of in-situ substrate heating versus ex-situ post-deposition heat-treatment, on development of mechanical and biocompatibility performance in Ti-Au films. A ∼250% increase in hardness is achieved for Ti-Au films compared to bulk Ti6Al4V and a ∼40% improvement from 8.8 GPa as-grown to 11.9 and 12.3 GPa with in-situ and ex-situ heat-treatment respectively, is corelated to changes in structural, morphological and chemical properties, providing insights into the origins of super-hardness in the Ti rich regions of these materials. X-ray diffraction reveals that as-grown films are in nanocrystalline states of Ti-Au intermetallic phases and thermal activation leads to emergence of mechanically hard Ti-Au intermetallics, with films prepared by in-situ substrate heating having enhanced crystalline quality. Surface morphology images show clear changes in grain size, shape and surface roughness following thermal activation, while elemental analysis reveals that in-situ substrate heating is better for development of oxide free Ti3Au ß-phases. All tested Ti-Au films are non-cytotoxic against L929 mouse fibroblast cells, while extremely low leached ion concentrations confirm their biocompatibility. With peak hardness performance tuned to >12 GPa and excellent biocompatibility, Ti-Au films have potential as a future coating technology for load bearing medical implants.

Cereal and Confectionary Packaging: Background, Application and Shelf-Life Extension.

In both public and private sectors, one can notice a strong interest in the topic of sustainable food and packaging. For a long time, the spotlight for optimization was placed on well-known examples of high environmental impacts, whether regarding indirect resource use (e.g., meat, dairy) or problems in waste management. Staple and hedonistic foods such as cereals and confectionary have gained less attention. However, these products and their packaging solutions are likewise of worldwide ecologic and economic relevance, accounting for high resource input, production amounts, as well as food losses and waste. This review provides a profound elaboration of the status quo in cereal and confectionary packaging, essential for practitioners to improve sustainability in the sector. Here, we present packaging functions and properties along with related product characteristics and decay mechanisms in the subcategories of cereals and cereal products, confectionary and bakery wares alongside ready-to-eat savories and snacks. Moreover, we offer an overview to formerly and recently used packaging concepts as well as established and modern shelf-life extending technologies, expanding upon our knowledge to thoroughly understand the packaging's purpose; we conclude that a comparison of the environmental burden share between product and packaging is necessary to properly derive the need for action(s), such as packaging redesign.

Evaluation of Bioactive Properties of Lipophilic Fractions of Edible and Non-Edible Parts of Nasturtium officinale (Watercress) in a Model of Human Malignant Melanoma Cells.

Watercress is an enriched source of phenethyl isothiocyanate (PEITC), among other phytochemicals, with an antioxidant capacity. The aim of this study was to (i) chemically characterize and (ii) biologically evaluate the profile of the main health-promoting compounds contained in edible (i.e., mixture of leaves and lateral buds) and non-edible (i.e., stems) parts of watercress in an in vitro model of malignant melanoma consisting of human malignant melanoma (A375), non-melanoma (A431) and keratinocyte (HaCaT) cells. The extraction of the main constituents of watercress was performed by subjecting the freeze-dried edible and non-edible samples through different extraction protocols, whereas their concentration was obtained utilizing analytical methodologies. In addition, cell viability was evaluated by the Alamar Blue assay, whereas levels of oxidative stress and apoptosis were determined by commercially available kits. The edible watercress sample contained a higher amount of various nutrients and phytochemicals in the hexane fraction compared to the non-edible one, as evidenced by the presence of PEITC, phenolics, flavonoids, pigments, ascorbic acid, etc. The cytotoxicity potential of the edible watercress sample in the hexane fraction was considerably higher than the non-edible one in A375 cells, whereas A431 and HaCaT cells appeared to be either more resistant or minimally affected, respectively. Finally, levels of oxidative stress and apoptotic induction were increased in both watercress samples, but the magnitude of the induction was much higher in the edible than the non-edible watercress samples. Herein, we provide further evidence documenting the potential development of watercress extracts (including watercress waste by-products) as promising anti-cancer agent(s) against malignant melanoma cells.

Chemical and Biological Characterization of the Anticancer Potency of Salvia fruticosa in a Model of Human Malignant Melanoma.

Malignant melanoma is one of the most aggressive types of skin cancer with an increasing incidence worldwide. Thus, the development of innovative therapeutic approaches is of great importance. Salvia fruticosa (SF) is known for its anticancer properties and in this context, we aimed to investigate its potential anti-melanoma activity in an in vitro model of human malignant melanoma. Cytotoxicity was assessed through a colorimetric-based sulforhodamine-B (SRB) assay in primary malignant melanoma (A375), non-malignant melanoma epidermoid carcinoma (A431) and non-tumorigenic melanocyte neighbouring keratinocyte (HaCaT) cells. Among eight (8) different fractions of S. fruticosa extracts (SF1-SF8) tested, SF3 was found to possess significant cytotoxic activity against A375 cells, while A431 and HaCaT cells remained relatively resistant or exerted no cytotoxicity, respectively. In addition, the total phenolic (Folin-Ciocalteu assay) and total flavonoid content of SF extracts was estimated, whereas the antioxidant capacity was measured via the inhibition of tert-butyl hydroperoxide-induced lipid peroxidation and protein oxidation levels. Finally, apoptotic cell death was assessed by utilizing a commercially available kit for the activation of caspases - 3, - 8 and - 9. In conclusion, the anti-melanoma properties of SF3 involve the induction of both extrinsic and intrinsic apoptotic pathway(s), as evidenced by the increased activity levels of caspases - 8, and - 9, respectively.

Prognostic Relevance of the Relative Presence of CD4, CD8 and CD20 Expressing Tumor Infiltrating Lymphocytes in Operable Non-small Cell Lung Cancer Patients.

BACKGROUND/AIM: Non-small cell lung cancer (NSCLC) is one of the most lethal tumors. Given the failure of conventional therapeutic strategies, immunotherapy has emerged as a promising treatment modality that may improve the survival of patients with operable and advanced disease. PATIENTS AND METHODS: We examined the relative presence of CD20+ B-cells, CD8+ cytotoxic, and CD4+ helper/regulatory T-cells in the tumor-infiltrating lymphocyte (TIL)-population in a series of surgically-treated NSCLCs, and assessed their role as prognostic indicators after surgery. RESULTS: A high percent of CD4+ and CD8+ TILs in the tumor stroma was linked with poor (p=0.003) and good prognosis (p=0.01), respectively. High CD4/CD8 ratio defined a significantly worst prognosis [median survival 22 months vs. undefined, p=0.0002, hazard ratio (HR) 0.3 vs. 3.0]. Statistically significant results were also noted when the analysis was focused on the invading tumor front. In a multivariate model, the CD4/CD8-ratio assessed in the tumor stroma and the stage of disease were independent prognostic variables (p=0.0001, HR=4.1 and p=0.001, HR=1.5, respectively). CONCLUSION: The balance between CD4+ and CD8+ lymphocytes infiltrating the tumor stroma is a crucial factor defining anti-tumor immune surveillance, has strong prognostic value, and may be tested as a predictive biomarker for immunotherapy in operable NSCLC.