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.

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.

Naturally Derived Phenethyl Isothiocyanate Modulates Induction of Oxidative Stress via Its N-Acetylated Cysteine Conjugated form in Malignant Melanoma.

Phenethyl isothiocyanate (PEITC) is a secondary metabolic product yielded upon the hydrolysis of gluconasturtiin and it is highly accumulated in the flowers of watercress. The aim of the current study was to assess the role of a naturally derived PEITC-enriched extract in the induction of oxidative stress and to evaluate its anti-melanoma potency through the regulation of its metabolism with the concurrent production of the N-acetyl cysteine conjugated by-product. For this purpose, an in vitro melanoma model was utilized consisting of human primary (A375) cells as well as metastatic (COLO-679) malignant melanoma cells together with non-tumorigenic immortalized keratinocytes (HaCaT). Cytotoxicity was assessed via the Alamar Blue assay whereas the antioxidant/prooxidant activity of PEITC was determined via spectrophotometric assays. Finally, kinetic characterization of the end-product of PEITC metabolism was monitored via UPLC coupled to a tandem mass spectrometry (MS/MS). Our results indicate that although PhEF showed very minor antioxidant activity in a cell-free system, in a cell-based system, it can modulate the activity of key enzyme(s) involved in cellular antioxidant defense mechanism(s). In addition, we have shown that PhEF induces lipid and protein oxidation in a concentration-dependent manner, while its cytotoxicity is not only dependent on PEITC itself but also on its N-acetylated cysteine conjugated form.

The Predictive Value of 8-Hydroxy-Deoxyguanosine (8-OHdG) Serum Concentrations in Irradiated Non-Small Cell Lung Carcinoma (NSCLC) Patients.

Ionizing radiation is strongly linked to direct or indirect DNA damage, as with the production of reactive oxygen species (ROS), which in turn produce DNA damage products, such as 8-hydroxy-2-deoxyguanosine (8-OHdG). In this study, we aimed to investigate the formation of 8-OHdG after irradiation in patients with non-small cell cancer (NSCLC) and its use as a biomarker. Sixteen patients with squamous and thirty-six patients with non-squamous pathology were included. An enzyme-linked-immunosorbent assay (ELISA) was performed before and after radiation. A dose-dependent relationship was confirmed: 8-OHdG plasma concentrations, increased in the total of NSCLC patients and specifically with a linear correlation in non-squamous pathology; in squamous histology, after an initial increase, a significant decrease followed after 20 Gy dose of irradiation. The pretreatment total irradiated tumor volume (cm3) was positively correlated with 8-OHdG levels in patients with squamous histology. When plotting the 8-OHdG plasma concentration at a 10 Gy irradiation dose to the baseline, the AUC was 0.873 (95% CI 0.614-0.984), p < 0.0001, with an associated criterion value of >1378 as a cutoff (sensitivity 72.7%, specificity 100%). When normalizing this ratio to BSA, the associated criterion cutoff value was >708 (sensitivity of 100%, specificity 80%). Lastly, 8-OHdG levels were closely related with the development of radiation-induced toxicities.